A phase I study involving six patients with inoperable lung cancer and an endobronchial lesion accessible by bronchoscopy was initiated to evaluate the feasibility, tolerance, and clinical effects using adenoviral delivery of the Escherichia coli lacZ marker gene driven by the RSV promoter; biopsy specimens of the tumor and surrounding mucosa in 5 patients were tested positive for b-galactosidase expression (Tursz et al, 1996).

B. Transfer of the luciferase and green fluorescent protein (GFP) reporter genes

A synthetic polyamino derivative was used by Goldman and coworkers (1997) to transfer the luciferase and b-galactosidase reporter genes in animal models bearing stereotactically implanted human glioma cell xenografts. The luciferase reporter gene was transferred in both newborn and adult rabbit lungs using polyethylenimine (Ferrari et al, 1997).

Thierry and coworkers (1995) have succeeded in sustaining the expression of the luciferase reporter gene in mice for up to three months using episomal vectors (Table 2).

GFP has been used as a reporter molecule for gene expression because it fluoresces green after blue-light excitation. However, many attempts by Hanazono et al (1997) to isolate stable retroviral producer cell clones secreting vectors containing GFP (after transfer of the neoR gene and selection in G418) have failed because stable GFP-clones were undergoing major rearrangements or other mutations which abrogated GFP expression and prevented vector production.

Additional studies using reporter gene transfer are summarized on Table 2.

DIVISION TWO: GENE THERAPY OF CANCER

XV. Cancer immunotherapy and tumor vaccines

A. The molecular basis of cancer immunotherapy

Many human tumors are nonimmunogenic or weakly immunogenic. The immune system, evolved to rid the body of unwanted intruders, could be instructed and reinforced to eliminate cancer cells. Increasing the immunogenicity of tumors by causing local cytokine production or by increase in the expression in MHC antigen can lead to local antitumor effect (Tepper et al, 1989; Fearon et al, 1990). Indeed, immune surveillance is of the major defense mechanisms against cancer; immunosuppressed individuals are more prone to cancer and nude mice, lacking immune response, are exploited in the lab to elicit tumors after injection of tumorigenic cells, a response that, in many cases, cannot be elicited in normal mice.

Cells undergoing malignant transformation are believed to be eliminated from the body by white blood cells including natural killer cells (NK), lymphokine-activated killer cells (LAK), cytotoxic T lymphocytes (CTL), tumor-infiltrating lymphocytes (TIL), and activated macrophages; since established cancers in the human body may escape this potential defense mechanism of immunologic surveillance, cancer patients have been treated with IL-2 to stimulate their cellular immune mechanisms to kill cancer cells; lengthy and complete remissions, however, were at a low rate and complications were encountered by the toxicity caused by the systemic administration of IL-2 (Rosenberg, 1992).

Transfection of the IL-2 gene into human melanoma cells increased cellular immune response (Uchiyama et al, 1993). This and similar approaches have established the foundation of the ex vivo cancer immunotherapy by transfer of autologous (cancer patient’s) cells after transduction in vitro with cytokine genes (see below). The ultimate goal is the activation of tumour-specific T lymphocytes capable of rejecting tumour cells from patients.

B. Cancer immunotherapy with tumor infiltrating lymphocytes (TILs)

Ex vivo approaches in immunotherapy have been aimed at isolating T cells directly from tumors (known as tumor infiltrating lymphocytes or TILs), stimulate TILs to proliferate in cell culture with IL-2 followed by their reintroduction into the blood stream of advanced cancer patients (Rosenberg et al, 1988). The adoptive transfer of TILs was 50-100 times more potent than that of lymphokine-activated killer (LAK) cells isolated from the patient's tumors. Treatment of 20 patients with TILs after their expansion in vitro, plus IL-2, resulted in objective regression of metastatic melanomas in lungs, liver, bone, skin, and subcutaneous sites which lasted for several months (Rosenberg et al, 1988).

TILs were also transfected in vitro with the bacterial neomycin-resistance gene and were reintroduced into patients with advanced cancer in order to follow their persistence in blood circulation with PCR methods (Aebersold et al, 1990). Such “gene marking” clinical protocols using TILs are numbers 1, 3, 9. 13, 57, and 169 in Appendix 1, pages 159-172. Having shown safety in the NeoR-modified TIL protocol, the gene for tumor necrosis factor (TNF) was added to the vector for therapy of malignant melanoma in advanced cancer patients; the first patient began treatment in January 1991. TNF, however, is effective as an anticancer agent in mice at 400 mg/kg body weight, but in humans, TNF is toxic at 8 mg/Kg and so far of no proven therapeutic value at this low concentration (reviewed by Anderson, 1992). In a similar approach, the TNF gene was replaced by the gene of interleukin-2 (IL-2) in order to develop locally high doses of IL-2 at the tumor site by immunization with TIL cells from the patient producing systemic antitumor immunity (Rosenberg et al, 1992).

TNF-a, (also IL-1b, IFN-g, and vitamin D3) after binding to their transmembrane receptors stimulate the production of the second messager ceramide from sphingomyelin in the plasma membrane by activating sphingomyelinase; this results in a cascade of signal transduction events that result in down regulation of c-myc and induction of apoptosis, to terminal differentiation, or to RB-mediated cell cycle arrest (see apoptosis further below).

IL-2 stimulates the differentiation of precursor lymphocytes into LAK cells and further stimulates LAK cell proliferation; LAK cells are probably produced by activation of NK cells or from activated T cells by IL-2. Administration of IL-2 plus amplified LAK cells into mice models led to marked regression of disseminated cancers and leukemia. LAK cells are able to destroy tumor cells that express only weakly histocompatibility antigens. IL-2, however, has several pleiotropic effects: stimulation of B cell proliferation; activation of HLA class II antigen expression on endothelial cells, TILs, and melanoma cells; and enhanced production and release of TNF-a, and IFN-g (see Cassileth et al, 1995 and the references cited therein).

However, the use of large numbers of adoptively transferred, broadly cytotoxic LAK cells in combination with IL-2 has been effective for only small subsets of cancer patients (reviewed by Wiltrout et al, 1995).

TILs, which could potentially kill tumor cells, are found in many tumors but remain suppressed or anergic; this anergy may arise from the absence of lymphokines which provide signals for TIL cell activation and stimulation to proliferation although ligands may be bound to the variable region of the T cell receptor; indeed, nonimmunogenic tumors are rejected by syngeneic mice upon transfection by IL-2 or IL-4 genes; IL-2 lymphokine production by the tumor cells bypasses T helper function in the generation of an antitumor response rendering the tumor cells immunogenic; nontransfected tumors are not rejected by the animal and grow causing its death (Tepper et al, 1989; Fearon et al, 1990).

Ex vivo gene therapy trials using cytokine gene transfer (see below) circumvent the problem of toxicity of IL-2 administration; for non-gene transfer therapies, white blood cells drawn from patients are fractionated, cultured, stimulated with IL-2 or other cytokines, and reintroduced in much higher numbers into the blood of the patient.

C. Cancer immunotherapy with cytokine genes

The combination of immunotherapy with conventional treatments such as radio- and chemotherapy may be necessary to eradicate minimal residual disease. Advanced therapies involve the transfection of lymphocytes in culture with cytokine genes followed by selection of the successfully transfected cells with a selectable marker such as the bacterial neomycin-resistance gene (Cassileth et al, 1995). Numerous phase I clinical trials employing either syngeneic genetically modified or allogenic tumor vaccines are in progress (see immunotherapy in Appendix 1, page 159-172). The development of tumor cells transduced with cytokine genes and their exploitation as tumor vaccines in patients with cancer is a very promising field (reviewed by Jaffee and Pardoll, 1997).

Cytokine genes used for cancer immunotherapy include those of IL-2, IL-4, IL-7, IL-12, IFNs, GM-CSF, TNF-a in combination with genes encoding co-stimulatory molecules, such as B7-I. The major goal of the use of immunostimulatory cytokines is the activation of tumour-specific T lymphocytes capable of rejecting tumour cells from patients with low tumour burden or to protect patients from a recurrence of the disease. As distant metastasis is the major cause for therapeutic failures in clinical oncology, treatment of patients having a low tumor volume with immunotherapy could protect the patient from recurrence of disease. Treatment of rodent tumor models with little or no intrinsic immunogenicity with this approach resulted in regression of preexisting tumors and cure of the animals from their disease; furthermore, in some instances cured animals had retained immunological memory and resisted a second challenge with the parental tumor cells (reviewed by Gilboa, 1996; Mackensen et al, 1997).

The transduction of the tumor cells of the patient with cytokine genes ex vivo and the development of tumor vaccines depends on the establishment of primary cell culture from the solid tumor. Although malignant melanomas are easy to culture, it is difficult to establish cell lines from most other primary human tumors using convenient methods; primary tumor cultures are being used (i) for the transduction of autologous cells from the cancer patient with cytokine genes to develop cancer vaccines after intradermal implantation to the patient; (ii) for characterization of tumor-specific cytotoxic T lymphocytes in order to identify specific antigens on the human primary culture; (iii) for extensive phenotypic characterization of the tumor in cell culture. The Cre/LoxP system (see recombinases in gene therapy) has been used to facilitate the establishment of primary cell lines from human tumors (Li et al, 1997).

Human gene therapy protocols 3 and 10 (Appendix 1) use immunization of cancer patients with autologous cancer cells transduced with the gene for tumor necrosis factor (TNF).

D. Cancer immunotherapy with the IL-2 gene

Active immunization with pancreatic tumor cells genetically engineered to secrete IL-2 were shown to inhibit pancreatic tumor growth in vivo; this was shown using a poorly immunogenic subcutaneous model of murine ductal pancreatic cancer by implanting tumor Panc02 cells in C57BL/6 mice; whereas 90% of animals vaccinated with irradiated parental Panc02 and subsequently challenged with parental Panc02 cells developed tumors by 48 days only 40% of animals vaccinated with irradiated Panc02 cells engineered to secrete IL-2 and challenged with parental Panc02 cells developed tumors by 48 days (Clary et al, 1997).

According to a RAC-approved clinical protocol the gene for human interleukin-2 (IL-2) was transduced into a cell line established from the neoplastic cells of a patient with malignant melanoma; this procedure established an IL-2-secreting cell line with integration of the IL-2 gene into genomic DNA. The IL-2-secreting cells were irradiated, in a manner sufficient to inactivate 100% of the cells but insufficient to completely inhibit IL-2 synthesis, and administered to 12 patients with metastatic malignant melanoma in a Phase I toxicity study. These cells have the capacity to induce an antimelanoma response as shown in animal studies (Das Gupta et al, 1997).

A significant number of RAC-approved clinical protocols use IL-2 cDNA transfer. These include protocols 11, 16, 19, 20, 46, 48, 50, 61, 71, 102, and 135, in Appendix 1 and protocols 190, 197, 198, 200, 204, 211, 213, 215, and 219 using cationic lipids for gene transfer (Table 4 in Martin and Boulikas, 1998, this volume, pages 203-206).

E. Cancer immunotherapy with the IL-3 gene

IL-3 was found to enhance the development of cytotoxic T lymphocytes; during antitumor response, macrophages could ingest whole tumor cells, cell fragments, or heat shock proteins complexed to antigenic peptides and then process the tumor antigens for presentation; IL-3 stimulated antigen-presenting cells (APCs), which are macrophage-like, within the tumor leading to generation of cytotoxic T lymphocytes (CTLs). This constitutes a plausible pathway for enhancement in tumor rejection by IL-3 stimulation (Pulaski et al, 1996).

IL-3 signaling proceeding either via the JAK-STAT or the Ras-Raf pathways, stimulates a number of genes such as the DUB-1 encoding a deubiquitinating enzyme the overexpression of which leads to G1 arrest (Zhu et al, 1996); deubiquitination might be an additional mechanism to couple extracellular signaling to cell growth. IL-3 signaling leads to stimulation in myeloid cell proliferation.

F. Cancer immunotherapy with the IL-7 gene

Primary cell cultures from 45 patients with malignant melanoma were transfected via electroporation with the gene encoding for human interleukin-7 (IL-7) resulting in the production of biologically active IL-7 without altering the expression of HLA class I and II, ICAM-1, and of a melanoma-associated antigen. Irradiation of the transfected cells with 10,000 cGy, which arrested tumor cell growth in vitro, did not affect the ability of the cells to secrete IL-7 in the culture medium; this approach, which does not use retroviruses, could be applicable in vaccination protocols for melanoma patients (Finke et al, 1997).

Transfer of the IL-7 cDNA for cancer immunotherapy is being used in a human clinical trial (protocol 70, Appendix 1).

G. Cancer immunotherapy with the IL-12 gene

IL-12 gene therapy is one of the more novel and promising approaches in cancer therapy. IL-12 is a heterodimeric cytokine composed of two subunits, p40 and p35, that requires the simultaneous expression of both the p35 and p40 chain genes from the same cell for production of biologically active IL-12. Coordinate expression of the IL-12 p40 and p35 genes in several solid tumor models has been found to induce strong and specific antitumor immune responses. A variety of biological functions have been attributed to IL-12 including the induction of IFN-g and the promotion of predominantly Th1-type immune responses to antigens (Tahara et al, 1996).

The local secretion of IL-12 achieved by gene transduction suppressed tumor growth and promoted the acquisition of specific antitumor immunity in mice. This was shown by intradermal inoculation of mice with NIH3T3 cells transduced with expression plasmids or a retroviral vector expressing the murine IL-12 gene admixed with murine melanoma BL-6 cells; CD4⁺ and CD8⁺ T cells, as well as NK cells, were responsible for the observed antitumor effects resulting from IL-12 paracrine secretion. Transduction of tumor cells with B7.1 gene enhanced the antitumor immune response (Tahara et al, 1996).

The antitumor effect of several transgene expression plasmids encoding the cytokines IL-2, IL-4, IL-6, IL-12, IFN-g, TNF-a, and GM-CSF was tested using the gene gun-mediated DNA delivery into the epidermis overlying an established intradermal murine tumor; this study showed that IL-12 gene therapy was much more effective than treatment with any other tested cytokine gene for induction of tumor regression as determined from the increased CD8⁺ T cell-mediated cytolytic activity in the draining lymph nodes of tumor-bearing mice; treated animals were able to eradicate not only the treated but also the untreated solid tumors at distant sites; elevated systemic levels of IFN-g, were found after IL-12 gene therapy. This approach is providing a safer alternative to IL-12 protein therapy for clinical treatment of cancers (Rakhmilevich et al, 1997).

Lieu et al (1997) have evaluated three IL-12 retroviral vector designs for their level of IL-12 expression in leukemia/lymphoma cells; these retroviral vectors were based on the murine stem cell virus (MSCV) which efficiently transduces functional genes into normal hematopoietic cells. MSCVpac-mlL-12 and MIPV-mIL-12 contained an encephalomyocarditis virus internal ribosome entry site for internal translation of bicistronic mRNA transcripts, while MDCVpac-mIL-12 carried an expression cassette in the U3 region of the 3' LTR. The MSCVpac-mIL-12 vector was more efficient and directed robust expression of both p40 and p35 IL-12 genes in several murine tumor cell lines of hematopoietic origin, including a T-cell lymphoma, a B-cell lymphoma, and a plasmacytoma/myeloma.

Adenoviral delivery of the IL-12 gene was effective against breast tumors (Bramson et al, 1996) or metastatic colon carcinoma (Caruso et al, 1996) in animal models: mice bearing breast tumors, injected intratumorally with a single dose of an adenovirus expressing IL-12 showed regressions in greater than 75% of the treated tumors; this effect was accompanied with a maximum expression of IL-12 within the tumor between 24 and 72 hr post-injection which lasted for 9 days and an elevation in IFN-g within the tumor; local production of IL-12 also stimulated IFN-g production in tumor-draining lymph node cells (Bramson et al, 1996). Whereas intratumoral adenoviral transfer of the HSV-tk and the murine IL-2 genes resulted in substantial hepatic tumor regression, induced an effective systemic antitumoral immunity in the host and prolonged the median survival time of the treated animals from 22 to 35 days a recombinant adenovirus expressing the murine IL-12 gene was much more effective: intratumoral administration of the IL-12 vector alone increased significantly survival time of the animals; 25% of the treated animals lived over 70 days (Caruso et al, 1996).

The immunological host response to syngeneic murine mammary carcinoma cell line variants, genetically modified to express B7-1 or secrete GM-CSF and IL-12, was examined by Aruga et al (1997). The mammary adenocarcinoma MT-901 subline was weakly immunogenic by immunization/challenge experiments and induced tumor-specific T-cell responses in lymph nodes draining progressive subcutaneous tumors; however, tumor clones from this cell line expressing B7-1 or secreting GM-CSF exhibited reduced tumorigenicity and resulted in significantly enhanced T-cell reactivity to tumor-draining lymph node (TDLN) cells as compared to wild-type TDLN cells. In contrast, transduction with the IL-12 gene led to complete tumor growth inhibition.

An adenovirus vector, AdIL12-B7-1, encoding the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 of adenovirus under control of the murine CMV promoter was used to treat mice tumors derived from a transgenic mouse mammary adenocarcinoma. A single intratumoral injection with a low dose (2.5 x10⁷ pfu/mouse) mediated complete regression in 70% of treated animals whereas a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated (Putzer et al, 1997).

Human peripheral blood lymphocytes (HuPBLs), injected s.c. in mixture with human lung tumor cells into severe combined immunodeficient (SCID) mice, engrafted and displayed antitumor cytotoxic activity; this antitumor activity was dependent upon both CD8⁺ T cells and CD56⁺ natural killer cells in the donor HuPBLs. IL-12 enhanced the human peripheral blood lymphocyte-mediated tumor suppression; this implies that transfer of the IL-12 gene has a prospect in this type of immunotherapy. This could be significant under the light of studies showing that PBLs isolated from a lung cancer patient also suppressed the growth of the patient's (autologous) tumor when coinjected s.c. with the tumor cells into SCID mice (Iwanuma et al, 1997).

Tumor cell vaccines were transduced with IL-12 or IL-2 genes and the antitumor response induced in mice bearing lung metastases of the BALB/c colon carcinoma C51 were compared by Rodolfo et al (1996). The cells used for transduction with the IL-12 or IL-2 genes were the histologically related, and antigenically cross-reacting C26 tumor cells which were irradiated and injected s.c. Vaccination with C26/IL12 cells cured 40% of mice, while vaccination with C26/IL2 cells reduced the number of metastatic nodules without affecting survival; both cell vaccination regimens showed similar antitumor CTL activation in mice. Both treatments induced antibodies directed against tumor-associated antigens, but only sera from mice treated with C26/IL12 contained antibodies that lysed tumor cells. The better therapeutic efficacy of vaccination with C26/IL12 was found to be associated, among other factors, with an early infiltration of the metastatic lungs by activated T lymphocytes (Rodolfo et al, 1996).

Transfer of the IL-12 cDNA for cancer immunotherapy is being used in human clinical trials (protocols 62, 111, 180, and 183, Appendix 1).

H. Adoptive immunotherapy with GM-CSF

1. Cell culture experiments

The human hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), is important in the management and gene therapy of a variety of malignant disorders of the human hematopoietic system. Infection of COS-1 monkey kidney cells with a recombinant AAV vector containing the GM-CSF gene resulted in the release of recombinant GM-CSF protein into the supernatant; the released GM-CSF was able to sustain the active proliferation of the GM-CSF-dependent human megakaryocytic leukemia cell line, M07e, (Luo et al, 1995).

2. Animal studies

The Dunning rat R3327-MatLyLu prostate tumor model (an anaplastic androgen-dependent, nonimmunogenic tumor that metastasizes to the lymph nodes and the lung) has been used for GM-CSF therapy; IL-2- or GM-CSF-secreting human tumor cell preparations (tumor vaccines) were used for the treatment of advanced human prostate cancer in rats. All animals with subcutaneously established tumors were cured; the cancer vaccine induced immunological memory that protected the animals from subsequent tumor challenge; GM-CSF was less effective than IL-2 (Vieweg et al, 1994). Using the Dunning rat prostate carcinoma model, animals with hormone refractory prostate cancer treated with irradiated prostate cancer cells genetically engineered to secrete human GM-CSF showed longer disease-free survival compared to untreated control rats.

To further test the clinical feasibility of the prostate cancer cell vaccine, cancer cells from patients with stage T2 prostate cancer undergoing radical prostatectomy were successfully transduced with MFG-GM-CSF, achieving a significant human GM-CSF secretion in each of 10 consecutive cases (Sanda et al, 1994).

Continuous secretion of GM-CSF and activation of macrophages may contribute to the antitumor effects of a recombinant vaccinia virus expressing the gene for murine GM-CSF injected to solid melanoma tumors twice weekly for 3 weeks; this injection regimen resulted in growth inhibition of the subcutaneous tumor and enhanced the survival of the animals (Ju et al, 1997).

A recent effort has been toward potentiation of T-lymphocyte-mediated antitumor effects. T-lymphocyte response incapacitation in the murine renal cancer model could arise from an impairment of critical nuclear transcription factors. A vaccine-oriented gene therapy approach used T cells and antigen-presenting dendritic cells which were recruited through the use of antigen, chemokines and GM-CSF and further potentiated by fibroblasts expressing IL-2, IL-4, IL-7, or IL-12; the goal of this approach was to optimize MHC class I- and class II-dependent pathways for induction of T-lymphocyte-mediated responses to cancer in animal models (Wiltrout et al, 1995).

Chen et al (1996) found that adenoviral delivery of a combination of HSV-tk, mouse IL-2, and mouse GM-CSF is much more effective for the treatment of metastatic colon carcinoma in the mouse liver than HSV-tk alone or HSV-tk combined only with IL-2; a fraction of the animals developed long-term antitumor immunity and survived for more than 4 months without tumor recurrence in the three gene combination regimen; thus, local expression of GM-CSF in the hepatic tumors and prolonged IL-2 expression were necessary to generate persistent antitumor immunity.

A gene gun device was used to accelerate and introduce gold particles coated with GM-CSF cDNA plasmids into mouse and human tumor cells. Transfected and irradiated murine B16 melanoma cells produced about 100 ng/ml murine GM-CSF/million cells per 24 hr in vitro for at least 10 days. Toward development of a tumor vaccine, irradiated B16 tumor cells expressing murine GM-CSF cDNA were then injected into mice. Subsequent challenge of these mice with nonirradiated, nontransfected B16 tumor cells showed that 58% of the animals were protected from the tumor by the prior vaccine treatment compared to only 2% of control animals inoculated with irradiated B16 cells transfected with the luciferase gene (Mahvi et al, 1996).

Human tumor tissue transfected within 4 hr of surgery produced significant levels of transgenic human GM-CSF protein in vitro. Human GM-CSF was readily detectable in serum and at the injection site following subcutaneous implantation of these transfected tumor cells into nude mice (Mahvi et al, 1996).

The autocrine secretion of GM-CSF by transduced tumor cells was found to serve as an effective immune adjuvant in the host response to a weakly immunogenic murine mammary carcinoma tumor: transfer of activated lymph node cells derived from mice inoculated with GM-CSF-secreting (240 ng/million cells/24 hours) murine mammary carcinoma cells resulted in the prolonged survival of animals with macroscopic metastatic disease; this was not evident utilizing lymph node cells from mice inoculated with wild-type tumor (Aruga et al, 1997).

3. Clinical trials

Autologous cells (sensitized T cells) genetically-modified to secrete GM-CSF have been used for adoptive immunotherapy on humans. GM-CSF has been used for the treatment of advanced melanoma or renal cell cancers (Chang et al, 1996). The steps included retrieval of tumor from the patient for use as a vaccine; the tumor cell line was transduced with a retroviral/GM-CSF vector; cells were reintroduced into the patient (tumor vaccination). Removal of draining lymph nodes after 7-10 days and activation of lymph node cells with a monoclonal antibody directed against CD3 and expansion of the cell population with IL-2 gave anti-CD3⁺/IL2-activated cells which were exquisitely tumor-specific and mediated the regression of established tumors in animal models (Figure 18).

According to a phase I clinical trial cancer patients are intradermally vaccinated with lethally-irradiated tumor cells that have been transfected by particle-mediated gene transfer with gold particles coated with human GM-CSF plasmid DNA; this is based on preclinical studies showing that vaccination of mice with irradiated, GM-CSF-transfected melanoma cells provided protection from subsequent challenges with non-irradiated, non-transfected tumor cells. Human tumor immunotherapy studies in course use patients' fresh specimens of melanoma or renal carcinoma; cells are dissociated, lethally-irradiated and transfected with GM-CSF plasmid DNA-coated gold particles resulting in the subsequent production of biologically active GM-CSF protein by the patient’s cells. Patient’s cells are used intradermally as a vaccine to elicit anti-tumor immune responses. Surgical excision of the vaccination sites will assess GM-CSF production and infiltration of immune effector cells; patients are being subjected to an intradermal injection in their opposite extremity of 5 million irradiated cryopreserved tumor cells taken from the patient at the time of vaccine preparation to asses immune reactions (DTH testing); if a positive reaction is noted on day 28 the DTH site will be surgically removed (Mahvi et al, 1997).

Figure 18. A clinical protocol for adoptive immunotherapy of advanced melanoma patients. Adapted from Chang et al (1996).

A number of RAC-approved human gene therapy protocols use GM-CSF cDNA transfer. These are protocols 35, 53, 63, 113, 149, 150, 162, and 181 in Appendix 1.

I. Cancer immunotherapy with the IFN-g gene

Solid tumors in nude mice have been successfully eradicated with treatment with tumor cell lines stably transfected with an IFN gene. A number of human tumor cell lines including 293, HeLa, K562, and Eskol (a malignant immunoblastic lymphoma) were infected with a rAAV carrying a synthetic type I interferon gene and the bacterial neomycin-resistant gene and geneticin-resistant cells were selected; when injected into nude mice, 293, K562, and Eskol cells failed to form tumors for a duration of up to 3 months; on the contrary, mice receiving nontransduced cells developed tumors within 7 to 10 days; in addition, treatment of an established Eskol tumor with transduced 293 cells resulted in tumor regression (Zhang et al, 1996).

Three RAC-approved human gene therapy protocols use IFN-g cDNA transfer. These are protocols 36, 54, and

71 in Appendix 1.

J. Immunotherapy with synthetic tumor peptide vaccines

Progress in the identification of tumor-specific antigens, that is proteins expressed at high levels by a specific tumor cell type such as prostate or breast cancer, most of which are surface glycoproteins easily recognizable by the immune system, as well as the deciphering of the mechanisms for enhancing the response of cytotoxic T cell lymphocytes have advanced the potential for developing cancer vaccines.

Cancer immunotherapies based on synthetic tumor peptide vaccines have been developed. Tumor-specific CD8⁺ cytotoxic T lymphocytes (CTLs) recognize short peptide epitopes presented by MHC class I molecules that are expressed on the surface of cancer cells. Bone marrow-derived dendritic cells, grown in vitro in media containing combinations of GM-CSF + IL-4, when pulsed with synthetic tumor peptides (which are loaded on the surface of the dendritic cells) became potent antigen-presenting cells (APCs) capable of generating a protective antitumor immune response. Injection of these cells into naive mice protected the mice against a subsequent lethal tumor challenge; in addition, treatment of mice bearing C3 sarcoma or 3LL lung carcinoma tumors with the same type of cells resulted in sustained tumor regression in over 80% of the animals (Mayordomo et al, 1995).

One of the obstacles of this method has been the difficulty in obtaining sufficient numbers of APCs; dendritic APCs have been isolated from CD34⁺ hematopoietic progenitor cells drawn from cord blood and expanded in cell culture in the presence of GM-CSF and TNF-a; TNF-a inhibits the differentiation of dendritic cells into granulocytes. Human peripheral blood mononuclear cells or mouse bone marrow cells depleted of lymphocytes could also yield dendritic cells when cultured in the presence of GM-CSF + IL-4 (Mayordomo et al, 1995).

K. DNA vaccines

Vaccines may be one of the first successful applications of foreign genes into mammalian cells under control of heterologous promoters and enhancers (Felgner and Rhodes, 1991; Thompson, 1992; Gilboa and Smith, 1994). Vaccination with DNA has been shown to be a promising approach for immunization against a variety of infectious diseases (Wang et al, 1993; Michel et al, 1995; Huygen et al, 1996; Kuhober et al, 1996). The method consists in introducing the gene of a viral or bacterial antigen which is uptaken and expressed by the host’s cells to elicit an antigen-specific immune response. DNA coding for an antigen can be directly injected into muscle or skin and stimulate an immune response against the expressed antigen; the gene can either code for surface molecules, which are often used for conventional peptide vaccines, or from internal microbial proteins.

During this approach the antigens are produced intracellularly where they are correctly folded and can be presented to the immune system to stimulate cytotoxic T cells; the method is safe and simple and has shown promising results on animals (reviewed by Moelling, 1997). For example, mice injected intramuscularly with an HIV-1 envelope DNA construct developed anti-HIV envelope immune responses (Wang et al, 1993); intramuscular injection of plasmid DNA expression vectors encoding the three envelope proteins of the hepatitis B virus (HBV) induced humoral responses in C57BL/6 mice specific to several antigenic determinants of the viral envelope (Michel et al, 1995). Immunization of mice with plasmid DNA constructs encoding one of the secreted components of Mycobacterium tuberculosis, antigen 85 gene induced substantial humoral and cell-mediated immune responses (Huygen et al, 1996).

Because immunization of cancer patients with tumor antigen proteins is a very promising approach used extensively in cancer therapy (e.g. Karanikas et al, 1997) many of these approaches could be transferred to the DNA level using the gene encoding the tumor antigen.

As an extension, this method could find application using human tumor antigen genes rather than bacterial/viral antigen genes, that is genes encoding for proteins expressed in tumor but not in normal cells leading to development of tumor vaccines (Graham et al, 1996; Okamoto et al, 1997); this method mimics the infection of the cell in the host by a pathogenic virus resulting in the intracellular processing of the viral proteins and their presentation on the cell surface. Human tumor antigens are, however, weakly immunogenic compared to microbial antigens a problem connected with polymorphism in the major histocompatibility complex proteins of the host and in antigen presentation.

Development of a fusigenic viral liposome vector was made possible using the HVJ (hemagglutinating virus of Japan, a Sendai virus) renowned for its cell fusion ability; plasmid DNA containing the human tumor antigen genes MAGE-1 and MAGE-3 was mixed with HMG-1 nonhistone protein (to increase nuclear import and expression of the plasmid after transfection) and was encapsulated into anionic liposomes (phosphatidylserine, phosphatidylcholine, cholesterol) followed by the addition of inactivated HVJ; intramuscular injection into mice resulted in production of MAGE-1 and -3 IgG antibodies (Okamoto et al, 1997).

XVI. Gene therapy of cancer and candidate genes

A. Mechanisms of carcinogenesis

Whereas for inborn errors of metabolism transfer of a single gene can correct the disorder, cancer is a complex disease involving mutations in a number of proto-oncogenes and tumor suppressor genes as well as an imbalance and disarray in phosphorylation events and regulatory circuits of the cell cycle. As a result of transformation, tumor cells acquire a proliferation advantage compared with normal cells, most of which are quiescent in the adult organism; cancer cells acquire partial independence from regulatory signals from neighboring cells for restricted cell growth. A crucial step in cancer development is the nonelimination of pre-cancer cells by apoptosis (usually a subsequence of a mutation in the p53 gene); such cells acquire a number of unrepaired damage in their DNA, such as strand breaks, which induce chromosomal translocations and result in clonal expansion of this cell population.

Tumor cells are able to survive after DNA damage, and display an increase in mutation rate; cancer cell populations are heterogenous with respect to translocations, loss of heterozygosity, point mutations and transpositions in various genes. Whenever the mutated cell acquires an advantage for rapid growth over other cells in the tumor mass, escaping cell cycle checkpoints, it may replace the original population, a phenomenon known as tumor progression; this may lead to appearance of a more malignant phenotype. As a result, tumor cells are of different genotypes and clones obtained from the same solid tumor may differ in the level of malignancy.

A number of candidate genes, when become mutated or overexpressed, may lead to tumor phenotype: p53, RB, and p21 appear to be the most important. The deregulation of other genes is connected to tumor progression whereas different groups of genes are associated with tumor cell metastasis. These facts make a single gene transfer approach to tumor cell mass to inhibit its growth or change its phenotype from malignant to normal very challenging.

B. Human clinical trials

The genes used for cancer gene therapy in human clinical trials include a number of tumor suppressor genes (p53, RB, BRCA1, E1A), antisense oncogenes (antisense c-fos, c-myc, K-ras), suicide genes (HSV-tk, in combination with ganciclovir, cytosine deaminase in combination with 5-fluorocytosine) which have been very effective in eradicating solid tumors in animals. Also the cytokine genes (IL-2, IL-7, IFN-g, GM-CSF) are being used for the ex vivo treatment of cancer cells isolated from human patients and are able to elicit an immunologic regression especially on immunoresponsive malignancies (melanomas, colorectal carcinomas, renal cell carcinomas) (Culver, 1996). Future directions might be toward use of genes involved in the control of tumor progression and metastasis. Discovery of new genes which are over- or under-expressed during transformation and metastasis is a promising approach for the identification of novel gene targets in cancer gene therapy (Georgiev et al, 1998, this volume).

Diseases amenable to therapy with gene transfer in clinical trials (Appendix 1 and Table 4 in Martin and Boulikas, this volume) include cancer (melanoma, breast, lymphoma, head and neck, ovarian, colon, prostate, brain, chronic myelogenous leukemia, non-small cell lung, lung adenocarcinoma, colorectal, neuroblastoma, glioma, glioblastoma, astrocytoma, and others), AIDS, cystic fibrosis, adenosine deaminase deficiency, cardiovascular diseases (restenosis, familial hypercholesterolemia, peripheral artery disease), Gaucher disease, Hunter syndrome, chronic granulomatous disease, PNP deficiency, a1-antitrypsin deficiency, leukocyte adherence deficiency, partial ornithine transcarbamylase deficiency, Cubital Tunnel syndrome, Canavan disease and rheumatoid arthritis. Several RAC-approved protocols use gene marking rather than gene therapy . An important number of protocols in cancer use ex vivo immunotherapy (Appendix 1, pages 159-172 & 203-206).

XVII. Gene therapy strategies based on p53

A. p53 as a tumor suppressor protein

The p53 has been a fascinating subject in cancer biology since its discovery (Lane and Crawford, 1979; Linzer and Levine, 1979). Originally assigned in the constellation of oncogenes was later shown to exert suppressive effects on cell growth (Finlay et al, 1989); indeed, the mutated p53 has many characteristics of an oncogene (Will and Deppert, 1998, this volume). Mutations in the p53 gene contribute to the emergence of the malignant phenotype (Diller et al., 1990; Baker et al., 1990). Alterations in the p53 tumor suppressor gene appear to be involved, directly or indirectly, in the majority of human malignancies (Vogelstein, 1990). For example, human lung cancer cell lines and specimens showed allelic loss for chromosome regions 3p and 17p (p53 is assigned to 17p13); these specimens displayed homozygous deletions of p53, DNA rearrangements involving the p53 gene, or expression of truncated p53 transcripts suggesting abnormal splicing, initiation, and termination arising from point or other mutations (Takahashi et al, 1989; Nigro et al, 1989).

An interesting approach to unravel the molecular mechanism of action of p53 in restricting cell growth and in inducing apoptosis was the cloning of genes induced by p53 before the onset of apoptosis; this led to the identification of a group of 14 genes (out of 7,202 transcripts examined) which were markedly increased in p53-expressing cells compared with control cells many of which were predicted to encode proteins that could generate oxidative stress or respond to oxidative stress (Polyak et al, 1997). Additional studies in this line have suggested that the induction of the apoptotic pathway by p53 involves (i) transcriptional induction of redox-related genes; (ii) formation of reactive oxygen species; and (iii) the oxidative degradation of mitochondrial components (Polyak et al, 1997).

p53 can inhibit transformation of rat embryo fibroblasts mediated by adenovirus E1A plus activated ras and can also suppress focus formation mediated by myc plus activated ras (Finlay et al, 1989; Eliyahu et al, 1989). Both alleles of p53 need to be mutated or altered for transformation. Introduction of a null mutation by homologous recombination in murine embryonic stem cells gave mice which appeared normal but were susceptible to a variety of neoplasms by 6 months of age (Donehower et al, 1992).

The tumor suppressive activity of p53 seems to involve at least six independent pathways: (i) induction by p53 of the p21/Waf-1/Cip-1 gene which causes growth arrest both via inhibition of cyclin-dependent kinases and via inactivation of PCNA; PCNA is the accessory molecule to DNA polymerases a and d and its absence causes arrest of DNA synthesis at the replication fork; (ii) induction of the death-promoting bax gene by p53 as a mechanism which eliminates oncogenic virus-infected and transformed cells; (iii) by a direct interaction of p53 with origins or replication preventing firing and initiation of DNA replication; (iv) via binding of p53 to a number of important molecules involved in transcription (TATA box-binding protein or TBP, TFIIH); (v) by the role of p53 in DNA repair via its patrolling the genome for small insertion deletion mismatches or free ends of DNA; (vi) p53 is able to attract RPA, an accessory to DNA polymerases a and d as well as TFIIH and RAD51 at the damaged DNA sites; TFIIH, RAD51, and RPA have a demonstrated role in DNA repair (Figure 19). Additional properties of p53 include the induction of Gadd45 involved in the arrest of the cell cycle and induction of Mdm2 which, after exceeding a threshold value in the cell associates with p53 to restrict its regulatory functions; thus, Mdm2 acts as a feedback loop for p53 to moderate its apoptotic and cell cycle restrictive functions (Figure 20).

B. Genes regulated by wild-type p53

Protein p53 appears to be a transcription factor able to recognize specific regulatory regions in a number of genes via its central DNA-binding domain; the DNA sequence-specific binding of wt p53 is regulated by the C-terminal domain of p53 and is activated by a variety of posttranslational modifications (Hupp et al, 1992). p53 is phosphorylated and is constitutively expressed at low levels in most normal tissues (Lane and Crawford, 1979; Linzer and Levine, 1979).

The sequence specificity of p53 has been determined using random synthetic oligonucleotides followed by selection by wtp53 and cloning; these studies revealed the 10 bp motif RRRCGYYY (where R is purine and Y pyrimidine) as the binding and recognition site of wtp53 recognition (El-Deiry et al., 1992); two such 10 bp motifs are required for p53 binding separated by up to 13 bp of random sequence. Since the 10 bp motif is a palindrome, the binding site of p53 comprises 4 copies of the half binding sites GYYY oriented in opposite directions, which suggested that p53 binds either as a dimer to two cruciforms or as a tetramer with each subunit interacting with one half site. The second possibility is favored since

the absence of transfection of these cells, a phenomenon known as "bystander effect".

Trigger genes in plasmids were made up of the DNA-binding domain of GAL4 (aa 1-147) fused in frame to a protein domain that could interact with p53; the p53-binding domain was the 84-708 aa region of SV40 T antigen or of the 305-393 aa TAD domain of p53 (which acts as a tetramer) and which is similar between wt and mutant p53 (mutations on p53 are within the DBD). The constructs included the E. coli (DeoD) gene which encodes the purine nucleoside phosphorylase (PNP) under control of the GAL4 response element (known as upstream activating sequence or UAS); the PNP gene can convert the 6-methylpurine deoxyribose (MeP-dR) prodrug into the diffusible, toxic 6-methylpurine (see page 69) and can become a powerful suicide gene under these conditions (Sorscher et al, 1994). Transfection of cells in culture with these constructs followed by treatment of the cells with MeP-dR resulted in the death of the cells (da Costa et al, 1996). The mechanism was based on the fact that most normal cells do not express the p53 gene and those who do express the wt p53 are destined for programmed cell death; therefore, cancer cells containing elevated amounts of mutant forms of p53 were amenable to this strategy.

The 55- kDa E1B protein of adenovirus binds to and inactivates the p53 gene; ONYX-015 is an E1B, 55-kDa gene-attenuated adenovirus unable to replicate and show cytopathogenicity in tumor cell lines which express a wt p53 such as in RKO and U20S carcinoma lines but can cause cytolysis in cell lines expressing a mutated form of p53; a wide range of human tumor cells, including numerous carcinoma lines with either mutant or normal p53 gene sequences (exons 5-9), were efficiently destroyed following intratumoral or intravenous administration of ONYX-015 to nude mouse-human tumor xenografts; furthermore, combination therapy with ONYX-015 plus chemotherapy (cisplatin, 5-fluorouracil) was significantly greater than with either agent alone. On the contrary, normal human cells were highly resistant to cytolysis by the adenovirus (Heise et al, 1997).

J. Transfer of the p53 gene in cell culture

Preclinical studies have shown that both viral and plasmid vectors able to mediate high efficiency delivery and expression of wild-type tumor suppressor p53 gene can cause regression in established human tumors, prevent the growth of human cancer cells in culture, or render malignant cells from human biopsies non-tumorigenic in nude mice. Inhibition in cell proliferation was observed in cell culture and in tumors after induction of p53 expression with adenovirus vectors (Bacchetti and Graham, 1993; Wills et al, 1994; Zhang et al, 1994).

Transfer of the wild-type p53 gene using a defective HSV vector into a human medulloblastoma cell line containing a mutant copy of p53 resulted in p53 expression, increased the levels of mdm2 proteins and induced cell cycle arrest of the majority of transduced cells (Rosenfeld et al, 1995).

Apoptosis can be induced in cultured NCI-H 596 human non-small cell lung cancer cells, which have a wild-type p53 gene, by EGF signaling in a p53-dependent manner; whereas treatment of these cells with EGF plus p53 sense oligonucleotides induced EGF-dependent and p53-dependent apoptosis within 8 hours, antisense p53 gene therapy suppressed the induction of apoptosis. A new nucleic acid drug was developed based on a mutated p53 antisense with a mutation at three bases immediately 5' and 3' from the CG dinucleotides which potentiated the induction of apoptosis and failed to suppress the induction of EGF-dependent apoptosis (Murayama and Horiuchi, 1997).

Infection of the androgen-independent human prostate Tsu-pr1 cell line lacking functional p53 alleles with recombinant adenovirus vectors (replication-deficient) carrying the p53 gene under control of the CMV promoter resulted in expression of p53 and induced striking morphological changes: the cells were detached from the substratum, condensed, and exhibited breakdown of the nuclear DNA into nucleosome-size fragments characteristic of apoptosis; whereas control cells were able to elicit tumors in nude mice, the AdCMV/p53-infected cells failed to form tumors (Yang et al, 1995).

K. Animal studies using p53 gene transfer

Intratracheal injection of a recombinant retrovirus containing the wt p53 gene was shown to inhibit the growth of lung tumors in mice nu/nu models inoculated intratracheally with human lung cancer H226Br cells whose p53 gene has a homozygous mutation at codon 254 (Fujiwara et al, 1994). A number of other studies have shown suppression in tumor cell growth and metastasis after delivery and expression of the wt p53 gene (Diller et al, 1990; Chen et al, 1991; Isaacs et al, 1991; Wang et al, 1993).

The safety of the adenovirus-mediated p53 transduction of the liver in normal rats and in 50%-hepatectomized animals subjected to asanguineous portal perfusion was examined by Drazan et al (1994). The gene transfer rate in whole liver and after hepatectomy ranged from 20% to 40%; liver regeneration and hepatocyte function were unaffected by overexpression of p53.

Delivery of the p53 gene to malignant human breast cancer cells in nude mice using DOTMA:DOPE 1:1 cationic liposomes (400 nmoles liposomes/35 mg DNA) resulted in regression (60% reduction in tumor cell volume) in 8 out of 15 animals treated; animals were receiving one injection every 10 days (Lesoon-Wood et al, 1995). It was thought that wild-type p53 expression (tumor cells were expressing mutant forms of p53) upregulated p21 gene to inhibit cell growth by inhibition in cyclin-dependent kinases but also via induction of apoptosis preferentially in cancer cells.

When a recombinant adenovirus encoding wild-type p53 under the control of the human CMV promoter was introduced into SK-OV-3 human ovarian carcinoma cells it increased by more than 50% the life span of nude mice injected with these cells; control animals in this highly aggressive ovarian xenograft model died between 25-45 days from injection time (Mujoo et al, 1996). Adenoviral transfer of a functional p53 gene into a radiation-resistant SCCHN cell line that harbors mutant p53 restored the G1 block and apoptosis in these cells in vitro and sensitized SCCHN-induced mouse xenografts to radiotherapy in vivo (Chang et al, 1997).

The efficacy of a replication-deficient p53 adenovirus construct was tested against three human breast cancer cell lines expressing mutant p53, MDA-MB-231, -468, and -435 and was found to be highly effective against 231 and 468 cells as well as their tumor xenografts in nude mice but not against 435 cells probably due to their low adenovirus transduction. 37% of growth inhibition of 231 cells was due to p53, while 49% was adenovirus-specific (Nielsen et al, 1997).

Cytotoxic T lymphocytes (CTLs) recognizing a murine wild-type p53 were able to discriminate between p53-overexpressing tumor cells and normal tissue and caused complete and permanent tumor eradication without damage to normal tissue after adoptive transfer into tumor-bearing p53^+/+ nude mice. CTLs, presented by the MHC class I molecule H-2Kb, were generated by immunizing p53 gene deficient (p53^-/-) C57BL/6 mice with syngeneic p53-overexpressing tumor cells (Vierboom et al, 1997).

L. Transfer of the p53 tumor suppressor gene to prostate cancer cells

Although primary prostate tumors have few mutations in the p53 gene (Voeller et al, 1994; Isaacs et al, 1994), specimens from advanced stages of the disease and metastases as well as their cell lines frequently display mutations or deletions at both alleles of the p53 gene (Chi et al, 1994; Dinjens et al, 1994). Three of five prostate cancer cell lines examined (TSUPr-1, PC3, DU145) and one out of two primary prostate cancer specimens were found to harbor mutations altering the amino acid sequence of the conserved exons 5-8 of the p53 gene; transduction of the p53-defective cell lines with the wt p53 gene using lipofectin showed reduction in tumorigenicity assayed from reduced colony formation and the cells became growth arrested (Isaacs et al, 1991).

Endocrine therapy is ineffective once the prostate cancer becomes androgen-independent; these cancers remain unresponsive to conventional chemotherapy. Androgen-independent and metastatic prostate cancers were established in athymic male mice by co-inoculation with the LNCaP human prostate cancer cell line and the MS human bone stromal cell line; these tumors became necrotic and were successfully eradicated by intratumoral injection of a recombinant p53/adenovirus; the p53 gene was driven by the CMV promoter and the SV40 poly(A) signal placed in the E1 region of Ad5 (Ko et al, 1996). It was suggested that in addition to the tumor suppressor, apoptotic, and antiangiogenesis function of p53, tumor necrosis was induced by a bystander effect or a general immune response which attracted immune cells to cause tumor cell killing (Ko et al, 1996).

M. Clinical trials using p53 gene transfer

A human clinical trial at M.D. Anderson Cancer Center uses transfer of the wild-type p53 gene, in patients suffering with non-small cell lung cancer and shown to have p53 mutations in their tumors, using local injection of an Ad5/CMV/p53 recombinant adenovirus at the site of tumor in combination with cisplatin (Roth, 1996; Roth et al, 1996; protocols #29 and 124, Appendix 1). A retroviral vector containing the wild-type p53 gene under control of a b-actin promoter was used for multiple percutaneous injections or direct thoracoscopic injections at the site of the tumor into nine patients, all in advanced stages, with non-small cell lung cancers. Patients whose conventional treatments failed were selected for a p53 mutation in the lung tumor. Reduction in tumor volume was achieved via apoptosis (assayed in posttreatment biopsies) in three patients, and arrest in tumor growth in three other patients (Roth et al, 1996).

RAC-approved clinical trials (Appendix 1) using p53 cDNA transfer are #29 (treatment of non-small cell lung cancer with p53 and antisense K-ras), #124 (intratumoral delivery of adenoviral p53 cDNA plus cisplatin), #130 (intratumoral injection of adenoviral p53 to treat head and neck squamous cell carcinoma), #131 (primary and metastatic malignant tumors of the liver), #147 (percutaneous injections of adenovirus p53 for hepatocellular carcinoma), #148 (advanced or recurrent adenocarcinoma of the prostate), #152 (intra-tumoral injections of ad5cmv-p53 to patients with recurrent squamous cell carcinoma of the head and neck), #153 (intralesional delivery of adenovirus p53 in combination with chemotherapy in breast cancer), #154 (intratumoral injection of adeno p53 to patients with advanced prostate cancer), #155 (intratumoral injection of adeno p53 to patients with advanced and metastatic bladder cancer), and #156 (adeno p53 for non-small cell lung cancer).

XVIII. p21 and p16 in cancer gene therapy

A. Molecular action of p21

p53 upregulates the p21/CIP1/WAF1 gene (simply called p21) (ElDeiry et al, 1993). Induction of the p21/Waf-1/Cip-1 gene causes growth arrest via inhibition of cyclin-dependent kinases (CDKs). CDKs are upregulated by cyclins which act as positive regulators of cell cycle progression. Cdk2, also called p33^cdk2, is the master regulator of the cell cycle at the G1/S transition point. Whereas cdk2 is expressed at constant levels throughout the cell cycle, its activation by phosphorylation is first detected a few hours before the onset of DNA synthesis; furthermore, antibodies directed against Cdk2 blocked mammalian cells from entering S phase. D1 cyclin associates with Cdk2, Cdk4, and Cdk5 to control the G1®S transition point; the genes of cyclins D1 and E are overexpressed or rearranged in malignancies and conditional overexpression of human cyclins D1 and E in Rat-1 fibroblasts causes a decrease in the length of G1 and an acceleration of the G1/S phase transition. D1 appears to be specialized in the emergence of cells from quiescence (Go®G1 transition) whereas cyclin E is more oriented toward control of the G1/S transition. Cdc2, a close relative of Cdk2 and whose pattern of phosphorylation is cell cycle-regulated, becomes associated with cyclin B to regulate the G2®M transition (see Boulikas, 1995a).

CDK activity is essential for the phosphorylation of RB at the G1/S checkpoint of the cell cycle resulting in the release of E2F transcription factor from RB-E2F complexes and in the up-regulation of genes required for DNA synthesis by the released E2F. p21 levels are reduced considerably in tumor cells that have lost the p53 protein or contain a nonfunctional mutated form of p53 (El-Deiry et al, 1993).

Induction of the p21/Waf-1/Cip-1 gene also causes growth arrest via inactivation of PCNA; indeed, the p21 inhibitor of cyclin-dependent kinases associates with PCNA, the accessory of DNA polymerases a and d , thus blocking its ability to activate these DNA polymerases; this could give rise to the abnormal control in DNA replication or to the loss of coordination between DNA replication and cell cycle progression seen in tumor cells (Li et al, 1994).

B. p21 and p16 gene transfer

Introduction of the wt p53 or of the p21 downstream mediator of p53-induced growth suppression into a mouse prostate cancer cell line, deficient in p53, led to an association of p21 with Cdk2; this interaction was sufficient to downregulate Cdk2 by 65% (Eastham et al, 1995). The p21 gene, driven by CMV promoter into an Adenovirus 5 vector, was more effective than the AD5CMV-p53 vector, (harboring the p53 gene under control of the same elements as p21), in reducing tumor volume in syngeneic male mice with established s.c. prostate tumors; tumors were induced by injection of 2 million cells in each animal. These studies suggested that p21 expression might have more potent growth suppressive effect than p53 in this tumor model and that p21 may be seriously be included in the constellation of anticancer arsenals.

Transfer of p21 is an effective tool to lead carcinoma cells with inactivated p53 into less malignant phenotypes. p53 is frequently inactivated by papilloma viruses in carcinomas of the uterine cervix. Transfer of the p21 gene to HeLa cells, a widely used uterine cervix cell line, resulted in a significant growth retardation by blockage of G1 to S transition, reduced anchorage-independent growth and attenuated telomerase activity (Yokoyama et al, 1997). Introduction of p21 with adenoviral vectors into malignant cells completely suppressed their growth in vivo and also reduced the growth of established pre-existing tumours (Yang et al, 1997).

Transfer of p21 was used to suppress neointimal formation in the balloon-injured porcine or rat carotid arteries in vivo (Yang et al, 1996; Ueno H et al, 1997a). A combination therapy in mice with simultaneous transfer of the p21 gene and of the murine MHC class I H-2Kb gene, which induces an immune response that stimulates tumor regression, was more effective than treatment with either gene alone (Ohno et al, 1997).

Malignant gliomas extensively infiltrate the surrounding normal brain and their diffuse invasion is one of the most important barriers to successful therapy; one of the most frequent abnormalities in the progression of gliomas is the inactivation of the tumor-suppressor gene p16, suggesting that loss of p16 is associated with acquisition of malignant characteristics. Restoring wild-type p16 activity into p16-null malignant glioma cells modified their phenotype. Adenoviral transfer of the p16/CDKN2 cDNA in p16-null SNB19 glioma cells significantly reduced invasion into fetal rat-brain aggregates and reduced expression of matrix metalloproteinase-2 (MMP-2), an enzyme involved in tumor-cell invasion (Chintala et al, 1997).

XIX. Gene therapies based on transfer of the retinoblastoma (RB) gene

A. RB and E2F proteins in the control of the cell cycle and apoptosis

Retinoblastoma protein is a transcription factor (Lee et al, 1987) involved in the regulation of cell cycle progression genes (reviewed by White, 1998, this volume). The role of RB on cell proliferation and tumor suppression arises (i) from its association with E2F, an association disrupted by RB phosphorylation at the G1/S checkpoint resulting in release of E2F and in the upregulation of a number of genes required for DNA replication; (ii) from the direct association of RB protein with a number of viral oncoproteins or key regulatory proteins including E1A of adenovirus (Whyte et al., 1988), SV40 large T (Ludlow et al., 1990) and the human papilloma virus E7 protein (Dyson et al., 1989). Normal cellular targets of RB, such as the transcription factor E2F (Bagchi et al., 1991; Chellapan et al., 1991) become dissociated from the RB protein in the presence of these viral proteins in the cell (E1A, T antigen, E7), leading to cell cycle progression. This constitutes a mechanism (also the interaction of viral proteins with p53, see above) viruses use to render infected cells continuously cycling.

(iii) RB is able to repress directly c-fos gene expression (Robbins et al., 1990) and has been proposed to have a similar effect on c-myc expression (Pietenpol et al., 1990). (iv) RB also suppresses cell growth by directly repressing transcription of the rRNA and tRNA genes by blocking the activity of RNA polymerase I transcription factor UBF (Cavanaugh et al, 1995; reviewed by White, 1998).

Hypophosphorylated RB, but not mutant RB, was associated with the nuclear matrix, particularly concentrated at the nuclear periphery and in nucleolar remnants, only during early G1; the peripheral matrix proteins lamin A and C bound RB in vitro. This association was thought to be important for the ability of RB to regulate cell cycle progression (Mancini et al, 1994). It is interesting that mutated p53 but not wtp53 interacts with specific types of MARs (Will et al, 1998); nuclear matrix is an essential structure for replication transcription recombination and repair processes intimately connected to mechanisms of carcinogenesis.

The tumor suppressor function of RB is believed to occur by complex formation between E2F and RB or the RB-related proteins p107 and p130, a complex that down-regulates the DNA-binding activities of E2F; the transcription activating capacity of E2F on the genes it regulates can be repressed by interaction with RB (Nevins, 1992). Cyclin A, believed to facilitate DNA replication, also associates with E2F; both types of complexes, E2F-RB and E2F-cyclin A, can be dissociated by the adenovirus E1A protein. The release of E2F by E1A results in cell cycling and this constitutes an additional mechanism of interference of adenoviruses with the proliferation of the infected cells; release of E2F from RB induced by E1A is critical for transformation of cells by E1A (for references see Hiebert et al, 1995).

The p107 protein with similarities in structure and DNA-binding properties to RB also binds cyclin A; whereas RB is complexed to E2F during G1 the p107-cyclin A complex interacted with E2F as cells entered S phase (Shirodkar et al., 1992).

E2F is a transcription factor that activates the adenovirus E2 gene and a number of cellular genes that respond to proliferation signals and that control the passage of the cell cycle through S phase such as myc and DHFR genes and contributes to the uncontrolled proliferation of adenovirus-transformed cells (Mudryj et al., 1991; see White, 1998, this volume). It has been speculated that the physiological function of RB (and also of its similar protein p107) in negatively-regulating cell growth and in acting as a tumor suppressor protein are exerted via its ability to down-regulate the activity of E2F (Shirodkar et al., 1992); this has been subsequently confirmed by numerous studies. RNA ligands that bind to E2F1 were selected from RNA libraries and were used to inhibit the induction of S phase in cultured cells (Ishizaki et al, 1996). Such molecules might find applications in cancer therapy because of the important role of E2F proteins in the regulation of cell cycling.

Retinoblastoma protein has a functional domain (the pocket) for binding to transcription factor E2F implicated in cell growth control. The same domain is responsible for the association of RB with the adenovirus E1A, the SV40 large T, and the human papilloma virus E7 proteins (Kaelin et al., 1992). Using an approach for screening lgt11 expression libraries, clones encoding for RB-binding proteins were identified; among those are RBAP-1 and 2, or retinoblastoma-associated proteins 1 and 2 (Kaelin et al., 1992) and RBP3 (Helin et al., 1992). RBAP-1 binds to the RB pocket, copurifies with E2F, contains a functional transactivation domain, and binds to E2F cognate sequences (Kaelin et al., 1992).

E2F contains a RB-binding domain in its C-terminus (Helin et al., 1992; Shan et al., 1992). RB binds directly to the activation domain of E2F1 and silences it, thereby preventing cells from entering S phase. To induce complete G1 arrest, RB requires the presence of the hbrm/BRG-1 proteins, which are components of the coactivator SWI/SNF complex. This cooperation was mediated through a physical interaction between RB and hbrm/BRG-1. RB can contact both E2F1 and hbrm at the same time, thereby targeting hbrm to E2F1 (Trouche et al, 1997).

E2F cooperates with p53 to induce apoptosis (Wu and Levine, 1994) and high levels of wild-type p53 potentiate E2F-induced apoptosis in fibroblasts (Qin et al, 1994). The physiological relevance of E2F in the apoptotic mechanism was thought to arise from the ability of E2F to act as a functional link between p53 and RB; p53 levels increase in response to high levels of E2F (DP is required for the association of E2F with RB); overexpression of both E2F-1 and DP-1 led to a rapid death of (IL-3)-dependent 32D.3 myeloid cells even in the presence of survival factors (Hiebert et al, 1995). Overexpression of exogenous E2F-1 using a tetracycline-controlled expression system in Rat-2 fibroblasts promoted S-phase entry and subsequently led to apoptosis (Shan and Lee, 1994).

B. Phosphorylation of RB: the TGF-b1, IL-1, and IL-6 connection

Work from several groups has shown that RB is un- or under-phosphorylated in G0/G1 and becomes phospho-rylated in its N-terminal domain during S and G2/M (Buchkovich et al., 1989; Chen et al., 1989; DeCaprio et al., 1989; Mihara et al., 1989). Only under-phosphorylated RB interacts with E2F (Chellappan et al., 1991). Treatment with TGF-b1 maintained RB protein in its active dephosphorylated form, thus providing a link between RB growth suppression and growth inhibition by TGF-b1.

Interleukin-6 (IL-6), known to mediate autocrine and paracrine growth of multiple myeloma (MM) cells and to inhibit tumor cell apoptosis was determined to exert this function via phosphorylation of RB protein; this finding could explain the abnormalities of RB protein and mutations of RB gene associated with up to 70% of MM patients and 80% of MM-derived cell lines. Culture of MM cells with RB antisense, but not RB sense, oligonucleotide triggered IL-6 secretion and proliferation in MM cells; phosphorylated pRB was constitutively expressed in MM cells and IL-6 shifted pRB from its dephosphorylated to its phosphorylated form (Urashima et al, 1996).

Interleukin-1 (IL-1) causes G0/G1 phase growth arrest in human melanoma cells, A375-C6 via hypophosphory-lation of RB protein. Exposure to IL-1 caused a time-dependent increase in hypo-phosphorylated RB that correlated with an accumulation of cells arrested in the G0/G1 phase; this was abrogated by the SV40 large T antigen which binds preferentially to hypo-phosphorylated RB, but not by the K1 mutant of the T antigen, which is defective in binding to RB (Muthukkumar et al, 1996).

C. Genes regulated by RB protein

RB represses a number of genes by sequestering or inactivating the positive transcription factor E2F and seems to activate some other genes by interacting with factors like Sp1 or ATF-2 (Rohde et al, 1996). RB protein is a master regulator of a complex network of gene activities defining the difference between dividing and resting or differentiated cells. Using the method of differential display Rohde et al (1996) detected a number of genes which were upregulated by ectopic expression of the RB gene in RB-deficient mammary carcinoma cells including the endothelial growth regulator endothelin-1 and the proteoglycans versican and PG40.

Introduction of the wild-type RB gene via retrovirus-mediated gene transfer has provided several RB-reconstituted retinoblastoma cell lines (Huang et al., 1988; Chen et al., 1992). These RB⁺ cell lines showed little difference in their growth rates in culture when compared to the parental or revertant RB^- cells; however, RB⁺ cells invariably lost their tumorigenicity in nude mice assays (Chen et al., 1992). RB protein down-regulates its own gene and this negative autoregulation is mediated by the transcription factor E2F; this was shown by inserting the promoter of the RB gene 5' of the bacterial CAT reporter gene followed by its transfection into RB⁺ and RB- retinoblastoma cells: RB promoter activity was signifi-cantly decreased in RB⁺ cells (Shan et al., 1994).

D. Transcription factors (TFs) that regulate the RB gene

Several mutations have been found in the promoter region of the RB gene, suggesting that inappropriate transcriptional regulation of this gene contributes to tumorigenesis. The presence of E2F recognition sites in promoters of a number of growth-related genes suggested that expression of these genes might be affected by RB. Understanding the nature and availability of TFs which regulate the RB gene in particular cell types is instructive for a successful gene therapy application involving transfer of RB.

An E2F recognition site lies within a region critical for RB gene transcription; binding of E2F-1 at this site transactivates the RB promoter; striking back, the resulting overexpression of RB suppresses E2F-1-mediated stimulation of RB promoter activity and, thus, the expression of RB is negatively autoregulated through E2F-1 (Shan et al, 1994). Up-regulation of the RB gene by E2F was shown by co-transfection of RB- osteosarcoma Saos2 cells in culture with a plasmid expressing E2F-1 under the control of the CMV immediate-early gene promoter-CAT construct: expression of E2F-1 stimulated RB promoter activity 10-fold under conditions where E2F-1 had little effect on c-jun, c-myc, and EGR-1 gene expression (Shan et al., 1994). The autoregulation of RB gene by RB may be accomplished via a direct protein-DNA complex formation, via protein-protein interaction regulating the activity of other transcription factors on the promoter of the RB gene, or both.

Two distinct DNA-binding factors, RBF-1 and ATF, play an important part in the transcription of the human RB gene. The promoter of the human RB gene and of the mouse RB1 gene (Zacksenhaus et al., 1993) contain binding sites for ATF, and a Sp1-like transcription factor (Mitchell and Tijan, 1989) where the RBF-1 (retinoblastoma binding factor 1) may bind (Sakai et al., 1991). Human RB gene is also regulated by AP-1 (Linardopoulos et al, 1993), as well by the early response transcription factor, nerve growth factor inducible A gene (NGFI-A) which is expressed in prostate cells and binds to the site GCGGGGGAG at -152 to -144 within the RB gene promoter (Day et al, 1993). The ATF site of the RB promoter is a responsive element during myogenic differentiation; RB promoter activity increased about 4-fold during differentiation and was reduced when a point mutation was designed in the ATF site (Okuyama et al, 1996).

pRB activates expression of the human transforming growth factor-b2 gene through ATF-2; the human RB gene promoter is autoregulated by RB protein via an ATF-2-like binding site at the carboxyl-terminal domain of pRB; overexpression of RB stimulates RB promoter activity through the ATF binding site in a variety of different cell types (Park et al, 1994).

The candidate oncoprotein Bcl-3, previously characterized as a member of the IkB family, activated transcription of the RB gene, whose promoter has no typical NF-kB sites, via binding to a DNA element identical to E4TF1/GABP site; Bcl-3 promoted tetramerization of E4TF1. Expression of the antisense bcl-3 RNA in myoblasts suppressed induction of RB and myogenic differentiation whereas transient expression of bcl-3 in myoblasts was shown to induce expression of the endogenous RB (Shiio et al, 1996).

Two oncogenic point mutations at the Sp1 and ATF sites of the RB gene promoter were identified in two separate hereditary RB families. The Sp1 consensus site mutation was blocking the action of RBF-1, recently identified as the human GABP/E4TF1, a transactivator from the adenovirus early-region 4 promoter. The human GABP/E4TF1 protein enhanced the core RB promoter activity, whereas it did not stimulate a mutant RBF-1 site and was proposed to be the most essential transcription factor for human RB gene activation (Clark et al, 1997).

Whereas binding of the Sp1 transcription factor is not significantly affected by methylation of the CpG dinucleotide within its binding site, 5'-GGGCGG (lower strand, 5'-CCGCCC) methylation of the outer C is inhibitory (mammalian cells also have the capacity to methylate cytosines at CpNpG sites) and in particular methylation of both cytosines ^mCp^mCpG inhibited binding by 95%; endogenous ^mCp^mCpG methylation of an Sp1 site in the CpG island promoter of the RB gene was identified by genomic sequencing in a proportion of retinoblastoma tumors which were extensively CpG methylated in the RB promoter (Clark et al, 1997).

E. RB gene transfer

Functional loss of the RB gene has been implicated in the initiation or progression of several human tumor types including cancer of the eye, bone, bladder, and prostate. The cancer suppressor activity of RB was directly demonstrated by the introduction of a normal RB gene into retinoblastoma cells that have lost the RB function (inability to be phosphorylated because of mutations at the appropriate sites) by mutation at both alleles; this led to the suppression of the neoplastic phenotype and loss of the tumorigenicity of RB cells in nude mice (Huang et al, 1988). Expression of the normal RB gene into the human prostate carcinoma cell line DU145, mediated by recombinant retrovirus integration, also resulted in loss of its tumorigenic ability in nude mice (Bookstein et al, 1990). Studies with tumor cells reconstituted with RB ex vivo and implanted into immunodeficient mice, as well as with germline transmission of a human RB transgene into tumor-prone Rb^+/- mice have demonstrated cancer suppression (see Riley et al, 1996).

DU145 cells express a shorter protein lacking 35 amino acids from exon 21 due to a 105 nucleotide in-frame deletion (Bookstein et al, 1990). The human bladder carcinoma cell line J82 contains a mutated RB protein with exactly these features (Horowitz et al, 1989); this 35 amino acid stretch is required for complexation with T antigen and E1A. However, the two cell lines have lost exon 21 of RB because of a different type of mutation: J82 cells have a point AG to GG mutation in the intron 20-splice acceptor site but the type of mutation in DU145 leading to exon 21 loss is different (Bookstein et al, 1990).

Intratumoral infection of spontaneous pituitary melanotroph tumors arising in immunocompetent Rb^+/- mice with a recombinant adenovirus carrying the RB cDNA inhibited the growth of tumors, re-established innervation by growth-regulatory dopaminergic neurons, and prolonged the life spans of treated animals (Riley et al, 1996).

Retrovirus-mediated gene transfer of RB to the breast carcinoma cell lines MDA-MB468 and BT549, both of which harbor partial RB gene deletions as well as point mutations of their p53 genes, restored its expression in cells, reduced their ability to grow in soft agar, and their tumorigenicity in nude mice, although it did not significantly altered growth rate in culture (Wang et al, 1993).

Future therapeutic approaches using the RB gene are directed toward inhibition in cell proliferation (such as to inhibit neointima formation and smooth muscle cell proliferation in arterial diseases, see Arterial injury below and Chang et al, 1995) rather that aggressive suppression and apoptosis of solid tumors; p53 is a better gene than RB for tumor eradication.

XX. Induction of apoptosis for cancer gene therapy

A. Apoptosis as an essential process

Apoptosis has become a basic tool in developing cancer research in establishing new anticancer strategies. The health of a multicellular organism depends both on the ability of the body to produce new cells but also on the ability of certain type of cells to perish, self-destruct, when they become superfluous or severely damaged. Apoptosis, or programmed cell death, is a biological process associated with pronounced morphological changes, chromatin condensation, drop in pH, and intranucleosomal DNA degradation by which a cell actively commits suicide. Virtually all tissues have apoptotic cells; salient examples in the adult are: the eye lenses which consist of apoptotic cells that replaced their cytoplasm with crystallin; intestinal wall cells which migrate to the tip of the finger-like projections over several days where they die; ineffectual T cells which mature in thymus and which would attack the body’s own tissues are eliminated by apoptosis before entering the bloodstream; skin cells migrate from the deepest layers to the surface where they commit suicide forming the outer layer of the skin. Apoptosis is an essential process during embryogenesis: mammals eliminate neuron cells as the nervous system is formed; tadpoles delete their tails by apoptosis (reviewed by Duke et al, 1996).

Virus-transformed as well as severely X-ray-damaged or UV-damaged cells are similarly eliminated from the tissue via apoptosis; if they are left they can form malignant cells. Initiated cancer cells may lead to tumor development only when a dysfunction in their apoptotic pathway takes place. Although the biochemical aspects of cell death are fraught with the problem of cause versus effect, the role of apoptosis in neoplasia and its regulation by a number of oncogenes and p53 has emerged. Apoptosis is essential for normal development and homeostasis; deregulation in the positive control of apoptosis is associated with cancer and autoimmune disease whereas deregulation in the negative control of apoptosis is associated with degenerative diseases (reviewed by White, 1993; Duke et al, 1996).

B. Molecular mechanisms for apoptosis: p53, Bax, Bcl-2, c-Myc and other proteins

Apoptosis is of special interest in gene therapy not only of cancer but of other diseases such as arterial disease. Apoptosis is a complex process involving a significant number of apoptotic and antiapoptotic mechanisms. The cytotoxic (killer) T lymphocytes of the immune system of the infected organism bind to virus-infected cells inflicting their eradication with two different type of proteins: Perforin is a transmembrane molecule transferred from the killer T cell to the membrane of the infected cells forming holes on the membrane of the target cell allowing uptake of proteases called granzymes that activate ICE-like proteases to induce apoptosis. A number of antiviral drug development strategies are based on blockage of the activity of antiapoptotic viral proteins.

Expression of a number of genes induce apoptosis; their protein products include adenovirus E1A (Debbas and White, 1993; Lowe and Rudley, 1993) and c-Myc (Hermeking and Eick, 1994; Wagner et al, 1994). A number of proteins when expressed at sufficient amounts block apoptosis; these include Bcl-2 and E1B 19 kDa protein of adenovirus (Debbas and White, 1993; Chiou et al, 1994). Exposure of cells to a variety of growth factors including IL-3, IL-6, and erythropoietin, acting as survival factors, inhibit induction of apoptosis (Johnson et al, 1993; Yonish-Rouach et al, 1993; Canman et al, 1995).

The role of p53 in these molecular processes has been discussed in previous pages in this review. The involvement of p53 in apoptosis is thought to occur via upregulation of bax and downregulation of bcl-2 genes by wt p53 but not by mutated p53 proteins; Bax protein induces apoptosis and its upregulation triggers the apoptotic mechanism in cells which display elevated levels of p53 as a result, for example, of DNA damage. Down-regulation of Bcl-2 has a similar effect on the induction of apoptosis. p53 may induce apoptosis independently of transcription, although the G1 arrest by p53 requires transcription of p53 targets (reviewed by Ko and Prives, 1996). Induction of the apoptotic pathway by p53 was proposed to involve: (i) transcriptional induction of redox-related genes; (ii) formation of reactive oxygen species; and (iii) the oxidative degradation of mitochondrial components (Polyak et al, 1997). The potential of p53 in cancer gene therapy is discussed above.

While p53 and E1A activate apoptosis, Bcl-2 and E1B 19k proteins inhibit apoptosis. All four protein molecules act upstream of Bax which is a potent inducer of apoptosis: both the cellular Bcl-2 and the 19 kDa protein E1B of adenovirus are able to interact with Bax inhibiting its involvement in induction of apoptosis (Han et al, 1996; Figure 1 on page 9). E1A acts upstream of p53 by increasing the half-life of p53 resulting in an accumulation of p53 molecules in the nucleus (Lowe and Ruley, 1993); increased levels of p53 are then believed to upregulate the bax gene (Figure 1). The survival factors IL-3 and IL-6 appear to prevent p53-dependent apoptosis (see White, 1993).

p53 induces apoptosis after exposure to UV irradiation (Ziegler et al, 1994) and hypoxia (Graeber et al, 1996); this acts as a protective mechanism for the removal of severely damaged cells from the body which could become initiated cancer cells and progress to tumors. Spontaneous or radiation-induced apoptosis mediated by p53 has been shown to act for the removal of cells from the gastrointestinal tract in mice (Merritt et al, 1994) and the skin after sunburn (Ziegler et al, 1994). Epidermal growth factor (EGF) has induced apoptosis in various cancer cell lines via a novel signal transduction pathway of EGF mediated through p53 (Murayama and Horiuchi, 1997).

c-myc expression, normally induced in proliferating hematopoietic cells by mitogens, drops dramatically by mitogen withdrawal leading to cell arrest in G1. During deregulated c-myc expression, c-myc levels were not down-regulated upon mitogen withdrawal; instead, DNA synthesis continued resulting in apoptosis but not in growth arrest. The transforming segment of c-Myc was responsible for induction of apoptosis (see White, 1993).

Pax5 is a repressor of expression of the p53 gene interacting directly with a regulatory region within exon 1 of the p53 gene. At early stages during pre-B cell development the levels of Pax5 are high and p53 is down-regulated; however, later in development Pax5 levels drop and the p53 gene is activated; this process was proposed to lead to the decision of B cells to enter apoptosis or differentiate into plasma cells (Stuart et al, 1995).

Down regulation of the Cu²⁺/Zn²⁺ superoxide dismutase (SOD1) induced oxidative stress and apoptosis (Troy et al, 1996). A great deal of oxidative damage during the procedures for ex vivo-modification of cells induces their apoptosis; transfer of the Cu²⁺/Zn²⁺ superoxide dismutase to ex vivo modified cells increased their survival after implantation (see Nakao et al, 1995). This demonstrates the importance of blocking apoptotic pathways during cell manipulation for successful ex vivo gene therapy.

Gene therapy for cancer could involve restoration of the apoptotic pathway in cancer cells leading to their suicidal death; this could be effected by overexpression of the bax gene, by suppression of the endogenous bcl-2 gene (see below), or by transfer of the wt p53 gene.

C. Role of tumor necrosis factor (TNF)

The tumor necrosis factor-a (TNF-a) is a cytokine produced by macrophages, monocytes, lymphoid cells, fibroblasts and other cell types in response to inflammation and infection. TNF-a is produced by lipopolysaccharide (LPS)-stimulated macrophages; the molecular pathways leading to TNF-a production in these specialized cells involves activation by LPS of several kinases including the extracellular-signal-regulated kinases 1 and 2 (ERK1 and ERK2), p38, Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), as well as activation of the immediate upstream MAPK activators MAPK/ERK kinases 1 and 4 (MEK1 and MEK4) and of MEK2, MEK3, and MEK6 (Swantek et al, 1997).

TNF-a binds to two type of specific receptors, TNFR1 and TNFR2, causing their trimerization and leading to activation of a number of kinases (ceramide-activated kinase, IkB kinase, Raf-1, Jun N-terminal kinases or JNKs, p38/Mpk2). Activation of Raf-1, JNK, and p38/Mpk2 contribute to the induction of AP-1 whereas activation of IkB kinase is leading to the activation of the transcription factors NF-kB. This activation leads further to upregulation of genes and induction of other cytokines, metalloproteinases, and immunoregulatory proteins (see Liu et al, 1996 and the references cited therein).

TNF can induce apoptotic death or necrosis in some tumor cells; this effect of TNF could be mediated by activation of sphingomyelinases and phospholipases, synthesis of metabolites of arachidonic acid, generation of free radicals, changes in intracellular calcium, generation of DNA strand breaks and activation of poly(ADP-ribosyl)ation, or activation of ICE-like proteases.

TNF-a, IL-1b, IFN-g, and vitamin D3 after binding to their transmembrane receptors stimulate the production of the second messager ceramide from sphingomyelin in the plasma membrane by activating sphingomyelinase; this results in a cascade of signal transduction events that result in down regulation of c-myc and induction of apoptosis, to terminal differentiation, or to RB-mediated cell cycle arrest (Figure 23).

IL-1 signaling leads to NF-kB activation and to protection against TNF-induced apoptosis. The IL-1R-associated kinase (IRAK) is homologous to Pelle of Drosophila. Two additional proximal mediators, both associating with the IL-1R signaling complex, were required for IL-1R-induced NF-kB activation: IRAK-2, a Pelle family member, and MyD88, an adaptor molecule containing a death domain (Muzio et al, 1997).

Treatment of different cell types with TNF-a results in the activation of the MEKK1 pathway of protein kinases ultimately resulting in AP-1 transcription factor activation and in the upregulation of several cytokine genes. TNF-a-stimulation also results in the activation of NF-kB and inhibition of apoptosis (Figure 24). A TNF-responsive

Figure 23. A pathway leading to the induction of growth arrest and apoptosis by the cytokines TNF-a, IL-1b, and IFN-g. The pathway is conserved between mammalian cells and yeast. Adapted from Nickels and Broach (1996). From Boulikas T (1997) Gene therapy of prostate cancer: p53, suicidal genes, and other targets. Anticancer Res 17, 1471-1506. Reproduced with the kind permission from Anticancer Research.

serine/threonine protein kinase termed GCK-related (GCKR) most likely signals via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase kinase 1 (MEKK1) to activate the SAPK pathway (Shi and Kehrl, 1997).

D. NF-kB as anti-apoptotic molecule and TNF-a signaling

Activation of NF-kB is believed to lead to the activation of antiapoptotic genes that have not been fully identified. The antiapoptotic role of NF-kB at the molecular level and the TNF-a connection consists of the following events; signaling by TNF-a induces trimerization of its receptors, an event causing three different cascades: (i) Activation of IkB kinase and activation of NF-kB, a pathway which prevents cell death. A key step for NF-kB activation leading to the activation of the stress-activated protein kinase (SAPK, also called c-Jun N-terminal kinase or JNK) is the recruitment to the TNF receptor of TNF receptor-associated factor 2 (TRAF2). (ii) induction of apoptosis via a different pathway involving activation of sphingomyelinase in plasma membrane and generation of ceramide leading to EGFR activation and induction of apoptosis; (iii) activation of MEKK1 and JNK protein kinases which is not linked to apoptotic death but to AP-1 activation (Figure 24). The antiapoptotic function of NF-kB may involve activation of the manganese superoxide dismutase and of the zinc finger protein A20; expression of these genes is induced by TNF and each of them provides protection against apoptosis (Liu et al, 1996).

bcl-2 upregulation during progression of prostate cancer was implicated in the acquisition of the androgen-independent growth; a strong antioxidant that interferes with activation of NF-kB in prostate carcinoma cells, potentiated TNF-a-stimulated apoptosis signaling through a bcl-2-regulated mechanism; based on these studies, modulation of the NF-kB survival signaling was proposed to be used to clinical advantage in the treatment of prostate cancer patients (Herrmann et al, 1997).

Transgenic mice lacking the p65 (RelA) subunit of NF-kB displayed increased apoptosis and degeneration in the liver providing further support to an apoptotic function of NF-kB (Beg et al, 1995). The TNF-induced death of mouse primary fibroblasts expressing deregulated c-Myc was inhibited by transient overexpression of the p65 subunit of NF-kB, which increased NF-kB activity in the cells (Klefstrom et al, 1997). Rel (a protooncogene, member of the NF-kB family) is implicated in both positive and negative regulation of GM-CSF expression in a variety of cell types (Gerontakis et al, 1996).

The elucidation of IL-1, TNF, IFN and other signaling pathways would lead to the discovery of new drugs causing specific inhibition; for example, members of the IL-1 signaling cascade may provide therapeutic targets for inhibiting IL-1-induced inflammation (Muzio et al, 1997).

Figure 24. TNF-a signaling via trimerization of its receptors (TNF-aR1), is causing: (i) activation of IkB kinase and activation of NF-kB, a pathway which prevents cell death via activation of the manganese superoxide dismutase and of the zinc finger protein A20. (ii) induction of apoptosis via second message ceramide (see Figure 23) and (iii) activation of JNK leading to AP-1 activation and up-regulation of cytokine genes.

E. Interleukin-1b converting enzyme (ICE) and apoptosis

The human interleukin-1b converting enzyme (ICE) is a cysteine-rich protease that can cleave the inactive 31 kDa precursor of IL-1b to generate the active cytokine; it has similarities to the C. elegans CED-3 protein. This protease plays a central role in apoptosis; the exact role and the involvement of IL-1b have not been elucidated; it is believed that signals from IL-1b, TNF-a, vitamin D3, and interferon-g, which induce an antiproliferative response, converge on sphingomyelin of plasma membrane activating a sphingomyelinase which generates a ceramide second messenger (Figure 23); in S. cerevisiae, this leads to signal transduction via activation both of a cytoplasmic protein phosphatase 2A and a protein kinase leading to down-regulation in c-myc expression and induction of apoptosis as well as RB-mediated cell cycle arrest via EGFR activation (Nickels and Broach, 1996).

At least 10 ICE-like proteases have been identified which mediate apoptotic death after their induction by a number of stimuli (see Martin and Green, 1995); these are divided into three families: (i) the ICE/CED3 family, including ICE itself; (ii) the CPP32/Yama family; and (iii) the Ich-1/Nedd2 family; they all contain the conserved QACRG pentapeptide in which the central cysteine participates in proteolytic catalysis (see Jänicke et al, 1996). Activation of these proteases by induction of apoptosis results in the cleavage of a large number of key regulatory proteins including among others poly(ADP-ribose) polymerase or PARP (Lazebnik et al, 1994), RB (Jänicke et al, 1996), PKCd (Emoto et al, 1995), Gas2 affecting microfilament reorganization (Brancolini et al, 1995), the DNA-dependent protein kinase (Casciola-Rosen et al, 1995), and the sterol regulatory element binding proteins (SREBPs) catalyzed by CPP32 ICE-like protease (PARP et al, 1996). Since cleavage of a single protein has not been shown to cause cell death it is not clear how many substrate protein molecules need to be cleaved. In addition different apoptotic pathways may exist and may operate in different cell types.

Expression of the murine ICE cDNA in Rat-1 cells induced programmed cell death and this phenomenon could be reversed by overexpression of the bcl-2 oncogene (Miura et al, 1993). Expression of members of the family of cysteine proteases related to ICE have been shown to be necessary for programmed cell death in a number of organisms (Yuan et al, 1993). Overexpression of murine ICE or of the ICE-like proteases NEDD-2/ICH-1 and Yama/apopain induced apoptosis (Miura et al, 1993). Mice lacking ICE were resistant to apoptosis induced by Fas antibody (Kuida et al, 1995).

F. Role of poly(ADP-ribose) polymerase (PARP)

PARP is a central mediator of genome integrity and transmits signals from DNA damage to recruit locally DNA repair activities (Zardo et al, 1998; Quesada, 1998, this volume). An additional role of PARP is its involvement in apoptosis causing suppression of an apoptotic endonuclease; PARP is cleaved by an ICE-like protease during TNF-induced apoptosis (Lazebnik et al, 1994). Cleavage of PARP would abort these pathways resulting in loss of recruitment of DNA repair enzymes at damaged sites but also in loss in the inhibitory function of poly(ADP-ribose) groups on key regulatory enzymes (DNA ligase, topoisomerase). It is unlikely that PARP proteolysis by an ICE-like protease is a primary event since PARP-deficient mice show normal resistance to DNA damaging agents (Wang et al, 1995).

G. Apoptosis in autoimmune disease and ischemic heart disease

T cells are produced by bone marrow and then migrate to the thymus gland where they mature. The cytotoxic or killer T cells directed against foreign bodies are released in the bloodstream; a specific apoptotic mechanism eliminates T cells directed against specific antigens on healthy cells. However, the body allows some mildly self-reactive lymphocytes to circulate; although harmless, exposure to a microbe or food antigen can stimulate them causing an expansion in their proliferation and resulting in a mild autoimmune disease. Such mild autoimmune reactions usually disappear when the stimulating antigen is cleared away; in more severe autoimmune disease, however, these lymphocytes survive longer inducing apoptosis and self-destruction in healthy cells in various tissues.

A number of classical diseases may originate by autoimmune mechanisms including initiation of atherosclerosis by apoptotic death of the epithelial cells in the arterial wall, diabetes by destruction of the pancreatic cells, lupus erythematosus, rheumatoid arthritis, and others. The mechanism via which T lymphocytes directed against self antigens defy apoptosis is not known; the mechanism might involve overexpression of the Bcl-2 gene in these lymphocytes or down-regulation of a gene encoding for the Fas ligand that sends a death message to the lymphocyte (Weih et al, 1996; reviewed by Duke et al, 1996).

Excessive necrotic death in cells of the coronary artery wall results by oxygen and glucose deprivation after blockage of a blood vessel feeding a segment of the heart (also the brain in stroke). Destructive free radicals are then produced during inflammation of the area which can cause apoptotic or necrotic death in cells in the surroundings. Since both brain and heart cells in the adult are not regenerated, Biotech Companies (for example Genentech) are focusing in developing drugs that block free radical formation, inhibit ICE-like proteases, or inhibit apoptosis via other mechanisms.

The progressive loss of neuron cells in senile or other brain diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophical lateral sclerosis may ensue by apoptosis. Etiologic factors may include excessive levels of neurotransmitters, low levels of NGF, free radical-mediated damage, and deregulation in the expression of genes encoding apoptotic regulators during aging. Deregulation in apoptosis may also have a share in the induction of osteoporosis.

XXI. Genes involved in the regulation of apoptosis as targets for gene therapy

Many of the molecular controllers of apoptosis including cytokine signaling pathways (TNF-a, IL-1b), tumor suppressor proteins (p53), viral proteins (E1A of adenovirus), cellular oncoproteins (Myc), proteins that control the cell cycle (E2F), apoptosis inducers (Bax) and antiapoptotic molecules (Bcl-2, NF-kB) could constitute potential targets for pharmacological intervention for the treatment not only of cancer but of other human disease. Although for cancer treatment it is desirable to induce apoptosis, the opposite effect, that is inhibition of apoptotic pathways is desirable in the gene therapy of heart disease and degenerative brain disease (see below).

A. Gene therapy that targets bcl-2

Bcl-2 protein is overexpressed in a variety of human leukemias because of translocation of its gene to the immunoglobulin locus; Bcl-2 is associated with the outer surface of the mitochondrion and appears to be involved in scavenging oxygen radicals. Overexpression of the bcl-2 gene in tumors is thought to be responsible for the poor response of the tumors to antineoplastic drugs and radiation therapy blocking apoptosis of the tumor cells. Bcl-2 can interact with members of the Bcl-2 family including Bax, Bcl-X-S, Bcl-X-L, and Mcl-1 but also with heterologous protein molecules including BAG-1, Raf-1, and R-Ras.

Introduction of the bcl-2 gene into human diploid breast epithelial MCF10A cells (containing the wild-type p53 gene) resulted in suppression in p21 gene expression although the level of expression of p53 was not affected; these studies suggested that Bcl-2 may inhibit the functional activity of p53 protein and might regulate the commitment of cells to commit suicide or proliferate (Upadhyay et al, 1995).

Overexpression of the bcl-2 gene in tumors is thought to be responsible for the poor response of the tumors to antineoplastic drugs and radiation therapy blocking apoptosis of the tumor cells; therefore, down-regulation of the bcl-2 gene specifically in tumor cells could induce apoptosis. Primary untreated human prostate cancers were found to express significant levels of this apoptosis-suppressing oncoprotein; this is a striking difference with normal prostate secretory epithelial tissue not expressing Bcl-2 (Raffo et al, 1995). Transfection of LNCaP human prostate cancer cells with a plasmid expressing bcl-2 rendered these cells highly resistant to a variety of apoptotic stimuli (serum starvation or treatment with phorbol ester) and induced earlier and larger tumors in nude mice. The ability of Bcl-2 to protect prostate cancer cells from apoptotic stimuli correlated with the ability of the cells to form hormone-refractory prostate tumors in nude mice (Raffo et al, 1995).

The Bcl-2 oncoprotein suppresses apoptosis and, when overexpressed in prostate cancer cells, makes these cells resistant to a variety of therapeutic agents, including hormonal ablation. Overexpression of BCL-2 is common in non-Hodgkin lymphoma leading to resistance to apoptosis and promoting tumorigenesis. Therefore, bcl-2 provides a strategic target for the development of gene knockout therapies to treat human prostate cancers (Dorai et al, 1997) and non-Hodgkin lymphomas (Webb et al, 1997).

Down-regulation of Bcl-2 can be accomplished with antisense. In patients with relapsing non-Hodgkin lymphoma, BCL-2 antisense therapy led to an improvement in symptoms; antisense oligonucleotides targeted at the open reading frame of the BCL-2 mRNA showed effectiveness against lymphoma grown in laboratory animals and has entered human clinical trials. The first study was conducted on nine patients with BCL-2-positive relapsed non-Hodgkin lymphoma using a daily subcutaneous infusion of 18-base, fully phosporothioated antisense oligonucleotide administered for 2 weeks (Webb et al, 1997). A local inflammation at the infusion site was noted. A reduction in tumour size was observed in two patients (one minor, one complete response) using computed tomography scans; in two other patients, the number of circulating lymphoma cells decreased during treatment. In four patients, serum concentrations of lactate dehydrogenase fell, and in two of these patients symptoms improved (Webb et al, 1997).

A divalent hammerhead ribozyme, constructed by recombining two catalytic RNA domains into an antisense segment of the coding region for human bcl-2 mRNA was able to rapidly degrade bcl-2 mRNA in vitro; it was then tested for its ability to eliminate bcl-2 expression from hormone-refractory prostate cancer cells. When this hammerhead ribozyme was directly transfected into cultured prostate cancer cells (LNCaP derivatives), it significantly reduced bcl-2 mRNA and protein levels within 18 hr of treatment and induced apoptosis in a low-bcl-2-expressing variant of LNCaP, but not in a high-bcl-2-expressing LNCaP line (Dorai et al, 1997).

B. Bcl-xs

Many cancers overexpress a member of the Bcl-2 family of inhibitors of apoptosis, such as Bcl-2 and Bcl-xL. Members of the Bcl-2 family were found to be essential for survival of cancer cells derived from solid tissues including breast, colon, stomach, and neuroblasts (Clarke et al, 1995). On the contrary, Bcl-xs is a dominant negative repressor of Bcl-2 and Bcl-xL; thus, Bcl-xs induces apoptosis. Transient overexpression of Bcl-xs in MCF-7 human breast cancer cells, which overexpress Bcl-xL, with a replication-deficient adenoviral vector induced apoptosis in vitro; intratumoral injection of the bcl-xs adenovirus on solid MCF-7 tumors in nude mice showed a 50% reduction in size with evident apoptotic cells at sites of injection (Ealovega et al, 1996).

An adenovirus vector expressing bcl-xs specifically and efficiently killed carcinoma cells arising from multiple organs including breast, colon, stomach, and neuroblasts even in the absence of an exogenous apoptotic signal such as x-irradiation. In contrast, normal hematopoietic progenitor cells and primitive cells capable of repopulating SCID mice were not killed by the bcl-xs adenovirus. Thus, transfer of the bcl-xs gene could be used in killing cancer cells contaminating the bone marrow of patients undergoing autologous bone marrow transplantation (Clarke et al, 1995).

C. E2F-1 and TNF-a gene transfer

E2F cooperates with p53 to induce apoptosis; high levels of wild-type p53 potentiate E2F-induced apoptosis in fibroblasts. The physiological relevance of E2F in the apoptotic mechanism is thought to arise from the ability of E2F to act as a functional link between p53 and RB; p53 levels increased in response to high levels of E2F. Targeted disruption of the E2F-1 gene yields transgenic animals with an excess of mature T cells due to a defect in lymphocyte apoptosis (Field et al, 1996).

Overexpression of the transcription factor E2F-1 could induce apoptosis in quiescent rat embryo fibroblasts in a p53-dependent manner; however, Hunt et al (1997) have shown that overexpression of the E2F-1 gene after adenoviral transfer can mediate apoptosis in the absence of wild-type p53: adenovirus-mediated transfer of the E2F-1 gene under control of the CMV promoter to human breast and ovarian carcinoma cell lines resulted in the induction of significant morphological changes in four of the five cell lines that had mutations in the p53 gene within 48 h of transduction characteristic of apoptosis.

Retroviral vector-mediated transfer of the TNF-a gene into the DNA of human tumor cells induced apoptosis in high- TNF-a-producing clones generated from a human lymphoma T-cell line (ST4); the apoptotic death of the cells was associated with a downregulation of the apoptosis-preventing gene, bcl-2, while the expression of bax and p53 genes persisted (Gillio et al, 1996).

D. E6, E7 of human papillomavirus (HPV)

E6 and E7 of HPV possess transforming ability, have been shown to interact with the cellular tumor suppressors p53 and RB (Werness et al, 1990; Dyson et al., 1989) and are believed to play a central role in HPV-induction of cervical carcinogenesis as well as in the maintenance of the malignant phenotype. Viruses have developed strategies to shut down protein synthesis in the host and subdue its protein synthesizing machinery to produce progeny virus when infecting cells. Because virus-infected cells commit suicide to protect the organism from further infection viruses have evolved mechanisms to prevent apoptosis of the host cell ensuring their propagation; E6 protein interacts with p53 to exclude p53 molecules from their apoptotic functions and to inhibit apoptosis in HPV-infected cells thus giving to HPV a proliferation advantage.

27-mer phosphorothioate oligodeoxynucleotides (oligos) targeting the ATG translational start region of HPV-16 E6 and E7 sequences showed antiproliferative effects in all HPV-16-positive cell lines tested and in primary cervical tumor explants while the endometrial and two ovarian primary tumors as well as the HPV-negative C33-A cell line and HPV-18-positive cell line HeLa were relatively insensitive to the HPV-16 oligos (Madrigal et al, 1997).

E. Prevention of apoptosis for gene therapy of heart disease and for ex vivo manipulations of therapeutic cells

As induction of apoptosis is the desired effect for the gene therapy of cancer, prevention of apoptosis by gene therapy can fight heart disease. Cardiomyocyte death results from heart ischemia proceeding via necrosis and from reperfusion which induces additional cardiomyocyte death by apoptosis; prevention of apoptosis would constitute an important target for fighting heart disease. Prevention of apoptosis should also solve a major problem in cell culture cells which are subject to oxidation damage during their manipulation for ex vivo gene transfer and most important during the step of reimplantation, encapsulation in biopolymer membranes for surgical implantation, and similar processes. Prevention of apoptosis could be effected by transfer and overexpression of the bcl-2 gene. Also prevention of oxidative damage during reimplantation of ex vivo-modified cells could be reduced by transfer and overexpression of the Cu/Zn superoxide dismutase gene (Nakao et al, 1995).

Overexpression of bcl-2 delayed onset of motor neuron disease and prolonged survival in a transgenic mouse model of familial amyotrophic lateral sclerosis (Kostic et al, 1997).

XXII. E1A and HER-2/neu (c-erbB-2) in cancer gene therapy

A. HER-2/neu

The human epidermal growth factor receptor-2 (HER2), a membrane tyrosine kinase highly expressed in many epithelial tumors, could be a target for cancer gene therapy. The HER-2/neu (also called c-erbB-2) proto-oncogene is overexpressed in many human cancer cells, including those of breast cancer and ovarian cancer correlating with lower survival rate in ovarian cancer patients; amplification or overexpression of HER-2/neu has also been observed in human lung cancer and has been correlated with poor prognosis and chemoresistance.

A reversible transformation of NIH3T3 fibroblasts by overexpression of the HER2/c-erbB2 receptor tyrosine kinase under control of a tetracycline-responsive promoter has been demonstrated in tissue culture; induction of HER2 expression resulted in cellular transformation in vitro and treatment of transformed cells with the effector anhydrotetracyline switched-off HER2 expression and induced morphological and functional changes characteristic for non-transformed cells (Baasner et al, 1996).

B. E1A-based gene therapy

E1A-based gene therapy approaches are now in clinical trials (see below); the molecular mechanism behind this approach is that the E1A protein of Adenovirus 5 represses HER-2/neu transcription and functions as a tumor suppressor gene in HER-2/neu-overexpressing cancer cells. Breast cancer cells that overexpress HER-2/neu are more resistant to chemotherapeutic agents such as paclitaxel (Taxol) and docetaxel (Taxotere) than those that do not overexpress HER-2/neu; paclitaxel sensitivity correlated with HER-2/neu expression level in a panel of mouse fibroblasts expressing different levels of HER-2/neu; downregulation of HER-2/neu expression by E1A sensitized the cells to paclitaxel. Transfer the E1A gene into two human breast cancer cell lines that overexpress HER-2/neu and E1A gene transfer sensitized these cells to the drug by repressing HER-2/neu expression (Ueno NT et al, 1997).

Increased HER-2/neu expression led to more severe ovarian malignancy and increased metastatic potential in animal models; the adenovirus 5 E1A gene repressed HER-2/neu gene expression and suppressed growth of human ovarian cancer SKOV-3 cells, which overexpress HER-2/neu, in cell culture (Yu et al, 1995). Intraperitoneal injection of SKOV-3 cells into female nu/nu mice elicited tumors and the animals died within 160 days of severe tumor symptoms; cationic liposome-mediated delivery of the E1A gene into adenocarcinomas that developed in the peritoneal cavity and on the mesentery of the mice significantly inhibited growth and dissemination of ovarian cancer cells; about 70% of the treated mice survived at least for 365 days (Yu et al, 1995).

Regulatory regions derived from the 5' flank of the human prostate-specific antigen (PSA) gene were inserted into adenovirus type 5 DNA to drive the expression of the E1A gene; infection of cells in culture with this recombinant adenovirus was able to drive the expression of the E1A gene only in cell lines which expressed PSA such as the human LNCaP cells but not in human DU145 cells which do not express PSA; the recombinant adenovirus destroyed large LNCaP tumors (1x10⁹ cells) and abolished PSA production in nu/nu mouse xenograft models after a single intratumoral injection (Rodriguez et al, 1997).

A replication-deficient adenovirus containing the E1A gene, Ad.E1A⁺, was used to transduce E1A into HER-2/neu-overexpressing and low expressing human lung cancer cell lines and shown a better therapeutic efficacy in HER-2/neu-overexpressing cells. The cell culture studies were then extended to animal studies: tumor-bearing mice established by intratracheal injection of lung cancer cells overexpressing HER-2/neu and treated by i.v. tail injections of Ad.E1A⁺ showed suppression of the intratracheal lung cancer growth. However, no significant tumor suppression effect was observed in mice bearing a low HER-2/neu-expressing cell line with the same regimen (Chang et al, 1996).

C. Clinical trials with E1A and c-Erb-B2

Liposome-mediated E1A gene transfer suppressed tumor development and prolonged survival of mice bearing human breast cancer cells overexpressing HER-2/neu. These studies resulted in the initiation of a phase I clinical trial using an E1A-liposome complex administered to patients with HER-2/neu-overexpressing breast or ovarian cancer (Protocol 205 in Table 4 of following article, pages 203-206). The principal investigators are Drs. Hortobagyi, Lopez-Berstein, and Hung at MD Anderson Cancer Center, Houston, Texas). The safety of this regimen was shown by intraperitoneal injection of E1A/liposomes in normal mice and at cumulative doses 5 to 40 times the DNA-lipid starting dose proposed for the phase I clinical trial (Xing et al, 1997). A Phase I multicenter study of intratumoral E1A gene therapy using cationic liposome gene transfer is also in course for patients with unresectable or metastatic solid tumors that overexpress HER-2 /neu (protocol 209, see page 205).

Delivery of an anti-erbB-2 single chain (sFv) antibody gene for previously treated ovarian and extraovarian cancer patients is in clinical trials using adenoviral gene delivery (protocol #133). A clinical trial for tumor vaccination with HER-2 /Neu using a B7 expressing tumor cell line prior to treatment with HSV-tk gene-modified cells is in phase I for ovarian cancer (protocol #96, page 165).

XXIII. Suicidal genes for cancer therapy (prodrug gene therapy)

A. Molecular mechanism of cell killing with HSV-tk gene and ganciclovir (GCV)

Expression of genes encoding prodrug-activating enzymes can increase the susceptibility of tumor cells to prodrugs, and may ultimately achieve a better therapeutic index than conventional chemotherapy (Table 3). Direct suppression of tumor growth by cytotoxic gene therapy is a successful gene transfer approach. This approach has promise for a variety of other applications where excess cell proliferation is detrimental and has also been used to restrict intimal hyperplasia of the arterial wall and smooth muscle cell growth to limit restenosis after artery angioplasty (see below).

Cancer cells can be induced to be conditionally sensitive to the antiviral drug ganciclovir after their transduction with the thymidine kinase (tk) gene from the herpes simplex virus (HSV); ganciclovir (GCV) is the 9-{[2-hydroxy-1-(hydroxymethyl)-ethoxy]methyl}guanine (Field et al, 1983); it is converted by HSV-tk into its monophosphate form which is then converted into its triphosphate form by cellular enzymes and is then incorporated into the DNA of replicating mammalian cells leading to inhibition in DNA replication and cell death (Moolten, 1986; Borrelli et al, 1988; Moolten and Wells, 1990). It is only viral TK, not the mammalian enzyme, that can use efficiently ganciclovir as a substrate and this drug has been synthesized to selectively inhibit herpes virus replication (Field et al, 1983); indeed, the mammalian TK has a very low affinity for this guanosine analog. The toxicity of ganciclovir is manifested only when cells undergo DNA replication and it is not harmful to normal nondividing cells. This treatment strategy has been used for hepatocellular carcinoma (Huber et al, 1991; Su et al, 1996), fibrosarcoma, glioma (Culver et al, 1992, see below), adenocarcinoma (Osaki et al, 1994), prostate cancer (Eastham et al, 1996) and many other cancers.

B. Treatment gliomas in rats with HSV-tk plus ganciclovir

Brain tumors have the privilege of escaping immunologic rejection; therefore brain tumors are inaccessible to cancer immunotherapy. Culver and cowor-

XXIX. Gene therapy of HIV

A. Mechanism of HIV-1 entry into T cells and macrophages

Targets of human immunodeficiency virus (HIV) are helper T cells and macrophages; macrophage-tropic HIV-1 isolates represent the most prevalent phenotype isolated from individuals shortly after seroconversion during the asymptomatic period of the disease; tropism is determined by specific sequences in the third variable loop (V3 domain) of gp120 coat protein of HIV-1. The CD4 receptor on both macrophages and T cells is the primary receptor mediating HIV-1 entry into the cell; however, HIV-1 was unable to infect CD4⁺ T cells of mice engineered to express human CD4 (reviewed by D’Souza and Harden, 1996). Thus, a second chemokine receptor was thought to be necessary for HIV entry into immortalized T cell lines.

The second receptor for entry of HIV-1 into T cells and macrophages is CCR-5, a b-chemokine receptor; CCR-5 is a seven transmembrane-domain glycoprotein of the chemokine superfamily of receptors related to the receptor of IL-8 (G protein-coupled proteins that transduce signals from the cell surface to the interior of cells). The b-chemokines RANTES, MIP-1a, and MIP-1b inhibited replication of M-tropic isolates of HIV-1 and were found to be major HIV-suppressive factors produced by CD8⁺ T cells. The V3 loop of gp120 determines interaction with the chemokine receptor. CD4, in addition to providing a docking surface for the gp120 glycoprotein of HIV-1 promotes exposure of the V3 domain on gp120 that can interact with the chemokine receptor CCR-5 (Scarlatti et al, 1997; reviewed by D’Souza and Harden, 1996).

A small number of individuals (1% in populations of European descent but much lower in non-Caucasian populations) remain uninfected despite multiple high risk exposures; such individuals have a homozygous defect in the CCR-5 receptor gene which consists of a 32-bp deletion in the region encoding the second extracellular loop of the receptor; the defective protein is not detected at the cell surface; this defect prevents the proper interaction and entry of HIV-1 into their T cells and macrophages (Liu et al, 1996). This defect precludes infection from HIV-1 from all routes. The disease progresses much slower in heterozygotes for the 32-bp deletion (18% in populations of European descent) who are not protected from HIV-1 infections. This finding offers the hope of reconstituting the immune system of HIV-infected individuals with CD34⁺ stem cells from fetal cord blood or stem cells and lymphoid tissue from individuals who carry the homozygous deletion in the CCR-5 gene, an approach to deal with the immune rejection problems in patients with heart and kidney transplants.

The identification of chemokine receptors and their role in HIV-1 infections has closed a major gap in AIDS research; transgenic animals can now be produced expressing both human CD4 and chemokine receptors to evaluate the efficacy of AIDS therapeutics and testing vaccines; new prophylactic or therapeutic vaccines can be designed by immunization with portions or the entirety of CCR-5 and/or gp120 to generate appropriate antibodies (D’Souza and Harden, 1996). Inactivation of the CCR-5 co-receptor to mimic the natural resistance of the CCR-5-defective individuals, in cultured lymphocytes, rendered them viable and resistant to macrophage-tropic HIV-1 infection (Yang et at, 1997).

B. Therapeutic strategies against HIV

A number of therapy strategies emerged soon after identification of HIV as the etiologic agent of AIDS. Virtually every stage in the viral life cycle and every viral gene product is a potential target. Albeit major efforts for the combat of HIV have focused on the development of antiviral drugs and preventive vaccines, a number of studies have been aimed at eliminating HIV with gene therapy. The intracellular immunization approach (Baltimore, 1988) has prompted the advent of molecular tactics for inhibiting replication and infection of HIV (Trono et al, 1989; Malim et al, 1989). Four main targets have been defined in HIV therapeutics: (i) viral RNAs using ribozymes and antisense RNAs; (ii) viral proteins using RNA decoys, trans-dominant viral proteins, intracellular single-chain antibodies, and soluble CD4; (iii) infected cells aimed at eliminating those with transfer and expression of suicide genes; and (iv) the immune system by in vivo immunization (see Corbeau et al, 1996). Such gene therapeutic approaches can be combined with potent antiretroviral drugs especially the potent reverse transcriptase and protease inhibitors (Junker et al, 1997).

The elucidation of the chemokine receptor mechanisms for entry of HIV-1 into T cells and macrophages provides new tactics for intervention at the level of interaction of gp120 with CCR-5. Inactivation of the CCR-5 gene might be achieved (i) with triplex oligonucleotide technologies once critical transcription factor binding sites in the regulatory region of the gene have been determined; (ii) with saturation of the blood of infected individuals with ligands, selected from peptide libraries, that block the extracellular domains of the CCR-5 receptor and preclude interaction with gp120; (iii) with antagonists of RANTES (see above) that lack chemotactic activity but can block HIV infections (Arenzana-Seisdedos et al, 1996).

Specificity for the ablation of HIV Tat-expressing cells has been achieved through the use of the promoter element from the long terminal repeat (LTR) of HIV (Venkatesh et al, 1990; Caruso et al, 1992).

C. Gene therapy against HIV in cell culture

Strategies for HIV gene therapy include the inactivation of the CCR-5 coreceptor which is accomplished by targeting a modified CC-chemokine to the endoplasmic reticulum to block the surface expression of newly synthesized CCR-5 (Yang et al, 1997). A different gene therapy strategy proposed is targeting Tat, an early regulatory protein that is critical for viral gene expression and replication and which transactivates the LTR of HIV-1 via its binding to the transactivation response element (TAR); Tat also superactivates the HIV-1 promoter via activation of NF-kB in a pathway involving protein kinase C and TNF-a; combinations of the NF-kB inhibitors, pentoxifylline and Go-6976, with a stably expressed anti-Tat single-chain intracellular antibody suppressed HIV-1 replication and LTR-driven gene expression (Mhashilkar et al, 1997). Production of recombinant retrovirus containing the HSV-tk gene coupled to the HIV-2 promoter and Tat responsive region (TAR) has been used on human and mouse cells in culture for the specific elimination of HIV Tat-expressing cells; since the HIV-2 promoter can sustain a considerable level of basal expression in the absence of its activator, Tat, a number of modifications were made to the HIV-2 promoter in order to minimize toxicity to non-infected cells.

Retroviruses export unspliced, intron-containing RNA to the cytoplasm of infected cells despite the fact that intron-containing cellular RNAs cannot be exported; this export pathway is a critical step in the HIV-1 life-cycle. In HIV-1 this is accomplished through an interaction between the viral regulatory Rev protein and the Rev response element (RRE) RNA. In the absence of Rev, these intron-containing HIV-1 RNAs are retained in the nucleus. The nuclear export sequence (NES) LQLPPLERLTL has been identified on Rev that is responsible for its export to the cytoplasm (see Boulikas, 1998, this volume for more details). Targeting of Rev has provided a framework for novel interventions to reduce virus production in the infected host. Because disruption of either Rev or the RRE will completely inhibit HIV-1 replication, an anti-HIV-1 intracellular immunization strategy was developed based on RRE region-specific hammerhead ribozymes and on the intracellular expression of an anti-HIV-1 Rev single chain variable fragment (Sfv), which specifically targets the Rev activation domain. This combination resulted in a potent inhibition of HIV-1 replication in cell culture that holds promise as a future therapeutic regimen (Duan et al, 1997). To disable Rev function, primary T cells or macrophages were transduced with a recombinant AAV carrying an anti-Rev single-chain immunoglobulin (SFv) gene or an RRE decoy gene or with combinations of the two genes to disrupt the interaction between Rev and the RRE; when the transfected cells were then challenged by either clinical or laboratory HIV-1 isolates, this genetic antiviral strategy effectively inhibited infection (Inouye et al, 1997).

A synergic effect of anti-Tat and anti-Rev molecules was found when the RRE sequence was cloned 3' to a tat transdominant negative mutant (tat22/37) gene; for this strategy Jurkat cells were transduced with the recombinant retroviruses containing the tat22/37 gene and an RRE decoy in different positions or the tat22/37 and the RevM10 transdominant negative mutant genes to produce monoclonal and polyclonal cultures expressing the integrated genes; none of these recombinant constructs inhibited virus replication at a high multiplicity of infection (MOI) and combination of tat and rev mutants was ineffective in inhibiting HIV-1 replication at both low and high MOIs; however, at a low MOI, two cell clones containing tat22/37 and the RRE decoy in 3' position showed a long lasting protection against virus replication and in two cell clones, expressing the RevM10 mutant alone, the HIV-1 replication was efficiently blocked (Caputo et al, 1997).

IL-16 is secreted by activated CD8⁺ T lymphocytes and acts on CD4⁺ T lymphocytes, monocytes and eosinophils. Recently, the C-terminal 130-amino acid portion of IL-16 was shown to suppress HIV-1 replication in vitro. HIV replication was inhibited by as much as 99% in HIV-1-susceptible CD4⁺ Jurkat cells following transfection and expression of the C-terminal 130-amino acid portion of IL-16; the mechanism of HIV-1 inhibition by IL-16 was not at the level of viral entry or reverse transcription, but at the expression of mRNA (Zhou et al, 1997).

The in vitro antiviral efficacy of two gene therapy strategies (trans-dominant RevM10 and Gag antisense RNA) were tested in combination with the clinically relevant reverse transcriptase inhibitors AZT and ddC or the protease inhibitor indinavir by Junker et al (1997). The combination of RevM10 or Gag antisense RNA with antiviral drugs inhibited HIV-1 replication 10-fold more effectively than the single antiviral drug regimen alone in retrovirally transduced human T cell lines after inoculation with high doses of HIV-1HXB3 in the presence or absence of inhibitors. The level of anti-HIV-1 activity of the psi-gag antisense sequence correlated with the length of the antisense transcript and maximal anti-HIV efficacy was observed with complementary sequence more than 1,000 nucleotides long, whereas transcripts less than 400 nucleotides long failed to inhibit HIV-1 replication in a T-cell line and in primary peripheral blood lymphocytes (Veres et al, 1996).

The HSV-1 and HSV-2 virion host shutoff gene (vhs), each of which encodes a protein that accelerates the degradation of mRNA molecules leading to inhibition of protein synthesis, was used as a suicide gene for HIV gene therapy to inhibit replication of HIV; an infectious HIV proviral clone was cotransfected into HeLa cells together with the vhs gene under control of the CMV IE promoter; HSV-1 vhs gene driven by the HIV LTR inhibited HIV replication more than 44,000-fold in comparison to a mutant vhs gene (Hamouda et al, 1997).

The specificity of the Vpr protein for the HIV-1 virus particle was exploited to develop an anti-HIV strategy targeting the events associated with virus maturation; nine cleavage sites of the Gag and Gag-Pol precursors were added to the C terminus of Vpr and the chimeric Vpr genes were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity; the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2 completely abolished virus infectivity (Serio et al, 1997).

D. Gene therapeutic strategies for AIDS

A gene therapy strategy to combat acquired immunodeficiency syndrome (AIDS) in individuals already infected with HIV-1 has been directed toward GM-CSF mobilized peripheral blood CD34⁺ cells isolated from HIV-1-infected individuals and transduced with retroviral vectors containing three different anti-HIV-1-genes: (i) the Rev binding domain of the RRE (RRE decoy) carrying also the Neo^R gene, (ii) a double hammerhead ribozyme vector targeted to cleave the tat and rev transcripts (L-TR/TAT-neo), and (iii) the RevM10 transdominant negative mutant gene. After selection with G418, transduced cultures displayed up to 1,000-fold inhibition of HIV-1 replication following challenge with HIV-1 (Bauer et al, 1997).

A safe strategy to gene therapy of AIDS aimed at reducing the virus load in HIV-1-infected individuals was developed by Nakaya et al (1997). The Rev protein shifts RNA synthesis to viral transcripts by binding to the RRE within the env gene. Anti-Rev chimeric RNA-DNA oligonucleotides, consisting of 29 or 31 nucleotides, were designed to inhibit the Rev regulatory function and as decoys on HIV-1 replication; anti-Rev oligonucleotides containing an RNA "bubble" structure of 13 oligonucleotides (that bound to Rev with high affinity) were found to reduce more than 90% of the HIV-1 production from infected human T-cell lines and from healthy donor-derived peripheral blood mononuclear cells; control oligonucleotides without the bubble structure, that bound to Rev with considerably less affinity, did not reduce HIV-1 production (Nakaya et al, 1997).

E. Gene therapy of HIV with ribozymes

Antisense, ribozyme, or RNA aptamers, must be efficiently transcribed, stabilized against rapid degradation, folded correctly, and directed to the part of the cell where they can be most effective. Among (i) antisense RNA, (ii) hairpin and hammerhead ribozymes, and (iii) RNA ligands (aptamers) for Tat and Rev RNA binding proteins, Rev-binding RNAs but not the others, efficiently blocked HIV-1 gene expression when tested in expression cassettes based on the human tRNA(met) and U6 snRNA promoters. In situ localization of both tRNA and U6 promoter transcripts revealed primarily punctate nuclear patterns (Good et al, 1997).

Isolation of an RNA aptamer that can bind with high affinity to Tat protein, including two TAR-like RNA motifs for higher-affinity binding to Tat peptides provided a novel therapeutic strategy against HIV (Yamamoto et al, 1998, this volume).

A hairpin ribozyme specific for simian immuno-deficiency virus (SIV) and HIV-2 was used to inhibit viral replication in T lymphocytes derived from transduced CD34⁺ progenitor cells. Retroviral transduction of rhesus macaque CD34⁺ progenitor cells with the SIV-specific ribozyme “gene” and the selectable marker neomycin phosphotransferase (Neo^R) gene, followed by expansion and selection with the neomycin analog G418, rendered CD4⁺ T cells (derived from the transduced CD34⁺ hematopoietic cells) highly resistant to challenge with SIV; CD4⁺ T cells exhibited up to a 500-fold decrease in SIV replication, even after high multiplicities of infection (Rosenzweig et al, 1997).

Monomeric (targeting one site) and multimeric (targeting nine highly conserved sites) hammerhead ribozyme genes both directed against the HIV-1 envelope (Env) mRNA were stably expressed in a human CD4⁺ T lymphocyte cell line; whereas the monomeric ribozyme caused a delay in HIV-1 replication, the multimeric ribozyme caused complete inhibition in HIV-1 replication for up to 60 days after infection (Ramezani et al, 1997).

A multitarget ribozyme of an unusually large size (3.7 kb) had a notable antiviral potential which may lead to a gene therapy approach; this ribozyme, directed against multiple sites within the gp120 coding region of HIV-1 RNA, co-localized with unspliced HIV-1 pre-mRNA and/or genomic HIV-1 RNA in the nucleus catalyzing the reduction of all spliced and unspliced HIV-1 RNAs; the same ribozyme functioned as a mRNA for a chimeric CD4/Env protein in the cytoplasm (Paik et al, 1997).

Antisense oligonucleotides for HIV (anti-TAT) coupled with the influenza HA-2 protein-derived N-terminal fusogenic peptide have improved 5- to 10-fold their antiviral potency (Bongartz et al, 1994).

F. Novel therapies based on HIV vectors

A major drawback in the gene therapy of HIV has been the poor efficiency of gene transfer in vivo; especially important for HIV, most recombinant retroviruses transduce poorly cells harboring HIV, such as monocytes and macrophages, which are nondividing. Transfection of a fibroblast cell line with a HIV vector, bearing a deletion of the major packaging sequence has provided an HIV-1 packaging cell line which produced a large amount of HIV-1 structural proteins and non-infectious mature particles with normal reverse transcriptase activity but lacked RNA. When this cell line was stably transfected with an HIV-1-based retroviral vector virions were produced capable of transducing CD4-positive cells with efficiencies up to 10⁵ cells per ml (Corbeau et al, 1996).

For more details see VI. HIV vectors for gene transfer.

E. Clinical trials involving HIV

Gene therapy approaches directed against viral targets have been successful at inhibiting HIV-1 replication in cultured human cells; however, clinical trials involving gene therapy directed at HIV-1 are still in their infancy (reviewed by Gottfredsson and Bohjanen, 1997). Treatment of HIV-1 infections using gene therapy for intracellular immunization strategies is currently being tested in clinical trials. The first RAC-approved phase I study on HIV was to evaluate the safety of cellular adoptive immunotherapy using genetically modified CD8⁺ HIV-specific T cells in HIV seropositive individuals (protocol #15, Appendix 1). The significant number of ongoing clinical trials directed against HIV can be found on protocols 24, 40, 47, 52, 55, 73, 78, 79, 81, 85, 86, 88, 91, 94, 105, 108, 116, 117, and 168 in Appendix 1.

XXX. Familial hypercholesterolemia (FH)

A. Molecular mechanisms for FH

FH is an autosomal dominant disorder caused by a defect in the low density lipoprotein (LDL) receptor gene. LDL receptor on hepatocytes clears LDL from the serum; patients with one abnormal LDL receptor allele suffer premature coronary disease and myocardial infarction whereas patients with two abnormal alleles have extraordinarily high levels of cholesterol and accelerated atherosclerosis developing life-threatening coronary artery disease in early childhood. One type of mutation in the LDL receptor gene has incurred by Alu-Alu recombination deleting several exons and thus producing a truncated receptor molecule with loss of function (Lehrman et al, 1987).

Treatment of patients with FH is accomplished through the administration of drugs that stimulate the expression of LDL receptor from the normal allele in order to lower the plasma level of LDL; however this regimen is not effective for the treatment of homozygous deficient patients, especially those that retain less than 2% of residual LDL-R activity. A more direct approach has been to correct the deficiency of hepatic LDL receptor by transplanting a liver that expresses normal levels of LDL receptor; three patients that survived this procedure normalized their serum LDL-cholesterol (see Wilson et al, 1992 for references).

B. Gene therapy of FH: experiments in cell culture

A transmissible retroviral vector containing a full-length human cDNA for the LDL-R was used to infect fibroblasts from the Watanabe heritable hyperlipidemic (WHHL) rabbit which expressed the human receptor efficiently, as indicated by RNA and ligand blotting studies (Miyanohara et al, 1988). The number of hepatocytes that could be transduced by retroviruses bearing the therapeutic gene was one of the limiting steps that could impair the success of this strategy; addition of human hepatocyte growth factor (HGF) to hepatocytes allowed marked increase in the transduction efficiency in mouse (up to 80%) and human (40%) hepatocytes (Pages et al, 1995). Transduction of the human LDL-R cDNA under the transcriptional control of the liver-type pyruvate kinase promoter allowed high and tissue specific expression of the gene in primary hepatocytes; a second vector with a housekeeping promoter corrected the LDL-R deficiency in fibroblasts from a FH patient (Pages et al, 1996b).

C. Gene therapy of FH: experiments on animals

Liver is the preferred target organ for gene transfer-mediated treatment of FH. The presence of unique receptors at the cellular membrane of hepatocytes forms the basis for transfer strategies based on receptor targeting (reviewed by Sandig and Strauss, 1996). An authentic animal model used in FH gene therapy is the Watanabe heritable hyperlipidemic rabbit which is homozygous for FH and has a deletion in a cysteine-rich region of the LDL receptor gene; this renders the receptor completely dysfunctional (Yamamoto et al, 1986); these animals display high levels of serum cholesterol, diffuse atherosclerosis, and die prematurely. Liver tissue was removed from such animals and the cultured hepatocytes were transduced with retroviruses carrying the rabbit LDL receptor gene; the genetically corrected cells were transplanted into the animal from which they were derived. This treatment resulted in a 30-40% reduction in serum cholesterol that lasted for at least 4 months (Chowdhury et al, 1991).

The portal vein has been used for liver targeting in New Zealand White (NZW) rabbits. Expression of lacZ was obtained in virtually all hepatocytes within 3 days (but was undetectable by 3 weeks) after transfer of the lacZ reporter gene under the control of different promoters using recombinant, replication-defective adenoviruses which were infused into the portal circulation. An adenovirus human LDL-R cDNA was then infused into the portal vein of rabbits deficient in LDL receptor and demonstrated human LDL receptor protein in the majority of hepatocytes that exceeded the levels found in human liver by at least 10-fold. Transgene expression diminished to undetectable levels within 3 weeks (Kozarsky et al, 1994).

To demonstrate feasibility of the ex vivo FH therapy, three baboons underwent a partial hepatectomy, their hepatocytes were isolated, cultured, transduced with a retrovirus containing the human LDL-R gene, and infused via a catheter that had been placed into the inferior mesenteric vein at the time of liver resection (Grossman et al, 1992). Gene replacement therapy of human LDL receptor gene into the murine model of FH transiently corrected the dyslipidaemia; long-term expression of the therapeutic gene was extinguished by humoral and cellular immune responses to LDL receptor which developed and possibly contributed to the associated hepatitis (Kozarsky et al, 1996).

As an alternative strategy, expression in the liver of the very low density lipoprotein (VLDL) receptor, which is homologous to the LDL receptor but has a different pattern of expression, using recombinant adenoviruses corrected the dyslipidaemia in the FH mouse; transfer of the VLDL receptor gene circumvented immune responses to the transgene leading to a higher duration of metabolic correction (Kozarsky et al, 1996).

Replication-defective adenovirus-mediated gene transfer of the very low density lipoprotein receptor (VLDL-R) driven by a cytomegalovirus promoter in LDL-R knockout mice by a single intravenous injection resulted in reduction in total cholesterol by approximately 50% at days 4 and 9 and returned toward control values on day 21. Lowering in total cholesterol was mediated by a marked reduction in the intermediate density lipoprotein/low density lipoprotein (IDL/LDL) fraction. In treated animals, there was also an approximately 30% reduction in plasma apolipoprotein (apo) E accompanied by a 90% fall in apoB-100 on day 4 of treatment. Thus, adenovirus-mediated transfer of the VLDLR gene induced high-level hepatic expression of the VLDL-R, resulted in a reversal of the hypercho-lesterolemia, and enhanced the ability of these animals to clear IDL (Kobayashi et al, 1996).

The human apolipoprotein E (apoE) gene driven by the cytochrome P450 1A1 promoter produced transgenic mice where robust expression of apoE depended upon injection of the inducer b-naphthoflavone; a transgenic line exhibiting basal expression of apoE in the absence of the inducer upon breeding with hypercholesterolemic apoE-deficient mice produced animals which were as hypercholesterolemic as their nontransgenic apoE-deficient littermates in the basal state. When injected with the inducer, plasma cholesterol levels of the transgenic mice decreased dramatically. The inducer could pass transplacentally and via breast milk from an injected mother to her suckling neonatal pups, giving rise to the induction of human apoE in neonate plasma (Smith et al, 1995).

Other potential approaches for the treatment of FH include transfer of the apolipoprotein (apo) B mRNA editing protein which is an essential catalytic component of the apoB mRNA editing enzyme complex. This enzyme deaminates a cytidine residue at nucleotide position 6666 in apoB mRNA, converting it to uridine leading to the production of apoB-48 in place of apoB-100. The editing protein exists as a homodimer and can be used as a therapeutic agent to reduce apoB-100; somatic gene transfer of the editing protein cDNA was highly effective in lowering plasma low density lipoproteins (Chan et al, 1996).

D. Clinical trials on FH

The first clinical trial for gene therapy in the liver, based on ex vivo gene delivery, has shown both the feasibility and the limits of the current technology. According to this protocol, cultured hepatocytes from patients homozygous for mutations in the LDL receptor gene were proposed to be transduced ex vivo with the LDL receptor gene and transplanted by infusion into the portal vein of the patient (Wilson et al, 1992). A 29 year old woman, with homozygous FH, was transplanted with autologous hepatocytes that were genetically corrected with recombinant retroviruses carrying the LDL receptor gene. She tolerated the procedures well and in situ hybridization of liver tissue four months after therapy revealed evidence for engraftment of transgene-expressing cells. The patient's LDL/HDL ratio declined from 10-13 before gene therapy to 5-8 following gene therapy, an improvement which remained stable for the 18-month duration of the treatment (Grossman et al, 1994).

Five patients, ranging in age from 7 to 41 years, with homozygous FH, underwent hepatic resection and placement of a portal venous catheter; primary hepatocyte cultures from the resected liver were transduced with the human LDL-R gene with a retrovirus and the cells were then transplanted into the liver through the portal venous catheter. The liver-directed ex vivo gene therapy was accomplished safely and, in a child patient, normalization of cholesterol levels took place; LDL-R expression was detected in a limited number of hepatocytes of liver tissue four months after hepatocyte implantation from all five patients whereas significant and prolonged reductions in LDL cholesterol were demonstrated in three of five patients. The major obstacle in this first trial was the level and duration of LDL-R expression which “precluded a broader application of liver-directed gene therapy without modifications supporting substantially greater gene transfer” (Grossman et al, 1995; Raper et al, 1996).

XXXI. Angiogenesis and human disease

A. Formation of new blood vessels (angiogenesis)

Blood vessels, named in anatomy on the basis of their luminar diameter, branching, position and organ supplied, are formed with their proper diameter in the embryo before the heart starts beating. During a complex developmental program leading to formation of the cardiovascular system angioblasts are derived from mesoderm; this process requires the action of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Angioblasts then differentiate into endothelial cells which undergo proliferation, migration, and morphologic organization in the context of their surrounding tissues to form the blood vessels (reviewed by Folkman and D’Amore, 1996).

Angiogenesis virtually never occurs physiologically in adult tissues except in the ovary, the endometrium and the placenta. During this process, which is also essential in pathological situations such as wound healing and inflammation, formation of new microvessels from parent microvessels takes place. The process involves remodeling of the basement membrane and interstitial extracellular matrix (ECM) using degrading proteases produced by the endothelial cells and other adjacent cells, and the synthesis of ECM. The endothelial cells are able to synthesize and secrete cytokines. The angiogenesis is strictly controlled by a redundancy of pro- and anti-angiogenic paracrine peptide molecules. The tumor suppressor p53 protein has been shown to control the expression and synthesis of two anti-angiogenic factors (reviewed by Norrby, 1997).

Blood vessels in embryogenesis are formed in two stages: (i) during vasculogenesis newly differentiated endothelial cells coassemble into tubules that further fuse forming the primary vasculature of the embryo; (ii) angiogenesis, involves sprouting of new capillary vessels from preexisitng vasculature, also occurring into initially avascular organs such as the brain and kidney, and remodeling of the primary vascular network into large and small vessels (Davis et al, 1996). During tumor angiogenesis endothelium gives rise to new vessels which requires (i) dissolution of the basement membrane, (ii) migration and (iii) proliferation of endothelial cells, (iv) formation of the vascular loop, and (v) formation of a new basement membrane; VEGF and PDGF act directly on endothelial cells and may also activate inflammatory cells to synthesize angiogenic factors such as SPARC (Secreted Protein Acidic and Rich in Cysteine) (reviewed by Jendraschak and Sage, 1996).

Additional angiogenic factors include bFGF, angiopoietin-1, and thymidine phosphorylase whereas naturally occurring inhibitors of angiogenesis, other than angiostatin and vasculostatin include thrombospondin (see below, reviewed by Bicknell and Harris, 1996). Tissue factor (TF), which is the principal cellular initiator of coagulation and its deregulated expression has been implicated in cancer and inflammation has a documented role in tumor-associated angiogenesis (reviewed by Carmeliet et al, 1997).

In the adult, angiogenesis accompanies the pathological processes of neovascularization during tumor growth, wound healing, and diabetic retinopathy and the normal processes of ovulation and placental development. There is a difference between vasculogenesis and angiogenesis: vasculogenesis occurs only during early embryogenesis resulting in the formation of the primordia of the heart and large vessels; on the other hand, angiogenesis is required for both the embryonic and postnatal tissues (for references see Davis et al, 1996). These processes involve two families of receptor tyrosine kinases, the Flt-1, Flk-1 receptors which interact with VEGF and the TIE (or Tek) receptor tyrosine kinases which are stimulated by angiopoietin-1 (see below).

B. Vascular endothelial growth factor (VEGF)

VEGF (also known as vascular permeability factor, VPF) is a secreted protein related to PDGF which has been cloned (Keck et al, 1989; Leung et al, 1989; Abraham et al, 1991). VEGF is a mitogen for endothelium required for the differentiation of mesoderm-derived angioblasts into endothelial cells which form de novo vessels during embryogenesis, and in pathological states such as cancer and wound healing. VEGF is required for both vasculogenesis and angiogenesis. Targeted disruption of the VEGF gene in mice resulted in delayed differentiation of endothelial cells, impairment of vasculogenesis and angiogenesis and death at 8.5 to 9 days of gestation (Carmeliet et al, 1996; Ferrara et al, 1996).

VEGF is a tumor secreted protein but also is synthesized in specialized endothelial and epithelial cells (primarily in alveolar walls of the lung, kidney glomeruli, heart, and adrenal gland, and secondarily in liver, spleen, and in autonomic nerves supplying the smooth muscle layers of the gastrointestinal tract) and in embryonic tissues; placenta expresses a related protein, placenta growth factor (PGF). The expression of VEGF in corpus luteum in primate ovaries is under hormonal control (reviewed by Senger et al, 1993). The positive staining for VEGF in normal corpus luteum is coincidental with the angiogenesis incurring concurrently with development and differentiation in this tissue; on the other hand, the expression of VEGF in lung, kidney, heart, GI tract, and adrenals, where angiogenesis is not normally occurring, might serve to maintain a certain density of endothelial cells in these tissues as well as for inducing and maintaining the base-line vascular permeability for plasma proteins, including antibodies and lipoproteins (reviewed by Senger et al, 1993).

VEGF is produced in four isoforms by alternative splicing giving polypeptides of 210, 189, 165, and 121 amino acid residues (Leung et al, 1989; Keck et al, 1989; Abraham et al, 1991); VEGF is overexpressed in about 70% of hepatocellular carcinomas and other tumors (Suzuki et al, 1996).

At least three pathophysiological roles for VEGF have been unraveled: (i) Primarily, VEGF induces angiogenesis (Plate et al, 1992; Shweiki et al, 1992), i.e. sprouting of capillaries from preexisting blood vessels, a process occurring during embryonic development but also under pathological conditions such as wound healing, eye disease, and tumor growth; VEGF expression is an early event during carcinogenesis and is also involved in metastasis (Liotta et al, 1991). (ii) VEGF is a mitogenic factor primarily for vascular endothelial cells stimulating phospholipase C after high affinity binding to the receptor. (iii) VEGF also stimulates migration of monocytes across the endothelial cell monolayer. Other endothelial growth factors with angiogenic activity are the platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) (reviewed by Folkman and Klagsbrun, 1987).

C. VEGF receptors

The potential targets of secreted VEGF are proximal cells expressing VEGF receptors. Three VEGF receptors have been identified:

(i) A fms-like tyrosine kinase, the Flt-1 protein, (also called VEGF-R1), which is a transmembrane receptor with seven immunoglobulin-like domains in the extracellular region, a single transmembrane-spanning region, and a tyrosine kinase sequence (de Vries et al, 1992); Flt-1 is expressed in endothelial cells but is not found in non-endothelial cells (Shweiki et al, 1992). Disruption of the flt-1 gene permits differentiation of endothelial cells but interferes with a later stage of vasculogenesis causing thin-walled blood vessels with larger diameter and the death of mouse embryos at day 9 (Fong et al, 1995).

(ii) The Flk-1, (also called VEGF-R2), also a tyrosine kinase receptor. Flk-1 is endothelial cell-specific, already expressed in the angioblasts of the blood islands in early mouse embryos, progenitors of vascular endothelial cells. The Flk-1, (fetal liver kinase-1) was cloned independently and had been suggested to be involved in hematopoietic stem cell renewal. Flk-1 exhibits a high affinity for VEGF (Kd=10^-10 M) and is a major regulator of angiogenesis and vasculogenesis (Millauer et al, 1993). Hybridization of a parasagittal section of mouse embryos, at 14.5 days of development, with a single-stranded antisense DNA probe comprising the Flk-1 extracellular domain showed high levels of hybridization in the endothelial lining of the atrium in heart, endothelial cells in the peribronchial capillaries (but not bronchial epithelium) in the lung, the menings, and at the inner surface of the atrium and the aorta; this expression seemed to be restricted to endothelial cells in blood vessels and capillaries. Flk-1 was also expressed in capillaries in brain at postnatal day 4 (Millauer et al, 1993).

(iii) The Flt-4 (or VEGF-R3) (for references see Suri et al, 1996).

Transgenic mouse embryos with targeted disruption of the VEGF-R1 and R2 genes have unraveled the different roles these receptors undertake during development: Flt-1 regulates normal endothelial cell-cell or cell-matrix interactions during vascular development (Fong et al, 1995) and Flk-1 is required for the formation of blood islands and vascularization of the embryo; disruption of the flk-1 gene interfered with differentiation of endothelial cells leading to death of embryos at day 8.5 to 9.5 (Shalaby et al, 1995 ).

The temporal and spatial expression of VEGF exactly correlated with the expression pattern of Flk-1 in all tissues and developmental stages in mice examined suggesting a pivotal role of the growth factor/receptor duet in development and differentiation of the vascular system; their expression was high during development and declined in adulthood (Millauer et al, 1993).

Although VEGF expression showed a moderate increase during tumor development in pancreatic islets of Langerhans, the expression of the flt-1 and flk-1 receptor genes remained unchanged during pancreatic carcinogenesis in an animal model (transgenic mice having a targeting expression of the SV40 T antigen gene under control of the insulin gene regulatory regions in b-cells of the pancreatic islets) (Christofori et al, 1995).

D. Angiopoietin and its receptors

Although the TIE1 and TIE2 receptor tyrosine kinases were found to be involved in vasculogenesis already in 1992, angiopoietin-1, the natural ligand of TIE2 was only recognized in 1996 by secretion-trap expression cloning; according to this method entire cDNA libraries were transfected into large numbers of cells and the cell that contained the desired cDNA was uniquely marked on its surface by expression of the desired ligand; this rare cell was isolated within a background of millions of cells and was expanded leading to the isolation of the ligand-encoding cDNA (Davis et al, 1996). This finding represents a significant milestone in the effort to understand the molecular mechanisms that govern formation of blood vessels with important implications for cancer targeting by inhibition of angiogenesis.

Targeted disruption of the angiopoietin-1 gene in mice (Suri et al, 1996) gave defects in the blood vessels reminiscent of those caused by disruption in its receptor TIE2 (Dumont et al, 1994); these defects were mainly in the endocardium and myocardium but also generalized defects in vascular complexity leading to the death of mice by day 12.5 of gestation. These studies have established the important role of angiopoietin in mediating reciprocal interactions between the endothelium and surrounding matrix and mesenchyme (Suri et al, 1996). However, angiopoietin-1 does not display the mitogenic activities of VEGF on endothelial cells. The efficacy of disrupting the binding of angiopoietin to its ligand in tumor cell growth remains to be established.

A missense mutation in the receptor tyrosine kinase TIE2, resulting in a Arg to Trp substitution at position 849 of the kinase domain, was found in patients from two unrelated families suffering with venous malformations; this disorder is characterized by the presence of veins with large lumens lined by a monolayer of endothelial cells but with thin walls because of a reduction in smooth muscle layers. Expression of the wild-type and mutant TIE2 in insect cells has shown that the mutation caused a 6 to 10-fold increase in autophosphorylation activity of TIE2; thus, this mutation causes a defect in vascular remodeling and the overproliferation of endothelial cells without a complementary increase in smooth muscle layers (Vikkula et al, 1996). This finding demonstrates the significance of the TIE2 signaling pathway for endothelial cell-smooth muscle cell communication in venous morphogenesis.

E. Involvement of VEGF in tumor angiogenesis

Important for tumor growth are alterations in extracellular matrix. Solid tumors are composed of the malignant cells and the supportive vascular and connective tissue stroma whose synthesis is induced by the malignant cells. Fibrin is an essential component of the connective tissue stroma upon which tumor cells depend for their oxygen/nutrient supply and waste disposal. Fibrin gel provides solid tumors with a matrix which favors the ingrowth of macrophages, fibroblasts, and endothelial cells; these compose, along with neoangiogenesis vessels and elements found in normal connective tissue, the mature tumor stroma. Fibrinogen and other plasma proteins are found in increased amounts in tumor stroma and in healing wounds compared with normal stroma of most tissues; deposition of fibrin at the extravascular site requires a previous increase of permeability of the microvasculature thought to be mediated by VEGF (Senger et al, 1993).

VEGF is the factor largely responsible for leakiness and hyperpermeability of tumor blood vessels; leakage of plasminogen (which is converted to plasmin) and of fibrinogen and clotting factors II, V, VII, X, and XIII (responsible for the formation of the extravascular fibrin gel) through postcapillary venules in tumor cells is responsible for generating the supporting matrix for fibroblast migration, angiogenesis, and fibroplasia (reviewed by Senger et al, 1993). Migration of macrophages and fibroblasts in the fibrin gel in tumors is determined by fibrinogen concentration and Factor XIII crosslinking of a and g chains. Tumor cells as well as inflammatory cells in healing wounds produce a higher concentration of degradative proteases for extracellular matrix but also protease inhibitors which explains the resistance toward degradation of the fibrin gel in solid tumors (reviewed by Senger et al, 1993).

The mechanism of overexpression of VEGF in solid tumors might involve its induction by hypoxia (Shweiki et al, 1992); the rapid proliferation of the cells in the center of the tumor induces an increase in the interstitial pressure and may lead to closure of capillaries by compression; inefficient vascular supply, including the compensatory development of collateral blood vessels in ischaemic tissues, leads to neovascularization via production of VEGF. A clustering of capillaries alongside VEGF-producing cells in a subset of glioblastoma cells immediately proximal to necrotic foci have been observed in intracranial brain neoplasms obtained from surgical specimens; this was thought to be the result of a local angiogenic response elicited by VEGF (Shweiki et al, 1992). VEGF expression by hypoxia was also induced in skeletal muscle myoblasts, in the fibroblast mouse L cell line, and in cells from rat heart muscle (Shweiki et al, 1992).

Targeting of VEGF gene leading to its transcriptional inactivation (e.g. via triplex-forming oligonucleotides or antisense vectors) is expected to limit growth in solid tumors via inhibition in neo-vascularization; the prolonged sustenance of hypoxia in the center of the tumor is also expected to induce p53. An important concept to understand is that tumor angiogenesis results from a balance between angiogenic and anti-angiogenic factors. Expression of VEGF₁₆₅in rat C6 glioma cells and subcutaneous injection of the transduced cells in athymic mice has shown that tumors from cells expressing VEGF grew slower than tumors developed from nontransduced C6 cells, were highly vascularized, and contained varying degrees of necrosis and eosinophilic infiltrate (Saleh, 1996).

VEGF plays an important role not only in carcinogenesis but also restenosis (see below). VEGF promotes endothelial cell proliferation to accelerate re-endothelialization of the artery reducing intimal thickening; up-regulation of VEGF is the desired effect for treatment of restenosis (Asahara et al, 1996; Isner et al, 1996a). The transfer of the VEGF gene demonstrates a special mission of gene therapy: how to treat one human disease by upregulating the expression of a specific gene while treating a different disease by downregulating the expression of the same gene. Targeting is important. Also, exploring the molecular mechanisms affected by the transfer and overexpression of the cDNA of a gene, in all aspects and at their entire spectrum, is essential for a successful gene therapy application.

F. Transfer of the VEGF gene in ischemia

VEGF gene transfer can improve blood supply to the ischaemic limb and is a promising approach for the treatment of acute limb ischemia. In a gene therapy approach for tissue ischemia, the VEGF₁₆₅cDNA under the transcriptional control of the HSV immediate-early 4/5 promoter was used to transduce BLK-CL4 fibroblasts resulting in the secretion of high levels of biologically active VEGF; when the transduced cells were resuspended in basement membrane extract (matrigel) and were injected subcutaneously into syngeneic C57BL/6 mice they showed a strong angiogenic response (Mesri et al, 1995). Regional angiogenesis was induced in nonischemic retroperitoneal adipose tissue by adenoviral VEGF gene transfer supporting a 123% increase in vessel number compared to control (Magovern et al, 1997).

Treatment of a 71 year-old patient with an ischaemic leg with 2 mg phVEGF₁₆₅ plasmid applied to the hydrogel polymer coating of an angioplasty balloon and reaching the distal popliteal artery resulted in an increase in collateral vessels at the knee, mid-tibial, and ankle levels, which persisted for 12-weeks (Isner et al, 1996a).

Ischemia, induced in the hindlimb of rats by excision of the femoral artery, was experimentally treated by transfer of the VEGF gene; therapeutic angiogenesis produced morphologically similar, but significantly more extensive, networks of collateral microvessels (Takeshita et al, 1997). Direct i.m. injection of naked VEGF₁₆₅ plasmid DNA into the ischemic thigh muscles in rabbits resulted in more angiographically recognizable collateral vessels at 30 days posttransfection (Tsurumi et al, 1996, 1997).

G. Cancer treatment with angiogenesis inhibitors

On November 20, 1997 the first exciting data on clinical trials using the TNP-470, a drug extracted from fungi which inhibits angiogenesis, were reported in a speech before the National Institutes of Health by Judah Folkman (Harvard University). A woman in Texas with cervical cancer and metastasis to lungs had been tumor-free for months after treated with TNP-470; and a young girl with a slow-growing bone tumor in her jaw was cancer-free after treatment with IFN-a. Both TNP-470 and IFN-a are relatively weak inhibitors of blood vessel formation compared with angiostatin (O'Reilly et al, 1994, 1996), endostatin (O'Reilly et al, 1997), and vasculostatin which eliminated tumors in small animals. A combination therapy with angiostatin and endostatin was even more effective in tumor eradication; furthermore, these drugs have no apparent side effects and there is virtually no resistance of tumors to these drugs (see below). However, the number of human patients treated with angiogenesis inhibitors is too small and a larger number of cases need to be examined.

H. Angiostatin and endostatin

Angiostatin is a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, which is generated by cancer-mediated proteolysis of plasminogen to a 38 kDa plasminogen fragment; angiostatin selectively instructs endothelium to become refractory to angiogenic stimuli (O'Reilly et al, 1994, 1996). A number of enzymes including metalloelastase, pancreas elastase, plasmin reductase, and plasmin collaborate in the conversion of plasminogen to angiostatin. Systemic administration of angiostatin, but not intact plasminogen, inhibited neovascularization in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases (O'Reilly et al, 1994; Gately et al, 1997); human angiostatin inhibited almost completely the growth of three human and three murine primary carcinomas in mice without detectable toxicity or resistance (O'Reilly et al, 1996); these studies have developed the “dormancy therapy” for cancer based on that malignant tumors are regressed by prolonged blockade of angiogenesis to microscopic dormant foci in which tumor cell proliferation is balanced by apoptosis (O'Reilly et al, 1996).

Human angiostatin, administered to mice with s.c. hemangioendothelioma and associated disseminated intravascular coagulopathy (Kasabach-Merritt syndrome), significantly reduced tumor volume and increased survival (Lannutti et al, 1997).

PC-3 human prostate carcinoma cells release uPA and free sulfhydryl donors that converted plasminogen to angiostatin; these two components were sufficient for angiostatin generation (Gately et al, 1997); reduction of one or more disulfide bonds in the serine proteinase, plasmin, by a reductase secreted by Chinese hamster ovary cells triggered proteolysis of plasmin, generating fragments with the domain structure of angiostatin; two reductases (protein disulfide isomerase and thioredoxin) although able to produce biologically active angiostatin from plasmin and to inhibit proliferation of human dermal microvascular endothelial cells, were not the reductases secreted by cultured cells; instead the plasmin reductase factor secreted was a different one requiring reduced glutathione for activity (Stathakis et al, 1997). Two members of the human matrix metalloproteinase (MMP) family, matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9), hydrolyzed human plasminogen to generate angiostatin fragments; 58-, 42- and 38-kDa angiostatin fragments were generated; these studies implicated MMP-7 and MMP-9 in regulation of new blood vessel formation by cleaving plasminogen and generating angiostatin molecules (Patterson and Sang, 1997).

Recent studies exploring the mechanism responsible for the in vivo production of angiostatin that inhibits growth and metastasis in Lewis lung carcinoma have shown that angiostatin is produced by tumor-infiltrating macrophages whose metalloelastase expression is stimulated by tumor cell-derived GM-CSF (Dong et al, 1997).

Endostatin is a 20 kDa C-terminal fragment of collagen XVIII produced by hemangioendotheliomas which specifically inhibits endothelial cell proliferation, angiogenesis and tumor growth (O'Reilly et al, 1997).

Reports on the transfer of the angiostatin cDNA for the treatment of malignancies are about to appear (Toshihide Tanaka, personal communication).

XXXII. Gene therapy of restenosis

A. Pathophysiology of restenosis

The pathological situation, described as recurrent narrowing of a blood vessel after a successful revascularization procedure, has been termed restenosis (from the Hellenic stenos=narrow); the most frequent revascularization procedure has been the percutaneous transluminal angioplasty (PTA) used to treat atherosclerotic obstructions in the coronary and peripheral vascular circulations; PTA is achieved using a tiny balloon mounted on a catheter which is advanced under x-ray guidance to the site of a blocked artery. The most frequent artery suffering restenosis is the superficial femoral artery (SFA)/popliteal artery of the leg and the iliac arteries.

One of the factors contributing to restenosis is the intimal hyperplasia of the arterial wall; among others, the mechanism for intimal hyperplasia involves increasing the tissue levels of TGF-b following injury; injection of antibodies directed against TGF-b has blocked restenosis in a rat model (reviewed by Border and Noble, 1995). Transfer of the TGF-b gene into porcine arteries caused restenosis (Nabel et al, 1993a,b). Arterial injury has pleiotropic effects at the molecular level; for example, injury of rat arteries led to an increase in FGF receptors in vascular smooth muscle cells.

Atherosclerosis and restenosis following balloon angioplasty are characterized by two steps: during the thrombotic phase in the arterial wall following injury fibrin networks are synthesized with platelet depositions; this phase is followed by smooth muscle cell proliferation. Both the synthesis of fibrin from fibrinogen, as well as the proliferation of platelet and smooth muscle cells are upregulated by the protease thrombin. Inhibition of the action of thrombin in the arterial wall is a potential target against arterial disease. The most potent and specific inhibitor of thrombin known today is the polypeptide hirudin, an anticoagulant derived from the medicinal leech, Hirudo medicinalis; especially important is a local delivery of hirudin to prevent thrombosis circumventing the systemic coagulopathy associated with systemic administration of the purified protein (for references see Rade et al, 1996).

The pathogenesis of both atherosclerosis and restenosis involves the migration of medial smooth-muscle cells across the internal elastic lamina to form a neointima; inflammatory reactions involving T cells and other leukocytes maintain smooth-muscle cell migration, proliferation and matrix deposition. The stenotic response involves the expression of HA (hyaluronan) receptors on both the infiltrating white cells and on smooth-muscle cell populations; exposure of injured arteries to high concentrations of HA in vivo resulted in significant inhibition of neointimal formation (Savani and Turley, 1995). Intervening with the HA and receptor genes could provide potential molecular targets for restenosis.

A number of drugs have been developed to inhibit neointima formation such as the drug CVT-313, identified from a purine analog library; CVT-313 is a specific and potent inhibitor of CDK2 reducing hyperphosphorylation of RB (Brooks et al, 1997). The angiotensin-converting enzyme inhibitor, cilazapril, also prevented myointimal proliferation after vascular injury (Powell et al, 1989). However, most of these drugs are toxic and need continuous administration to the artery. Gene therapy could result in stable transfection of the arterial wall cells with the gene of a therapeutic protein circumventing these problems.

Significant progress has been made in the area of coronary restenosis, particularly in identifying target genes to reduce neointima formation in vein grafts used in coronary bypass surgery. Targets of gene therapy include the prevention of postangioplasty restenosis, postbypass atherosclerosis, peripheral atherosclerotic vascular disease and thrombus formation (reviewed by Malosky and Kolansky, 1996; Ylä-Herttuala, 1996).

B. VEGF gene transfer for restenosis

Thickening of the arterial intima, composed of smooth muscle cells, is an important area for intervention by gene transfer to alleviate the syptoms of restenosis following balloon treatment. Gene therapy for restenosis may be achieved following transfer of the gene encoding VEGF; this therapy has been applied to animal models and has now entered clinical trials (Isner et al, 1996a). Previous studies using administration of recombinant, 165 amino acid, VEGF protein to rabbits in vivo has shown a significant augmentation in collateral vessel development by angiography after excision of the ipsilateral femoral artery in the animal to induce severe hind limb ischemia (Takeshita et al, 1994a). The rationale behind this approach is that VEGF promotes endothelial cell proliferation (Leung et al, 1989) to accelerate re-endothelialization of the artery reducing intimal thickening and thrombogenicity (Asahara et al, 1996); the inner lining of the blood vessels is made up of endothelial cells which are important in preventing the formation of blood clots or atherosclerotic plaques. Animal studies have shown that endothelial cells require a relatively long time for growth after injury. "Naked" plasmid encoding the 165 amino acid VEGF isoform is being delivered using a hydrogel /polymer-coated balloon angioplasty catheter without use of liposomes or recombinant viruses to humans (Isner et al, 1996a).

Ylä-Herttuala and coworkers (Laitinen et al, 1997a) have examined and compared the efficiency of lacZ delivery to the rabbit carotid artery (using a collar method) with (i) plasmid/liposome complexes (Lipofectin), (ii) replication-deficient Moloney murine leukemia virus (MMLV)-derived retroviruses, (iii) pseudotyped vesicular stomatitis virus protein G (VSV-G)-containing retroviruses and (iv) adenoviruses. Transfer of the gene to the adventitia took place with all gene transfer systems tested except MMLV; the adenovirus gave the highest gene transfer efficiency and up to 10% of the cells displayed b-galactosidase activity compared with 0.05% of cells using VSV-G retrovirus, 0.05% with Lipofectin, and less than 0.01% with MMLV retrovirus (Figure 31).

During the collar application procedure extravascular gene transfer took place without intravascular manipulation; the model is suited for gene transfer studies involving difussible or secreted gene products (such as VEGF, see Figure 32) that act primarily on the endothelium; effects on medial smooth muscle cell (SMC) proliferation and even endothelium can be achieved from the adventitial side of the artery (Laitinen et al, 1997a).

Transfer of the VEGF gene was performed on rabbits using a silicone collar inserted around the carotid arteries. The collar acted as an agent that caused intimal SMC growth and as a reservoir for the VEGF gene; the model preserved the integrity of endothelial cells and permitted extravascular gene transfer without intravascular manipulation. A plasmid carrying the mouse VEGF₁₆₄ gene under control of the CMV promoter in a mixture with Lipofectin was injected under anesthesia by gently opening the collar (Laitinen et al, 1997b). As a control, arteries were injected with the lacZ cDNA; animals after 3, 7, or 14 days from the operation were sacrificed and the arteries were examined by electron microscopy using SMC-specific immunostaining (Figure 32A,B). One week after VEGF transfer with cationic liposomes there was a significant reduction in intimal thickening (Figure 32B) compared to control arteries (A). When the nitric oxide synthase inhibitor L-NAME was administered to the animals it abolished the therapeutic efficacy of VEGF and there was no VEGF-induced reduction in intimal thickening of the arteries (Laitinen et al, 1997b).

C. Transfer of other genes against restenosis and arterial injury

A number of vascular disorders can be treated by arterial gene transfer (Nabel et al, 1990, 1993a-c; Ohno et al, 1994; Takeshita et al, 1994b; Chang et al, 1995a,b). One of the drawbacks has been the low percentage of the cells transfected which were in the order of 1% to less than 0.1% using naked plasmid delivery with a balloon catheter (e.g. Isner et al, 1996a), cationic liposomes (Nabel et al, 1990, 1993a-c), or retroviruses (Flugelman et al, 1992). A variety of genes have been transferred into arterial wall cells. Transfer of the human growth hormone gene, a secreted protein, into rabbit arterial organ culture using lipofectin permitted determination of hGH levels in the culture medium using a sensitive immunoassay method (Takeshita et al, 1994b). The ADA gene has been transferred efficiently to vascular smooth muscle cells in rats (Lynch et al, 1992). Transfer of the fibroblast growth factor-1 gene (Nabel et al, 1993b), of the platelet-derived growth factor B gene (Nabel et al, 1993c), or of the transforming growth factor-b1 gene (Nabel et al, 1993a,b) into animal arteries promoted intimal hyperplasia and angiogenesis.

The therapeutic induction of angiogenesis in ischemic tissues using recombinant cytokines is also promising for clinical application (Norrby, 1997).

In vivo suppression of injury-induced vascular smooth muscle cell (VSMC) accumulation is a widely used approach (Ohno et al, 1994). Other than VEGF gene transfer (Isner et al, 1996a, see above), additional approaches for the treatment of restenosis after injury-induced VSMC accumulation is via delivery of the HSV-tk gene followed by ganciclovir treatment in order to kill preferentially the smooth muscle cells (Guzman et al, 1994; Ohno et al, 1994); by transfer of the cytosine deaminase (CD) gene the product of which is capable of metabolizing 5-fluorocytosine (5-FC) to 5-fluorouracil in a rabbit femoral artery model of balloon-induced injury (Harrell et al, 1997); by transfer of the RB (Chang et al, 1995a; Smith et al, 1997), or p21 genes (Chang et al, 1995b); by transfer of ras (Indolfi et al, 1995), TGFb gene (Grainger et al, 1995); and, by transfer of the nitric oxide synthase gene (von der Leyen et al, 1995).

At the molecular level, arterial injury results in exposure of vascular smooth muscle cells (VSMC) and fibroblasts to multiple growth factors that activate second messengers and induce expression of immediate-early genes within minutes to hours after stimulation resulting in the exit of VSMC from the quiescent G0 state. A series of CDKs are activated and tumor suppressor genes need to be down-regulated for VSMC proliferation including p53, p21, p16, and RB (reviewed by Muller, 1997). Therapeutic strategies to restrict neointima formation in the injured artery include (i) inhibition in the expression of proto-oncogenes (c-myc); (ii) transfer of suicide genes (HSV-tk, CD); (iii) use of molecular decoys or drugs to block specific steps required for cell cycle progression, (iv) transfer of tumor suppressor genes (p21, RB); (v) treatment with antisense oligonucleotides to down regulate genes required for cell proliferation or DNA synthesis (antisense cyclin G1, PCNA); (vi) transfer of a number of unrelated genes such as of gax, TGF-b, hirudin, PKCd, b-interferon (Table 7). It is worth considering that delivery of recombinant adenoviruses themselves causes (i) a pronounced infiltration of T cells throughout the artery wall; (ii) upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in arterial smooth muscle cells; (iii) neointimal hyperplasia (Newman et al, 1995).

Inhibition in proliferation of the smooth muscle cells has also been achieved by an antibody against basic fibroblast growth factor (Lindner and Reidy, 1991), by antisense oligodeoxynucleotides to c-myc applied in a pluronic gel to the arterial adventitia (Bennettet al, 1994), and by antisense phosphorothioate oligonucleotides to PCNA in a rat carotid artery injury model (Simons et al, 1994). Transfer of the protein kinase Cd gene to a rat clonal VSMC inhibited the proliferation of vascular smooth muscle cells by suppressing G1 cyclin expression and arrested the cells in the G0/G1 phase of the cell cycle; overexpression of PKCd caused reduction in the expression of cyclins D1 and E and RB phosphorylation, and increased the protein levels of p27 (Fukumoto et al, 1997).

RB is implicated in the control of the cell cycle via its interaction with E2F (see above); a phosphorylation competent, amino-terminal-truncated RB protein (Rb56) was a more potent inhibitor of E2F-mediated transcription relative to the full-length Rb construct (Rb110); adenoviral transfer of either Rb56 or Rb110 inhibited neointima formation after balloon injury in the rat carotid artery (Smith et al, 1997).

On the other hand, overexpression of the fibroblast growth factor-1 gene (Nabel et al, 1993b), platelet-derived growth factor B gene (Nabel et al, 1993c), and transforming growth factor b1 gene (Nabel et al, 1993a,b) into the arteries induced intimal hyperplasia in vivo.

p21 protein is a negative regulator of mammalian cell cycle progression that functions both by inhibiting cyclin dependent kinases (CDKs) required for the initiation of S phase and by binding to and inhibiting PCNA. p21 gene transfer has been used to inhibit vascular smooth muscle cell (VSMC) proliferation after arterial injury for the treatment of atherosclerosis and restenosis. Over-expression of human p21 inhibited growth factor-stimulated VSMC proliferation and neointima formation in the rat carotid artery (a model of balloon angioplasty) by arresting VSMCs in the G1 phase of the cell cycle. p21-associated cell cycle arrest was associated both with a significant inhibition of the phosphorylation of RB (see above) and with the formation of complexes between p21 and PCNA in VSMCs (Chang et al, 1995b). Transfer of p21 was also used by others to suppress neointimal formation in the balloon-injured porcine or rat carotid arteries in vivo; vascular endothelial and smooth muscle cell growth was arrested through the ability of p21 to inhibit progression through the G1 phase of the cell cycle (Yang et al, 1996; Ueno et al, 1997a).

Gax is a growth arrest gene which regulates proliferation of VSMC; gax is an homeobox gene whose expression in the adult is largely confined to cardiovascular tissues. In contrast to a number of genes which are up-regulated following balloon injury and/or angioplasty, such as the early response genes c-myc and c-fos, gax is down-regulated within hours of balloon injury; gax may be required to maintain the gene expression of proteins in VSMC that are associated with the nonproliferative or contractile phenotype. Gax is also rapidly down-regulated in cultured VSMC upon stimulation by serum or platelet-derived growth factor (PDGF); like the genes in the gas and gadd families, gax is expressed at its highest levels in quiescent cells and is down-regulated following mitogen activation (Weir et al, 1995). Percutaneous gax adenovirus-mediated gene transfer into normal and atherosclerotic rabbit iliac arteries suggested prevention of neointimal formation (Maillard and Walsh, 1996). The gax-induced growth inhibition correlated with a p53-independent up-regulation of the cyclin-dependent kinase inhibitor p21; gax overexpression also led to an association of p21 with cdk2 complexes and a decrease in cdk2 activity and, thus, gax overexpression inhibited cell proliferation in a p21-dependent manner.

Ras proteins are key transducers of mitogenic signals from the membrane to the nucleus. DNA vectors expressing ras transdominant negative mutants, which interfere with ras function, reduced neointimal formation after injury in rats in which the common carotid artery was subjected to balloon injury (Indolfi et al, 1995). An adenoviral vector, expressing a potent dominant-negative mutated form of c-H-ras, in which tyrosine replaced aspartic acid at residue 57, completely inhibited serum-stimulated activation of mitogen-activated protein kinase, and abolished the DNA synthesis in response to serum mitogens in infected smooth muscle cells in culture. Application of the adenoviral vector into balloon-injured rat carotid arteries from inside the lumen resulted in a significant reduction in neointima formation (Ueno H et al, 1997b).

Nitric oxide-generating vasodilators inhibit vascular smooth muscle cell proliferation. S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide-releasing agent, inhibited the activity of cyclin-dependent kinase 2 (Cdk2) and the phosphorylation of RB but it did not inhibit the activities of cyclin D-associated kinases Cdk4 and Cdk6 (Ishida et al, 1997). Suppression of injury-induced vascular smooth muscle cell accumulation was also achieved by transfer of the endothelial cell nitric oxide synthase gene (von der Leyden et al, 1995). Based on the fact that administration of the nitric oxide (NO) synthase inhibitor L-NAME to rabbit carotids eliminated the difference in intimal thickening between VEGF and mock-transfected (lacZ) arteries it was proposed that VEGF may reduce smooth muscle cell proliferation via VEGF-induced NO production from the endothelium (Laitinen et al, 1997).

D. Atherosclerosis

The atherotic plaque is formed by a complex mechanism initiated by the accumulation of lipid, macrophages and T cells at artery lesions leading to smooth muscle cell proliferation (Ross, 1993). TGF-b is involved in atherosclerosis via its activation by plasmin and via its inhibition by atherogenic lipoprotein deposited on the arterial wall. Inhibition of TGF-b would lead to smooth muscle cell proliferation; patients with advanced coronary disease have decreased serum TGF-b levels; furthermore, transgenic mice overexpressing lipoprotein (a) exhibit decreased levels of TGF-b at sites of lipoprotein (a) accumulation such as in the aortic wall (Grainger et al, 1995). As prolonged overproduction of TGF-b may lead to tissue fibrosis by overproduction of extracellular cell matrix (Border and Noble, 1995) a balanced overexpression of TGF-b might be of great therapeutic value for heart disease.

The plasminogen system, via its triggers, t-PA and u-PA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), has been implicated in thrombosis, arterial neointima formation, and atherosclerosis (reviewed by Carmeliet et al, 1997).

A number of animal models have been used to induce atherosclerotic lesions such as the iliac arteries of New Zealand white rabbits fed with cholesterol. Substantial progress in vector development and the demonstration of efficacy in relevant animal models will be required before gene therapy for atherosclerosis becomes a clinical reality (Rader, 1997).

E. Acidic and basic fibroblast growth factors (aFGF, bFGF)

Acidic and basic fibroblast growth factors and their receptors are involved in many fundamental biological processes but also in pathological processes (Webster and Donoghue, 1998, this volume).

Whereas VEGF is a regulator of vascular permeability and an endothelial cell growth factor the acidic and basic fibroblast growth factor (aFGF, bFGF) and placenta growth factor (PGF) polypeptides are endowed with endothelial cell growth-promoting activity; however, FGFs have not been reported to be expressed in blood vessels in vivo. aFGF and bFGF seem to act as mitogens for a large number of different cell types; in situ hybridization analysis has shown that there is no expression of FGF receptors 1 and 2 (Flg and Bek) in capillary endothelial cells during embryonic development; it is only VEGF and PGF which are known to be specific for endothelial cells (for references see Millauer et al, 1993).

Many of the studies that have demonstrated therapeutic efficacy using fibroblast (aFGF, bFGF, FGF-5), endothelial (VEGF) and other types of factors using the purified peptides can potentially be transferred to the gene level. This will not require repeated administration of the drug, especially whenever the somatic cell targets are transfected efficiently and the expression of the transgene lasts.

Liposome-mediated gene transfer of antisense-oriented bFGF or fibroblast growth factor receptor-1 (FGFR-1) cDNAs in episomal vectors into human melanomas, grown as subcutaneous tumors in nude mice caused complete arrest or regression of the tumors as a result of blocked intratumoral angiogenesis and subsequent necrosis (Wang and Becker, 1997). Inhibition of bFGF synthesis in vivo using an antisense RNA strategy significantly inhibited intimal thickening after arterial balloon injury (Hanna et al, 1997).

Infection of human umbilical vein endothelial cell cultures with a bFGF-expressing recombinant adenovirus enhanced the proliferation rate and tubular formation of these cells on reconstituted basement membrane (Takahashi et al, 1997). Low level expression of bFGF upregulated Bcl-2 and delayed apoptosis in NIH3T3 cells; on the other hand cells expressing from 8-15 times background levels of bFGF became phenotypically transformed (gave dense foci at confluence, had decreased adherence to tissue culture plates and grew colonies in soft agar) (Wieder et al, 1997). Blood vessels of spontaneously hypertensive rats were shown to be associated with subphysiological amounts of bFGF; transfer of the bFGF gene corrected hypertension, restored the physiological levels of bFGF in the vascular wall, and ameliorated endothelial-dependent responses to vasoconstrictors (Cuevas et al, 1996).

F. Wound healing and plasminogen

A number of diseases including cancer, cancer metastasis, atherosclerosis, arthritis, hepatitis, dermatitis, inflammatory bowel disease, sickle cell anemia, and autoimmune disease result in severe tissue damage. Plasminogen has a profound importance in wound healing and might play a central role in many of these diseases (Rømer et al, 1996). Plasminogen is an inactive precursor-protease synthesized and secreted by the liver and converted into plasmin (trypsin-like serine protease) by the action of two different proteases: (i) tissue-type plasminogen activator (tPA) and (ii) urokinase-type plasminogen activator (uPA); in addition to uPA- and tPA-regulation, the activity of plasminogen is also controlled by plasminogen-specific cell surface receptors, by inhibitors of plasminogen activation (PAI-1 and PAI-2), and by the receptor of uPA (uPAR). Angiostatin is a 38 kDa plasminogen fragment generated by cancer-mediated proteolysis of plasminogen (O'Reilly et al, 1994, 1996, see angiostatin). Endostatin is a 20 kDa C-terminal fragment of collagen XVIII (O'Reilly et al, 1997); both angiostatin and endostatin inhibit angiogenesis and tumor growth.

Plasmin passes to extravascular fluids and stimulates proteolytic activity in the extracellular matrix (such as degradation of fibrin) but also contributes to the activation of growth factors and other proteases (see Rømer et al, 1996). Fibrin is an important component of the wound healing and reepithelization process and is formed from fibrinogen by the action of the protease thrombin (see Rade et al, 1996). These processes take place during wound healing but also during the process of atherosclerosis, restenosis, response to vascular injury, and in tumorigenesis during formation of the tumor stroma.

Plasminogen-deficient mice (transgenic animals produced by targeted disruption in the plasminogen gene) completed embryonic development and survived to adulthood but were predisposed to spontaneous thrombotic lesions in many tissues and displayed fibrin deposition in the liver (Bugge et al, 1995). These animals showed severe impairment in the healing of skin wounds; thus, plasminogen plays a central role in extracellular matrix degradation during wound healing in rodents in vivo and most likely also in humans (Rømer et al, 1996). Fibrin dissolution allows keratinocyte migration from incisional wound edges; detriment in fibrin degradation slows down wound repair by the limited ability of epidermis cells to dissect their way through the extracellular matrix beneath the wound crust (Rømer et al, 1996). Fibrin is a major component of the extracellular matrix in wounds and solid tumors but not in normal embryonic or adult tissue.

G. TGF-b in injury and wound healing

Transforming growth factor-b (TGF-b) is a cytokine implicated in the pathogenesis of impaired wound healing but also in autoimmune disease, malignancy, and atherosclerosis (Grainger et al, 1995). TGF-b has gained interest for the treatment of autoimmune disease, multiple sclerosis (autoimmune encephalomyelitis), and arthritis; however, prolonged overproduction of TGF-b may lead to tissue fibrosis by overproduction of extracellular cell matrix affecting kidney, liver, lung and other organs. TGF-b1 is involved in the pathogenesis of fibrosis by its matrix-inducing effects on stromal cells such as in activation of the pulmonary fibrotic process (Sime et al, 1997). Overexpression of TGF-b1 in the heart is thought to contribute to the development of cardiac hypertrophy and fibrosis (Villarreal et al, 1996). TGF-b can activate and then suppress T cells, macrophages, and leukocytes; transgenic mice with targeted disruption of the TGF-b gene die with autoimmune symptoms (reviewed by Border and Noble, 1995).

The preservation and architectural design of the extracellular matrix depends on the action of cytokine polypeptides. TGF-b controls the mitogenic action of platelet-derived growth factor (PDGF); in response to injury or disease, the production of TGF-b and PDGF are increased stimulating extracellular matrix production; this is accomplished by inhibition of proteases and stimulation of synthesis of extracellular matrix proteins by TGF-b. Failure of cells to produce enough TGF-b has been proposed to result in impaired wound healing in the elderly, glucocorticoid-treated individuals, and in diabetics; a single injection of TGF-b has been shown to accelerate wound healing (reviewed by Border and Noble, 1995).

TGF-b inhibits epithelial and smooth cell proliferation; it is believed that one of the factors contributing to the unrestricted growth of cancer cells is their nonresponsiveness to TGF-b because of loss of functional TGF-b receptors; microsatellite instability in colon cancer cells inactivates the type II TGF-b receptors (Markowitz et al, 1995). Tamoxifen, an anticancer drug, stimulates TGF-b thus inhibiting cancer cell proliferation. Restoration of the TGF-b receptor genes might constitute a strategy for treating human cancers (Border and Noble, 1995).

Transfer of the cDNA of porcine TGF-b1 to rat lung induced prolonged and severe interstitial and pleural fibrosis characterized by extensive deposition of the extracellular matrix (ECM) proteins collagen, fibronectin, and elastin (Sime et al, 1997). Particle-mediated delivery of mutant porcine constitutively active TGF-b1 cDNA to rat skin at the site of skin incisions increased tensile strength up to 80% for 14-21 days (Benn et al, 1996). Neurodegeneration associated with Alzheimer's disease is believed to involve toxicity to b-amyloid and related peptides; this neurotoxicity was significantly attenuated by single treatments with TGF-b1 and was prevented by repetitive treatments, a process associated with a preservation of mitochondrial potential and function (Prehn et al, 1996); this implies a potential avenue of TGF-b1 gene transfer for Alzheimer's disease.

Transfer of TGF-b1 cDNA in vivo suppresses local T cell immunity and prolonged cardiac allograft survival in mice; TGF-b1 gene transfer may become a new type of immunosuppressant avoiding the systemic toxicity of conventional immunosuppression (Qin et al, 1996).

GM-CSF overexpression induced TGF-b1 gene expression and secretion from macrophages purified from bronchoalveolar lavage fluid 7 days after GM-CSF gene transfer; these findings implicate GM-CSF in pulmonary fibrogenesis (Xing et al, 1997).

XXXIII. Cystic fibrosis (CF)

A. Molecular mechanism of CF pathogenesis

CF is a lethal recessive hereditary disorder characterized by abnormalities of the airway epithelium; it affects 1 in 2,000 Caucasians. Inflicted individuals show secretion of thick mucus and chronic colonization of the lung epithelium with pathogens such as Pseudomonas aeruginosa. The defect arises from mutations in the 250-kb gene encoding a 12-transmembrane domain glycoprotein (1480 amino acids), called cystic fibrosis transmembrane conductance regulator (CFTR), that modulates the permeability of Cl- in response to elevation of intracellular cAMP. The defect is caused by deletion of three base pairs eliminating a single phenylalanine residue at the center of the first nucleotide-binding domain of the CFTR protein (Riordan et al, 1989).

Much of the mortality seen in CF is related to chronic infection of the respiratory tract with Pseudomonas aeruginosa; Pseudomonas colonization has been attributed to increased numbers of specific cell-surface receptors and to the presence of mucus. Adherence of P. aeruginosa directly to the cell surface of CF airway epithelium (from noncultured nasal epithelial cells isolated from CF patients) was significantly increased over that in cells from healthy donors. Liposome-mediated CFTR gene transfer resulted in a significant reduction in the numbers of bacteria bound to ciliated CF epithelial cells (Davies et al, 1997).

The architecture of the lung and the terminal differentiation of the defective cells imposes a serious hurdle for ex vivo gene therapy for CF: the epithelial cells on the airway surface cover the successively branching structures of the lung making impossible their removal and reimplantation (Yoshimura et al, 1992).

Successful introduction of the entire 250 kb human CFTR gene locus and adjacent sequences into Chinese hamster ovary-K1 (CHO) cells which lack endogenous CFTR was achieved using yeast artificial chromosomes (YACs); integration of the human CFTR-containing YACs into the CHO genome took place on the order of one copy per genome; functional human CFTR was expressed from subclones and human CFTR expression in CHO cells was unexpectedly high (Mogayzel et al, 1997). This type of studies are very useful as a number of DNA control elements for CFTR may be “hidden” throughout the gene locus, including enhancers, ORIs, silencers, MARs, that participate in the tissue- and developmental stage-specific CFTR gene expression.

The airway epithelium is in the process of injury and regeneration in CF; regenerating poorly differentiated cells of human airway epithelium in culture were efficiently transfected with CFTR cDNA using adenoviral vectors; CFTR expression and cAMP-regulated stimulation of the cell membrane chloride ion secretion took place on these cells as determined by light fluorescence microscopy and scanning laser confocal microscopy (Dupuit et al, 1997).

B. CFTR gene transfer in animal models

Direct transfer of the human CFTR gene was achieved using a replication-defective adenovirus vector by intratracheal instillation into cotton rat lungs; the presence of human CFTR mRNA transcripts was detected by in situ hybridization with a cRNA (antisense) probe as well as by immunohistochemical evaluation using antibodies directed against the CFTR protein (Rosenfeld et al, 1992).

First generation adenovirus-mediated gene transfer of CFTR to the mouse lung resulted in the expression of viral proteins leading to the elimination of the therapeutic cells expressing CFTR by cellular immune responses; second generation E1-deleted viruses displayed substantially longer recombinant gene expression and induced a lower inflammatory response (Yang et al, 1994).

Adenoviral vector constructs with an E1-E3+E4ORF6+ backbone encoding CFTR (or b-galactosidase) produced declining levels of expression while a similar vector with an E1-E3+E4+ backbone gave rise to sustained, long-term reporter gene expression in the lung in nude mice; CTLs directed against either adenoviral proteins or b-galactosidase reduced expression in nude mice stably expressing b-galactosidase from the E4+ vector (Kaplan et al, 1997).

Aerosol delivery of an adenoviral vector encoding CFTR to non-human primates showed human CFTR mRNA in lung tissue from all treated animals on days 3, 7, and 21 post-exposure; other than some rather mild complications on individual animals ranging from an increase in lavage lymphocyte numbers to bronchointerstitial pneumonia, the treatment was rather safe (McDonald et al, 1997).

Adenoviruses elicit am immune response. Effective gene therapy for CF would ideally be accomplished with a vector capable of long-term expression of the CFTR in the absence of a host inflammatory response; in this respect AAV might be better suited. Administration of single doses of AAV-CFTR vector to 10 rhesus macaques by fiberoptic bronchoscopy to the right lower lobe of lungs showed that transfer of the CFTR gene occurred in bronchial epithelial cells of each animal by in situ DNA PCR; vector mRNA was detectable for 180 days after administration as detected by RT-PCR and there was no evidence of inflammation (Conrad et al, 1996).

Transfer of CFTR gene was also achieved using retroviral vectors; sodium butyrate treatment of murine retrovirus packaging cells producing the vector increased the production of the retrovirus vector between 40- and 1,000-fold (Olsen and Sechelski, 1995).

Yoshimura and coworkers (1992) have transduced airway epithelial cells in mice by intratracheal instillation of a plasmid carrying the CFTR gene under control of the Rous sarcoma virus promoter with cationic liposomes. Use of liposomes have successfully transferred the CFTR gene to epithelia and to alveoli deep in the lung leading to correction of the ion conductance defects found in the trachea of transgenic mice (Hyde et al, 1993).

Complexes of cationic polymers and cationic lipids with adenovirus enhanced gene transfer to the nasal epithelium of cystic fibrosis mice in vivo (Fasbender et al, 1997). The novel cationic lipid EDMPC (1,2-dimyristoylsn-glycero-3-ethylphosphocholine, chloride salt) mediated efficient intralobar DNA delivery of CFTR to rodents; there was no correlation between DNA-EDMPC formulations that delivered DNA most efficiently in vitro and those that worked best in vivo (Gorman et al, 1997). The structures of several novel cationic lipids that were effective for CFTR gene delivery to the lungs of mice were investigated; an amphiphile (lipid ^#67) consisting of a cholesterol anchor linked to a spermine head-group in a "T-shape" configuration was shown to sustain a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and was greater than 100-fold more active than other cationic lipids and comparable to that of adenoviral vectors (Lee et al, 1996).

C. Clinical trials on cystic fibrosis patients

A significant number of clinical trials on CF have received RAC approval (Appendix 1 and Table 4 in Martin and Boulikas, following article). The clinical protocols approved use adenoviral delivery of CFTR (#118-123, 125, 128, 129) or cationic lipids (#193, 203, 212, and 214). The only two protocols using AAV in clinical trials are for CF (#165, 166) whereas no retroviral protocol has been approved for CF as of December 1997.

Results of clinical trials have been reported and more will become available in the near future. A single dose of 400 mg pCMV-CFTR in complex with 2.4 mg DOTAP, administered to the nasal epithelium of eight CF patients resulted in partial, sustained correction of CFTR-related functional changes toward normal values in two treated patients; transgene DNA was detected in seven of eight treated patients for up to 28 days after treatment; vector derived CFTR mRNA was detected in two of the seven patients at 3 and 7 days from treatment using PCR (Porteous et al, 1997). Complexes of plasmids with DOTAP liposomes rendered their DNA resistant to DNaseI something relevant to clinical trials for gene therapy of CF, in which patients are normally removed from treatment with DNase before receiving administration of DNA (Crook et al, 1997).

A formulation of plasmid encoding CFTR (pCF1-CFTR) was at least as effective as complexes of DNA with lipid in partially correcting the Cl- transport defect in CF patients by administering complexes of DNA-lipid to one nostril and DNA alone to the other nostril in a randomized, double-blind study (Zabner et al, 1997).

The safety of the nebulised cationic lipid formulation (GL-67/DOPE/DMPE-PEG5000) to be used for the transfer of CFTR to CF patients was first tested on 15 healthy volunteers in the absence of plasmid DNA; no adverse clinical events were seen (Chadwick et al, 1997).

XXXIV. Rheumatoid arthritis (RA)

A. Molecular mechanisms for development of RA

RA is a systemic autoimmune disease caused by genetic and environmental factors; chronic inflammation and hyperactivation of synovial cells in the joints is the salient feature of RA resulting in the thickening (hyperplasia) of the synovial membrane lining the interior surface of the joint capsule. The mechanism involves synovial cell proliferation, infiltration by leukocytes, and excessive extracellular cell matrix deposition. The activated synovial cells in the hypertrophied synovium produce inflammatory cytokines and degradative enzymes that invade and erode the articular cartilage leading to partial or complete destruction of cartilage and bones. The inflamed synovium in RA is infiltrated by lymphocytes and monocytes, a process mediated by the enhanced binding of the very late antigen-4 (VLA-4) to vascular cell adhesion molecule-1 (VCAM-1) (Chen et al, 1995).

Several well-characterized murine models of arthritis closely resemble RA immunologically, genetically, and histopathologically and have been developed to study RA. Collagen-induced arthritis in DBA/1 mice is one model of RA; the animals exhibit marked synovitis and erosions. The disease can be adoptively transferred to SCID mice using arthritogenic splenocytes from DBA/1 mice injected with bovine collagen type II (Chernajovsky et al, 1997).

Bacterial cell wall-induced arthritis in rats is another model (Makarov et al, 1996).

B. Approaches to gene therapy of RA

The current emphasis for RA gene therapy is on transferring genes encoding secreted proteins which possess antiarthritic properties. Genes may be delivered locally to individual diseased joints or systemically to extra-articular sites where the secreted gene products may enter the circulation. Gene transfer to the synovium would ensure local production of anti-inflammatory gene products directly in the articular space where they could exert a down-regulatory effect on the autoimmune process. Although adenoviral delivery appeared best suited for gene delivery to synovium, induction of an inflammatory response resulting in loss of gene expression may take place (Evans and Robbins, 1996).

High efficiency lacZ gene transfer and expression was achieved in both type A and type B synoviocytes throughout the articular and periarticular synovium of the rabbit knee by Roessler et al (1993). Intra-articular administration of an E1a-E3-deleted adenoviral (Ad5) vector expressing the lacZ transgene into mouse joints showed lacZ expression in the articular synovium for at least 14 days. However, a gradual loss of transgene expression was caused by a predominantly neutrophilic, inflammatory response. Pretreatment with the anti-T cell receptor monoclonal antibody (mAb) H57 resulted in a significant reduction in lymphocytic infiltration and in persistence of transgene expression. Thus, anti-T cell mAbs may be useful in inhibiting adenovirus-induced immune responses that lead to the loss of therapeutically transduced cells (Sawchuk et al, 1996).

Many new therapeutic approaches are currently being developed, including the use of soluble receptors to IL-1 or TNF, monoclonal antibodies to TNF-a, and a specific IL-1 receptor antagonist. A number of studies have assessed the impact of gene transfer on inflammatory and chondrodestructive effects during the acute phase of antigen-induced arthritis in RA joints. A promising therapy for RA involves delivery of the TNF-a and IL-1 proteins to the joints to inhibit the activity of proinflammatory cytokines (Bandara et al, 1993; Arend and Dayer, 1995).

Angiogenesis is not only essential for the growth and metastatic spread of solid tumors but in diseases such as rheumatoid arthritis, psoriasis, liver cirrhosis and diabetic retinopathy (Norrby, 1997). Future approaches for the gene therapy of RA may thus include anti-angiogenesis approaches to the inflamed joints.

C. Ex vivo gene therapy of RA using IL-1Ra-transduced cells

Degradation of cartilage in RA in vitro is stimulated by IL-1, a proinflammatory cytokine, which is released from RA synovial fibroblasts (RA-SF). Synovial cells were surgically removed from joints of animals with experimental arthritis, cultured and transduced with the naturally occurring inhibitor of IL-1, IL-1-receptor antagonist (IL-1Ra) protein gene and reimplanted into the respective donors by intra-articular injection (Bandara et al, 1993). Retroviral transfer of the IL-1Ra gene to RA-SF which were then coimplanted with normal human cartilage in SCID mice protected the cartilage from chondrocyte-mediated degradation; the IL-1Ra-transduced RA-SF continued to secrete IL-1Ra over a 60-day period (Muller-Ladner et al, 1997a,b). Transfer the human IL-1Ra gene to rabbits' knees produced a marked chondroprotective effect although the anti-inflammatory effect was milder (Otani et al, 1996).

Ex vivo retroviral delivery of the secreted human IL-1Ra cDNA to primary synoviocytes followed by engraftment in ankle joints of rats with recurrent bacterial cell wall-induced arthritis significantly suppressed the severity of recurrence of arthritis as assessed by measuring joint swelling and by the gross-observation score; this ex vivo approach attenuated but did not abolish erosion of cartilage and bone; the level of locally expressed IL-1Ra was about four orders of magnitude higher than that attained from systemically administered recombinant IL-1Ra protein (Makarov et al, 1996). These findings provide experimental evidence for the feasibility of antiinflammatory gene therapy for arthritis.

Retroviral transduction of hematopoietic stem cells with human IL-1Ra cDNA was also used for the treatment of RA; HSCs were subsequently injected into lethally irradiated mice; all of the mice survived and over 98% of the white blood cells in these mice were arising from the transduced HSCs (donor type) from 2-13 months after transplantation; the animals had the human IL-1Ra protein in their sera for at least 15 months. These results demonstrated that systemic production of biologically active human IL-1Ra can be obtained by retrovirus-mediated gene transfer to hematopoietic stem cells which could be useful in the treatment of chronic diseases such as rheumatoid arthritis as well as bone degeneration caused by aging (Boggs et al, 1995).

Characterization of the interleukin-1/interleukin-1 receptor antagonist pathways in RA resulted in the first gene therapy trial in animals and humans for RA (Evans et al, 1996; reviewed by Evans and Robbins, 1996; Muller-Ladner et al, 1997a). Protocol #56 (page 162) involves removal of autologous synovial cells from the patient, their retroviral transduction with the IL-1Ra cDNA followed by injection of the transduced cells into the metacarpal phalangeal joints of RA patients.

Infection of arthritogenic splenocytes from DBA/1 mice transferred to SCID mice with a recombinant retrovirus carrying the TGF-b1 gene was effective in lowering inflammation of joints with already established arthritis and inhibiting the spreading of the disease to other joints in mice (Chernajovsky et al, 1997).

D. Direct gene delivery for RA

A retroviral vector based on a murine leukemia virus was used to deliver the human growth hormone and lacZ genes to the synovium of the rabbit knee to test the efficacy of gene transfer and to develop an approach for the gene therapy of RA (Ghivizzani et al, 1997).

An effective treatment of arthritis is via eliminating most or all of the activated synovial cells. The death factor Fas/Apo-1 and its ligand (FasL) play pivotal roles in maintaining self-tolerance and immune privilege; Fas is expressed constitutively in most tissues and is dramatically upregulated at the site of inflammation. Unlike Fas, however, the levels of FasL expressed in the arthritic joints are extremely low, and most activated synovial cells survive despite high levels of Fas expression. Delivery of the FasL gene via a replication-defective adenovirus by injection into inflamed joints conferred high levels of FasL expression, induced apoptosis of synovial cells, and ameliorated collagen-induced arthritis in DBA/1 mice (Zhang et al, 1997).

A strategy was developed for inhibiting T lymphocyte retention and activation within the rheumatoid synovium (Chen et al, 1995). The inflamed synovium in RA is infiltrated by lymphocytes and monocytes, a process mediated by the enhanced binding of the very late antigen-4 (VLA-4) to vascular cell adhesion molecule-1 (VCAM-1) expressed on microvascular endothelial cells; VLA-4 binding appears to play a role in T cell retention and activation within the inflamed synovial membrane. Therefore, blocking of VLA-4 binding by utilizing a soluble congener of the VCAM-1 molecule might be of therapeutic efficiency for RA. Adenoviral infection of human synoviocytes carrying the cDNA for a secreted form of VCAM-1 (sVCAM-1) showed secretion of transgenic sVCAM-1 by ELISA of tissue culture supernatants. In vivo, transgenic sVCAM-1 expression was determined by immunohistochemical analysis and in situ hybridization of synovial tissue, and secretion of transgenic sVCAM-1 was demonstrated by ELISA of tidal knee lavage fluid. The results showed that recombinant adenovirus can mediate the expression of a biologically active sVCAM-1 by synoviocytes in vivo and suggested that this strategy may be useful for inhibiting T lymphocyte retention and activation within rheumatoid synovium. Ex vivo studies using this strategy have not been reported.

XXXV. Adenosine deaminase (ADA) deficiency and severe combined immunodeficiency (SCID)

Severe combined immunodeficiency is secondary to the deficiency in adenosine deaminase; the enzyme is involved in purine catabolism. The syndrome is characterized by defective B and T cell function caused by the large amounts of deoxyadenosine which is preferentially converted into the toxic compound deoxyadenosine triphosphate in T cells disabling the immune system (see Blaese et al, 1995 and the references cited therein). Affected individuals experience recurrent infections and the disease is usually fatal unless affected children are kept in isolation. One therapeutic approach has been to partially reconstitute the immune system by bone marrow transplantation from a human leukocyte antigen (HLA)-identical sibling donor (Hirschorn et al, 1981). An enzyme replacement therapy consisted of introducing bovine ADA enzyme conjugated with polyethylene glycol (PEG-ADA) in order to increase the circulation time of the ADA enzyme in the blood and other extracellular fluids (Hershfield et al, 1987).

The first person to be treated ex vivo was a 4-year-old suffering with ADA deficiency in 1990. Protocol #2 treating ADA deficiency with autologous lymphocytes transduced ex vivo with the ADA gene was the second RAC-approved protocol. Because of this innovative work, the US Patent Office has issued in 1995 a patent covering all ex vivo gene therapy to French Anderson, Steven Rosenberg, and Michael Blaese.

From 1990-1992, a clinical trial was initiated using retrovirus mediated transfer of the 1.5 kb ADA gene cDNA to T cells from two children with severe combined immunodeficiency following multiple transplantations of ex vivo modified blood cells; the integrated ADA gene was expressed for long periods (Blaese et al, 1995; Bordignon et al, 1995). The success of this ex vivo approach probably arose from that the ADA gene-corrected T cells acquired a survival advantage compared with uncorrected cells when transplanted into immunodeficient but ADA normal BNX mice (Ferrari et al, 1991) and humans (Kohn et al, 1995).

Three neonates with ADA deficiency have been successfully treated later with hematopoietic stem CD34⁺ cells, isolated from their umbilical cord blood, transduced with the ADA gene under control of the LTR of the MoMuLV using retroviral vectors, followed by autologous transplantation (Kohn et al, 1995).

Ex vivo studies have shown correction of the severe combined immunodeficiency in ADA-deficient mice by transfer of human peripheral blood lymphocytes transduced with a retroviral vector in cell culture carrying the ADA gene; the injected human cells survived for long times in mice and restored the immune functions (presence of human immunoglobulin and antigen-specific T cells) (Ferrari et al, 1991). Ex vivo correction of the defect in T cells from ADA-deficient patients with retroviral vectors gave to these cells an advantage for cell division against a background of slowly dividing uncorrected T cells after their transplantation (Karlsson, 1991).

XXXVI. Gaucher disease, lysosomal storage disease, and mucopolysaccharidosis VII

Many mutations affecting the glucocerebrosidase gene have been defined as causes of the glycolipid storage disorder, Gaucher disease; disease symptoms are a result of macrophage engorgement secondary to this enzyme deficiency. The recombinant from of glucocerebrosidase imiglucerase protein is effective in treating the disease as a replacement therapy (reviewed by Beutler, 1997).

An amphotropic producer cell line that synthesized viral particles carrying a fusion of the selectable MDR1 cDNA encoding P-glycoprotein (P-gp) and the human glucocerebrosidase gene was constructed; complete restoration of glucocerebrosidase deficiency in Gaucher fibroblasts was achieved using this retrovirus; selection of the transduced Gaucher fibroblasts in colchicine (MDR1 function) raised their glucocerebrosidase activity from nearly undetectable to normal levels; combination of much lower concentrations of colchicine and inhibitors of the P-gp pump (verapamil) allowed to select for high-level expression of MDR1 and glucocerebrosidase; this regimen, in clinical use for the treatment of multidrug-resistant malignancies, may find application for high level selection of a nonselectable gene such as glucocerebrosidase (Aran et al, 1996; see also Migita et al, 1995).

Allogenic bone marrow transplantation (Parkman, 1986) or intravenous infusion of glucocerebrosidase (enzyme replacement therapy) in a patient with Gaucher's disease (Barton et al, 1990), although has partially corrected deficiencies in lysosomal enzymes, was not amenable to brain cells because of the brain barrier and cannot alleviate symptoms in the central nervous system. Protocols #38, 39, and 51 use glucocerebrosidase cDNA to transduce CD34⁺ autologous peripheral blood cells followed by intravenous injection of the transduced cells into patients with Gaucher's disease. CD34⁺ cells obtained from G-CSF mobilized peripheral blood stem cells or from bone marrow (#51) are being transduced ex vivo and reinfused into the patient at the National Institutes of Health and Children’s Hospital of Los Angeles (Dunbar and Kohn, 1996).

Type II Glycogen Storage Disease, a deficiency of acid a-glucosidase (GAA), results in the abnormal accumulation of glycogen in skeletal and cardiac muscle lysosomes; this can have devastating effects ultimately leading to death; the wild-type enzyme was produced in deficient myoblasts after gene transfer with a retroviral vector carrying the cDNA for GAA. The transduced cells secreted GAA that was endocytosed via the mannose-6-phosphate receptor into lysosomes of deficient cells and digested glycogen; thus, the transduced cells provided phenotypic correction to distant cells in the culture by secretion. Figure 33 shows that the GAA-transduced myoblasts (red fluorescence) were able to fuse with deficient myoblasts (green fluorescence) and provide enzyme activity mediating phenotypic correction to neighboring GAA-deficient cells (Zaretsky et al, 1997).

Aspartylglucosaminuria (AGU, appearance of aspartylglucosamine in the urine) is the only known human disease caused by an amidase deficiency, in this case by deficiency of the enzyme aspartylglucosaminidase (AGA) in virtually all cell types of patients; AGA deficiency fails to perform the final breakdown of asparagine-linked glycoproteins leading to the intraly-sosomal accumulation of uncleaved glycoasparagines and to their abnormal urinary excretion. AGU patients display progressive psychomotor retardation starting in early childhood. The most common mutation in the Finnish population responsible for AGA deficiency is a point mutation resulting in the amino acid substitution Cys-163 to Ser in the AGA gene (Ikonen et al, 1991).

Retrovirus-mediated gene transfer was successfully used to correct the AGA gene in cultured human primary fibroblasts and lymphoblasts from AGU patients as a prelude to ex vivo human gene therapy; enzyme correction

Figure 33. Immunofluorescent detection of in vitro fusion of a-glucosidase (GAA)-deficient muscle cells with myoblasts transduced with the GAA gene using a retroviral vector. The GAA-transduced myoblasts (red fluorescence) were able to fuse (arrows) with deficient myoblasts (green fluorescence) and provide enzyme activity. From Zaretsky JZ, Candotti F, Boerkoel C, Adams EM, Yewdell JW, Blaese RM, Plotz PH (1997) Retroviral transfer of acid a-glucosidase cDNA to enzyme-deficient myoblasts results in phenotypic spread of the genotypic correction by both secretion and fusion. Hum Gene Ther 8, 1555-1563. Reproduced with the kind permission of the authors and Mary Ann Liebert, Inc.

further took place by cell-to-cell interaction between transduced and nontransduced cells in culture suggesting that only partial cell transduction might be sufficient to correct AGA deficiency in vivo (Enomaa et al, 1995).

Mucopolysaccharidosis type VII (Sly syndrome or MPS VII) is a result of an inherited deficiency of b-glucuronidase in humans, mice, and dogs (see Wolfe et al, 1992 for references). The symptoms are progressive degeneration in several tissues resulting from storage in lysosomes of undegraded glycosaminoglycans affecting the spleen, liver, brain, cornea, kidney, and skeletal muscle. Affected individuals display a reduced life-span which is 5 months in mice. Retroviral vectors have successfully treated mucopolysaccharidosis VII by somatic cell gene transfer in mouse models (Wolfe et al, 1992) or by implantation of ex vivo modified mouse skin fibroblasts (Moullier et al, 1993).

Retrovirus-mediated transfer of the iduronate-2-sulfatase cDNA into lymphocytes is being applied for the clinical treatment of mild Hunter syndrome (mucopo-lysaccharidosis type II, protocol ^#65 on page 163).

XXXVII. Hereditary a1-antitrypsin (a1-AT) deficiency

Destruction of components of the extracellular matrix of the lung by neutrophil elastase (NE) is believed to be a critical event in the development of obstructive lung disease. a1-antitrypsin (also known as a1-proteinase inhibitor) is an antiprotease that protects the lung from destruction by the powerful protease NE. The hereditary a1-antitrypsin deficiency is caused by mutations in the coding region of the 12.2 kb-gene resulting in decreased serum and lung levels of a1-antitrypsin; affected individuals develop emphysema at age 30-40 (Crystal, 1990). Lung-derived epithelial cells have the capacity to synthesize functional a1-antitrypsin but also to increase the rate of its production when stimulated by specific inflammatory mediators, including oncostatin M, IL-1, and dexamethasone (Cichy et al, 1997).

The respiratory epithelium has been a potential site for somatic gene therapy because of the possibility of direct delivery of a functional gene by tracheal instillation. A drawback for retrovirus-mediated gene transfer arises from that the majority of alveolar and airway epithelial cells are terminally differentiated and only a small fraction of these cells are proliferating and amenable to recombinant retrovirus infection; however, lung epithelial cells are prone to adenovirus infections because host cell replication is not required for expression of adenoviral proteins (see Rosenfeld et al, 1991).

The adenovirus major late promoter was linked to a human a1-antitrypsin gene for its transfer to lung epithelia of cotton rat respiratory pathway as a model for the treatment of a1-antitrypsin deficiency; the cotton rat is an animal commonly used to evaluate the pathogenesis of adenoviral respiratory tract infections. Both in vitro and in vivo infections have shown production and secretion of a1-antitrypsin by the lung cells: cells, removed by brushing the epithelial surface of the tracheobronchial tree from the lungs of cotton rats demonstrated the possibility of their infection in culture and secretion of the therapeutic protein; following direct infection of the animals in vivo, demonstrated that the protein was synthesized and secreted in the epithelial lining fluid of the lung for over 1 week (Rosenfeld et al, 1991).

i.v. administration of a full-length cDNA encoding human a1-antitrypsin (100 ng/mouse) encapsulated in small liposomes resulted in expression in liver parenchymal cells as shown on immunohistochemical liver sections; this effect remained for at least 2 weeks and some enzyme could be detected in plasma (Aliño et al, 1996). The human a1-antitrypsin gene was transferred to lungs of rabbits; immunohistochemical staining showed a1-AT protein in the pulmonary endothelium following intravenous administration, in alveolar epithelial cells following aerosol administration, and in the airway epithelium by either route (Canonico et al, 1994). A recombinant adenovirus vector was also used for a1-AT cDNA transfer (Gilardi et al, 1990). a1-AT cDNA in an adenoviral vector was administered by retrograde ductal instillation to the submandibular glands of male rats; transient expression took place in salivary glands (Kagami et al, 1996).

Guo et al (1996) have evaluated the transcriptional activities of 5 viral and cellular enhancer/promoter elements, showing either high-level or hepatocyte-specific expression following transient transfection into hepatoma cells using recombinant adenoviruses expressing human a1-antitrypsin; the human elongation factor 1a gene promoter produced 2 mg/ml serum level of human a1-antitrypsin, which is physiologic in humans and will be therapeutic for patients with a1-antitrypsin deficiency.

XXXVIII. Approaches to the gene therapy of Parkinson’s disease (PD)

A. Etiology and mechanisms of destruction of neurons in PD

First described by James Parkinson in 1817 this neurodegenerative disorder is characterized by resting tremor, postural instability and bradykinesia (slow movement); surviving neurons display intracytoplasmic inclusions known as Lewy bodies. PD symptoms ensue when the pars compacta region of the substantia nigra (black substance) at the base of the brain loses neurons that normally issue motion-controlling signals (dopamine) to the striatum (divided into caudate nucleus and putamen). The death of neurons is believed to be caused by oxygen free radical damage; brain contains unusually low levels of antioxidants. This damage might be caused by a decline in the activity of the mitochondrial complex I.

PD can be induced in experimental animals by selective destruction of the dopaminergic neurons of the substantia nigra by the neurotoxic drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) through inhibition of complex I of the mitochondrial respiratory chain (see Polymeropoulos et al, 1996 and the references cited therein). MPTP was found as an impurity in heroin and explained some earlier observations of addicts who became almost completely immobile after making use of the drug, a symptom characteristic of severe PD. MPTP crosses the brain-blood barrier and is converted by mitochondrial monoamine oxidase B into a reactive molecule that inhibits the complex I enzyme resulting in energy deficit and increase in free radicals in the cell (reviewed by Youdim and Riederer, 1997).

There is substantial evidence which implicates immune mechanisms in the destruction of neurons. The substantia nigra of Parkinson’s patients contains active microglia which, after stimulation by cytokines, could produce the free radical nitric oxide which can penetrate the cell membrane of vicinal neurons, inhibit the complex I mitochondrial enzyme and activate signal transduction pathways. Furthermore, NO with superoxide, emitted by hyperactive microglia, can free iron ions from intracellular stores which can oxidize dopamine into neuromelanin, a molecule that acts as an oxidant when complexed with transition metals. These oxidative stress mechanisms could trigger apoptosis in neurons. Excessive release of the neurotransmitter glutamate (known to occur in stroke) into the striatum and substantia nigra could induce a similar cascade of NO and free radical damage. These mechanisms suggest that excessive stressful conditions in predisposed individuals might precipitate the onset of PD symptoms.

B. Drug treatment of PD

The first medicament in the mid-1900s included extracts of the deadly nightshade plant which inhibited the activity of acetylcholine in the striatum; acetylcholine overexcites striatal neurons that projected to higher motor regions of the brain, an effect normally counteracted by dopamine. Later in 1960s L-DOPA, which is converted into dopamine, proved valuable for treatment of PD patients; dopamine itself cannot cross the blood-brain barrier (a network of specialized blood vessels that control which substances are allowed to pass from the blood into the central nervous system). Drugs that mimic the actions of dopamine (agonists) have also been used. Selegiline (also called deprenyl), an inhibitor of monoamine oxidase B, the enzyme that breaks down dopamine in the astrocytes and microglia, is of therapeutic potential (reviewed by Youdim and Riederer, 1997).

Amantadine is used to block the effects of glutamate in substantia nigra. Antioxidants able to cross the brain-blood barrier could have a protective effect on the destruction of neurons. Unfortunately, the first indications show that vitamin E in the low doses tested, which can cross to some extent the brain-blood barrier, is ineffective; however, the effect of higher doses of vitamin E need to be investigated. The efficacy of glial-derived neurotrophic factor (GDNF) injected into the brain of PD patients is in trials. Rasagiline, which could activate neuronal growth factors in the brain is under investigation on humans. Also in clinical trials are strategies of direct implantation of dopamine-producing cells into the brain of patients (Youdim and Riederer, 1997).

Ex vivo and in vivo gene therapy strategies for PD have a promising future (see below).

C. Candidate genes for PD

A susceptibility gene for PD has been mapped to chromosome 4q21-q23 by genotyping genomic DNA from a large family in Contursi in the Salemo province of Southern Italy where 60 individuals out of 592 members are affected by PD at an average age of 46. A total of 140 genetic markers were typed in the pedigree and only those associated with this chromosomal region were altered showing recombination events in PD patients in this family; this type of recombination does not involve expansions of the CAG trinucleotide repeat (Polymeropoulos et al, 1996).

The neurologic abnormalities associated with PD were thought to result from a severe reduction in L-DOPA as a consequence of degeneration of dopaminergic neurons of the nigrostriatal pathway. L-DOPA is synthesized from tyrosine by the enzyme tyrosine hydroxylase (TH); L-DOPA is then converted into the neurotransmitter dopamine by a decarboxylase. Besides TH, other genes whose malfunction has been linked to PD include glutathione peroxidase, a brain-derived neurotrophic factor, catalase, amyloid precursor protein, Cu/Zn superoxide dismutase, and debrisoquine 4-hydroxylase; however, none of these candidate genes are found in the 4q21-q23 region to be linked as etiologic agents of PD; instead, candidate genes in the 4q21-q23 region include alcohol dehydrogenase, formaldehyde dehydrogenase, synuclein, and UDP-N-acetylglycosamine phosphotransferase (Polymero-poulos et al, 1996).

A mutation was identified in the a-synuclein gene, which codes for a presynaptic protein thought to be involved in neuronal plasticity, in the Italian kindred with autosomal dominant inheritance for the PD phenotype (Polymeropoulos et al, 1997). The missense mutation in the a-synuclein gene suggested that at least some fraction of familial PD with diffuse Lewy bodies is the result of an abnormal protein that interferes with normal protein degradation leading to the development of inclusions and ultimately neuronal cell death. Furthermore, a peptide fragment of a-synuclein is known to be a constituent of Alzheimer's disease plaques; there may be common pathogenetic mechanisms involved in a-synuclein mutations in PD and b-amyloid and presenilin gene mutations in Alzheimer's disease (Nussbaum and Polymeropoulos, 1997).

D. Gene and cell therapy for PD

1. Grafting of dopamine neurons

Transplantation of human embryonic dopamine neurons have been performed on patients with Parkinson's disease but the amelioration of the symptoms is transient; death of therapeutic cells was thought to arise from hypoxia, oxidative stress, and trauma during preparation and grafting of the cells. Grafting of dopamine neurons into transgenic mice overexpressing the Cu/Zn superoxide dismutase increased 4-fold the survival of the transplanted cells providing a direct support to the free radical-mediated death of dopaminergic neurons in brain tissue grafts (Nakao et al, 1995).

Cells transduced with tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells transduced with aromatic L-amino acid decarboxylase into the 6-hydroxydopamine-denervated rat striatum; it was concluded that there is sufficient aromatic L-amino acid decarboxylase near striatal grafts producing L-DOPA and that the close proximity of L-amino acid decarboxylase to TH-producing cells is detrimental for optimal dopamine production (Wachtel et al, 1997).

2. Tyrosine hydroxylase (TH)

Since adult brain cells are nonproliferative, they are refractory to retroviral infection that could deliver the TH gene to the brain to alleviate degeneration at the nigrostriatal pathway. Gene therapy of PD has been approached ex vivo using PD animal models with TH deficiency. Unilateral destruction of dopaminergic nigrostriatal neurons in PD animal models with 6-hydroxydopamine and administration of apomorphine causes PD rats to turn contralaterally (7-15 rotations/min).

XLIII. Prospects

A. Gene discovery: novel horizons in gene therapy

By the year 2005 the human genome project will be completed and by the end of 1998 the entire cDNA repertoire of the 125,000 human genes will be sequenced completely (Incyte Pharmaceuticals, Palo Alto, CA). Every single open reading frame of the human genome will become known.

Genes implicated in human disease are being identified by mapping the mutation to a chromosomal locus after examining the DNA from a number of patients; candidate genes residing in this locus are then examined in a large number of patients for inactivating mutations (e.g. Polymeropoulos et al, 1996, 1997). A number of other classical techniques are aimed at identifying novel tumor suppressor genes or genes involved in metastasis, adding new weapons to the fight against cancer. The elucidation of many of the pathways implicated in the regulation of the cell cycle, signaling pathways with cytokines, and activation and action of transcription factors on the regulatory regions of genes lead to the discovery of new drugs interfering with those pathways. The elucidation of a number of players in apoptosis provides also targets not only for cancer treatment but for a number of neurodegenerative diseases. All these studies will ultimately provide new targets and genes for gene therapy.

B. Mutations in DNA and human disease

A number of human disorders have been linked to mutations in specific genes that result in loss of function of a specific protein in all somatic cells of the body. In the majority of cases known today the mutation is at the coding region of the gene resulting in one amino acid substitution at an important domain of the encoded protein, in amino acid deletions, or in protein truncation. For example, deletion of three nucleotides resulting in deletion of a single phenylalanine at the protein level of the CFTR molecule is responsible for cystic fibrosis (Riordan et al, 1989); an A to T transversion leading to a premature stop at amino acid 337 in one allele and a C to T transition triggering an erroneous splice event and to frameshift in the other allele are associated with mutations in the ERCC6 helicase in Cockayne's syndrome (Troelstra et al, 1992).

Mutations could result in failure of the protein to interact with DNA (mutated p53), with other regulatory proteins, or in enzymatic dysfunction of the molecule. Defects at the nuclear localization signal of a nuclear protein resulting in its cytoplasmic retention have been identified in cancer cells (Chen et al, 1995). Mutations in regulatory regions (promoters, enhancers) of genes, poorly understood but expected to play an important role in human disease, could result in down-regulation of the gene they dictate; mutation in the DNA-binding or transactivation domains of transcription factors are expected to down-regulate the expression of their target genes. Most important, mutations in genes involved in DNA repair are expected to have a domino effect on the appearance of mutations in other regions of the genome, since it is these genes that are responsible for removal of premutagenic lesions incurring by a number of xenobiotics by patrolling the human genome (Boulikas, 1996c).

C. Regulatory regions and the MAR project

The identification of the regulatory regions from the human genome should also become a first priority. Regulatory regions will provide new DNA control elements for the tissue-specific expression, episomal replication, insulation, and silencing of genes in gene therapy protocols but also targets, using small oligonucleotides (e.g. triplex) to abort transcription of specific genes. Regulatory regions include enhancers (ENHs, at least two for each gene), promoters (about 125,000 total, a significant fraction of which might be known because of their proximity to the 5' end), origins of replication (ORIs, about 50,000 have been estimated), silencers, locus control regions (LCRs, perhaps several thousand), and matrix-attached regions (seem to coincide with enhancers and ORIs). 445,000 is a modest estimate of the total number of regulatory regions in the human genome. Their size ranges from 100-500 bp but much more for LCRs and some ORIs. Identification of strong regulatory regions from the human genome is expected to provide strong promoter and enhancer sequences for the universal and cell type-specific expression of transgenes in gene therapy but also strong human ORIs able to sustain extrachromosomal replication of plasmids loaded with therapeutic genes in human and animal model tissues.

Transcription factor recognition sequence databases can be used in conjuction with other software methods (DNA curvature, inverted repeats, triplex DNA, Z-DNA, phased nucleosomes) to predict regulatory regions from the large DNA sequence information arising from the human genome project (Boulikas, 1995b; Bode et al, 1998, this volume).

A technology developed in our laboratory based on isolation and cloning of matrix-attached regions, shown to harbor a large fraction of regulatory regions from the human and other genomes, is being applied for identifying human regulatory regions (MAR project). MAR libraries include tissue-specific and tumor-specific regulatory regions. One particular MAR clone that has been extensively characterized (Boulikas et al, in preparation) represents the ORI, enhancer, and MAR of the human choline acetyltransferase gene, of crucial importance in neurological disorders including Alzheimer's disease. When a subfragment of only 513 bp of this MAR/ORI/ENH was placed at the flanks of the luciferase gene it was able to sustain episomal replication in human culture cells (K562 erythroleukemia) for more than 4 months. The actual 3.6 kb ChAT ORI region comprises a 1.2 kb silencer whose presence inhibits the ORI function; thus, mammalian origins of replication are much more sophisticated than viral ORIs and contain a number of control elements, including silencers, for the cell type and developmental stage-specific regulation. Identification and elimination of silencers from human ORIs is of importance in the exploitation of ORI fragments in the episomal replication of therapeutic genes.

MAR sequences sorted out into MAR/ORI, MAR/enhancer and MAR/insulators can be used to promote extrachromosomal replication, to enhance the transcription of genes or to insulate genes from position effects from chromatin surroundings after integration. A number of studies show that MARs act as insulators of genes shielding them from position effect variegation from neighboring chromatin domains in transgenic studies; this shielding results in a 2 to 1000-fold increase in the expression level of transgenes when MARs are included on both sides of the foreign gene (see Boulikas 1995b).

Identification of tumor-specific MARs, such as identification of the MARs of the carcinoembryonic antigen (CEA) gene, the breast cancer/ovarian cancer BRCA1 gene, and others can lead to the development of plasmid vectors able to drive the expression of therapeutic genes in specific tumor cell types. In the postgenomic era, identification of a reasonable fraction of regulatory regions will revolutionarize our approaches to human disease.

D. What is next on gene therapy?

Theoretically, most human disorders could constitute targets for gene therapy, aimed at correcting the defect either by transferring the wild-type gene in all somatic cells of the body or to those specific cell types responsible mainly for the synthesis of the particular protein (e.g. factor IX gene in liver cells of hemophilia B patients).

Nuclear localization signal (NLS) peptides hooked to triplex-oligonucleotides or to plasmids, or complexation of plasmids with nuclear proteins possessing multiple NLSs are expected to increase nuclear localization and enhanced expression of foreign genes.

A significant number of discoveries in molecular biology of human diseases have opened doors to the development of strategies for gene therapy. New genes whose mutations are responsible for human disease, from mild to life threatening, are being discovered and the molecular mechanisms are being unraveled. Many pieces of the puzzle aimed at elucidating mechanisms leading to human disease and the genes implicated have been solved and lie as scattered pieces of knowledge in various publications, lab notebooks, or patent applications. Preexisting Biotech Companies redefine their missions and new Biotech Companies are being founded to explore new discoveries and develop new drugs; to win the race in the fight against human disease, especially cancer and AIDS, we need to gather the right components into a successful assemble.

Retroviruses, adenoviruses, AAV, HSV, naked plasmid delivery, and liposomes all have a good share as delivery vehicles for genes and it seems that they will be developed independently, each with its own strengths and limitations for particular gene therapy protocols. For example, liposomes have a distinct advantage over other systems for the delivery of oligonucleotides, stealth liposomes could prove their strength in the systemic delivery of genes by intravenous injection, retroviruses and adenoviruses for their high transfection efficiency, AAV for not stimulating inflammation, HSV as a vehicle for gene therapy to the nervous system, HIV and HSV vectors for their high payload capacity. Furthermore, adenoviruses, AAV, HSV-1, HIV-1 vectors can transduce nondividing cells (Table 1 on page 29).

A lot has been learned about the involvement of the tumor suppressor p53 protein in cancer etiology. The current view is that an initiated tumor cell in the body, having mutations in one or more oncogenes needs to acquire loss in function in both alleles of p53 or other tumor suppressor gene in order to expand into the tumor cell mass. Expression of the wild-type (non-mutated form) of p53 arrests the proliferation in tumor cells and induces apoptosis (suicidal programmed death) by boosting the expression of the genes of p21, bax, and Gadd45 and by repressing the bcl-2 gene. Transfer of the p53 gene with adenovirus or retrovirus after intratumoral injection has successfully led to eradication of tumors in animal models and in human patients at advanced stages of non small cell lung cancer. Intratumoral injection, however, is not expected to be applicable to metastases very frequently associated with advanced stages of cancer. Stealth liposomes might offer a solution to this problem.

Anti-angiogenesis therapy, both drug-mediated and gene therapy, would bring important ammunition in the fight against cancer.

Improvements in oligonucleotide delivery in vivo, a very promising field that is in its infancy at the delivery level, will advance the field of pharmacogenomics by providing triplex-forming oligonucleotide drugs to inhibit the transcription of specific genes or ribozyme drugs to lower the mRNA level of a specific target protein.

We expect the final victory of the human race on cancer to be accomplished over the next 10 years. Gene therapy would, no doubt, have an important role to play. It is likely that a combination of gene therapy (p53, HSV-tk, angiostatin) along with the already existing antineoplastic drugs (doxorubicin, cisplatin) but at subtoxic doses and at much lower concentrations than those used today, as well as less severe doses of radiation, would become routine regimens in hospitals for the eradication of all cancer types. In this respect, the emerging priority in gene therapy is to improve the efficiency of tissue targeting and gene delivery.

Acknowledgements

I am grateful to all authors who have kindly provided their permission and original photos to use in this article. Special thanks to François Meyer, Max Birnstiel, Brian Johnston, Marc Vasseur, Claude Hélène, Frank Martin, Joe Vallner, Douglas Jolly, Jeff Seilhamer, Demetrios Spandidos, Kaz Taira, Ernst Wagner, Peter Dehlinger, Ko Kurachi, Dan Maneval, Jerzy Jurkat, Ed Trifonov, and Emile Zuckerkandl for stimulating discussions. I am also grateful to past and present members of my group for their contributions to the ongoing projects related to the subjects discussed here in particular Linda Hsie, CF Kong, Dawn Brooks, John Costouros, and Jie Hu, as well as to my collaborators Maria Zannis-Hadjopoulos, Jürgen Bode, and Wolfgang Deppert.

References

Abdallah (1996) A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: polyethylenimine. Hum Gene Ther 7, 1947-1954.

Abdel-Mageed A, Agrawal KC (1997) Antisense down-regulation of metallothionein induces growth arrest and apoptosis in human breast carcinoma cells. Cancer Gene Ther 4, 199-207.

Aebersold P, Kasid A, Rosenberg S (1990) Selection of gene-marked tumor infiltrating lymphocytes from post-treatment biopsies: A case study. Hum Gene Ther 1, 373-384.

Agoff SN, Hou J, Linzer DIH, Wu B (1993) Regulation of the human hsp70 promoter by p53. Science 259, 84-87.

Ahima RS, Dushay J, Flier SN, Prabakaran D, Flier JS (1997) Leptin accelerates the onset of puberty in normal female mice. J Clin Invest 99, 391-395.

Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, Maratos-Flier E, Flier JS (1996) Role of leptin in the neuroendocrine response to fasting. Nature 382, 250-252.

Akkina RK, Walton RM, Chen ML, Li QX, Planelles V, Chen IS (1996) High-efficiency gene transfer into CD34⁺ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G. J Virol 70, 2581-2585.

Ali RR, Reichel MB, Thrasher AJ, Levinsky RJ, Kinnon C, Kanuga N, Hunt DM, Bhattacharya SS (1996) Gene transfer into the mouse retina mediated by an adeno-associated viral vector. Hum Mol Genet 5, 591-594.

Aliño SF, Bobadilla M, Crespo J, and Lejarreta M (1996) Human a1-antitrypsin gene transfer to in vivo mouse hepatocytes. Hum Gene Ther 7, 531-536.

Allay J, Dumenco L, Koc ON, LiumL, and Gerson SL (1995) Williams DA, Hsieh K, Desilva A, and Mulligan RC (1987) Protection of bone marrow transplant recipients from lethal doses of methotrexate by the generation of methotrexate resistant bone marrow. J Exp Med 166, 210-218.

Allen, GC., Hall, GE Jr, Childs LC, Weissinger AK, Spiker S, and Thompson WF (1993) Scaffold attachment regions increase reporter gene expression in stably transformed plant cells. Plant Cell 5, 603-613.

Alon T, Hemo I, Itin A, Pe'er J, Stone J, Keshet E (1995) Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nature Med 1, 1024-1028.

Alton EWFW, Middleton PG, Caplen NJ, Smith SN, Steel DM, Munkonge FM, Jeffery PK, Geddes DM, Hart SL, Williamson R, Fasold KI, Miller AD, Dickinson P, Stevenson BJ, McLachlan G, Dorin JR, Porteous DJ (1993) Non-invasive liposome-mediated gene delivery can correct the ion transport defect in cystic fibrosis mutant mice. Nature Genet 5, 135-142.

Anderson WF (1992) Human gene therapy. Science 256: 808-813.

Antony AC (1992) The biological chemistry of folate receptors. Blood 79, 2807-2820.

Antony AC, Briddell RA, Brandt JE, Straneva JE, Verma RS, Miller ME, Kalasinski LA and Hoffman R (1991) Megaloblastic hematopoiesis in vitro. Interaction of anti-folate receptor antibodies with hematopoietic progenitor cells leads to a proliferative response independent of megaloblastic changes. J Clin Invest 87, 313-325.

Aoki K, Yoshida T, Matsumoto N, Ide H, Hosokawa K, Sugimura T, Terada M (1997) Gene therapy for peritoneal dissemination of pancreatic cancer by liposome-mediated transfer of herpes simplex virus thymidine kinase gene. Hum Gene Ther 8, 1105-1113.

Aoki K, Yoshida T, Sugimura T, Terada M(1995) Liposome-mediated in vivo gene transfer of antisense K-ras construct inhibits pancreatic tumor dissemination in the murine peritoneal cavity. Cancer Res 55, 3810-3816.

Aran JM, Licht T, Gottesman MM, Pastan I (1996) Complete restoration of glucocerebrosidase deficiency in Gaucher fibroblasts using a bicistronic MDR retrovirus and a new selection strategy. Hum Gene Ther 7, 2165-2175.

Arcasoy SM, Latoche JD, Gondor M, Pitt BR, Pilewski JM (1997) Polycations increase the efficiency of adenovirus-mediated gene transfer to epithelial and endothelial cells in vitro. Gene Ther 4, 32-38.

Arend WP, Dayer JM (1995) Inhibition of the production and effects of interleukin-1 and tumor necrosis factor a in rheumatoid arthritis. Arthritis Rheum 38, 151-160.

Arenzana-Seisdedos F, Virelizier JL, Rousset D, Clark-Lewis I, Loetscher P, Moser B, Baggiolini M (1996) HIV blocked by chemokine antagonist. Nature 383, 400-400.

Armentano D, Zabner J, Sacks C, Sookdeo CC, Smith MP, St George JA, Wadsworth SC, Smith AE, Gregory RJ (1997) Effect of the E4 region on the persistence of transgene expression from adenovirus vectors. J Virol 71, 2408-2416.

Arteaga CL and Holt JT (1996) Tissue-targeted antisense c-fos retroviral vector inhibits established breast cancer xenografts in nude mice. Cancer Res 56, 1098-1103.

Arthur JF, Butterfield LH, Roth MD, Bui LA, Kiertscher SM, Lau R, Dubinett S, Glaspy J, McBride WH, Economou JS (1997) A comparison of gene transfer methods in human dendritic cells. Cancer Gene Ther 4, 17-25.

Aruga E, Aruga A, Arca MJ, Lee WM, Yang NS, Smith JW 2nd, Chang AE (1997) Immune responsiveness to a murine mammary carcinoma modified to express B7-1, interleukin-12, or GM-CSF. Cancer Gene Ther 4, 157-166.

Asahara T, Chen D, Tsurumi Y, Kearney M, Rossow S, Passeri J, Symes JF, Isner JM (1996) Accelerated restitution of endothelial integrity and endothelium-dependent function after phVEGF165 gene transfer. Circulation 94, 3291-3302.

Austin EA and Huber BH (1993) A first step in the development of gene therapy for colorectal carcinoma: Cloning, sequencing, and expression of Escherichia coli cytosine deaminase. Mol Pharmacol 43, 380-387.

Baasner S, von Melchner H, Klenner T, Hilgard P, Beckers T (1996) Reversible tumorigenesis in mice by conditional expression of the HER2/c-erbB2 receptor tyrosine kinase. Oncogene 13, 901-911.

Bacchetti S and Graham FL (1993) Inhibition of cell proliferation by an adenovirus vector expressing the human wild-type p53 protein. Int J Oncol 3, 781-788.Baltimore D (1988) Gene therapy. Intracellular immunization. Nature 335, 395-396.

Bagchi, S., Raychaudhuri, P. and Nevins, J.R. (1990) Adenovirus E1A proteins can dissociate heteromeric complexes involving the E2F transcription factor: a novel mechanism for E1A trans-activation. Cell 62, 659-669.

Bagchi, S., Weinmann, R. and Raychaudhuri, P. (1991) The retinoblastoma protein copurifies with E2F-I, and E1A-regulated inhibitor of the transcription factor E2F. Cell 65, 1063-1072.

Baker SJ, Markowitz S, Fearon ER, Willson JKV, Vogelstein B (1990) Suppression of human colorectal carcinoma cell growth by wild-type p53. Science 249, 912-915.

Baldwin HS, Mickanin C, and Buck C (1997) Adenovirus-mediated gene transfer during initial organogenesis in the mammalian embryo is promoter-dependent and tissue-specific. Gene Ther 4, 1142-1149.

Balicki D and Beutler E (1997) Histone H2A significantly enhances in vitro DNA transfection. Mol Med 3, 782-787.

Ballay A, Levrero M, Buendia MA, Tiollais P, Perricaudet M (1985) In vitro and in vivo synthesis of the hepatitis B virus surface antigen and of the receptor for polymerized human serum albumin from recombinant human adenoviruses. EMBO J 4, 3861-3865.

Bandara G, Mueller GM, Galea-Lauri J, Tindal MH, Georgescu HI, Suchanek MK, Hung GL, Glorioso JC, Robbins PD, Evans CH (1993) Intraarticular expression of biologically active interleukin 1-receptor-antagonist protein by ex vivo gene transfer. Proc Natl Acad Sci USA 90, 10764-10768.

Banerjee S, Livanos E, Vos JM (1995) Therapeutic gene delivery in human B-lymphoblastoid cells by engineered non-transforming infectious Epstein-Barr virus. Nat Med 1, 1303-1308.

Barak Y, Juven T, Haffner R, Oren M (1993) mdm2 expression is induced by wild typep53 activity. EMBO J 12: 461-468.

Bargonetti J, Friedman PN, Kern SE, Vogelstein B, Prives C (1991) Wild-type but not mutant p53 immunopurified proteins bind to sequences adjacent to the SV40 origin of replication. Cell 65, 1083-1091.

Barr E, Leiden JM (1991) Systemic delivery of recombinant proteins by genetically modified myoblasts. Science 254, 1507-1509.

Barry MA, Dower WJ, Johnston SA (1996) Toward cell-targeting gene therapy vectors: Selection of cell-binding peptides from random peptide-presenting phage libraries. Nature Med 2, 299-305.

Barton NW, Furbish FS, Murray GJ, Garfield M, Brady RO (1990) Therapeutic response to intravenous infusions of glucocerebrosidase in a patient with Gaucher disease. Proc Natl Acad Sci USA 87, 1913-1916.

Baudard M, Flotte TR, Aran JM, Thierry AR, Pastan I, Pang MG, Kearns WG, Gottesman MM (1996) Expression of the human multidrug resistance and glucocerebrosidase cDNAs from adeno-associated vectors: efficient promoter activity of AAV sequences and in vivo delivery via liposomes. Hum Gene Ther 7, 1309-1322.

Bauer G, Valdez P, Kearns K, Bahner I, Wen SF, Zaia JA, Kohn DB (1997) Inhibition of human immunodeficiency virus-1 (HIV-1) replication after transduction of granulocyte colony-stimulating factor-mobilized CD34⁺ cells from HIV-1-infected donors using retroviral vectors containing anti-HIV-1 genes. Blood 89, 2259-2267.

Beal PA and Dervan PB (1991) Second structural motif for recognition of DNA by oligonucleotide-directed triple-helix formation. Science 251, 1360-1363.

Beg AA, Sha WC, Bronson RT, Ghosh S, and Baltimore D (1995) Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kB. Nature 376, 167-170.

Behr J-P (1994) Gene transfer with synthetic cationic amphiphiles; prospects for gene therapy. Bioconjugate Chem 5, 382-389.

Behr J-P, Demeneix B, Loeffler J-P, and Perez-Mutul J (1989) Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc Natl Acad Sci USA 86, 6982-6986.

Benn SI, Whitsitt JS, Broadley KN, Nanney LB, Perkins D, He L, Patel M, Morgan JR, Swain WF, Davidson JM (1996) Particle-mediated gene transfer with transforming growth factor-b1 cDNAs enhances wound repair in rat skin. J Clin Invest 98, 2894-2902.

Bennett MR, Anglin S, McEwan JR, Jagoe R, Newby AC, Evan GI (1994) Inhibition of vascular smooth muscle cell proliferation in vitro and in vivo by c-myc antisense oligodeoxynucleotides. J Clin Invest 93, 820-828.

Bergemann J, Kuhlcke K, Fehse B, Ratz I, Ostertag W, Lother H (1995) Excision of specific DNA-sequences from integrated retroviral vectors via site-specific recombination. Nucleic Acids Res 23, 4451-4456.

Berns KI, Linden RM (1995) The cryptic life style of adeno-associated virus. Bioessays 17, 237-245.

Beutler E (1997) Gaucher disease. Curr Opin Hematol 4, 19-23.

Bi W, Kim YG, Feliciano ES, Pavelic L, Wilson KM, Pavelic ZP, Stambrook PJ (1997) An HSVtk-mediated local and distant antitumor bystander effect in tumors of head and neck origin in athymic mice. Cancer Gene Ther 4, 246-252.

Bianchi, M.E., Beltrame, M. and Paonessa, G. (1989) Specific recognition of cruciform DNA by nuclear protein HMG1. Science 243, 1056-1059.

Bicknell R and Harris AL (1996) Mechanisms and therapeutic implications of angiogenesis. Curr Opin Oncol 8, 60-65.

Blaese RM, Culver KW, Miller AD, Carter CS, Fleisher T, Clerici M, Shearer G, Chang L, Chiang Y, Tolstoshev P, Greenblatt JJ, Rosenberg SA, Klein H, Berger M, Mullen CA, Ramsey WJ, Muul L, Morgan RA, and Anderson WF (1995) T lymphocyte-directed gene therapy for ADA-SCID: Initial trial results after 4 years. Science 270, 475-480.

Block A, Chen SH, Kosai K, Finegold M, Woo SL (1997) Adenoviral-mediated herpes simplex virus thymidine kinase gene transfer: regression of hepatic metastasis of pancreatic tumors. Pancreas 15, 25-34.

Bloom BR (1996) A perspective on AIDS vaccines. Science 272, 1888-1890.

Blumenthal R, Seth P, Willingham MC, Pastan I (1986) pH-dependent lysis of liposomes by adenovirus. Biochemistry 25, 2231-2237.

Bode J, Bartsch J, Boulikas T, Iber M, Mielke C, Schübeler D, Seibler J, and Benham C (1998) Transcription-promoting genomic sites in mammalia: their elucidation and architectural principles. Gene Ther Mol Biol 1, 551-580.

Bodnar, J.W., Hanson, P.I., Polvino-Bodnar, M., Zempsky, W., and Ward, D.C. (1989) The terminal regions of adenovirus and minute virus of mice DNAs are preferentially associated with the nuclear matrix in infected cells. J Virol. 63, 4344-4353.

Bohenzky RA, LeFebvre RB, Berns KI (1988) Sequence and symmetry requirements within the internal palindromic sequences of the adeno-associated virus terminal repeat. Virology 166, 316-327.

Bohn MC and Choi-Lundberg DL (1998) Neurotrophic factor gene therapy for neurodegenerative diseases. Gene Ther Mol Biol 1, 265-277.

Boletta A, Benigni A, Lutz J, Remuzzi G, Soria MR, Monaco L (1997) Nonviral gene delivery to the rat kidney with polyethylenimine. Hum Gene Ther 8, 1243-1251.

Bongartz J-P, Aubertin A-M, Milhaud PG, and Lebleu B (1994) Improved biological activity of antisense oligonucleotides conjugated to a fusogenic peptide. Nucleic Acids Res 22, 4681-4688.

Bookstein R, Shew J-Y, Chen P-L, Scully P, and Lee W-H (1990) Suppression of tumorigenicity of human prostate carcinoma cells by replacing a mutated RB gene. Science 247, 712-715.

Border WA, Noble NA (1995) Targeting TGF-b for treatment of disease. Nature Med 1, 1000-1001.

Bordignon C, Notarangelo LD, Nobili N, Ferrari G, Casorati G, Panina P, Mazzolari E, Maggioni D, Rossi C, Servida P, Ugazio AG, and Mavilio F (1995) Gene therapy in peripheral blood lymphocytes and bone marrow for ADA- immunodeficient patients. Science 270, 470-475.

Boris-Lawrie KA and Temin HM (1993) Recent advances in retrovirus vector technology. Curr Opin Genet Dev 3, 102-109.

Bornstein SR, Uhlmann K, Haidan A, Ehrhart-Bornstein M, Scherbaum WA (1997) Evidence for a novel peripheral action of leptin as a metabolic signal to the adrenal gland: leptin inhibits cortisol release directly. Diabetes 46, 1235-1238.

Borrelli E, Heyman R, Hsi M, and Evans RM (1988) Targeting of an inducible toxic phenotype in animal cells. Proc Natl Acad Sci USA 85, 7572-7576.

Boulikas T (1994) A compilation and classification of DNA binding sites for protein transcription factors from vertebrates. Crit. Rev. Euk. Gene Expression 4, 117-321.

Boulikas T (1995a) Phosphorylation of transcription factors and the control of the cell cycle. Crit Rev Eukar. Gene Expression 5, 1-77.

Boulikas T (1995b) Chromatin domains and prediction of MAR sequences. Int Rev Cytol. 162A, 279-388.

Boulikas T (1996a) Cancer gene therapy and immunotherapy. Int J Oncol 9, 941-954.

Boulikas T (1996b) Gene therapy to human diseases: ex vivo and in vivo studies. Int J Oncol 9, 1239-1251.

Boulikas T (1996c) DNA lesion-recognizing proteins and the p53 connection. Anticancer Res 16, 225-242.

Boulikas T (1996d) Liposome DNA delivery and uptake by cells. Oncol Rep. 3, 989-995.

Boulikas T (1996e) Common structural features of replication origins in all life forms J. Cell. Biochem. 60, 297-316.

Boulikas T (1997) Gene therapy of prostate cancer: p53, suicidal genes, and other targets. Anticancer Res 17, 1471-1506.

Boulikas T (1998) Nucleocytoplasmic trafficking: implications for the nuclear import of plasmid DNA during gene therapy. Gene Ther Mol Biol 1, 713-740.

Boussif (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 92, 7297-7301.

Boussif O, Zanta MA, Behr JP (1996) Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold. Gene Ther 3, 1074-1080.

Bowles NE, Eisensmith RC, Mohuiddin R, Pyron M, Woo SL (1996) A simple and efficient method for the concentration and purification of recombinant retrovirus for increased hepatocyte transduction in vivo. Hum Gene Ther 7, 1735-1742.

Brady HJM, Miles CG, Pennington DJ, Dzierzak EA (1994) Specific ablation of human immunodeficiency virus Tat-expressing cells by conditionally toxic retroviruses. Proc Natl Acad Sci USA 91, 365-369.

Braithwaite AW, Sturzbecher H-W, Addison C, Palmer C, Rudge K, Jenkins JR (1987) Mouse p53 inhibits SV40 origin-dependent DNA replication. Nature 329, 458-460.

Bramson JL, Hitt M, Addison CL, Muller WJ, Gauldie J, Graham FL (1996) Direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and long-lasting immunity that is associated with highly localized expression of interleukin-12. Hum Gene Ther 7, 1995-2002.

Brancolini C, Benedetti M, Schneider C (1995) Microfilament reorganization during apoptosis: the role of Gas2, a possible substrate for ICE-like proteases. EMBO J 14, 5179-5190.

Brewster SF, Simons JW (1994) Gene therapy in urological oncology: Principles, strategies and potential. Eur Urol 25, 177-182.

Breyne P, Van Montagu M, Depicker A, and Gheysen G. (1992) Characterization of a plant scaffold attachment region in a DNA fragment that normalizes transgene expression in tobacco. Plant Cell 4, 463-471.

Bridgen A and Elliott RM (1996) Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs. Proc Natl Acad Sci USA 93, 15400-15404.

Brooks EE, Gray NS, Joly A, Kerwar SS, Lum R, Mackman RL, Norman TC, Rosete J, Rowe M, Schow SR, Schultz PG, Wang X, Wick MM, Shiffman D (1997) CVT-313, a specific and potent inhibitor of CDK2 that prevents neointimal proliferation. J Biol Chem 272, 29207-29211.

Brooks, A.R., Nagy, B.P., Taylor, S., Simonet, W.S., Taylor, J.M., and Levy-Wilson, B. (1994) Sequences containing the second-intron enhancer are essential for transcription of the human apolipoprotein B gene in the livers of transgenic mice. Mol Cell Biol 14, 2243-2256.

Buchkovich, K., Duffy, L.A. and Harlow, E. (1989) The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell 58, 1097-1105.

Budker V, Zhang G, Knechtle S, Wolff JA (1996) Naked DNA delivered intraportally expresses efficiently in hepatocytes. Gene Ther 3, 593-598.

Bugge TH, Flick MJ, Daugherty CC, Degen JL (1995) Plasminogen deficiency causes severe thrombosis but is compatible with development and reproduction. Genes Dev 9, 794-807.

Bullough PA, Hughson FM, Skehel JJ, Wiley DC (1994) Structure of influenza haemagglutinin at the pH of membrane fusion. Nature 371, 37-43.

Burfeind P, Chernicky CL, Rininsland F, Ilan J, Ilan J (1996) Antisense RNA to the type I insulin-like growth factor receptor suppresses tumor growth and prevents invasion by rat prostate cancer cells In vivo. Proc Natl Acad Sci USA 93, 7263-7268.

Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK (1993) Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci USA 90, 8033-8037.

Bushman FD and Miller MD (1997) Tethering human immunodeficiency virus type 1 preintegration complexes to target DNA promotes integration at nearby sites. J Virol 71, 458-464.

Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P (1995) Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 269, 546-549.

Canman CE, Gilmer TM, Coutts SB, and Kastan MB (1995) Growth factor modulation of p53-mediated growth arrest versus apoptosis. Genes Dev 9, 600-611.

Cannon PM, Kim N, Kingsman SM, Kingsman AJ (1996) Murine leukemia virus-based Tat-inducible long terminal repeat replacement vectors: a new system for anti-human immunodeficiency virus gene therapy. J Virol 70, 8234-8240.

Canonico AE, Conary JT, Meyrick BO, Brigham KL (1994) Aerosol and intravenous transfection of human a1-antitrypsin gene to lungs of rabbits. Am J Respir Cell Mol Biol 10, 24-29.

Cao L, Zheng Z-C, Zhao Y-C, Jiang Z-H, Liu Z-G, Chen S-D, Zhou C-F, and Liu X-Y (1995) Gene therapy of Parkinson disease model rat by direct injection of plasmid DNA-lipofectin complex. Hum Gene Ther 6, 1497-1501.

Capaccioli S, Di Pasquale G, Mini E, Mazzei T, Quattrone A (1993) Cationic lipids improve antisense oligonucleotide uptake and prevent degradation in cultured cells and in human serum. Biochem Biophys Res Commun 197, 818-825.

Caputo A, Rossi C, Bozzini R, Betti M, Grossi MP, Barbanti-Brodano G, Balboni PG (1997) Studies on the effect of the combined expression of anti-tat and anti-rev genes on HIV-1 replication. Gene Ther 4, 288-295.

Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435-439.

Carmeliet P, Moons L, Dewerchin M, Mackman N, Luther T, Breier G, Ploplis V, Muller M, Nagy A, Plow E, Gerard R, Edgington T, Risau W, Collen D (1997) Insights in vessel development and vascular disorders using targeted inactivation and transfer of vascular endothelial growth factor, the tissue factor receptor, and the plasminogen system. Ann N Y Acad Sci 811, 191-206.

Carreau M, Quilliet X, Eveno E, Salvetti A, Danos O, Heard J-M, Mezzina M, and Sarasin A (1995) Functional retroviral vector for gene therapy of xeroderma pigmentosum group D patients. Hum Gene Ther 6, 1307-1315.

Caruso M, Pham-Nguyen K, Kwong YL, Xu B, Kosai KI, Finegold M, Woo SL, Chen SH (1996) Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma. Proc Natl Acad Sci USA 93, 11302-11306.

Casalini P, Menard S, Malandrin SM, Rigo CM, Colnaghi MI, Cultraro CM, Segal S (1997) Inhibition of tumorigenicity in lung adenocarcinoma cells by c-erbB-2 antisense expression. Int J Cancer 72, 631-636.

Casciola-Rosen LA, Anhalt GJ, Rosen A (1995) DNA-dependent protein kinase is one of a subset of autoantigens specifically cleaved early during apoptosis. J Exp Med 182, 1625-1634.

Cassileth PA, Podack E, Sridhar K, Savaraj N, and Hanlon J (1995) Phase I study of transfected cancer cells expressind the interleukin-2 gene product in limited stage small cell lung cancer. Hum Gene Ther 6, 369-383.

Cavanaugh AH, Hempel WM, Taylor LJ, Rogalsky V, Todorov G, Rothblum LI (1995) Activity of RNA polymerase I transcription factor UBF blocked by Rb gene product. Nature 374, 177-180.

Cesano A, Visonneau S Jeglum KA, Owen J, Wilkinson K, Carner K, Reese L, Santoli D (1996) Phase I clinical trial with a major histocompatibility complex nonrestricted cytotoxic T-cell line (TALL-104) in dogs with advanced tumors. Cancer Res 56, 3021-3029.

Chadwick SL, Kingston HD, Stern M, Cook RM, O'Connor BJ, Lukasson M, Balfour RP, Rosenberg M, Cheng SH, Smith AE, Meeker DP, Geddes DM, Alton EW (1997) Safety of a single aerosol administration of escalating doses of the cationic lipid GL-67/DOPE/DMPE-PEG5000 formulation to the lungs of normal volunteers. Gene Ther 4, 937-942.

Chambers R, Gillespie GY, Soroceanu L, Andreansky S, Chatterjee S, Chou J, Roizman B, Whitley RJ (1995) Comparison of genetically engineered herpes simplex viruses for the treatment of brain tumors in a scid mouse model of human malignant glioma. Proc Natl Acad Sci USA 92, 1411-1415.

Chan L, Teng BB, Lau P (1996) Apolipoprotein B mRNA editing protein: a tool for dissecting lipoprotein metabolism and a potential therapeutic gene for hypercholesterolemia. Z Gastroenterol 34 Suppl 3, 31-32.

Chan YJ, Chiou CJ, Huang Q, Hayward GS (1996) Synergistic interactions between overlapping binding sites for the serum response factor and ELK-1 proteins mediate both basal enhancement and phorbol ester responsiveness of primate cytomegalovirus major immediate-early promoters in monocyte and T-lymphocyte cell types. J Virol 70, 8590-8605.

Chang AE, Sondak VK, Bishop DK, Nickoloff BJ, Mulligan RC, and Mule JJ (1996). Clinical protocol. Adoptive immunotherapy of cancer with activated lymph node cells primed in vivo with autologous tumor cells transduced with the GM-CSF gene. Hum Gene Ther 7, 773-792.

Chang EH, Jang YJ, Hao Z, Murphy G, Rait A, Fee WE Jr, Sussman HH, Ryan P, Chiang Y, Pirollo KF (1997) Restoration of the G1 checkpoint and the apoptotic pathway mediated by wild-type p53 sensitizes squamous cell carcinoma of the head and neck to radiotherapy. Head Neck Surg 123, 507-512.

Chang JY, Xia W, Shao R, Hung MC (1996) Inhibition of intratracheal lung cancer development by systemic delivery of E1A. Oncogene 13, 1405-1412.

Chang MW, Barr E, Lu MM, Barton K, Leiden JM (1995b) Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest 96, 2260-2268.

Chang MW, Barr E, Seltzer J, Jiang Y-Q, Nabel GJ, Nabel EG, Parmacek MS, and Leiden JM (1995a) Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product. Science 267, 518-522.

Chang MW, Barr E, Seltzer J, Jiang Y-Q, Nabel GJ, Nabel EG, Parmacek MS, and Leiden JM (1995) Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product. Science 267, 518-522.

Chang YN, Jeang KT, Chiou CJ, Chan YJ, Pizzorno M, Hayward GS (1993) Identification of a large bent DNA domain and binding sites for serum response factor adjacent to the NFI repeat cluster and enhancer region in the major IE94 promoter from simian cytomegalovirus. J Virol 67, 516-529.

Chao J, Jin L, Chen L-M, Chen VC, and Chao L (1996) Systemic and portal vein delivery of human kallikrein gene reduces blood pressure in hypersensitive rats. Hum Gene Ther 7, 901-911.

Chatterjee PK and Flint SJ (1986) Partition of E1A proteins between soluble and structural fractions of adenovirus-infected and -transformed cells. J. Virol. 60, 1018-1026.

Chatterjee S Johnson PR, and Wong KK (1992) Dual tasrget inhibition of HIV-1 in vitro by means of an adeno-associated virus antisense vector. Science 258, 1485-1488.

Chellappan S, Hiebert S, Mudryj M, Horowitz J, Nevins J (1991) The E2F transcription factor is a cellular target for the RB protein. Cell 65, 1053-1061.

Chen BP, Fraser C, Reading C, Murray L, Uchida N, Galy A, Sasaki D, Tricot G, Jagannath S, Barlogie B, et al (1995) Cytokine-mobilized peripheral blood CD34⁺Thy-1⁺Lin^- human hematopoietic stem cells as target cells for transplantation-based gene therapy. Leukemia 9 Suppl 1:S17-S25.

Chen C, Parangi S, Tolentino MJ, Folkman J (1995) A strategy to discover circulating angiogenesis inhibitors generated by human tumors. Cancer Res 55, 4230-4233.

Chen C-Y, Oliner JD, Zhan Q, Fornace Jr AJ, Vogelstein B, Kastan MB (1994) Interactons between p53 and MDM2 in a mammalian cell cycle checkpoint pathway. Proc Natl Acad Sci USA 91, 2684-3688.

Chen DS, Zhu NL, Hung G, Skotzko MJ, Hinton DR, Tolo V, Hall FL, Anderson WF, Gordon EM (1997) Retroviral vector-mediated transfer of an antisense cyclin G1 construct inhibits osteosarcoma tumor growth in nude mice. Hum Gene Ther 8, 1667-1674.

Chen G, Koyama K, Yuan X, Lee Y, Zhou YT, O'Doherty R, Newgard CB, Unger RH (1996) Disappearance of body fat in normal rats induced by adenovirus-mediated leptin gene therapy. Proc Natl Acad Sci USA 93, 14795-14799.

Chen J, Stickles RJ, Daichendt KA (1994) Galactosylated histone-mediated gene transfer and expression. Hum Gene Ther 5, 429-435.

Chen LM, Chao L, Chao J (1997) Adenovirus-mediated delivery of human kallistatin gene reduces blood pressure of spontaneously hypertensive rats. Hum Gene Ther 8, 341-347.

Chen PL, Chen Y, Shan B, Bookstein R, Lee WH (1992) Stability of retinoblastoma gene expression determines the tumorigenicity of reconstituted retinoblastoma cells. Cell Growth Differ 3, 119-125

Chen SH, Kosai K, Xu B, Pham-Nguyen K, Contant C, Finegold MJ, Woo SL (1996) Combination suicide and cytokine gene therapy for hepatic metastases of colon carcinoma: sustained antitumor immunity prolongs animal survival. Cancer Res 56, 3758-3762.

Chen SJ, Wilson JM, Vallance DK, Hartman JW, Davidson BL, Roessler BJ (1995) A recombinant adenoviral vector expressing a soluble form of VCAM-1 inhibits VCAM-1/VLA-4 adhesion in transduced synoviocytes. Gene Ther 2, 469-480.

Chen ST, Iida A, Guo L, Friedmann T, Yee JK (1996) Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system. Proc Natl Acad Sci USA 93, 10057-10062.

Chen X, Farmer G, Zhu H, Prywes R, Prives C (1993) Cooperative DNA binding of p53 with TFIID (TBP): A possible mechanism for transcriptional activation. Genes Dev 7, 1837-1849.

Chen Y, Chen C-F, Riley DJ, Allred DC, Chen P-L, Von Hoff D, Osborne CK, and Lee W-H (1995) Aberrant subcellular localization of BRCA1 in breast cancer. Science 270, 789-791.

Chen YM, Chen PL, Arnaiz N, Goodrich D, Lee WH (1991) Expression of wild-type p53 in human A673 cells suppresses tumorigenicity but not growth rate. Oncogene 6, 1799-1805.

Chen P-L , Scully, P., Shew, J.-Y., Wang, J.Y.J. and Lee, W.-H. (1989) Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell 58, 1193-1198.

Chernajovsky Y, Adams G, Triantaphyllopoulos K, Ledda MF, Podhajcer OL (1997) Pathogenic lymphoid cells engineered to express TGF b 1 ameliorate disease in a collagen-induced arthritis model. Gene Ther 4, 553-559

Chi SG, de Vere White RW, Meyers FJ,. Siders DB, Lee F, and Gumerlock PH (1994) p53 in prostate cancer: frequent expressed transition mutations. J Natl Cancer Inst 86, 926-933.

Chin K-V, Ueda K, Pastan I, Gottesman MM (1992) Modulation of activity of the promoter of the human MDR1 gene by Ras and p53. Science 255, 459-462.

Chintala SK, Fueyo J, Gomez-Manzano C, Venkaiah B, Bjerkvig R, Yung WK, Sawaya R, Kyritsis AP, Rao JS (1997) Adenovirus-mediated p16/CDKN2 gene transfer suppresses glioma invasion in vitro. Oncogene 15, 2049-2057.

Chiou S, Rao L, and White E (1994) Bcl-2 blocks p53-dependent apoptosis. Mol Cell Biol 14, 1556-1563.

Choate KA and Khavari PA (1997) Direct cutaneous gene delivery in a human genetic skin disease. Hum Gene Ther 8, 1659-1665.

Chowdhury JR, Grossman M, Gupta SJ, Chowdhury NR, Baker Jr JR, Wilson JM (1991) Long term improvement of hypercholesterolemia after ex vivo gene therapy in LDLR-deficient rabbits. Science 254, 1802-1805.

Christofori G, Naik P, and Hanahan D (1995) Vascular endothelial growth factor and its receptors, flt-1 and flk-1, are expressed in normal pancreatic islets and throughout islet cell tumorigenesis. Mol Endocrinol 9, 1760-1770.

Chuah MKL, Vandendriessche T, and Morgan RA (1995) Development and analysis of retroviral vectors expressing human factor VIII as a potential gene therapy for hemophilia A. Hum Gene Ther 6, 1363-1377.

Cichy J, Potempa J, Travis J (1997) Biosynthesis of a1-proteinase inhibitor by human lung-derived epithelial cells. J Biol Chem 272, 8250-8255.

Clark KR, Voulgaropoulou F, Fraley DM, and Johnson PR (1995) Cell lines for the production of recombinant adeno-associated virus. Hum Gene Ther 6, 1329-1341.

Clark KR, Voulgaropoulou F, Johnson PR (1996) A stable cell line carrying adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors. Gene Ther 3, 1124-1132.

Clark SJ, Harrison J, Molloy PL (1997) Sp1 binding is inhibited by ^mCp^mCpG methylation. Gene 195, 67-71.

Clarke MF, Apel IJ, Benedict MA, Eipers PG, Sumantran V, Gonzalez-Garcia M, Doedens M, Fukunaga N, Davidson B, Dick JE, et al (1995) A recombinant bcl-xs adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells. Proc Natl Acad Sci USA 92, 11024-11028.

Clary BM, Coveney EC, Philip R, Blazer DG 3rd, Morse M, Gilboa E, Lyerly HK (1997) Inhibition of established pancreatic cancers following specific active immunotherapy with interleukin-2 gene-transduced tumor cells. Cancer Gene Ther 4, 97-104.

Cleat PH and Hay RT (1989) Co-operative interactions between NFI and the adenovirus DNA binding protein at the adenovirus origin of replication. EMBO J. 8, 1841-1848.

Coenjaerts FEJ, De Vries E, Pruijn GJM, van Driel W. Bloemers SM, van der Lugt MT, and van der Vliet PC (1991) Enhancement of DNA replication by transcription factors NFI and NFIII/Oct-1 depends critically on the positions of their binding sites in the adenovirus origin of replication. Biochim. Biophys. Acta 1090, 61-69.

Colak A, Goodman JC, Chen S-H, Woo SLC, Grossman RG, Shine HD (1995) Adenovirus-mediated gene therapy in an experimental model of breast cancer metastatic to the brain. Hum Gene Ther 6, 1317-1322.

Coll JL, Wagner E, Combaret V, Metchler K, Amstutz H, Iacono-Di-Cacito I, Simon N, Favrot MC (1997) In vitro targeting and specific transfection of human neuroblastoma cells by chCE7 antibody-mediated gene transfer. Gene Ther 4, 156-161.

Conary JT, Parker RE, Christman BW, Raulks RD, King GA, Meyrick BO, Brigham KL (1994) Protection of rabbit lungs from endotoxin injury by in vivo hyperexpression of the prostaglandin G/H synthase gene. J Clin Invest 93, 1834-1840.

Connelly S and Kaleko M (1998) Hemophilia A: current treatment and future gene therapy. Gene Ther Mol Biol 1, 279-292.

Conrad CK, Allen SS, Afione SA, Reynolds TC, Beck SE, Fee-Maki M, Barrazza-Ortiz X, Adams R, Askin FB, Carter BJ, Guggino WB, Flotte TR (1996) Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung. Gene Ther 3, 658-668.

Considine RV, Considine EL, Williams CJ, Nyce MR, Magosin SA, Bauer TL, Rosato EL, Colberg J, Caro JF (1995) Evidence against either a premature stop codon or the absence of obese gene mRNA in human obesity. J Clin Invest 95, 2986-2988.

Cooney M, Czernuszewicz G, Postel EH, Flint SJ, Hogan ME (1988) Site-specific oligonucleotide binding represses transcription of the human c-myc gene in vitro. Science 241, 456-459.

Corbeau P, Kraus G, and Wong-Staal F (1996) Efficient gene transfer by a human immunodeficiency virus type 1 (HIV-1)-derived vector utilizing a stable HIV packaging cell line. Proc Natl Acad Sci USA 93, 14070-14075.

Corey CA, Desilva AD, Holland CA, and Williams DA (1990) Serial transplantation of MTX resistant bone marrow; protection of murine recipients from drug toxicity by progeny of transduced stem cells. Blood 76, 337-343.

Cornetta K and Anderson WF (1989) Protamine sulfate as an effective alternative to polybrene in retroviral-mediated gene-transfer: implications for human gene therapy. J Virol Methods 23, 187-194.

Cotten M, Wagner E, Zatloukal K, Phillips S, Curiel DT, and Birnstiel ML (1992) High efficiency receptor-mediated delivery of small and large (48 kilobase) gene constructs using the endosome disruption activity of defective or chemically inactivated adenovirus particles. Proc Natl Acad Sci USA 89, 6094-6098.

Cox LS, Hupp T, Midgley CA, Lane DP (1995) A direct effect of activated human p53 on nuclear DNA replication. EMBO J 14, 2099-2105.

Crook K, McLachlan G, Stevenson BJ, Porteous DJ (1997) Plasmid DNA molecules complexed with cationic liposomes are protected from degradation by nucleases and shearing by aerosolisation. Gene Ther 3, 834-839.

Cross GAM (1987) Eukaryotic protein modification and membrane attachment via phosphatidylinositol. Cell 48, 179-181.

Crystal RG (1990) a1-antitrypsin deficiency, emphysema, and liver disease. J Clin Invest 85, 1343-1352.

Cuevas P, Garcia-Calvo M, Carceller F, Reimers D, Zazo M, Cuevas B, Munoz-Willery I, Martinez-Coso V, Lamas S, Gimenez-Gallego G (1996) Correction of hypertension by normalization of endothelial levels of fibroblast growth factor and nitric oxide synthase in spontaneously hypertensive rats. Proc Natl Acad Sci USA 93, 11996-12001.

Culver KW (1996) in “Gene Therapy: a primer for physicians”. Second Edition. Mary Ann Liebert, Inc. Publications NY. pp. 1-198.

Culver KW, Ram Z, Wallbridge S, Ishii H, Oldfield EH, and Blaese RM (1992) In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 256, 1550-1552.

Curiel DT (1994) High-efficiency gene transfer employing adenovirus-polylysine-DNA complexes. Nat Immun 13, 141-164.

Curiel DT, Agarwal S, Wagner E, and Cotten M (1991) Adenovirus enhancement of transferrrin-polylysine-mediated gene delivery. Proc Natl Acad Sci USA 88, 8850-8854.

Cwirla SE, Peters EA, Barrett RW, and Dower WJ (1990) Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Acad Sci USA 87, 6378-6382.

Czubayko F, Downing SG, Hsieh SS, Goldstein DJ, Lu PY, Trapnell BC, and Wellstein A (1997) Adenovirus-mediated transduction of ribozymes abrogates HER-2/neu and pleiotrophin expression and inhibits tumor cell proliferaation. Gene Ther 4, 934-949.

D’Souza MP and Harden VA (1996) Chemokines and HIV-1 second receptors. Confluence of two fields generates optimism in aids research. Nat Med 2, 1293-1300.

da Costa LT, Jen J, He T-C, Chan TA, Kinzler KW, and Vogelstein B (1996) Converting cancer genes into killer genes. Proc Natl Acad Sci USA 93, 4192-4196.

Dai Y, Roman M, Naviaux RK, and Verma IM (1992) Gene therapy via primary myoblasts: Long term expression of factor IX protein following transplantation in vivo. Proc Natl Acad Sci USA 89, 10892-10895.

Dai Y, Schwarz EM, Gu D, Zhang W-W, Sarvetnick N, and Verma IM (1995) Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: Tolerization of factor IX and vector antigens allows for long-term expression. Proc Natl Acad Sci USA 92, 1401-1405.

Dameron KM, Volpert OV, Tainsky MA, Bouck N (1994) Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 265, 1582-1584.

Das Gupta TK, Cohen EP, Richards JM (1997) Phase I evaluation of interleukin-2-transfected irradiated allogeneic melanoma for the treatment of metastatic melanoma: appendix 1: protocol. Hum Gene Ther 8, 1701-1714.

Davies JC, Stern M, Dewar A, Caplen NJ, Munkonge FM, Pitt T, Sorgi F, Huang L, Bush A, Geddes DM, Alton EW (1997) CFTR gene transfer reduces the binding of Pseudomonas aeruginosa to cystic fibrosis respiratory epithelium. Am J Respir Cell Mol Biol 16, 657-663.

Davis S, Aldrich TH, Jones PF, Acheson A, Compton DL, Jain V, Ryan TE, Bruno J, RadziejewskiC, Maisonpierre PC, and Yancopoulos GD (1996) Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell 87, 1161-1169.

Day ML, Wu S, Basler JW (1993) Prostatic nerve growth factor inducible A gene binds a novel element in the retinoblastoma gene promoter. Cancer Res 53, 5597-5599.

de los Santos C, Rosen M, Patel D (1989) NMR studies of DNA (R⁺)_n.(Y^-)_n.(Y⁺)_n triple helices in solution: imino and amino proton markers of T.A.T and C.G.C⁺ base-triple formation. Biochemistry 28, 7282-7289.

de Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, and Williams LT (1992) The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science 255, 989-991.

Deb S, Jackson CT, Subler MA, and Martin DW (1992) Modulation of cellular and viral promoters by mutant human p53 proteins found in tumor cells. J Virol 66, 6164-6170.

Deb SP, Muñoz RM, Brown DR, Subler MA, and Deb S (1994) Wild-type human p53 activates the human epidermal growth factor receptor promoter. Oncogene 9, 1341-1349.

Debbas, M. and White, E. (1993) Wild-type p53 mediates apoptosis by E1A, which is inhibited by E1B. Genes Dev. 7, 546-554.

DeCaprio, J.A. , Ludlow, J.W., Lynch, D., Furukawa, Y., Griffin, J., Piwnica-Worms, H., Huang, C.-M. and Livingston, D.M. (1989) The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell 58, 1085-1095.

Deglon N, Heyd B, Tan SA, Joseph JM, Zurn AD, Aebischer P (1996) Central nervous system delivery of recombinant ciliary neurotrophic factor by polymer encapsulated differentiated C2C12 myoblasts. Hum Gene Ther 7, 2135-2146.

Delort JP and Capecchi MR (1996) TAXI/UAS: a molecular switch to control expression of genes in vivo. Human Gene Ther 7, 809-820.

DeLuca NA, and Schaffer PA (1987) Activities of herpes simplex virus type 1 (HSV-1) ICP4 genes specifying nonsense peptides. Nucleic Acids Res 15, 4491-4511.

Dematteo RP, McClane SJ, Fisher K, Yeh H, Chu G, Burke C, Raper SE (1997) Engineering tissue-specific expression of a recombinant adenovirus: selective transgene transcription in the pancreas using the amylase promoter. J Surg Res 72, 155-161.

Deppert W (1994) The yin and yang of p53 in cellular proliferation. Sem Cancer Biol 5, 187-202.

Devlin JJ, Panganiban LC, Devlin PE (1990) Random peptide libraries: A source of specific binding protein molecules. Science 249, 404-406.

Dhawan J, Pan LC, Pavlath GK, Travis MA, Lanctot AM, Blau HM (1991) Systemic delivery of human growth hormone by injection of genetically engineered myoblasts. Science 254, 1509-1512.

Diller L, Kassel J, Nelson CE, Gryka MA, Litwak G, Gebhardt M, Bressac B, Ozturk M, Baker SJ, Vogelstein B, Friend SH (1990) p53 functions as a cell cycle control protein in osteosarcomas. Mol Cell Biol 10, 5772-5781.

Dimaio JM, Clary BM, Via DF, Coveney E, Papas TN, and Lyerly HK (1994) Directed enzyme pro-drug gene therapy for pancreatic cancer in vivo. Surgery 116, 205-213.

Dinjens WN, van der Weiden MM, Schroeder FH, Bosman FT, and Trapman J (1994) Frequency and characterization of p53 mutations in primary and metastatic human prostate cancer. Int J Cancer 56, 630-633.

Dion LD, Goldsmith KT, Strong TV, Bilbao G, Curiel DT, Garver RI Jr (1996) E1A RNA transcripts amplify adenovirus-mediated tumor reduction. Gene Ther 3, 1021-1025.

Dittmer D, Pati S, Zambetti G, Chu S, Teresky AK, Moore M, Finlay C, Levine AJ: Gain of function mutations in p53. Nature Genet 4: 42-45, 1993.

Doll RF, Crandall JE, Dyer CA, Aucoin JM, Smith FI (1996) Comparison of promoter strengths on gene delivery into mammalian brain cells using AAV vectors. Gene Ther 3, 437-447.

Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CAJr, Butel JS, Bradley A (1992) Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215-221.

Dong JY, Fan PD, Frizzell RA (1996) Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther 7, 2101-2112.

Dong Z, Kumar R, Yang X, Fidler IJ (1997) Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma. Cell 88, 801-810.

Doorbar J, Winter G (1994) Isolation of a peptide antagonist to the thrombin receptor using phage display. J Mol Biol 244, 361-369.

Dorai T, Olsson CA, Katz AE, Buttyan R (1997) Development of a hammerhead ribozyme against bcl-2. I. Preliminary evaluation of a potential gene therapeutic agent for hormone-refractory human prostate cancer. Prostate 32, 246-258.

Dougherty JP, Wisniewski R, Yang SL, Rhode BW, Temin HM (1989) New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors. J Virol 63, 3209-3212.

Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H, Brose K, Jackson V, Hamada H, Pardoll D, Mulligan R (1993) Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci USA 90, 3539-3543.

Drazan KE, Shen XD, Csete ME, Zhang WW, Roth JA, Busuttil RW, Shaked A (1994) In vivo adenoviral-mediated human p53 tumor suppressor gene transfer and expression in rat liver after resection. Surgery 116, 197-203.

Duan L, Zhu M, Bagasra O, Pomerantz RJ (1995) Intracellular immunization against HIV-1 infection of human T lymphocytes: Utility of anti-rev single-chain variable fragments. Hum Gene Ther 6, 1561-1573.

Duan L, Zhu M, Ozaki I, Zhang H, Wei DL, Pomerantz RJ (1997) Intracellular inhibition of HIV-1 replication using a dual protein- and RNA-based strategy. Gene Ther 4, 533-543.

Duke RC, Ojcius DM, and Young JD-E (1996) Cell suicide in health and disease. Scientifc Amer 275, 80-87.

Dumont DJ, Gradwohl G, Fong GH, Puri MC, Gertsenstein M, Auerbach A, Breitman ML (1994) Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev 8, 1897-1909.

Dunbar C and Kohn D (1996) Retroviral mediated transfer of the cDNA for human glucocerebrosidase into hematopoietic stem cells of patients with Gaucher disease. A phase I study. Hum Gene Ther 7, 231-253.

Dunlap DD, Maggi A, Soria MR, Monaco L (1997) Nanoscopic structure of DNA condensed for gene delivery. Nucleic Acids Res 25, 3095-3101.

Dupuit F, Chinet T, Zahm JM, Pierrot D, Hinnrasky J, Kaplan H, Bonnet N, Puchelle E (1997) Induction of a cAMP-stimulated chloride secretion in regenerating poorly differentiated airway epithelial cells by adenovirus-mediated CFTR gene transfer. Hum Gene Ther 8, 1439-1450.

During MJ, Naegele JR, O'Malley KL, Geller AI (1994) Long-term behavioral recovery in Parkinsonian rats by an HSV Vector expressing tyrosine hydroxylase. Science 266, 1399-1403.

Dutta A, Ruppert JM, Aster JC and Winchester E (1993) Inhibition of DNA replication factor RPA by p53. Nature 365: 79-82.

Dwarki VJ, Belloni P, Nijjar T, Smith J, Couto L, Rabier M, Clift S, Berns A, and Cohen LK (1995) Gene therapy for hemophilia A: Production of therapeutic levels of human factor VIII in vivo in mice. Proc Natl Acad Sci USA 92, 1023-1027.

Dyson, N., Howley, P.M., Münger, K. and Harlow, E. (1989) The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243, 934-937.

Ealovega MW, McGinnis PK, Sumantran VN, Clarke MF, Wicha MS (1996) bcl-xs gene therapy induces apoptosis of human mammary tumors in nude mice. Cancer Res 56, 1965-1969.

Eastham JA, Chen S-H, Sehgal I, Yang G, Timme TL, Hall SJ, Woo SLC, and Thompson TC (1996) Prostate cancer gene therapy: herpes simplex virus thymidine kinase gene transduction followed by ganciclovir in mouse and human prostate cancer models. Hum Gene Ther 7, 515-523.

Eastham JA, Hall SJ, Sehgal I, Wang J, Timme TL, Yang G, Connell-Crowley L, Elledge SJ, Zhang W-W, Harper JW, and Thompson TC (1995) In vivo gene therapy with p53 or p21 adenovirus for prostate cancer. Cancer Res 55, 5151-5155.

Ebbinghaus SW, Vigneswaran N, Miller CR, Chee-Awai RA, Mayfield CA, Curiel DT, Miller DM (1996) Efficient delivery of triplex forming oligonucleotides to tumor cells by adenovirus-polylysine complexes. Gene Ther 3, 287-297.

Eckert HG, Stockschlader M, Just U, Hegewisch-Becker S, Grez M, Uhde A, Zander A, Ostertag W, Baum C (1996) High-dose multidrug resistance in primary human hematopoietic progenitor cells transduced with optimized retroviral vectors. Blood 88, 3407-3415.

Einerhand MP, Antoniou M, Zolotukhin S, Muzyczka N, Berns KI, Grosveld F, Valerio D (1995) Regulated high-level human b-globin gene expression in erythroid cells following recombinant adeno-associated virus-mediated gene transfer. Gene Ther 2, 336-343.

El Ouahabi A, Thiry M, Pector V, Fuks R, Ruysschaert JM, Vandenbranden M (1997) The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids. FEBS Lett 414, 187-192.

El-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B (1992) Definition of a consensus binding site for p53. Nature Genet 1, 45-49.

El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817-825.

Eliyahu D, Michalovitz D, Eliyahu S, Pinhasi-Kimhi O, Oren M (1989) Wild-type p53 can inhibit oncogene-mediated focus formation. Proc Natl Acad Sci USA 86, 8763-8767.

Elshami AA, Cook JW, Amin KM, Choi H, Park JY, Coonrod L, Sun J, Molnar-Kimber K, Wilson JM, Kaiser LR, Albelda SM (1997) The effect of promoter strength in adenoviral vectors containing herpes simplex virus thymidine kinase on cancer gene therapy in vitro and in vivo. Cancer Gene Ther 4, 213-221.

Emoto Y, Manome Y, Meinhardt G, Kisaki H, Kharbanda S, Robertson M, Ghayur T, Wong WW, Kamen R, Weichselbaum R, et al (1995) Proteolytic activation of protein kinase C d by an ICE-like protease in apoptotic cells. EMBO J 14, 6148-6156.

Endicott JA and Ling V (1989) The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu Rev Biochem 58, 137-171.

Enomaa N, Danos O, Peltonen L, and Jalanko A (1995) Correction of deficient enzyme activity in a lysosomal storage disease, aspartylglucosaminuria, by enzyme replacement and retroviral gene transfer. Hum Gene Ther 6, 723-731.

Evans CH and Robbins PD (1996) Pathways to gene therapy in rheumatoid arthritis. Curr Opin Rheumatol 8, 230-234.

Evans CH, Robbins PD, Ghivizzani SC, Herndon JH, Kang R, Bahnson AB, Barranger JA, Elders EM, Gay S, Tomaino MM, Wasko MC, Watkins SC, Whiteside TL, Glorioso JC, Lotze MT, Wright TM (1996) Clinical trial to assess the safety, feasibility, and efficacy of transferring a potentially anti-arthritic cytokine gene to human joints with rheumatoid arthritis. Hum Gene Ther 7, 1261-1280.

Fang B, Eisensmith RC, Wang H, Kay MA, Cross RE, Landen CN, Gordon G, Bellinger DA, Read MS, Hu PC, Brinkhous KM, and Woo SLC (1995) Gene therapy for hemophilia B: Host immunosuppression prolongs the therapeutic effect of adenovirus-mediated factor IX expression. Hum Gene Ther 6, 1039-1044.

Fardel O, Escande F, Drenou B, Le Bescot J, Rault B, and Fauchet R (1995) Expression of P-glycoprotein in multidrug-resistant human leukemia K562 cells during erythroid differentiation. Int J Oncol 7, 377-381.

Farmer G, Bargonetti J, Zhu H, Friedman P, Prywes R, Prives C (1992) Wild-type p53 activates transcription in vitro. Nature 358, 83-86.

Fasbender A, Zabner J, Chillon M, Moninger TO, Puga AP, Davidson BL, Welsh MJ (1997) Complexes of adenovirus with polycationic polymers and cationic lipids increase the efficiency of gene transfer in vitro and in vivo. J Biol Chem 272, 6479-6489.

Fassati A, Wells DJ, Walsh FS, Dickson G (1995) Efficiency of in vivo gene transfer using murine retroviral vectors is strain-dependent in mice. Hum Gene Ther 6, 1177-1183.

Fearon ER, Pardoll DM, Itaya T, Golumbek P, Levitsky HI, Simons JW, Karasuyama H, Vogelstein B, Frost P (1990) Interleukin-2 production by tumor cells bypasses T helper function in the generation of an antitumor response. Cell 60, 397-403.

Federspiel MJ, Swing DA, Eagleson B, Reid SW, Hughes SH (1996) Expression of transduced genes in mice generated by infecting blastocysts with avian leukosis virus-based retroviral vectors. Proc Natl Acad Sci USA 93, 4931-4936.

Felgner JH, Kumar R, Sridhar CN, Wheeler CJ, Tsai YJ, Border R, Ramsey P, Martin M, Felgner PL (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol Chem 269, 2550-2561.

Felgner PL and Ringold GM (1991) Cationic liposome-mediated transfection. Nature 337, 387-388.

Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, and Danielsen M (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA 84, 7413-7417.

Felgner PL, Rhodes G (1991) Gene therapeutics. Nature 349, 351-352.

Fender P, Ruigrok RW, Gout E, Buffet S, Chroboczek J (1997) Adenovirus dodecahedron, a new vector for human gene transfer. Nat Biotechnol 15, 52-56.

Feng M, Jackson WH, Goldman CK, Rancourt C, Wang M, Dusing SK, Siegal G, and Curiel DT (1997) Stable in vivo gene transduction via a novel adenoviral/retroviral chimeric vector. Nature Biotechnol 15, 866-870.

Fenjves ES, Gordon DA, Persing LK, Williams DL, Taichman LB (1989) Systemic distribution of apolipoprotein E secreted by grafts of epidermal keratinocytes: Implications for epidermal function and gene therapy. Proc Natl Acad Sci USA 86, 8803-8807.

Fernex C, Dubreuil P, Mannoni P, Bagnis C (1997) Cre/loxP-mediated excision of a neomycin resistance expression unit from an integrated retroviral vector increases long terminal repeat-driven transcription in human hematopoietic cells. J Virol 71, 7533-7540.

Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O'Shea KS, Powell-Braxton L, Hillan KJ, Moore MW (1996) Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380, 439-442.

Ferrari G, Rossini S, Giavazzi R, Maggioni D, Nobili N, Soldati M, Ungers G, Mavilio F, Gilboa E, Bordignon C (1991) An in vivo model of somatic cell gene therapy for human severe combined immunodeficiency. Science 251, 1363-1366.

Ferrari S, Moro E, Pettenazzo A, Behr J-P, Zacchello F, and Scarpa M (1997) ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo. Gene Therapy 4, 1100-1106.

Field AK, Davies ME, DeWitt, Perry HC, Liou R, Germershausen J, Karkas JD, Ashton WT, Johnston DBR, Tolman RL (1983) 9-{[2-Hydroxy-1-(hydroxymethyl) ethoxy]methyl} guanine: A selective inhibitor of herpes group virus replication. Proc Natl Acad Sci USA 80, 4139-4143.

Field SJ, Tsai F-Y, Kuo F, Zubiaga AM, Kaelin WGJr, Livingston DM, Orkin SH, and Greenberg ME (1996) E2F-1 functions in mice to promote apoptosis and suppresses proliferation. Cell 85, 549-561.

Fields S and Jang SK (1990) Presence of a potent transcription activating sequence in the p53 protein. Science 249: 1046-1049.

Filion MC and Phillips NC (1997) Toxicity and immunomodulatory activity of liposomal vectors formulated with cationic lipids toward immune effector cells. Biochim Biophys Acta 1329, 345-356.

Finke S, Trojaneck B, Moller P, Schadendorf D, Neubauer A, Huhn D, Schmidt-Wolf IG (1997) Increase of cytotoxic sensitivity of primary human melanoma cells transfected with the interleukin-7 gene to autologous and allogeneic immunologic effector cells. Cancer Gene Ther 4, 260-268.

Finlay CA, Hinds PW, Levine AJ (1989) The p53 proto-oncogene can act as a suppressor of transformation. Cell 57, 1083-1093.

Fisher KJ, Choi H, Burda J, Chen SJ, Wilson JM (1996) Recombinant adenovirus deleted of all viral genes for gene therapy of cystic fibrosis. Virology 217, 11-22.

Fisher KJ, Jooss K, Alston J, Yang Y, Haecker SE, High K, Pathak R, Raper SE, Wilson JM (1997) Recombinant adeno-associated virus for muscle directed gene therapy. Nat Med 3, 306-312.

Fisher LJ, Jinnah HA, Kale LC, Higgins GA, Gage FH (1991) Survival and function of intrastriatally grafted primary fibroblasts genetically modified to produce L-dopa. Neuron 6, 371-380.

Flamme I, von Reutern M, Drexler HC, Syed-Ali S, Risau W (1995) Overexpression of vascular endothelial growth factor in the avian embryo induces hypervascularization and increased vascular permeability without alterations of embryonic pattern formation. Dev Biol 171, 399-414.

Flotte TR, Barraza-Ortiz X, Solow R, Afione SA, Carter BJ, Guggino WB (1995) An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction. Gene Ther 2, 29-37.

Flugelman MY, Jaklitch MT, Newman KD, Casscells W, Bratthauer GL, and Dichek DA (1992) Low level in vivo gene transfer into the arterial wall through a perforated balloon catheter. Circulation 85, 1110-1117.

Folkman J and D’Amore PA (1996) Blood vessel formation: what is its molecular basis? Cell 87, 1153-1155.

Folkman J and Klagsbrun M (1987) Angiogenic factors. Science 235, 442-447.

Fong G, Rossant J, Gertsenstein M, and Breitman ML (1995) Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 376, 66-70.

Forrester WC, van Genderen C, Jenuwein T, and Grosschedl R (1994) Dependence of enhancer-mediated transcription of the immunoglobulin m gene on nuclear matrix attachment regions. Science 265, 1221-1225.

Foster BJ and Kern JA (1997) HER2-targeted gene transfer. Hum Gene Ther 8, 719-727.

Frederich RC, Hamann A, Anderson S, Lollmann B, Lowell BB, Flier JS (1995) Leptin levels reflect body lipid content in mice: evidence for diet-induced resistance to leptin action. Nat Med 1, 1311-1314.

Fredman JN and Engler JA (1993) Adenovirus precursor to terminal protein interacts with the nuclear matrix in vivo and in vitro. J. Virol. 67, 3384-3395.

Friedman PN, Kern SE, Vogelstein B, Prives C (1990) Wild-type but not mutant p53 proteins inhibit the replication activities of simian virus 40 large tumor antigen. Proc Natl Acad Sci USA 87, 9275-9279.

Friedmann T (1992) Gene therapy of cancer through restoration of tumor-suppressor functions? Cancer 70, 1810-1817.

Friedmann T, Yee J-K (1995) Pseudotyped retroviral vectors for studies of human gene therapy. Nature Med 1, 275-277.

Fritz JD, Herweijer H, Zhang G, and Wolff JA (1996) Gene transfer into mammalian cells using histone-condensed plasmid DNA. Hum Gene Ther 7, 1395-1404.

Fujiwara T, Cai DW, Georges RN, Mukhopadhyay T, Grimm EA, and Roth JA (1994) Therapeutic effect of a retroviral wild-type p53 expression vector in an orthotopic lung cancer model. J Natl Cancer Inst 86, 1458-1462.

Fukumoto S, Nishizawa Y, Hosoi M, Koyama H, Yamakawa K, Ohno S, Morii H (1997) Protein kinase C d inhibits the proliferation of vascular smooth muscle cells by suppressing G1 cyclin expression. J Biol Chem 272, 13816-13822.

Galipeau J, Benaim E, Spencer HT, Blakley RL, Sorrentino BP (1997) A bicistronic retroviral vector for protecting hematopoietic cells against antifolates and P-glycoprotein effluxed drugs. Hum Gene Ther 8, 1773-1783.

Gannon JV, Lane DP (1987) p53 and DNA polymerase a compete for binding to SV40 T antigen. Nature 329, 456-458.

Gao X, Huang L (1991) A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochem Biophys Res Commun 179, 280-285.

Gassmann M, Donoho G, and Berg P (1995) Maintenance of an extrachromosomal plasmid vector in mouse embryonic stem cells. Proc Natl Acad Sci USA 92, 1292-1296.

Gately S, Twardowski P, Stack MS, Cundiff DL, Grella D, Castellino FJ, Enghild J, Kwaan HC, Lee F, Kramer RA, Volpert O, Bouck N, Soff GA (1997) The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin. Proc Natl Acad Sci USA 94, 10868-10872.

Gavaghan H (1995) A shot in the arm for gene therapy company. Nature Med 1, 392.

Geller AI and Freese A (1990) Infection of cultured central nervous system neurons with a defective herpes simplex virus 1 vector results in stable expression of Escherichia coli b-galactosidase. Proc Natl Acad Sci USA 87, 1149-1153.

Georgiev GP, Kiselev SL and Lukanidin EM (1998) Genes involved in the control of tumor progression and their possible use for gene therapy. Gene Ther Mol Biol 1, 381-398.

Gerontakis S, Strasser A, Metcalf D, Grigoriadis G, Scheerlinck J-PY, and Grumont RJ (1996) Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor. Proc Natl Acad Sci USA 93, 3405-3409.

Gerrard AJ, Hudson DL, Brownlee GG, Watt FM (1993) Towards gene therapy for haemophilia B using primary human keratinocytes. Nature Genet 3, 180-183.

Ghitescu L, Fixman A, Simionescu M and Simionescu N (1986) Specific binding sites for albumin restricted to plasmalemmal vesicles of continuous capillary endothelium: Receptor-mediated transcytosis. J Cell Biol 102, 1304-1311.

Ghivizzani SG, Lechman ER, Tio C, Mulé KM, Chada S, McCormack JE, Evans CH, and Robbins PD (1997) Direct retrovirus-mediated gene transfer to the synovium of the rabbit knee: implications for arthritis gene therapy. Gene Ther 4, 977-982.

Gilardi P, Courtney M, Pavirani A, Perricaudet M (1990) Expression of human a 1-antitrypsin using a recombinant adenovirus vector. FEFS Lett 267, 60-62.

Gilboa E (1996) Immunotherapy of cancer with genetically modified tumor vaccines. Semin Oncol 23, 101-107.

Gilboa E and Smith G (1994) Gene therapy for infectious diseases: The AIDS model. Trends Genet 10, 139-144.

Gilgenkrantz H, Duboc D, Juillard V, Couton D, Pavirani A, Buillet JG, Briand P, Kahn A (1995) Transient expression of genes transferred in vivo into heart using first-generation adenoviral vectors: Role of the immune response. Hum Gene Ther 6, 1265-1274.

Gillio Tos A, Cignetti A, Rovera G, Foa R (1996) Retroviral vector-mediated transfer of the tumor necrosis factor a gene into human cancer cells restores an apoptotic cell death program and induces a bystander-killing effect. Blood 87, 2486-2495.

Gilmour SK, Verma AK, Madara T, and O'Brien TG (1987) Regulation of ornithine decarboxylase gene expression in mouse epidermis and epidermal tumors during two stage tumorigenesis. Cancer Res 47, 1221-1225.

Ginsberg D, Mechta F, Yaniv M, Oren M (1991) Wild-type p53 can down-modulate the activity of various promoters. Proc Natl Acad Sci USA 88: 9979-9983.

Giordano FJ, Ping P, McKirnan MD, Nozaki S, DeMaria AN, Dillmann WH, Mathieu-Costello O, Hammond HK (1995) Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. Nature Med 2, 534-539.

Giovannangeli C, Perrouault L, Escude C, Gryaznov S, Hélène C (1996) Efficient inhibition of transcription elongation in vitro by oligonucleotide phosphoramidates targeted to proviral HIV DNA. J Mol Biol 261, 386-398.

Goldman CK, Soroceanu L, Smith N, Gillespie GY, Shaw W, Burgess S, Bilbao G, Curiel DT (1997) In vitro and in vivo gene delivery mediated by a synthetic polycationic amino polymer. Nat Biotechnol 15, 462-466.

Good PD, Krikos AJ, Li SX, Bertrand E, Lee NS, Giver L, Ellington A, Zaia JA, Rossi JJ, Engelke DR (1997) Expression of small, therapeutic RNAs in human cell nuclei. Gene Ther 4, 45-54.

Gorman CM, Aikawa M, Fox B, Fox E, Lapuz C, Michaud B, Nguyen H, Roche E, Sawa T, Wiener-Kronish JP (1997) Efficient in vivo delivery of DNA to pulmonary cells using the novel lipid EDMPC. Gene Ther 4, 983-992.

Gottesman MM amd Pastan I (1988) The multidrug transporter, a double-edged sword. J Biol Chem 263, 12163-12166.

Gottfredsson M, Bohjanen PR (1997) Human immunodeficiency virus type I as a target for gene therapy. Front Biosci 2, D619-D634.

Gottschalk S, Christiano RJ, Smith LC, and Woo SLC (1993) Folate receptor mediated DNA delivery into tumor cells: Potosomal disruption results in enhanced gene expression. Gene Ther 1, 185-191.

Gözükara EM, Parris CN, Weber CA, Salazar EP, Seidman MM, Watkins JF, Prakash L , Kraemer KH (1994) The human DNA repair gene, ERCC2 (XPD), corrects ultraviolet hypersensitivity and ultraviolet hypermutability of a shuttle vector replicated in xeroderma pigmentosum group D cells. Cancer Res 54, 3837-3844.

Graeber TG, Osmanian C, Jacks T, Housman DE, Koch CJ, Lowe SW, and Giaccia AJ (1996) Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature 379, 88-91.

Graham RA, Burchell JM, Beverley P, Taylor-Papadimitriou J (1996) Intramuscular immunisation with MUC1 cDNA can protect C57 mice challenged with MUC1-expressing syngeneic mouse tumour cells. Int J Cancer 65, 664-670.

Greber UF, Webster P, Weber J, and Helenius A (1996) The role of the adenovirus protease in virus entry into cells. EMBO J 15, 1766-1777.

Green NK, Youngs DJ, Neoptolemos JP, Friedlos F, Knox RJ, Springer CJ, Anlezark GM, Michael NP, Melton RG, Ford MJ, Young LS, Kerr DJ, Searle PF (1997) Sensitization of colorectal and pancreatic cancer cell lines to the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) by retroviral transduction and expression of the E. coli nitroreductase gene. Cancer Gene Ther 4, 229-238.

Grossman M, Rader DJ, Muller DW, Kolansky DM, Kozarsky K, Clark BJ 3rd, Stein EA, Lupien PJ, Brewer HB Jr, Raper SE, et al (1995) A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolaemia. Nat Med 1, 1148-1154.

Grossman M, Raper SE, Kozarsky K, Stein EA, Engelhardt JF, Muller D, Lupien PJ, Wilson JM (1994) Successful ex vivo gene therapy directed to liver in a patient with familial hypercholesterolaemia. Nat Genet 6, 335-341.

Grossman M, Raper SE, Wilson JM (1992) Transplantation of genetically modified autologous hepatocytes into nonhuman primates: feasibility and short-term toxicity. Hum Gene Ther 3, 501-510.

Guieysse AL, Praseuth D, Hélène C (1997) Identification of a triplex DNA-binding protein from human cells. J Mol Biol 267, 289-298.

Gunzburg WH, Salmons B (1996) Development of retroviral vectors as safe, targeted gene delivery systems. J Mol Med 74, 171-182.

Guo ZS, Wang LH, Eisensmith RC, Woo SL (1996) Evaluation of promoter strength for hepatic gene expression in vivo following adenovirus-mediated gene transfer. Gene Ther 3, 802-810.

Guzman RJ, Hirskowitz EA, Brody SL, Crystal RG, Epstein SE, and Finkel T (1994) In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci USA 91, 10732-10736.

Haffe HA, Danel C, Longenecker G, Metzger M, Setoguchi Y et al, (1992) Adenovirus-mediated in vivo gene transfer and expression in normal rat liver. Nature Genet 1, 372-378.

Haj-Ahmad Y and Graham FL (1986) Development of a helper-independent human adenovirus vector and its use in the transfer of the herpes simplex virus thymidine kinase gene. J Virol 57, 267-274.

Halaas JL, Boozer C, Blair-West J, Fidahusein N, Denton DA, Friedman JM (1997) Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Proc Natl Acad Sci USA 94, 8878-8883.

Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM (1995) Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269, 543-546.

Halbert CL, Standaert TA, Aitken ML, Alexander IE, Russell DW, Miller AD (1997) Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration. J Virol 71, 5932-5941.

Haluska, F.G., Tsujimoto, Y. and Croce, C.M. (1987) Oncogene activation by chromosome translocation in human malignancy. Annu. Rev. Genetics 21, 321-347.

Hammes H-P, Brownlee M, Jonczyk A, Sutter A, Preissner KT (1995) Subcutaneous injection of a cyclic peptide antagonist of vitronectin receptor-type integrins inhibits retinal neovascularization. Nature Med 2, 529-533.

Hamouda T, McPhee R, Hsia SC, Read GS, Holland TC, King SR (1997) Inhibition of human immunodeficiency virus replication by the herpes simplex virus virion host shutoff protein. Virol 71, 5521-5527.

Han J, Sabbatini P, Perez D, Rao L, Modha D, and White E (1996) The E1B 19K protein blocks apoptosis by interacting with and inhibiting the p53-inducible and death-promoting Bax protein. Genes Dev 10, 461-477.

Hanazono Y, Yu JM, Dunbar CE, Emmons RV (1997) Green fluorescent protein retroviral vectors: low titer and high recombination frequency suggest a selective disadvantage. Hum Gene Ther 8, 1313-1319.

Hanna AK, Fox JC, Neschis DG, Safford SD, Swain JL, Golden MA (1997) Antisense basic fibroblast growth factor gene transfer reduces neointimal thickening after arterial injury. J Vasc Surg 25, 320-325.

Hansson JH, Nelson-Williams C, Suzuki H, Schild L, Shimkets R, Lu Y, Canessa C, Iwasaki T, Rossier B, Lifton RP (1995) Hypertension caused by a truncated epithelial sodium channel g subunit: genetic heterogeneity of Liddle syndrome. Nat Genet 11, 76-82.

Harms JS, Splitter GA (1995) Intergeron-g inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter. Hum Gene Ther 6, 1291-1297.

Harrell RL, Rajanayagam S, Doanes AM, Guzman RJ, Hirschowitz EA, Crystal RG, Epstein SE, Finkel T (1997) Inhibition of vascular smooth muscle cell proliferation and neointimal accumulation by adenovirus-mediated gene transfer of cytosine deaminase. Circulation 96, 621-627.

Hart Sl (1994) Cell binding and internalization by filamentous phage displaying a cyclic Arg-Gly-Asp-containing peptide. J Biol Chem 269, 12468-12474.

Hay JG, McElvaney NG, Herena J, Crystal RG (1995) Modification of nasal epithelial potential differences of individuals with cystic fibrosis consequent to local administration of a normal CFTR cDNA adenovirus gene transfer vector. Hum Gene Ther 6, 1487-1496.

Hay, R.T. (1985) Origin of adenovirus DNA replication. Role of the Nuclear Factor I binding site in vivo. J. Mol. Biol. 186, 129-136.

Hayday, A.,C., Gillies, S.D., Saito, H., Wood, C., Wiman, K., Hayward, W.S. and Tonegawa, S. (1984) Activation of a translocated human c-myc gene by an enhancer in the immunoglobulin heavy-chain locus. Nature 307, 334-340.

Hayward WS, Neel BG, Astrin SM (1981) Activation of a cellular onc gene by promoter insertion in ALV-induced lymphoid leukosis. Nature 290, 475-480.

Hazinski TA, Ladd PA, DeMatteo CA (1991) Localization and induced expression of fusion genes in the rat lung. Am J Respir Cell Mol Biol 4, 206-209.

Heise C, Sampson-Johannes A, Williams A, McCormick F, Von Hoff DD, Kirn DH (1997) ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Nat Med 3, 639-645.

Helin K, Lees JA, Vidal M, Dyson N, Harlow E, Fattaey A (1992) A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell 70, 337-350.

Hermeking H and Eick D (1994) Mediation of c-Myc-induced apoptosis by p53. Science 265, 2091-2093.

Hermonat PL, and Muzyczka N (1984) Use of adeno-associated virus as a mammalian DNA cloning vector: Transduction of neomycin resistance into mammalian tissue culture cells. Proc Natl Acad Sci USA 81, 6466-6470.

Hermonat PL, Quirk JG, Bishop BM, Han L (1997) The packaging capacity of adeno-associated virus (AAV) and the potential for wild-type-plus AAV gene therapy vectors. FEBS Lett 407, 78-84.

Herrmann JL, Beham AW, Sarkiss M, Chiao PJ, Rands MT, Bruckheimer EM, Brisbay S, McDonnell TJ (1997) Bcl-2 suppresses apoptosis resulting from disruption of the NF-kB survival pathway. Exp Cell Res 237, 101-109.

Hershfield MS, Buckley RH, Greenberg ML, Melton AL, Schiff R, Hatem C, Kurtzberg J, Markert ML, Kobayashi RH, Kobayashi AL, et al (1987) Treatment of adenosine deaminase deficiency with polyethylene glycol-modified adenosine deaminase. N Engl J Med 316, 589-596.

Herz J and Gerard RD (1993) Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice. Proc Natl Acad Sci USA 90, 2812-2816.

Herzog RW, Hagstrom JN, Kung SH, Tai SJ, Wilson JM, Fisher KJ, High KA (1997) Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc Natl Acad Sci USA 94, 5804-5809.

Hiebert SW, Packham G, Strom DK, Haffner R, Oren M, Zambetti G, and Cleveland JL (1995) E2F-1:DP-1 induces p53 and overrides survival factors to trigger apoptosis. Mol Cell Biol 15, 6864-6874.

Hirschhorn R, Roegner-Maniscalco V, Kuritsky L, Rosen FS (1981) Bone marrow transplantation only partially restores purine metabolites to normal in adenosine deaminase-deficient patients. J Clin Invest 68, 1387-1393.

Hoeben RC (1998) Gene therapy for haemophilia. Gene Ther Mol Biol 1, 293-300.

Hofland HEJ, Shephard L, Sullivan SM (1996) Formation of stable cationic lipid/DNA complexes for gene transfer. Proc Natl Acad Sci USA 93, 7305-7309.

Hofmann C, Lehnert W, and Strauss M (1997) The baculovirus vector system for gene delivery into hepatocytes. Gene Ther Mol Biol 1,

Hollenberg NK (1996) Genes, hypertension, and intermediate phenotypes. Curr Opin Cardiol 11, 457-463.

Hollstein M, Sidransky D ,Vogelstein B, Harris CC (1991) p53 mutations in human cancers. Science 253, 49-53.

Holt JT, Arteaga CB, Robertson D, Moses HL (1996) Gene therapy for the treatment of metastatic breast cancer by in vivo transduction with breast-targeted retroviral vector expressing antisense c-fos RNA. Hum Gene Ther 7, 1367-1380.

Horellou P, Mallet J (1997) Gene therapy for Parkinson's disease. Mol Neurobiol 15, 241-256.

Horn NA, Meedi JA, Budahazi G, Marquet M (1995) Cancer gene therapy using plasmid DNA: Purification of DNA for human clinical trials. Hum Gene Ther 6, 565-573.

Horowitz JM, Yandell DW, Park S-H, Canning S, Whyte P, Buchkovich K, Harlow E, Weinberg RA, Dryja TP (1989) Point mutational inactivation of the retinoblastoma antioncogene. Science 243, 937-940.

Hu E, Kim JB, Sarraf P, and Spiegelman BM (1996) Inhibition in adipogenesis through MAP kinase-mediated phosphoryl;ation of PPARg. Science 274, 2100-2103.

Huang H-JS, Yee J-K, Shew J-Y, Chen P-L, Bookstein R, Friedmann T, Lee EYHP, Lee W-H (1988) Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science 242, 1563-1566.

Huber BE, Austin EA, Good SS, Knick VC, Tibbels S, Richards CA (1993) In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinaoma cells genetically modified to express cytosine deaminase. Cancer Res 53, 4619-4626.

Huber BE, Austin EA, Richards CA, Davis ST, Good SS (1994) Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: Significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. Proc Natl Acad Sci USA 91, 8302-8306.

Huber BE, Richards CA, Krenitsky TA (1991) Retroviral-mediated gene therapy for the treatment of hepatocellular carcinaoma: An innovative approach for cancer therapy. Proc Natl Acad Sci USA 88, 8039-8043.

Hunger, S.P., Ohyashiki, K., Toyama, K. and Cleary, M.L. (1992) Hlf, a novel hepatic bZIP protein, shows altered DNA-binding properties following fusion to E2A in t(17;19) acute lymphoblastic leukemia. Genes Dev. 6, 1608-1620.

Hunt KK, Deng J, Liu TJ, Wilson-Heiner M, Swisher SG, Clayman G, Hung MC (1997) Adenovirus-mediated overexpression of the transcription factor E2F-1 induces apoptosis in human breast and ovarian carcinoma cell lines and does not require p53. Cancer Res 57, 4722-4726.

Hupp TR, Meek DW, Midgley CA, and Lane DP (1992) Regulation of the specific DNA binding function of p53. Cell 71, 875-886.

Huygen K, Content J, Denis O, Montgomery DL, Yawman AM, Deck RR, DeWitt CM, Orme IM, Baldwin S, D'Souza C, Drowart A, Lozes E, Vandenbussche P, Van Vooren JP, Liu MA, Ulmer JB (1996) Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nature Med 2, 893-898.

Hyde SC, Gill DR, Higgins CF, Trezise AEO, MacVinish LJ, Cuthbert AW, Ratcliff R, Evans MJ, Colledge WH (1993) Correction of the ion transport defect in cystic fibrosis transgenic mice by gene therapy. Nature 362, 250-255.

Iida A, Chen ST, Friedmann T, Yee JK (1996) Inducible gene expression by retrovirus-mediated transfer of a modified tetracycline-regulated system. J Virol 70, 6054-6059.

Ikonen E, Aula P, Gron K, Tollersrud O, Halila R, Manninen T, Syvanen A-C, Pertonen L (1991) Spectrum of mutations in aspartylglucosaminuria. Proc Natl Acad Sci USA 88, 11222-11226.

Indolfi C, Avvedimento EV, Rapacciuolo A, Di Lorenzo E, Esposito G, Stabile E, Feliciello A, Mele E, Giuliano P, Condorelli G, et al (1995) Inhibition of cellular ras prevents smooth muscle cell proliferation after vascular injury in vivo. Nat Med 1, 541-545.

Inouye RT, Du B, Boldt-Houle D, Ferrante A, Park IW, Hammer SM, Duan L, Groopman JE, Pomerantz RJ, Terwilliger EF (1997) Potent inhibition of human immunodeficiency virus type 1 in primary T cells and alveolar macrophages by a combination anti-Rev strategy delivered in an adeno-associated virus vector. J Virol 71, 4071-4078.

Isaacs WB, Bova GS, Morton RA, Bussemakers MJG, Brooks JD, and Ewing CM (1994) Molecular biology of prostate cancer. Semin Oncol 21, 514-521.

Isaacs WB, Carter BS, and Ewing CM (1991) Wild-type p53 suppresses growth of human prostate cancer cells containing mutant p53 alleles. Cancer Res 51, 4716-4720.

Ishida A, Sasaguri T, Kosaka C, Nojima H, Ogata J (1997) Induction of the cyclin-dependent kinase inhibitor p21(Sdi1/Cip1/Waf1) by nitric oxide-generating vasodilator in vascular smooth muscle cells. J Biol Chem 272, 10050-10057.

Ishizaki J, Nevins JR, Sullenger BA (1996) Inhibition of cell proliferation by an RNA ligand that selectively blocks E2F function. Nat Med 2, 1386-1389.

Isner JM, Pieczek A, Schainfeld R, Blair R, Haley L, Asahara T, Rosenfield K, Razvi S, Walsh K, Symes JF (1996b) Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb. Lancet 348, 370-374.

Isner JM, Walsh K, Rosenfield K, Schainfeld R, Asahara T, Hogan K, and Pieczek A (1996a) Clinical protocol. Arterial gene therapy for restenosis. Hum Gene Ther 7, 989-1011.

Iwanuma Y, Chen FA, Egilmez NK, Takita H, Bankert RB (1997) Antitumor immune response of human peripheral blood lymphocytes coengrafted with tumor into severe combined immunodeficient mice. Cancer Res 57, 2937-2942.

Jaaskelainen I, Monkkonen J, Urtti A (1994) Oligonucleotide-cationic liposome interactions. A physicochemical study. Biochim Biophys Acta 1195, 115-123.

Jaffee EM, Pardoll DM (1997) Considerations for the clinical development of cytokine gene-transduced tumor cell vaccines. Methods 12, 143-153.

Janicek MF, Sevin BU, Nguyen HN, Averette HE (1995) Combination anti-gene therapy targeting c-myc and p53 in ovarian cancer cell lines. Gynecol Oncol 59, 87-92.

Jänicke RU, Walker PA, Lin XY, and Porter AG (1996) Specific cleavage of the retinoblastoma protein by an ICE-like protease in apoptosis. EMBO J 15, 6969-6978.

Jendraschak E and Sage EH (1996) Regulation of angiogenesis by SPARC and angiostatin: implications for tumor cell biology. Semin Cancer Biol 7, 139-146.

Jessberger R, Heuss D, Doerfler W (1989) Recombination in hamster cell nuclear extracts between adenovirus type 12 DNA and two hamster preinsertion sequences. EMBO J 8, 869-878.

Ji W and St CW (1997) Inhibition of hepatitis B virus by retroviral vectors expressing antisense RNA. J Viral Hepat 4, 167-173.

Jiao S, Gurevich V, and Wolff JA (1993) Long-term correction of rat model of Parkinson's disease by gene therapy. Nature 362, 450-453.

Jin L, Zhang JJ, Chao L, Chao J (1997) Gene therapy in hypertension: adenovirus-mediated kallikrein gene delivery in hypertensive rats. Hum Gene Ther 8, 1753-1761.

Johnson P, Chung S, and Benchimol S (1993) Growth suppression of Friend virus-transformed erythroleukemia cells by p53 protein is accompanied by hemoglobin production and is sensitive to erythropoietin. Mol Cell Biol 13, 1456-1463.

Johnston BH (1988) The S1-sensitive form of d(C-T)n.d(A-G)n: chemical evidence for a three-stranded structure in plasmids. Science 241, 1800-1804.

Joki T, Nakamura M, and Ohno T (1995) Activation of the radiosensitive EGR-1 promoter induces expression of the herpes simplex virus thymidine kinase gene and sensitivity of human glioma cells to ganciclovir. Hum Gene Ther 6, 1507-1513.

Jomary C, Vincent KA, Grist J, Neal MJ, Jones SE (1997) Rescue of photoreceptor function by AAV-mediated gene transfer in a mouse model of inherited retinal degeneration. Gene Ther 4, 683-690.

Jones, K.A., Kadonaga, J.T., Rosenfeld, P.J., Kelly, T.J. and Tjian, R. (1987) A cellular DNA-binding protein that activates eukaryotic transcription and DNA replication. Cell 48, 79-89.

Joshi RL, Lamothe B, Cordonnier N, Mesbah K, Monthioux E, Jami J, and Bucchini D (1996) Targeted disruption of the insulin receptor gene in the mouse results in neonatal lethality. EMBO J 15, 1542-1547.

Ju DW, Cao X, Acres B (1997) Intratumoral injection of GM-CSF gene encoded recombinant vaccinia virus elicits potent antitumor response in a mixture melanoma model. Cancer Gene Ther 4, 139-144.

Junker U, Baker J, Kalfoglou CS, Veres G, Kaneshima H, Bohnlein E (1997) Antiviral potency of drug-gene therapy combinations against human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 13, 1395-1402.

Kaelin WG Jr, Krek W, Sellers WR, DeCaprio JA, Ajchenbaum F, Fuchs CS, Chittenden T, Li Y, Farnham PJ, Blanar MA, et al (1992) Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell 70, 351-364.

Kafri T, Blomer U, Peterson DA, Gage FH, Verma IM (1997) Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat Genet 17, 314-317.

Kagami H, O'Connell BC, Baum BJ (1996) Evidence for the systemic delivery of a transgene product from salivary glands. Hum Gene Ther 7, 2177-2184.

Kahn P (1995) From genome to proteome: Looking at a cell's proteins. Science 270, 369-370.

Kamata H, Yagisawa H, Takahashi S, Hirata H (1994) Amphiphilic peptides enhance the efficiency of liposome-mediated DNA transfection. Nucleic Acids Res 22, 536-537.

Kamohara S, Burcelin R, Halaas JL, Friedman JM, Charron MJ (1997) Acute stimulation of glucose metabolism in mice by leptin treatment. Nature 389, 374-377

Kamps, M.P., Look, A.T. and Baltimore, D. (1991) The human t(1;19) translocation in pre-B ALL produces multiple nuclear E2A-Pbx1 fusion proteins with differing transforming potentials. Genes Dev. 5, 358-368.

Kaneda Y, Iwai K, and Uchida T (1989) Increased expression of DNA cointroduced with nuclear protein in adult rat liver. Science 243, 375-378.

Kaneda Y, Morishita R, Dzau VJ (1997) Prevention of restenosis by gene therapy. Ann N Y Acad Sci 811, 299-308.

Kaplan JM, Armentano D, Sparer TE, Wynn SG, Peterson PA, Wadsworth SC, Couture KK, Pennington SE, St George JA, Gooding LR, Smith AE (1997) Characterization of factors involved in modulating persistence of transgene expression from recombinant adenovirus in the mouse lung. Hum Gene Ther 8, 45-56.

Karanikas V, Hwang LA, Pearson J, Ong CS, Apostolopoulos V, Vaughan H, Xing PX, Jamieson G, Pietersz G, Tait B, Broadbent R, Thynne G, McKenzie IFC (1997) Antibody and T cell responses of patients with adenocarcinoma immunized with mannan-MUC1 fusion protein. J Clin Invest 100, 2783-2792.

Karet FE, Lifton RP (1997) Mutations contributing to human blood pressure variation. Recent Prog Horm Res 52, 263-276.

Karlsson S (1991) Treatment of genetic defects in hematopoietic cell function by gene transfer. Blood 78, 2481-2492.

Kasahara N, Dozy AM, Kan YW (1994) Tissue-specific targeting of retroviral vectors through ligand-receptor interactions. Science 266, 1373-1376.

Kastan MB, Zhan Q, El-Deiry WS, Carrier F, Jacks T, Walsh WV, Plunkett BS, Vogelstein B and Fornace AJ (1992) A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 71, 587-597.

Kato K, Nakanishi M, Kaneda Y, Uchida T, Okada Y (1991) Expression of hepatitis B virus surface antigen in adult rat liver. Co-introduction of DNA and nuclear protein by a simplified liposome method. J Biol Chem 266, 3361-3364.

Kato T, Iwamoto K, Ando H, Asakawa N, Tanaka I, Kikuchi J, Murakami Y (1996) Synthetic cationic amphiphile for liposome-mediated DNA transfection with less cytotoxicity. Biol Pharm Bull 19, 860-863.

Kawabata K, Takakura Y, Hashida M (1995) The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake. Pharm Res 12, 825-830.

Kay MA, Landen CN, Rothenberg SR, Taylor LA, Leland F, Wiehle S, Fang B, Bellinger D, Finegold M, Thompson AR, Read M, Brinkhous KM, Woo SLC (1994) In vivo hepatic gene therapy: Complete albeit transient correction of factor IX deficiency in hemophilia B dogs Proc Natl Acad Sci USA 91, 2353-2357.

Kay MA, Rothenberg S, Landen CN, Bellinger DA, Leland F, Toman C, Finegold M, Thompson AR, Read MS, Brinkhous KM, and Woo SLC (1993) In vivo gene therapy of hemophilia B: Sustained partial correction in factor IX-deficient dogs. Science 262, 117-119.

Kazazian HH Jr, Wong C, Youssoufian H, Scott AF, Phillips DG, and Antonarakis S (1988) Haemophilia A resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man. Nature 332, 164-166.

Kearns WG, Afione SA, Fulmer SB, Pang MC, Erikson D, Egan M, Landrum MJ, Flotte TR, Cutting GR (1996) Recombinant adeno-associated virus (AAV-CFTR) vectors do not integrate in a site-specific fashion in an immortalized epithelial cell line. Gene Ther 3, 748-755.

Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, and Connolly DT (1989) Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 246, 1309-1312.

Kern SE, Kinzler KW, Bruskin A, Jarosz D, Friedman P, Prives C, Vogelstein B (1991) Identification of p53 as a sequence-specific DNA-binding protein. Science 252: 1708-1711.

Kern SE, Pietenpol JA, Thiagalingam S, Seymour A, Kinzler KW, Vogelstein B (1992) Oncogenic forms of p53 inhibit p53-regulated gene expression. Science 256, 827-830.

Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ (1996) Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Proc Natl Acad Sci USA 93, 14082-14087.

Kim JS and Pabo CO (1997) Transcriptional repression by zinc finger peptides. Exploring the potential for applications in gene therapy. J Biol Chem 272, 29795-29800.

Kim M, Wright M, Deshane J, Accavitti MA, Tilden A, Saleh M, Vaughan WP, Carabasi MH, Rogers MD, Hockett RD Jr, Grizzle WE, Curiel DT (1997) A novel gene therapy strategy for elimination of prostate carcinoma cells from human bone marrow. Hum Gene Ther 8, 157-170.

Kimura H, Sakamoto T, Cardillo JA, Spee C, Hinton DR, Gordon EM, Anderson WF, and Ryan SJ (1996) Retrovirus-mediated suicide gene transduction in the vitreous cavity of the eye: Feasibility in prevention of proliferative vitreoretinopathy. Hum Gene Ther 7, 799-808.

Kirchgessner CU, Patil CK, Evans JW, Cuomo CA, Fried LM, Carter T, Oettinger MA, Brown JM (1995) DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science 267, 1178-1183.

Kitsis RN, Buttrick PM, McNally EM, Kaplan ML, Leinwand LA (1991) Hormonal modulation of a gene injected into rat heart in vivo. Proc Natl Acad Sci USA 88, 4138-4142.

Klefstrom J, Arighi E, Littlewood T, Jäättelä M, Saksela E, Evan GI, Alitalo K (1997) Induction of TNF-sensitive cellular phenotype by c-Myc involves p53 and impaired NF-kB activation. EMBO J 16, 7382-7392.

Ko LJ and Prives C (1996) p53: puzzle and paradigm. Genes Dev 10, 1054-1072.

Ko S-C, Gotoh A, Thalmann GN, Zhau HE, Johnston DA, Zhang W-W, Kao C, and Chung LWK (1996) Molecular therapy with recombinant p53 adenovirus in an androgen-independent, metastatic human prostate cancer model. Hum Gene Ther 7, 1683-1691.

Kobayashi K, Oka K, Forte T, Ishida B, Teng B, Ishimura-Oka K, Nakamuta M, Chan L (1996) Reversal of hypercholesterolemia in low density lipoprotein receptor knockout mice by adenovirus-mediated gene transfer of the very low density lipoprotein receptor. J Biol Chem 271, 6852-6860.

Koc ON, Allay JA, Lee K, Davis BM, Reese JS, Gerson SL (1996) Transfer of drug resistance genes into hematopoietic progenitors to improve chemotherapy tolerance. Semin Oncol 23, 46-65.

Koeberl DD, Alexander IE, Halbert CL, Russell DW, Miller AD (1997) Persistent expression of human clotting factor IX from mouse liver after intravenous injection of adeno-associated virus vectors. Proc Natl Acad Sci USA 94, 1426-1431.

Kohn DB, Weinberg KI, Nolta JA, Heiss LH, Lenarsky C, Crooks GM, Hanley ME, Annett G, Brooks JS, El-Khoureiy A, Lawrence K, Wells S, Moen RC, Bastian J, Williams-Herman DE, Elder M, Wara D, Bowen T, Hershfield MS, Mullen CA, Blaese RM, Parkman R (1995) Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency. Nature Med 1, 1017-1023.

Kohwi Y and Kohwi-Shigematsu T (1988) Magnesium ion-dependent triple-helix structure formed by homopurine-homopyrimidine sequences in supercoiled plasmid DNA. Proc Natl Acad Sci USA 85, 3781-3785.

Kordower JH, Winn SR, Liu Y-T, Mufson EJ, Sladek Jr JR , Hammang JP, Baetge EE, Emerich DF (1994) The aged monkey basal forebrain: Rescue and sprouting of axotomized basal forebrain neurons after grafts of encapsulated cells secreting human nerve growth factor. Proc Natl Acad Sci USA 91, 10898-10902.

Korhonen J, Lahtinen I, Halmekyto M, Alhonen L, Janne J, Dumont D, Alitalo K (1995) Endothelial-specific gene expression directed by the tie gene promoter in vivo. Blood 86, 1828-1835.

Kostic V, Jackson-Lewis V, de Bilbao F, Dubois-Dauphin M, Przedborski S (1997) Bcl-2: prolonging life in a transgenic mouse model of familial amyotrophic lateral sclerosis. Science 277, 559-562.

Kotin RM, Linden RM, Berns KI (1992) Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination. EMBO J 11, 5071-5078.

Kotin RM, Siniscalco M, Samulski RJ, Zhu X, Hunter L, Laughlin CA, McLaughlin S, Muzyczka N, Rocchi M, and Berns KI (1990) Site-specific integration by adeno-associated virus. Proc Natl Acad Sci USA 87, 2211-2215

Kovesdi, I., Reichel, R. and Nevins, J.R. (1987) Role of an adenovirus E2 promoter binding factor in E1A-mediated coordinate gene control. Proc. Natl. Acad. Sci. USA 84, 2180-2184.

Kozarsky KF, Jooss K, Donahee M, Strauss JFIII, and Wilson JM (1996) Effective treatment of familial hypercholesterolaemia in the mouse model using adenovirus-mediated transfer of the VLDL receptor gene. Nature Genet 13, 54-62.

Kozarsky KF, McKinley DR, Austin LL, Raper SE, Stratford-Perricaudet LD, Wilson JM (1994) In vivo correction of low density lipoprotein receptor deficiency in the Watanabe heritable hyperlipidemic rabbit with recombinant adenoviruses. J Biol Chem 269, 13695-13702.

Kruse CA, Roper MD, Kleinschmidt-DeMasters BK, Banuelos SJ, Smiley WR, Robbins JM, Burrows FJ (1997) Purified herpes simplex thymidine kinase Retrovector particles. I. In vitro characterization, in situ transduction efficiency, and histopathological analyses of gene therapy-treated brain tumors. Cancer Gene Ther 4, 118-128.

Kuhober A, Pudollek HP, Reifenberg K, Chisari FV, Schlicht HJ, Reimann J, Schirmbeck R (1996) DNA immunization induces antibody and cytotoxic T cell responses to hepatitis B core antigen in H-2b mice. J Immunol 156, 3687-3695.

Kuida K, Lippke JA, Ku G, Harding MW, Livingston DJ, Su MS-S, and Flavell RA (1995) Altered cytokine export and apoptosis in mice deficient in interleukin-1b converting enzyme. Science 267, 2000-2003.

Lafarge-Frayssinet C, Duc HT, Frayssinet C, Sarasin A, Anthony D, Guo Y, Trojan J (1997) Antisense insulin-like growth factor I transferred into a rat hepatoma cell line inhibits tumorigenesis by modulating major histocompatibility complex I cell surface expression. Cancer Gene Ther 4, 276-285.

Laitinen M, Pakkanen T, Donetti E, Baetta R, Luoma J, Lehtolainen P, Viita H, Agrawal R, Miyanohara A, Friedmann T, Risau W, Martin JF, Soma M, Ylä-Herttuala S (1997a) Gene transfer into the carotid artery using an adventitial collar: comparison of the effectiveness of the plasmid-liposome complexes, retroviruses, pseudotyped retroviruses, and adenoviruses. Hum Gene Ther 8, 1645-1650.

Laitinen M, Zachary I, Breier G, Pakkanen T, Hakkinen T, Luoma J, Abedi H, Risau W, Soma M, Laakso M, Martin JF, Ylä-Herttuala S (1997b) VEGF gene transfer reduces intimal thickening via increased production of nitric oxide in carotid arteries. Hum Gene Ther 8, 1737-1744.

Lalwani AK, Walsh BJ, Reilly PG, Muzyczka N, Mhatre AN (1996) Development of in vivo gene therapy for hearing disorders: introduction of adeno-associated virus into the cochlea of the guinea pig. Gene Ther 3, 588-592.

Lane DP, Crawford LV (1979) T antigen is bound to a host protein in SV40-transformed cells. Nature 278: 261-263.

Lannutti BJ, Gately ST, Quevedo ME, Soff GA, Paller AS (1997) Human angiostatin inhibits murine hemangioendothelioma tumor growth in vivo. Cancer Res 57, 5277-5280.

Lappalainen K, Miettinen R, Kellokoski J, Jaaskelainen I, Syrjanen S (1997) Intracellular distribution of oligonucleotides delivered by cationic liposomes: light and electron microscopic study. Histochem Cytochem 45, 265-274.

Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, and Earnshaw WC (1994) Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 371, 346-347.

Le Gal La Salle F, Robert JJ, Berrard S, Ridoux V, Stratford-Perricaudet LD, Perricaudet M, Mallet J (1993) An adenovirus vector in gene transfer into neurons and glia in the brain. Science 259, 988-990.

Le M, Okuyama T, Cai SR, Kennedy SC, Bowling WM, Flye MW, Ponder KP (1997) Therapeutic levels of functional human factor X in rats after retroviral-mediated hepatic gene therapy. Blood 89, 1254-1259.

Lechanteur C, Princen F, Bue SL, Detroz B, Fillet G, Gielen J, Bours V, and Merville M-P (1997) HSV-1 thymidine kinase gene therapy for colorectal adenocarcinoma-derived peritoneal carcinomatosis. Gene Ther 4, 1189-1194.

Ledley FD (1995) Nonviral gene therapy: The promise of genes as pharmaceutical products. Hum Gene Ther 6, 1129-1144.

Lee CGL, Vieira W, Pastan I, and Gottesman MM (1998) Delivery Systems for the MDR1 gene. Gene Ther Mol Biol 1, 241-251.

Lee ER, Marshall J, Siegel CS, Jiang C, Yew NS, Nichols MR, Nietupski JB, Ziegler RJ, Lane MB, Wang KX, Wan NC, Scheule RK, Harris DJ, Smith AE, Cheng SH (1996) Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung. Hum Gene Ther 7, 1701-1717.

Lee RJ, Low PS (1995) Folate-mediated tumor cell targeting of liposome-entrapped doxorubicin in vitro. Biochim Biophys Acta 1233, 134-144.

Lee, W.-H., Bookstein, R., Hong, F., Young, L.-J., Shew, J.-Y. and Lee, E.Y-H.P (1987) Human retinoblastoma susceptibility gene: cloning, identification and sequence. Science 235, 1394-1399.

Lehrman MA, Goldstein JL, Russell DW, Brown MS (1987) Duplication of seven exons in LDL receptor gene caused by Alu-Alu recombination in a subject with familial hypercholesterolemia. Cell 48, 827-835.

Lesoon-Wood LA, Kim WH, Kleinman HK, Weintraub BD, and Mixson AJ (1995) Systemic gene therapy with p53 reduces growth and metastases of a malignant human breast cancer in nude mice. Hum Gene Ther 6, 395-405.

Leung DW, Cachianes G, Kuang W-J, Goeddel DV, and Ferrara N (1989) Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246, 1306-1309.

Levine AJ (1993) The tumor suppressor genes. Annu Rev Biochem 62: 623-651.

Levy MY, Barron LG, Meyer KB, Szoka FC Jr (1996) Characterization of plasmid DNA transfer into mouse skeletal muscle: evaluation of uptake mechanism, expression and secretion of gene products into blood. Gene Ther 3, 201-211.

Lew D, Parker SE, Latimer T, Abai AM, Kuwahara-Rundell A, Doh SG, Yang Z-Y, Laface D, Gromkowski SH, Nabel GJ, Manthorpe M, and Norman J (1995) Cancer gene therapy using plasmid DNA: pharmacokinetic study of DNA following injection in mice. Hum Gene Ther 6, 553-564.

Lewis EB (1950) The phenomenon of position effect. Adv Genet 3, 73-115.

Lewis JG, Lin KY, Kothavale A, Flanagan WM, Matteucci MD, DePrince RB, Mook RA Jr, Hendren RW, Wagner RW (1996) A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA. Proc Natl Acad Sci USA 93, 3176-3181.

Li LP, Schlag PM, Blankenstein T (1997) Transient expression of SV 40 large T antigen by Cre/LoxP-mediated site-specific deletion in primary human tumor cells. Hum Gene Ther 8, 1695-1700.

Li R and Botchan MR (1993) The acidic transcriptional activation domains of VP16 and p53 bind the cellular replication protein A and stimulate in vitro BPV-1 DNA replication. Cell 73: 1207-1221.

Li R, Waga S, Hannon GJ, Beach D, Stillman B (1994) Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature 371, 534-537.

Li S and Huang L (1997) In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes. Gene Ther 4, 891-900.

Li Z, Shanmugam N, Katayose D, Huber B, Srivastava S, Cowan K, Seth P (1997) Enzyme/prodrug gene therapy approach for breast cancer using a recombinant adenovirus expressing Escherichia coli cytosine deaminase. Cancer Gene Ther 4, 113-117.

Lidner V, Reidy MA (1991) Proliferation of smooth muscle cells after vascular injury if inhibited by an antibody against basic fibroblast growth factor. Proc Natl Acad Sci USA 88, 3739-3743.

Lieber A, He CY, Kirillova I, Kay MA (1996) Recombinant adenoviruses with large deletions generated by Cre-mediated excision exhibit different biological properties compared with first-generation vectors in vitro and in vivo. J Virol 70, 8944-8960.

Lieu FH, Hawley TS, Fong AZ, Hawley RG (1997) Transmissibility of murine stem cell virus-based retroviral vectors carrying both interleukin-12 cDNAs and a third gene: implications for immune gene therapy. Cancer Gene Ther 4, 167-175.

Lin KF, Chao J, Chao L (1995) Human atrial natriuretic peptide gene delivery reduces blood pressure in hypertensive rats. Hypertension 26, 847-853.

Lin KF, Chao L, Chao J (1997) Prolonged reduction of high blood pressure with human nitric oxide synthase gene delivery. Hypertension 30, 307-313.

Linardopoulos S, Papadakis E, Delakas D, Theodosiou V, Cranidis A, Spandidos DA (1993) Human lung and bladder carcinoma tumors as compared to their adjacent normal tissue have elevated AP-1 activity associated with the retinoblastoma gene promoter. Anticancer Res 13, 257-262.

Linzer DP and Levine AJ (1979) Characterization of a 54k dalton cellular SV40 tumor antigen present in SV40 transformed cells and uninfected embryonal carcinoma cells. Cell 17, 43-52.

Liotta LA, Steeg PS, and Stetler-Stevenson WG (1991) Review. Cancer metastasis and angiogenesis: An imbalance of positive and negative regulation. Cell 64, 327-336.

Litzinger DC, Brown JM, Wala I, Kaufman SA, Van GY, Farrell CL, Collins D (1996) Fate of cationic liposomes and their complex with oligonucleotide in vivo. Biochim Biophys Acta 1281, 139-149.

Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, MacDonald ME, Stuhlmann H, Koup RA, Landau NR (1996) Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86, 367-377.

Liu X, Miller CW, Koeffler PH, Berk AJ (1993) The p53 activation domain binds the TATA box-binding polypeptide in holo-TFIID, and a neighboring p53 domain inhibits transcription. Mol Cell Biol 13: 3291-3300.

Liu Z-g, Hsu H, Goeddel DV, and Karin M (1996) Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kB activation prevents cell death. Cell 87, 565-576.

Lowe S and Ruley HE (1993) Stabilization of the p53 tumor suppressor is induced by adenovirus-5 E1A and accompanies apoptosis. Genes Dev 7, 535-545.

Lowe SW, Jacks T, Housman DE, and Ruley EH (1994) Abrogation of oncogene-associated apoptosis allows transformation of p53-deficient cells. Proc Natl Acad Sci USA 91, 2026-2030.

Lubovy M, McCune S, Dong JY, Prchal JF, Townes TM, Prchal JT (1996) Stable transduction of recombinant adeno-associated virus into hematopoietic stem cells from normal and sickle cell patients. Biol Blood Marrow Transplant 2, 24-30.

Ludlow, J.W., Shon, J., Pipas, J.M., Livingston, D.M. and DeCaprio, J.A. (1990) The retinoblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation and binding to and release from SV40 large T. Cell 60, 387-396.

Luhrs CA, Raskin CA, Durbin R, Wu B, Sadasivan E, McAllister W and Rothenberg SP (1992) Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium. J Clin Invest 90, 840-847.

Luo F, Zhou SZ, Cooper S, Munshi NC, Boswell HS, Broxmeyer HE, Srivastava A (1995) Adeno-associated virus 2-mediated gene transfer and functional expression of the human granulocyte-macrophage colony-stimulating factor. Exp Hematol 23, 1261-1267.

Lynch C, Israel DI, Kaufman RJ, Miller AD (1993) Sequences in the coding region of clotting FVIII act as dominant inhibitors of RNA accumulation and protein production. Hum Gene Ther 4, 259-272.

Lynch CM, Clowes MM, Osborne WRA, Clowes AW, Miller AD (1992) Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: a model for gene therapy. Proc Natl Acad Sci USA 89, 1138-1142.

Mack DJ, Vartikar J, Pipas JM, Laimins LA (1993) Specific repression of TATA-mediated but not initiator-mediated transcription by wild-type p53. Nature 363: 281-283.

Mackensen A, Lindemann A, Mertelsmann R (1997) Immunostimulatory cytokines in somatic cells and gene therapy of cancer. Cytokine Growth Factor Rev 8, 119-128.

Madrigal M, Janicek MF, Sevin BU, Perras J, Estape R, Penalver M, Averette HE (1997) In vitro antigene therapy targeting HPV-16 E6 and E7 in cervical carcinoma. Gynecol Oncol 64, 18-25.

Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H, Kim S, Lallone R, Ranganathan S, et al (1995) Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med 1, 1155-1161.

Maffei M, Stoffel M, Barone M, Moon B, Dammerman M, Ravussin E, Bogardus C, Ludwig DS, Flier JS, Talley M, et al (1996) Absence of mutations in the human OB gene in obese/diabetic subjects. Diabetes 45, 679-682.

Magovern CJ, Mack CA, Zhang J, Rosengart TK, Isom OW, Crystal RG (1997) Regional angiogenesis induced in nonischemic tissue by an adenoviral vector expressing vascular endothelial growth factor. Hum Gene Ther 8, 215-227.

Maher J, Colonna F, Baker D, Luzzatto L, Roberts I (1994) Retroviral-mediated gene transfer of a mutant H-ras gene into normal human bone marrow alters myeloid cell proliferation and differentiation. Exp Hematol 22, 8-12.

Maher LJ 3d, Dervan PB, Wold B (1992) Analysis of promoter-specific repression by triple-helical DNA complexes in a eukaryotic cell-free transcription system. Biochemistry 31, 70-81.

Mahvi DM, Burkholder JK, Turner J, Culp J, Malter JS, Sondel PM, Yang NS (1996) Particle-mediated gene transfer of granulocyte-macrophage colony-stimulating factor cDNA to tumor cells: implications for a clinically relevant tumor vaccine. Hum Gene Ther 7, 1535-1543.

Mahvi DM, Sondel PM, Yang NS, Albertini MR, Schiller JH, Hank J, Heiner J, Gan J, Swain W, Logrono R (1997) Phase I/IB study of immunization with autologous tumor cells transfected with the GM-CSF gene by particle-mediated transfer in patients with melanoma or sarcoma. Hum Gene Ther 8, 875-891.

Maillard L and Walsh K (1996) Growth-arrest homeobox gene Gax: a molecular strategy to prevent arterial restenosis. Schweiz Med Wochenschr 126, 1721-1726.

Makarov SS, Olsen JC, Johnston WN, Anderle SK, Brown RR, Baldwin AS Jr, Haskill JS, Schwab JH (1996) Suppression of experimental arthritis by gene transfer of interleukin 1 receptor antagonist cDNA. Proc Natl Acad Sci USA 93, 402-406.

Malik P, McQuiston SA, Yu XJ, Pepper KA, Krall WJ, Podsakoff GM, Kurtzman GJ, Kohn DB (1997) Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line. J Virol 71, 1776-1783.

Malim MH, Böhnlein S, Hauber J, Cullen BR (1989) Functional dissection of the HIV-1 Rev trans-activator--Derivation of a trans-dominant repressor of Rev function. Cell 58, 205-214.

Malosky S, Kolansky DM (1996) Gene therapy for ischemic heart disease. Curr Opin Cardiol 11, 361-368.

Mamounas M, Leavitt M, Yu M, Wong-Staal F (1995) Increased titer of recombinant AAV vectors by gene transfer with adenovirus coupled to DNA-polylysine complexes. Gene Ther 2, 429-432.

Mancini MA, Shan B, Nickerson JA, Penman S, Lee WH (1994) The retinoblastoma gene product is a cell cycle-dependent, nuclear matrix-associated protein. Proc Natl Acad Sci USA 91, 418-422.

Mandel RJ, Spratt SK, Snyder RO, Leff SE (1997) Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson's disease in rats. Proc Natl Acad Sci USA 94, 14083-14088.

Manome Y, Wen PY, Dong Y, Tanaka T, Mitchell BS, Kufe DW, Fine HA (1995) Viral vector transduction of the human deoxycytidine kinase cDNA sensitizes glioma cells to the cytotoxic effects of cytosine arabinoside in vitro and in vivo. Nature Med 2, 567-573.

Marie J-P, Zittoun R and Sikic BI (1991) Multidrug resistance (mdr1) gene expression in adult acute leukemias: correlations with treatment outcome and in vitro drug sensitivity. Blood 78, 586-592.

Mariman, E.C.M, van Eekelen, C.A.G., Reinders, R.J., Berns, A.J.M. and van Venrooij, W.J. (1982) Adenoviral heterogeneous nuclear RNA is associated with the host nuclear matrix during splicing. J Mol Biol 154, 103.

Marini III FC, Cannon JP, Belmont JW, Shillitoe EJ, LaPeyre J-N (1995) In vivo marking of spontaneous or vaccine-induced fibrosarcomas in the domestic house cat, using an adenoviral vector containing a bifunctional fusion protein, GAL-TEK. Hum Gene Ther 6, 1215-1223.

Markowitz D, Hesdorffer C, Ward M, Goff S, Bank A (1990) Retroviral gene transfer using safe and efficient packaging cell lines. Ann N Y Acad Sci 612, 407-414.

Markowitz S, Wang J, Myeroff L, Parsons R, Sun L, Lutterbaugh J, Fan RS, Zborowska E, Kinzler KW, Vogelstein B, et al (1995) Inactivation of the type II TGF-b receptor in colon cancer cells with microsatellite instability. Science 268, 1336-1338.

Martin F and Boulikas T (1998) The challenge of liposomes in gene therapy. Gene Ther Mol Biol 1, 173-214.

Martin PA (1997) Editorial: Bridging the “commercialization gap” in Europe. Gene Ther 4, 881-882.

Martin SJ and Green DR (1995) Protease activation during apoptosis: death by a thousand cuts? Cell 82, 349-352.

Mayordomo, JI, Zorina T, Storkus WJ, Zitvogel L, Celluzzi C, Falo LD, Melief CJ, Ildstad ST, Kast WM, Deleo AB, Lotze MT (1995) Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity. Nature Med 1, 1297-1302.

Maze R, Hanenberg H, Williams DA (1997) Establishing chemoresistance in hematopoietic progenitor cells. Mol Med Today 3, 350-358.

McDonald RJ, Lukason MJ, Raabe OG, Canfield DR, Burr EA, Kaplan JM, Wadsworth SC, St George JA (1997) Safety of airway gene transfer with Ad2/CFTR2: aerosol administration in the nonhuman primate. Hum Gene Ther 8, 411-422.

McKnight, R.A., Shamay, A., Sankaran, L., Wall, R.J., and Hennighausen, L. (1992) Matrix-attacment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice. Proc. Natl. Acad. Sci. 89, 6943-6947.

Mercer WE, Shields MT, Lin D, Appella, Ullrich SJ (1991) Growth supprssion induced by wild-type p53 protein is accompanied by selective down-regulation of proliferating-cell nuclear antigen expression. Proc Natl Acad Sci USA 88, 1958-1962.

Merritt AJ, Potten CS, Kemp CJ, Hickman JA, Balmain A, Lane DP, and Hall PA (1994) The role of p53 in spontaneous and radiation-induced apoptosis in the gastrointestinal tract of normal and p53-deficient mice. Cancer Res 54, 614-617.

Mesri EA, Federoff HJ, Brownlee M (1995) Expression of vascular endothelial growth factor from a defective herpes simplex virus type 1 amplicon vector induces angiogenesis in mice. Circ Res 76, 161-167.

Mhashilkar AM, Biswas DK, LaVecchio J, Pardee AB, Marasco WA (1997) Inhibition of human immunodeficiency virus type 1 replication in vitro by a novel combination of anti-Tat single-chain intrabodies and NF-kB antagonists. J Virol 71, 6486-6494.

Michel ML, Davis HL, Schleef M, Mancini M, Tiollais P, Whalen RG (1995) DNA-mediated immunization to the hepatitis B surface antigen in mice: aspects of the humoral response mimic hepatitis B viral infection in humans. Proc Natl Acad Sci USA 92, 5307-5311

Midoux P, Mendes C, Legrand A, Raimond J, Mayer R, Monsigny M, Roche AC (1993) Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells. Nucleic Acids Res 21, 871-878.

Migita M, Medin JA, Pawliuk R, Jacobson S, Nagle JW, Anderson S, Amiri M, Humphries RK, Karlsson S (1995) Selection of transduced CD34⁺ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors. Proc Natl Acad Sci USA 92, 12075-12079.

Mihara, K., Cao, X.-R., Yen, A., Chandler, S., Driscoll, B., Murphree, A.L., T'ang, A. and Fung, Y.-K.T. (1989) Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science 246, 1300-1303.

Millauer B, Wizigmann-Voos S, Schnürch H, Martinez R, Møller NPH, Risau W, and Ullrich A (1993) High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 72, 835-846.

Miller DG, Adam MA, Miller AD (1990) Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 10, 4239-4242.

Miller SD, Farmer G, Prives C (1995) p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner. Mol Cell Biol 15, 6554-6560.

Mirkin SM, Lyamichev VI, Drushlyak KN, Dobrynin VN, Filippov SA, Frank-Kamenetskii MD (1987) DNA H form requires a homopurine-homopyrimidine mirror repeat. Nature 330, 495-497.

Mitchell PJ and Tjian R (1989) Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245, 371-378

Miura M, Zhu H, Rotello R, Hartwieg EA, and Huan J (1993) Induction of apoptosis in fibroblasts by IL-1b-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 75, 653-660.

Miyake K, Tohyama T, Shimada T (1996) Two-step gene transfer using an adenoviral vector carrying the CD4 gene and human immunodeficiency viral vectors. Hum Gene Ther 7, 2281-2286.

Miyanohara A, Sharkey MF, Witztum JL, Steinberg D, Friedmann T (1988) Efficient expression of retroviral vector-transduced human low density lipoprotein (LDL) receptor in LDL receptor-deficient rabbit fibroblasts in vitro. Proc Natl Acad Sci USA 85, 6538-6542.

Miyashita T , Reed JC (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293-299.

Miyashita T, Harigai M, Hanada M, Reed JC (1994b) Identification of p53-dependent negative response element in the bcl-2 gene. Cancer Res 54, 3131-3135.

Miyashita T, Krajewski S, Krajewska M, Wang HG, Lin HK, Liebermann DA, Hoffman B, Reed JC (1994a) Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 9, 1799-1805.

Miyatake S, Martuza RL, Rabkin SD (1997) Defective herpes simplex virus vectors expressing thymidine kinase for the treatment of malignant glioma. Cancer Gene Ther 4, 222-228.

Miyoshi H, Takahashi M, Gage FH, Verma IM (1997) Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc Natl Acad Sci USA 94, 10319-10323.

Moelling K (1997) DNA for genetic vaccination and therapy. Cytokines Cell Mol Ther 3, 127-135.

Moffatt M, Cookson W (1995) Naked DNA: New shots for allergy? Nature Med 2, 515-516.

Mogayzel PJ Jr, Henning KA, Bittner ML, Novotny EA, Schwiebert EM, Guggino WB, Jiang Y, Rosenfeld MA (1997) Functional human CFTR produced by stable Chinese hamster ovary cell lines derived using yeast artificial chromosomes. Hum Mol Genet 6, 59-68.

Momand J, Zambetti GP, Olson DC, George D, Levine AJ (1992) The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell 69, 1237-1245.

Momparler RL, Eliopoulos N, Bovenzi V, Letourneau S, Greenbaum M, Cournoyer D (1996) Resistance to cytosine arabinoside by retrovirally mediated gene transfer of human cytidine deaminase into murine fibroblast and hematopoietic cells. Cancer Gene Ther 3, 331-338.

Moolten FL (1986) Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: Paradigm for a prospective cancer control strategy. Cancer Res 46, 5276-5281.

Moolten FL, Wells JM (1990) Curability of tumors bearing herpes thymidine kinase genes transferred by retroviral vectors. J Natl Cancer Inst 82, 297-300.

Morgan JR, Barrandon Y, Green H, Mulligan RC (1987) Expression of an exogenous growth hormone gene by transplantable human epidermal cells. Science 237, 1476-1479.

Morgenstern JP and Land H (1990) Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res 18, 3587-3596.

Morishita R, Gibbons GH, Ellison KE, Nakajima M, von der Leyen H, Zhang L, Kaneda Y, Ogihara T, Dzau VJ (1994) Intimal hyperplasia after vascular injury is inhibited by antisense cdk 2 kinase oligonucleotides. J Clin Invest 93, 1458-1464.

Morishita R, Gibbons GH, Ellison KE, Nakajima M, Zhang L, Kaneda Y, Ogihara T, Dzau VJ (1993) Single intraluminal delivery of antisense cdc2 kinase and proliferating-cell nuclear antigen oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci USA 90, 8474-8478.

Morozov VA, Noguiez-Hellin P, Laune S, Tamboise E, Salzmann JL, Klatzmann D (1997) Plasmovirus: replication cycle of a novel nonviral/viral vector for gene transfer. Cancer Gene Ther 4, 286-293.

Morris MC, Vidal P, Chaloin L, Heitz F, Divita G (1997) A new peptide vector for efficient delivery of oligonucleotides into mammalian cells. Nucleic Acids Res 25, 2730-2736.

Morsy MA, Alford EL, Bett A, Graham FL, and Caskey CT (1993) Efficient adenoviral-mediated ornithine transcarmylase expression in deficient mouse and human hepatocytes. J Clin Invest 92, 1580-1586.

Moser HE and Dervan PB (1987) Sequence-specific cleavage of double helical DNA by triple helix formation. Science 238, 645-650.

Moullier P, Bohl D, Heard J-M, Danos O (1993) Correction of lysosomal storage in the liver and spleen of MPS VII mice by implantation of genetically modified skin fibroblasts. Nature Genet 4, 154-159.

Mounzih K, Lu R, Chehab FF (1997) Leptin treatment rescues the sterility of genetically obese ob/ob males. Endocrinology 138, 1190-1193.

Mudryj, M., Devoto, S.H., Hiebert, S.W., Hunter, T., Pines, J. and Nevins, J.R. (1991) Cell cycle regulation of the E2F transcription factor involves an interaction with cyclin A. Cell 65, 1243-1253

Mudryj, M., Hiebert, S.W. and Nevins, J.R. (1990) A role for the adenovirus inducible E2F transcription factor in a proliferation dependent signal transduction pathway. EMBO J. 9, 2179-2184.

Mujoo K, Maneval DC, Anderson SC, Gutterman JU (1996) Adenoviral-mediated p53 tumor suppressor gene therapy of human ovarian carcinoma. Oncogene 12, 1617-1623.

Mukhopadhyay T, Tainsky M, Cavender AC, Roth JA (1991) Specific inhibition of K-ras expression and tumorigenicity of lung cancer cells by antisense RNA. Cancer Res 51, 1744-1748.

Mul YM, Verrijzer CP, and van der Vliet PC (1990) Transcription factors NFI and NFIII/Oct-1 function independently, employing different mechanisms to enhance adenovirus DNA replication J. Virol. 64, 5510-5518.

Mullen CA, Kilstrup M, Blaese RM (1992) Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: A negative selection system. Proc Natl Acad Sci USA 89, 33-37.

Muller DW (1997) The role of proto-oncogenes in coronary restenosis. Prog Cardiovasc Dis 40, 117-128.

Muller-Ladner U, Gay RE, Gay S (1997a) Cellular pathways of joint destruction. Curr Opin Rheumatol 9, 213-220

Muller-Ladner U, Roberts CR, Franklin BN, Gay RE, Robbins PD, Evans CH, Gay S (1997b) Human IL-1Ra gene transfer into human synovial fibroblasts is chondroprotective. J Immunol 158, 3492-3498.

Mummenbrauer T, Janus F, Müller B, Wiesmüller L, Deppert W, and Grosse F (1996) p53 protein exhibits 3'-to-5' exonuclease activity. Cell 85, 1089-1099.

Murata M, Takahashi S, Kagiwada S, Suzuki A, Ohnishi S-I (1992) pH-dependent membrane fusion and vesiculation of phospholipid large unilamellar vesicles induced by amphiphilic anionic and cationic peptides. Biochemistry 31, 1986-1992.

Murayama Y and Horiuchi S (1997) Antisense oligonucleotides to p53 tumor suppressor suppress the induction of apoptosis by epidermal growth factor in NCI-H 596 human lung cancer cells. Antisense Nucleic Acid Drug Dev 7, 109-114.

Muthukkumar S, Sells SF, Crist SA, Rangnekar VM (1996) Interleukin-1 induces growth arrest by hypophosphorylation of the retinoblastoma susceptibility gene product RB. J Biol Chem 271, 5733-5740.

Muzio M, Ni J, Feng P, Dixit VM (1997) IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. Science 278, 1612-1615.

Muzzin P, Eisensmith RC, Copeland KC, Woo SL (1996) Correction of obesity and diabetes in genetically obese mice by leptin gene therapy. Proc Natl Acad Sci USA 93, 14804-14808.

Nabel EG, Plautz G, and Nabel GJ (1990) Site-specific gene expression in vivo by direct gene transfer into arterial wall. Science 249, 1285-1288.

Nabel EG, Shum L, Pompili VJ, Yang Z-Y, San H, Shu HB, Liptay S, Gold L, Gordon D, Derynck R, Nabel GJ (1993a) Direct transfer of transforming growth factor b1 gene into arteries stimulates fibrocellular hyperplasia. Proc Natl Acad Sci USA 90, 10759-10763.

Nabel EG, Yang Z, Liptay S, San H, Gordon D, Haudenschild CC, et al. (1993c) Recombinbant platelet-derived growth factor B gene expression in porcine arteries induces intimal hyperplasia in vivo. J Clin Invest 91, 1822-1829.

Nabel EG, Yang Z, Plautz G, Forough R, Zhan X, Haudenschild CC, et al (1993b) Recombinant fibroblast growth factor-1 promotes intimal hyperplasia and angiogenesis in arteries in vivo. Nature 362, 844-846.

Nabel GJ, Nabel EG, Yang Z-Y, Fox BA, Plautz EG, Gao X, Huang L, Shu S, Gordon D, Chang AE (1993d) Direct gene transfer with DNA-liposome complexes in melanoma: Expression, biologic activity, and lack of toxicity in humans. Proc Natl Acad Sci USA 90, 11307-11311.

Nakao N, Frodl EM, Widner H, Carlson E, Eggerding FA, Epstein CJ, Brundin P (1995) Overexpressing Cu/Zn superoxide dismutase enhances survival of transplanted neurons in a rat model of Parkinson's disease. Nature Med 1, 226-231.

Nakaoka T, Gonda K, Ogita T, Otawara-Hamamoto Y, Okabe F, Kira Y, Harii K, Miyazono K, Takuwa Y, Fujita T (1997) Inhibition of rat vascular smooth muscle proliferation In vitro and In vivo by bone morphogenetic protein-2. J Clin Invest 100, 2824-2832.

Nakaya T, Iwai S, Fujinaga K, Sato Y, Otsuka E, Ikuta K (1997) Decoy approach using RNA-DNA chimera oligonucleotides to inhibit the regulatory function of human immunodeficiency virus type 1 Rev protein. Antimicrob Agents Chemother 41, 319-325.

Naviaux RK and Verma IM (1992) Retroviral vectors for persistent expression in vivo. Curr Opin Biotechnol 3, 540-547.

Neri D, Carnemolla B, Nissim A, Leprini A, Querze G, Balza E, Pini A, Tarli L, Halin C, Neri P, Zardi L, Winter G (1997) Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform. Nat Biotechnol 15, 1271-1275.

Nevins JR (1992) E2F: a link between the RB tumor suppressor protein and viral oncoproteins. Science 258, 424-429.

Newman KD, Dunn PF, Owens JW, Schulick AH, Virmani R, Sukhova G, Libby P, Dichek DA (1995) Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation, and neointimal hyperplasia. J Clin Invest 96, 2955-2965.

Nguyen DM, Wiehle SA, Koch PE, Branch C, Yen N, Roth JA, Cristiano RJ (1997b) Delivery of the p53 tumor suppressor gene into lung cancer cells by an adenovirus/DNA complex. Cancer Gene Ther 4, 191-198.

Nguyen DM, Wiehle SA, Roth JA, Cristiano RJ (1997a) Gene delivery into malignant cells in vivo by a conjugated adenovirus/DNA complex. Cancer Gene Ther 4, 183-190.

Nickels JT and Broach JR (1996) A ceramide-activated protein phosphatase mediates ceramide-induced G1 arrest of Saccharomyces cerevisiae. Genes Dev 10, 382-394.

Nielsen LL, Dell J, Maxwell E, Armstrong L, Maneval D, Catino JJ (1997) Efficacy of p53 adenovirus-mediated gene therapy against human breast cancer xenografts. Cancer Gene Ther 4, 129-138.

Nigro JM, Baker SJ, Preisinger AC, Jessup JM, Hostetter R, Cleary K, Bigner SH, Davidson N, Baylin S, Devilee P, Glover T, Collins FS, Weston A, Modali R, Harris CC, Vogelstein B (1989) Mutations in the p53 gene occur in diverse human tumour types. Nature 342, 705-708.

Nilson BH, Morling FJ, Cosset FL, Russell SJ (1996) Targeting of retroviral vectors through protease-substrate interactions. Gene Ther 3, 280-286.

Norrby K (1997) Angiogenesis: new aspects relating to its initiation and control. APMIS 105, 417-437

Nowak R (1995) Entering the postgenome era. Science 270, 368-371.

Nussbaum RL and Polymeropoulos MH (1997) Genetics of Parkinson's disease. Hum Mol Genet 6, 1687-1691.

O'Neill, E.A. Fletcher, C., Burrow, C.R., Heintz, N., Roeder, R.G., Kelly, T.J. (1988) Transcription factor OTF-1 is functionally identical to the DNA replication factor NF-III. Science 241, 1210-1213.

O'Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J (1997) Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277-285.

O'Reilly MS, Holmgren L, Chen C, Folkman J (1996) Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat Med 2, 689-692.

O'Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS, Cao Y, Sage EH, Folkman J (1994) Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79, 315-328.

Ohno T, Gordon D, San H, Pompili VJ, Imperiale MJ, Nabel GJ, and Nabel EG (1994) Gene therapy for vascular smooth muscle cell proliferation after areterial injutry. Science 265, 781-784.

Ohno T, Yang Z, Ling X, Jaffe M, Nabel EG, Normolle D, Nabel GJ (1997) Combination gene transfer to potentiate tumor regression. Gene Ther 4, 361-366.

Okada H, Miyamura K, Itoh T, Hagiwara M, Wakabayashi T, Mizuno M, Colosi P, Kurtzman G, Yoshida J (1996) Gene therapy against an experimental glioma using adeno-associated virus vectors. Gene Ther 3, 957-964.

Okamoto K and Beach D (1994) Cyclin G is a transcriptional target of the p53 tumor suppressor protein. EMBO J 13, 4816-4822.

Okamoto T, Kaneda Y, Yuzuki D, Huang SKS, Chi DDJ, and Hoon DSB (1997) Induction of antibody response to human tumor antigens by gene therapy using a fusigenic viral liposome vaccine. Gene Ther 4, 969-976.

Okuyama Y, Sowa Y, Fujita T, Mizuno T, Nomura H, Nikaido T, Endo T, Sakai T (1996) ATF site of human RB gene promoter is a responsive element of myogenic differentiation. FEBS Lett 397, 219-224.

Oliner JD Kinzler KW, Meltzer PS, George DL, Vogelstein B (1992) Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature 358, 80-83.

Olsen JC, Sechelski J (1995) Use of sodium butyrate to enhance production of retroviral vectors expressing CFTR cDNA. Hum Gene Ther 6, 1195-1202.

Ono T, Fujino Y, Tsuchiya T, Usuda M (1990) Plasmid DNAs directly injected into mouse brain with lipofectin can be incorporated and expressed by brain cells. Neurosci Lett 117, 259-263.

Ory DS, Neugeboren BA, Mulligan RC (1996) A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc Natl Acad Sci USA 93, 11400-11406.

Osaki T, Tanio Y, Tachibana I, Hosoe S, Kumagai T, Kawase I, Oikawa S, Kishimoto T (1994) Gene therapy for carcinoembryonic antigen-producing human lung cancer cells by cell type-specific expression of herpes simplex virus thymidine kinase gene. Cancer Res 54, 5258-5261.

Otani K, Nita I, Macaulay W, Georgescu HI, Robbins PD, Evans CH (1996) Suppression of antigen-induced arthritis in rabbits by ex vivo gene therapy. J Immunol 156, 3558-3562.

Pages J-C, Andreoletti M, Bennoun M, Vons C, Elcheroth J, Lehn P, Houssin D, Chapman J, Briand P, Benarous R, Franco D, Weber A (1995) Efficient retroviral-mediated gene transfer into primary culture of murine and human hepatocytes: expression of the LDL receptor. Hum Gene Ther 6, 21-30.

Pages J-C, Loux N, Bellusci S, Farge D, Bennoun M, Vons C, Jouanneau J, Franco D, Briand P, Weber A (1996a) Hepatocyte growth factor expressed by a retrovirus-producing cell line enhances retroviral transduction of primary hepatocytes: implications for in vivo gene transfer. Biochem Biophys Res Commun 222, 726-731.

Pages JC, Andreoletti M, Loux N, Vons C, Mahieu D, Bargy F, Chapman J, Briand P, Franco D, Weber A (1996b) Towards gene therapy in familial hypercholesterolemia (in french) C R Seances Soc Biol Fil 190, 53-65.

Paik SY, Banerjea A, Chen CJ, Ye Z, Harmison GG, Schubert M (1997) Defective HIV-1 provirus encoding a multitarget-ribozyme inhibits accumulation of spliced and unspliced HIV-1 mRNAs, reduces infectivity of viral progeny, and protects the cells from pathogenesis. Hum Gene Ther 8, 1115-1124.

Paleyanda RK, Velander WH, Lee TK, Scandella DH, Gwazdauskas FC, Knight JW, Hoyer LW, Drohan WN, and Lubon H (1997) Transgenic pigs produce functional human factor VIII in milk. Nature Biotech 15, 971-975.

Palmer TD, Hock RA, Osborne WRA, Miller AD (1987) Efficient retrovirus-mediated transfer and expression of a human adenosine deamnase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human. Proc Natl Acad Sci USA 84, 1055-1059.

Palmer TD, Rosman GJ, Osborne WRA, Miller AD (1991) Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes. Proc Natl Acad Sci USA 88, 1330-1334.

Pang S, Taneja S, Dardashti K, Cohan P, Kaboo R, Sokoloff M, Tso C-L, Dekernion JB, and Belldegrun AS (1995) Prostate tissue specificity of the prostate-specific antigen promoter isolated from a patient with prostate cancer. Hum Gene Ther 6, 1417-1426.

Papathanasiou MA, Kerr NCK, Robbins JH, McBride OW, Alamo Jr I, barrett SF, Hickson ID, Fornace Jr AJ (1991) Induction by ionizing radiation of the gadd45 gene in cultured human cells: Lack of mediation by protein kinase C. Mol Cell Biol 11, 1009-1016.

Park K, Choe J, Osifchin NE, Templeton DJ, Robbins PD, Kim SJ (1994) The human retinoblastoma susceptibility gene promoter is positively autoregulated by its own product. J Biol Chem 269, 6083-6088.

Parker SE, Vahlsing HL, Serfilippi LM, Franklin CL, Doh SG, Gromkowski SH, Lew D, Manthorpe M, Norman J (1995) Cancer gene therapy using plasmid DNA: Safety evaluation in rodents and non-human primates. Hum Gene Ther 6, 575-590.

Parker WB, King SA, Allan PW, Bennett LLJr, Secrist JAIII, Montgomery JA, Gilbert KS, Waud WR, Wells AH, Gillespie GY, and Sorscher EJ (1997) In vivo gene therapy of cancer with E. coli purine nucleoside phosphorylase. Hum Gene Ther 8, 1637-1644.

Parkman R (1986) The application of bone marrow transplantation to the treatment of genetic diseases. Science 232, 1373-1378.

Patterson BC and Sang QA (1997) Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9). J Biol Chem 272, 28823-28825.

Pennacchio LA et al (1996) Mutations in the gene encoding cystatin B in progressive myoclonous epilepsy (EPM1). Science 271, 1731-1734.

Perrouault L, Asseline U, Rivalle C, Thuong NT, Bisagni E, Giovannangeli C, Le Doan T, Hélène C (1990) Sequence-specific artificial photo-induced endonucleases based on triple helix-forming oligonucleotides. Nature 344, 358-360.

Perry ME, Piette J, Zawadzki JA, Harvey D and Levine AJ (1993) The mdm-2 gene is induced in response to UV light in a p53-dependent manner. Proc Natl Acad Sci USA 90, 11623-11627.

Peters C, Rommerskirch W, Modaressi S, von Figura K (1991) Restoration of arylsulphatase B activity in human mucopolysaccharidosis-type-VI fibroblasts by retroviral-vector-mediated gene transfer. Biochem J 276,499-504.

Peters K-R, Carley WW and Palade GE (1985) Endothelial plasmalemmal vesicles have a characteristic striped bipolar surface structure. J Cell Biol 101, 2233-2238.

Peterson KR, Clegg CH, Li Q, Stamatoyannopoulos G (1997) Production of transgenic mice with yeast artificial chromosomes. Trends Genet 13, 61-66.

Phillips MI (1997) Antisense inhibition and adeno-associated viral vector delivery for reducing hypertension. Hypertension 29, 177-187.

Phillips MI, Mohuczy-Dominiak D, Coffey M, Galli SM, Kimura B, Wu P, Zelles (1997) Prolonged reduction of high blood pressure with an in vivo, nonpathogenic, adeno-associated viral vector delivery of AT1-R mRNA antisense. Hypertension 29, 374-380.

Pietenpol JA, Holt JT, Stein RW, Moses HL (1990) Transforming growth factor b 1 suppression of c-myc gene transcription: role in inhibition of keratinocyte proliferation. Proc Natl Acad Sci USA 87, 3758-3762.

Plank C, Mechtler K, Szoka FC Jr, Wagner E (1996) Activation of the complement system by synthetic DNA complexes: a potential barrier for intravenous gene delivery. Hum Gene Ther 7, 1437-1446.

Plate KH, Breier G, Weich HA, and Risau W (1992) Vascular endothelial growth factor is a potential tumor angiogenesis factor in human gliomas in vivo. Nature 359, 845-848.

Pochon NA-M, Heyd B, Déglon N, Joseph J-M, Zurn AD, Baetge EE, Hammang JP, Goddard M, Lysaght M, Kaplan F, Kato AC, Schluep M, Hirt L, Regli F, Porchet F, and de Tribolet N (1996) Clinical Protocol: Gene therapy for amyotrophic lateral sclerosis (ALS) using a polymer-encapsulated xenogenic cell line engineered to secrete hCNTF. Hum Gene Ther 7, 851-860.

Podda S, Ward M, Himelstein A, Richardson C, De La Flor-Weiss E, Smith L, Gottesman M, Pastan I, Bank A (1992) Transfer and expression of the human multiple drug resistance gene into live mice. Proc Natl Acad Sci USA 89, 9676-9680.

Polyak K, Xia Y, Zweier JL, Kinzler KW, and Vogelstein B (1997) A model for p53-induced apoptosis. Nature 389, 300-305.

Polymeropoulos MH, Higgins JJ, Golbe LI, Johnson WG, Ide SE, Di Iorio G, Sanges G, Stenroos ES, Pho LT, Schaffer AA, Lazzarini AM, Nussbaum RL, and Duvoisin RC (1996) Mapping of a gene for Parkinson’s Disease to chromosome 4q21-q23. Science 274, 1197-1199.

Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B, Root H, Rubenstein J, Boyer R, Stenroos ES, Chandrasekharappa S, Athanassiadou A, Papapetropoulos T, Johnson WG, Lazzarini AM, Duvoisin RC, Di Iorio G, Golbe LI, Nussbaum RL (1997) Mutation in the a-synuclein gene identified in families with Parkinson's disease. Science 276, 2045-2047.

Pombo A, Ferreira J, Bridge E, and Carmo-Fonseca M (1994) Adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells. EMBO J. 13, 5075-5085.

Ponnazhagan S, Erikson D, Kearns WG, Zhou SZ, Nahreini P, Wang XS, Srivastava A (1997a) Lack of site-specific integration of the recombinant adeno-associated virus 2 genomes in human cells. Hum Gene Ther 8, 275-284.

Ponnazhagan S, Yoder MC, Srivastava A (1997b) Adeno-associated virus type 2-mediated transduction of murine hematopoietic cells with long-term repopulating ability and sustained expression of a human globin gene in vivo. J Virol 71, 3098-3104.

Porteous DJ, Dorin JR, McLachlan G, Davidson-Smith H, Davidson H, Stevenson BJ, Carothers AD, Wallace WA, Moralee S, Hoenes C, Kallmeyer G, Michaelis U, Naujoks K, Ho LP, Samways JM, Imrie M, Greening AP, Innes JA (1997) Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Ther 4, 210-218.

Powell JS, Clozel JP, Muller RK, Kuhn H, Hefti F, Hosang M, Baumgartner HR (1989) Inhibitors of angiotensin-converting enzyme prevent myointimal proliferation after vascular injury. Science 245, 186-188.

Praseuth D, Perrouault L, Le Doan T, Chassignol M, Thuong N, Hélène C (1988) Sequence-specific binding and photocrosslinking of a and b oligodeoxynucleotides to the major groove of DNA via triple-helix formation. Proc Natl Acad Sci USA 85, 1349-1353.

Prehn JH, Bindokas VP, Jordan J, Galindo MF, Ghadge GD, Roos RP, Boise LH, Thompson CB, Krajewski S, Reed JC, Miller RJ (1996) Protective effect of transforming growth factor-b1 on b-amyloid neurotoxicity in rat hippocampal neurons. Mol Pharmacol 49, 319-328.

Prives C (1994) How loops, B sheets, and helices help us to understand p53. Cell 78: 543-546.

Prives C and Manfredi JJ (1993) The p53 tumor suppressor protein: meeting review. Genes Dev 7, 529-534.

Pruijn, G.J.M., van Driel, W. and van der Vliet, P.C. (1986) Nuclear factor III, a novel sequence-specific DNA-binding protein from HeLa cells stimulating adenovirus DNA replication. Nature 322, 656-659.

Pulaski BA, Yen K-Y, Shastri N, Maltby KM, Penney DP, Lord EM, and Frelinger JG (1996) Interleukin 3 enhances cytotoxic T lymphocyte development and class I major histocompatibility complex "re-presentation" of exogenous antigen by tumor-infiltrating antigen-presenting cells. Proc Natl Acad Sci USA 93, 3669-3674.

Putzer BM, Hitt M, Muller WJ, Emtage P, Gauldie J, Graham FL (1997) Interleukin 12 and B7-1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression. Proc Natl Acad Sci USA 94, 10889-10894.

Qin L, Ding Y, Bromberg JS (1996) Gene transfer of transforming growth factor-b1 prolongs murine cardiac allograft survival by inhibiting cell-mediated immunity. Hum Gene Ther 7, 1981-1988.

Qin XQ, Livingston DM, Kaelin WG Jr, Adams PD (1994) Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis. Proc Natl Acad Sci USA 91, 10918-10922.

Quesada P (1998) Poly (ADP-ribosyl)ation as one of the molecular events that accompany mammalian spermatogenesis. Gene Ther Mol Biol 1, 681-699.

Rade JJ, Schulick AH, Virmani R, Dichek DA (1996) Local adenoviral-mediated expression of recombinant hirudin reduces neointima formation after arterial injury. Nature Med 2, 293-298.

Rader DJ (1997) Gene therapy for atherosclerosis. Int J Clin Lab Res 27, 35-43.

Raffo AJ, Perlman H, Chen M-W, Day ML, Streitman JS, Buttyan R (1995) Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo. Cancer Res 55, 4438-4445.

Rajagopal P and Feigon J (1989) Triple-strand formation in the homopurine:homopyrimidine DNA oligonucleotides d(G-A)4 and d(T-C)4. Nature 339, 637-640.

Rak J, Mitsuhashi Y, Bayko L, Filmus J, Shirasawa S, Sasazuki T, Kerbel RS (1995) Mutant ras oncogenes upregulate VEGF/VPF expression: Implications for induction and inhibition of tumor angiogenesis. Cancer Res 55, 4575-4580.

Rakhmilevich AL, Janssen K, Turner J, Culp J, Yang NS (1997) Cytokine gene therapy of cancer using gene gun technology: superior antitumor activity of interleukin-12. Hum Gene Ther 8, 1303-1311.

Ramesh N, Shin YK, Lau S, Osborne WRA (1995) High-level expression from a cytomegalovirus promoter in macrophage cells. Hum Gene Ther 6, 1323-1327.

Ramesh R, Marrogi AJ and Freeman SM (1998) Tumor killing using the HSV-tk suicide gene. Gene Ther Mol Biol 1, 253-263.

Ramezani A, Ding SF, Joshi S (1997) Inhibition of HIV-1 replication by retroviral vectors expressing monomeric and multimeric hammerhead ribozymes. Gene Ther 4, 861-867.

Rapaport D, Ovadia M, Shai Y (1995) A synthetic peptide corresponding to a conserved heptad repeat domain is a potent inhibitor of Sendai virus-cell fusion: An emerging similarity with functional domains of other viruses. EMBO J 14, 5524-5531.

Raper SE, Grossman M, Rader DJ, Thoene JG, Clark BJ 3rd, Kolansky DM, Muller DW, Wilson JM (1996) Safety and feasibility of liver-directed ex vivo gene therapy for homozygous familial hypercholesterolemia. Ann Surg 223, 116-126.

Raycroft L, Wu H, Lozano G (1990) Transcriptional activation by wild-type but not transforming mutants of the p53 anti-oncogene. Science 249: 1049-1051.

Reddy JC, Hosono S, Licht JD (1995) The transcriptional effect of WT1 is modulated by choice of expression vector. J Biol Chem 270, 29976-29982.

Richards CA, Austin EA, and Huber BE (1995) Transcriptional regulatory sequences of carcinoembryonic antigen: identification and use with cytosine deaminase for tumor-specific gene therapy. Hum Gene Ther 6, 881-893.

Richman DD (1996) HIV Therapeutics. Science 272, 1886-1887.

Riley DJ, Nikitin AY, Lee WH (1996) Adenovirus-mediated retinoblastoma gene therapy suppresses spontaneous pituitary melanotroph tumors in Rb^+/- mice. Nat Med 2, 1316-1321.

Riordan JE, Rommens JM, Kerem B-S, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou J-L Drumm MI, Iannuzzi MC, Collins FS, Tsui L-C (1989) Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 245, 1066-1073.

Robbins, P.D., Horowitz, J.M. and Mulligan, R.C. (1990) Negative regulation of human c-fos expression by the retinoblastoma gene product. Nature 346, 668-671.

Rodolfo M, Zilocchi C, Melani C, Cappetti B, Arioli I, Parmiani G, Colombo MP (1996) Immunotherapy of experimental metastases by vaccination with interleukin gene-transduced adenocarcinoma cells sharing tumor-associated antigens. Comparison between IL-12 and IL-2 gene-transduced tumor cell vaccines. J Immunol 157, 5536-5542.

Rodriguez R, Schuur ER, Lim HY, Henderson GA, Simons JW, Henderson DR(1997) Prostate attenuated replication competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells. Cancer Res 57, 2559-2563.

Roessler BJ and Davidson BL (1994) Direct plasmid mediated transfection of adult murine brain cells in vivo using cationic liposomes. Neurosci Lett 167, 5-10.

Roessler BJ, Allen ED, Wilson JM, Hartman JW, Davidson BL (1993) Adenoviral-mediated gene transfer to rabbit synovium in vivo. J Clin Invest 92, 1085-1092

Rohde M, Warthoe P, Gjetting T, Lukas J, Bartek J, Strauss M (1996) The retinoblastoma protein modulates expression of genes coding for diverse classes of proteins including components of the extracellular matrix. Oncogene 12, 2393-2401.

Rolling F, Nong Z, Pisvin S, Collen D (1997) Adeno-associated virus-mediated gene transfer into rat carotid arteries. Gene Ther 4, 757-761.

Rømer J, Bugge TH, Pyke C, Lund LR, Flick MJ, Degen JL, Dano K (1996) Impaired wound healing in mice with a disrupted plasminogen gene. Nat Med 2, 287-292.

Rommerskirch W, Fluharty AL, Peters C, von Figura K, Gieselmann V (1991) Restoration of arylsulphatase A activity in human-metachromatic-leucodystrophy fibroblasts via retroviral-vector-mediated gene transfer. Biochem J 280, 459-461.

Roninson IB, Chin JE, Choi K, Gros P, Housman DE, Fojo A, Shen D-W, Gottesman MM, Pastan I (1986) Isolation of human mdr DNA sequencs amplified in multidruf-resistant KB carcinoma cells. Proc Natl Acad Sci USA 83, 4538-4542.

Rosenberg SA (1992) The immunotherapy and gene therapy of cancer. J Clin Oncol 10, 180-199.

Rosenberg SA, Packard BS, Aebersold PM, Solomon D, Topalian SL, Toy ST, Simon P, Lotze MT, Yang JC, Seipp CA, Simpson C, Carter C, Bock S, Schwartzentruber D, Wei JP, and White DE (1988) Use of tumor infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. N Engl J Med 319, 1676-1680.

Rosenberg, SA, Anderson WF, Blaese MR, Ettinghausen SE, Hwu P, Karp SE, Kasid A, Mule JJ, Parkinson DR, Salo JC, Schwartzentruber DJ, Topalian Sl, Weber JS, Yannelli JR, Yang JC, and Linehan WM (1992) Initial proposal of clinical research project. Immunization of cancer patients using autologous cancer cells modified by insertion of the gene for interleukin-2. Hum Gene Ther 3, 75-90.

Rosenecker J, Zhang W, Hong K, Lausier J, Geppetti P, Yoshihara S, Papahdjopoulos D, Nadel JA (1996) Increased liposome extravasation in selected tissues: Effect of substance P. Proc Natl Acad Sci USA 93, 7236-7241.

Rosenfeld MA, Siegfried W, Yoshimura K, Yoneyama K, Fukayama M, Stier LE, Pääkkö PK, Gilardi P, Stratford-Perricaudet L, Perricaudet M, Jallat S, Pavirani A, Lecocq J-P, and Crystal RG (1991) Adenovirus-mediated transfer of recombinant a1-antitrypsin gene to the lung epithelium in vivo. Science 252, 431-434.

Rosenfeld MA, Yoshimura K, Trapnell BC, Yoneyama K, Rosenthal ER, Dalemans W, Fukayama M, Bargon J, Stier LE, Stratford-Perricaudet L, Perricaudet M, Guggino WB, Pavirani A, Lecocq J-P, and Crystal RG (1992) In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell 68, 143-155.

Rosenfeld MR, Bergman I, Schramm L, Griffin JA, Kaplitt MG, Meneses PI (1997) Adeno-associated viral vector gene transfer into leptomeningeal xenografts. J Neurooncol 34, 139-144.

Rosenfeld MR, Meneses P, Dalmau J, Drobnjak M, Cordon-Cardo C, Kaplitt MG (1995) Gene transfer of wild-type p53 results in restoration of tumor-suppressor function in a medulloblastoma cell line. Neurology 45, 1533-1539.

Rosenzweig M, Marks DF, Hempel D, Heusch M, Kraus G, Wong-Staal F, Johnson RP (1997) Intracellular immunization of rhesus CD34⁺ hematopoietic progenitor cells with a hairpin ribozyme protects T cells and macrophages from simian immunodeficiency virus infection. Blood 90, 4822-4831.

Ross G, Erickson R, Knorr D, Motulsky AG, Parkman R, Samulski J, Straus SE, and Smith BR (1996) Gene therapy in the United States: a five-year status report. Hum Gene Ther 7, 1781-1790.

Ross R (1993) The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362, 801-809.

Ross R (1995) Genetically modified mice as models of transplant atherosclerosis. Nature Med 2, 527-528.

Roth JA (1996) Clinical protocol. Modification of tumor suppressor gene expression and induction of apoptosis in non-small cell lung cancer (NSCLC) with an adenovirus vector expressing wild-type p53 and cisplatin. Hum Gene Ther 7, 1013-1030.

Roth JA, Nguyen D, Lawrence DD, Kemp BL, Carrasco CH, Ferson DZ, Hong WK, Komaki R, Lee JJ, Nesbitt JC, Pisters KMW, Putnam JB, Schea R, Shin DM, Walsh GL, Dolormente MM, Han C-I, Martin FD, Yen N, Xu K, Stephens LC, McDonnell TJ, Mukhopadhyay T, and Cai D (1996) Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer. Nature Med 2, 985-991.

Rubenstein RC, McVeigh U, Flotte TR, Guggino WB, Zeitlin PL (1997) CFTR gene transduction in neonatal rabbits using an adeno-associated virus (AAV) vector. Gene Ther 4, 384-392.

Russ AP, Friedel C, Grez M, von Melchner H (1996) Self-deleting retrovirus vectors for gene therapy. J Virol 70, 4927-4932.

Russell DW, Alexander IE, and Miller AD (1995) DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus. Proc Natl Acad Sci USA 92, 5719-5723.

Russell DW, Alexander IE, Miller AD (1995) DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors. Proc Natl Acad Sci USA 92, 5719-5723.

Russell SJ (1996) Peptide-displaying phages for targeted gene delivery. Nature Med 2, 276-277.

Rutledge EA, Russell DW (1997) Adeno-associated virus vector integration junctions. J Virol 71, 8429-8436.

Sabbatini P, Chiou S-K, Rao L, White E (1995) Modulation of p53-mediated transcriptional repression and apoptosis by the adenovirus E1B 19K protein. Mol Cell Biol 15, 1060-1070.

Safa AR, Glover CJ, Meyers MB, Biedler JL, Felsted RL (1986) Vinblastine photoaffinity labeling of a high molecular weight surface membrane glycoprotein specific for multidrug-resistant cells. J Biol Chem 261, 6137-6140.

Saijo Y, Perlaky L, Wang H, Busch H (1994) Pharmacokinetics, tissue distribution, and stability of antisense oligodeoxynucleotide phosphorothioate ISIS 3466 in mice. Oncol Res 6, 243-249.

Sakai T, Ohtani N, McGee TL, Robbins PD, Dryja TP (1991) Oncogenic germ-line mutations in Sp1 and ATF sites in the human retinoblastoma gene. Nature 353, 83-86.

Saleh M (1996) Ectopic expression of vascular endothelial growth factor (VEGF) by C6 glioma cells does not increase tumour growth in vivo despite an increase in angiogenesis. Int J Oncol 9, 33-41.

Saleh MN, Khazaeli MB, Wheeler RH, Bucy RP, Liu T, Everson MP, Munn DH, Schlom J, LoBuglio AF (1995) Phase II trial of murine monoclonal antibody D612 combined with recombinant human monocyte colony-stiumlating factor (rhM-CSF) in pateints with metastatic gastrointestinal cancer. Cancer Res 55, 4339-4346.

Salvetti A, Moullier P, Cornet V, Brooks D, Hopwood JJ, Danos O, Heard J-M (1995) In vivo delivery of human a-L-Iduronidase in mice implanted with neo-organs. Hum Gene Ther 6, 1153-1159.

Sanda MG, Ayyagari SR, Jaffee EM, Epstein JI, Clift SL, Cohen LK, Dranoff G, Pardoll DM, Mulligan RC, Simons JW (1994) Demonstration of a rational strategy for human prostate cancer gene therapy. J Urol 151, 622-628.

Sandig V, Hofmann C, Steinert S, Jennings G, Schlag P, Strauss M (1996) Gene transfer into hepatocytes and human liver tissue by baculovirus vectors. Hum Gene Ther 7, 1937-1945.

Sandig V and Strauss M (1996) Liver-directed gene transfer and application to therapy. J Mol Med 74, 205-212.

Santoro C, Mermod N, Andrews PC, and Tjian R.(1988) A family of human CCAAT-box-binding proteins active in transcription and DNA replication: cloning and expression of multiple cDNAs. Nature 334, 218-224.

Sargiacomo M, Sudol M, Tang ZL and Lisanti MP (1993) Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J. Cell Biol. 122, 789-807.

Sarnow P, Ho YS, Williams J, Levine AJ (1982) Adenovirus E1b-58kd tumor antigen and SV40 large tumor antigen are physically associated with the same 54 kd cellular protein in transformed cells. Cell 28, 387-394.

Savani RC and Turley EA (1995) The role of hyaluronan and its receptors in restenosis after balloon angioplasty: development of a potential therapy. Int J Tissue React 17, 141-151.

Sawchuk SJ, Boivin GP, Duwel LE, Ball W, Bove K, Trapnell B, Hirsch R (1996) Anti-T cell receptor monoclonal antibody prolongs transgene expression following adenovirus-mediated in vivo gene transfer to mouse synovium. Hum Gene Ther 7, 499-506.

Scarlatti G, Tresoldi E, Bjorndal A, Fredriksson R, Colognesi C, Deng HK, Malnati MS, Plebani A, Siccardi AG, Littman DR, Fenyo EM, Lusso P (1997) In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression. Nat Med 3, 1259-1265.

Schaack, J., Ho, W. Y.-W., Freimuth, P., and Shenk, T. (1990) Adenovirus terminal protein mediates both nuclear matrix association and efficient transcription of adenovirus DNA Genes Dev 4, 1197-1208.

Scharfmann R, Axelrod JH, and Verma IM (1991). Long-term in vivo expression of retrovirus-mediated gene transfer in mouse fibroblast implants. Proc Natl Acad Sci USA 88, 4626-4630.

Schild L (1996) The ENaC channel as the primary determinant of two human diseases: Liddle syndrome and pseudohypoaldosteronism. Nephrologie 17, 395-400.

Schlake T, Klehr-Wirth D, Yoshida M, Beppu T, and Bode J (1994) Gene expression within a chromatin domain: the role of core histone hyperacetylation. Biochemistry 33, 4197-4206.

Schwartz B, Benoist C, Abdallah B, Rangara R, Hassan A, Scherman D, Demeneix BA (1996) Gene transfer by naked DNA into adult mouse brain. Gene Ther 3, 405-411.

Schwartz B, Benoist C, Abdallah B, Scherman D, Behr J-P, and Demeneix BA (1995) Lipospermine-based gene transfer into the newborn mouse brain is optimized by a low lipospermine/DNA charge ratio. Hum Gene Ther 6, 1515-1524.

Schwarzenberger P, Spence SE, Gooya JM, Michiel D, Curiel DT, Ruscetti FW, Keller JR (1996) Targeted gene transfer to human hematopoietic progenitor cell lines through the c-kit receptor. Blood 87, 472-478.

Seibel P, Trappe J, Villani G, Klopstock T, Papa S, and Reichman H: Transfection of mitochondria: strategy towards a gene therapy of mitochondrial DNA diseases. Nucleic Acids Res 23: 10-17, 1995.

Senger DR, van de Water L, Brown LF, Nagy JA, Yeo K-T, Yeo T-K, Berse B, Jackman RW, Dvorak AM, and Dvorak HF (1993) Vascular permeability factor (VPF, VEGF) in tumor biology. Cancer Metastasis Rev 12, 303-324.

Serio D, Rizvi TA, Cartas M, Kalyanaraman VS, Weber IT, Koprowski H, Srinivasan A (1997) Development of a novel anti-HIV-1 agent from within: effect of chimeric Vpr-containing protease cleavage site residues on virus replication. Proc Natl Acad Sci USA 94, 3346-3351.

Seth P (1994) Adenovirus-dependent release of choline from plasma membrane vesicles at an acidic pH is mediated by the penton base protein. J Virol 68, 1204-1206.

Seth P, FitzGerald D, Ginsberg H, Willingham M and Pastan I (1984) Evidence that the penton base of adenovirus is involved in potentiation of toxicity of Pseudomonas exotoxin conjugated to epidermal growth factor. Mol Cell Biol 4, 1528-1533.

Seto E, Usheva A, Zambetti GP, Momand J, Horikoshi N, Weinmann R, Levine AJ, Shenk T (1992) Wild-type p53 binds to the TATA-binding protein and represses transcription. Proc Natl Acad Sci USA 89, 12028-12032.

Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu X, Breitman ML, and Schuh AC (1995) Failure of blood island formation and vasculogenesis in Flk-1-defficient mice. Nature 376, 62-66.

Shan B and Lee WH (1994) Deregulated expression of E2F-1 induces S-phase entry and leads to apoptosis. Mol Cell Biol 14, 8166-8173.

Shan B, Chang CY, Jones D, Lee WH (1994) The transcription factor E2F-1 mediates the autoregulation of RB gene expression. Mol Cell Biol 14, 299-309.

Shan B, Zhu X, Chen PL, Durfee T, Yang Y, Sharp D, Lee WH (1992) Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F. Mol. Cell. Biol. 12, 5620-5631.

Sharma S, Cantwell M, Kipps TJ, Friedmann T (1996) Efficient infection of a human T-cell line and of human primary peripheral blood leukocytes with a pseudotyped retrovirus vector. Proc Natl Acad Sci USA 93, 11842-11847.

Shen F, Ross JF, Wang X, and Ratnam M (1994) Identification of a novel folate receptor, a truncated receptor, and receptor type b in hematopoietic cells: cDNA cloning, expression, immunoreactivity, and tissue specificity. Biochemistry 33, 1209-1215.

Shen Y and Shenk T (1994) Relief of p53-mediated transcriptional repression by the adenovirus E1B 19-kDa protein or the cellular Bcl-2 protein. Proc Natl Acad Sci USA 91, 8940-8944.

Shevelev A, Burfeind P, Schulze E, Rininsland F, Johnson TR, Trojan J, Chernicky CL, Hélène C, Ilan J, Ilan J (1997) Potential triple helix-mediated inhibition of IGF-I gene expression significantly reduces tumorigenicity of glioblastoma in an animal model. Cancer Gene Ther 4, 105-112.

Shi CS and Kehrl JH (1997) Activation of stress-activated protein Kinase/c-Jun N-terminal kinase, but not NF-kB, by the tumor necrosis factor (TNF) receptor 1 through a TNF receptor-associated factor 2- and germinal center kinase related-dependent pathway. J Biol Chem 272, 32102-32107.

Shih CC, Stoye JP, Coffin JM (1988) Highly preferred targets for retrovirus integration. Cell 53, 531-537.

Shiio Y, Sawada J, Handa H, Yamamoto T, Inoue J (1996) Activation of the retinoblastoma gene expression by Bcl-3: implication for muscle cell differentiation. Oncogene 12, 1837-1845.

Shiio Y, Yammoto T, Yamaguchi N (1992) Negative regulation of Rb expression by the p53 gene product. Proc Natl Acad Sci USA 89, 5206-5210.

Shimkets RA, Warnock DG, Bositis CM, Nelson-Williams C, Hansson JH, Schambelan M, Gill JR Jr, Ulick S, Milora RV, Findling JW, et al (1994) Liddle's syndrome: heritable human hypertension caused by mutations in the b subunit of the epithelial sodium channel. Cell 79, 407-414.

Shirodkar, S., Ewen, M., DeCaprio, J.A., Morgan, J., Livingston, D.M. and Chittenden, T. (1992) The transcription factor E2F interacts with the retinoblastoma product and a p107-cyclin A complex in a cell cycle-regulated manner. Cell 68, 157-166.

Shivakumar CV, Brown DR, Deb S, Deb SP (1995) Wild-type human p53 transactivates the human proliferating cell nuclear antigen promoter. Mol Cell Biol 15, 6785-6793.

Shweiki D, Itin A, Soffer D, and Keshet E (1992) Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359, 843-845.

Sime PJ, Xing Z, Graham FL, Csaky KG, Gauldie J (1997) Adenovector-mediated gene transfer of active transforming growth factor-b1 induces prolonged severe fibrosis in rat lung. J Clin Invest 100, 768-776.

Simons M, Edelman ER, Rosenberg RD (1994) Antisense proliferating cell nuclear antigen oligonucleotides inhibit intimal hyperplasia in a rat carotid artery injury model. J Clin Invest 93, 2351-2356.

Singh D and Rigby PW (1996) The use of histone as a facilitator to improve the efficiency of retroviral gene transfer. Nucleic Acids Res 24, 3113-3114.

Smith JD, Wong E, Ginsberg M (1995) Cytochrome P450 1A1 promoter as a genetic switch for the regulatable and physiological expression of a plasma protein in transgenic mice. Proc Natl Acad Sci USA 92, 11926-11930.

Smith ML, Chen I-T, Zhan Q, Bae I, Chen C-Y, Gilmer TM, Kastan MB, O'Connor PM, Fornace Jr AJ (1994) Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science 266: 1376-1380.

Smith RC, Branellec D, Gorski DH, Guo K, Perlman H, Dedieu JF, Pastore C, Mahfoudi A, Denefle P, Isner JM, Walsh K (1997) p21CIP1-mediated inhibition of cell proliferation by overexpression of the gax homeodomain gene. Genes Dev 11, 1674-1689.

Smith RC, Wills KN, Antelman D, Perlman H, Truong LN, Krasinski K, Walsh K (1997) Adenoviral constructs encoding phosphorylation-competent full-length and truncated forms of the human retinoblastoma protein inhibit myocyte proliferation and neointima formation. Circulation 96, 1899-1905.

Smith TAG, Mehaffey MG, Kayda DB, Saunders JM, Yei S, Trapnell BC, McClelland A, Kaleko M (1993) Adenovirus mediated expression of therapeutic plasma levels of human factor IX in mice. Nature Genet 5, 3970-402.

Snyder RO, Miao CH, Patijn GA, Spratt SK, Danos O, Nagy D, Gown AM, Winther B, Meuse L, Cohen LK, Thompson AR, Kay MA (1997) Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. Nat Genet 16, 270-276.

Somia NV, Zoppe M, Verma IM (1995) Generation of targeted retroviral vectors by using single-chain variable fragment: an approach to in vivo gene delivery. Proc Natl Acad Sci USA 92, 7570-7574.

Song S, Wang Y, Bak SY, Lang P, Ullrey D, Neve RL, O'Malley KL, Geller AI (1997) An HSV-1 vector containing the rat tyrosine hydroxylase promoter enhances both long-term and cell type-specific expression in the midbrain. J Neurochem 68, 1792-1803.

Song YK, Liu F, Chu S, Liu D (1997) Characterization of cationic liposome-mediated gene transfer in vivo by intravenous administration. Hum Gene Ther 8, 1585-1594.

Sorgi FL, Bhattacharya S, Huang L (1997) Protamine sulfate enhances lipid-mediated gene transfer. Gene Ther 4, 961-968.

Sorrentino BP, Brandt SJ, Bodine D, Gottesman M, Pastan I, Cline A, Nienhuis AW (1992) Selection of drug-resistant bone marrow cells in vivo after retroviral transfer of human MDR1. Science 257, 99-103.

Sorscher EJ, Peng S, Bebok Z, Allan PW, Bennett LLJ, and Parker WB (1994) Tumor cell bystander killing in colonic carcinoma utilizing the Escherichia coli DeoD gene to generate toxic purines. Gene Ther 1, 233-238.

Sowa Y, Shiio Y, Fujita T, Matsumoto T, Okuyama Y, Kato D, Inoue J, Sawada J, Goto M, Watanabe H, Handa H, Sakai T (1997) Retinoblastoma binding factor 1 site in the core promoter region of the human RB gene is activated by hGABP/E4TF1. Cancer Res 57, 3145-3148.

Spaete, R.R. and Frenkel, N. (1982) The herpes simplex virus amplicon: a new eucaryotic defective-virus cloning-amplifying vector. Cell 30, 295-304.

Spiegelman BM and Flier JS (1996) Adipogenesis and obesity: rounding out the big picture. Cell 87, 377-389.

Stahl F (1996) Meiotic recombination in yeast: coronation of the double-strand-break repair model. Cell 87, 965-968.

Stathakis P, Fitzgerald M, Matthias LJ, Chesterman CN, Hogg PJ (1997) Generation of angiostatin by reduction and proteolysis of plasmin. Catalysis by a plasmin reductase secreted by cultured cells. J Biol Chem 272, 20641-20645.

Stenger JE, Mayr GA, Mann K, Tegtmeyer P (1992) Formation of stable p53 homotetramers and multiples of tetramers. Mol Carcinog 5, 102-106.

Stephan D, San H, Yang ZY, Gordon D, Goelz S, Nabel GJ, Nabel EG (1997) Inhibition of vascular smooth muscle cell proliferation and intimal hyperplasia by gene transfer of b-interferon. Mol Med 3, 593-599.

Stinchcomb DT (1995) Constraining the cell cycle: Regulating cell division and differentiation by gene therapy. Nature Med 1, 1004-1006.

Stuart E, Haffner R, Oren M, Gruss P (1995) Loss of p53 function through PAX-mediated transcriptional repression. EMBO J 14, 5638-5645.

Stürzbecher H-W, Donzelmann B, Henning W, Knippschild U, Buchhop S (1996) p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction. EMBO J 15, 1992-2002.

Su H, Chang JC, Xu SM, and Kan YW (1996) Selective killing of AFP-positive hepatocellular carcinoma cells by adeno-associated virus transfer of ther herpes simplex virus thymidine kinase gene. Hum Gene Ther 7, 463-470.

Suchi M, Dinur T, Desnick RJ, Gatt S, Pereira L, Gilboa E, Schuchman EG (1992) Retroviral-mediated transfer of the human acid sphingomyelinase cDNA: Correction of the metabolic defect in cultured Niemann-Pick disease cells. Proc Natl Acad Sci USA 89, 3227-3231.

Sugimoto Y, Aksentijevich I, Murray G, Brady RO, Pastan I, Gottesman MM (1995) Retroviral coexpression of a multidrug resistance gene (MDR1) and human a-galactosidase A for gene therapy of fabry disease. Hum Gene Ther 6, 905-915.

Sun TQ, Fernstermacher DA, Vos JM (1994) Human artificial episomal chromosomes for cloning large DNA fragments in human cells. Nat Genet 8, 33-41.

Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN, and Yancopoulos GD (1996) Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87, 1171-1180.

Suzuki K, Hayashi N, Miyamoto Y, Yamamoto M, Ohkawa K, Ito Y, Sasaki Y, Yamaguchi Y, Nakase H, Noda K, Enomoto N, Arai K, Yamada Y, Yoshihara H, Tujimura T, Kawano K, Yoshikawa K, and Kamada T (1996) Expression of vascular permeability factor/vascular endothelial factor in human hepatocellular carcinoma. Cancer Res 56, 3004-3009.

Svensson U and Persson R (1984) Entry of adenovirus 2 into HeLa cells. J Virol 51, 687-694.

Swantek JL, Cobb MH, Geppert TD (1997) Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is required for lipopolysaccharide stimulation of tumor necrosis factor a (TNF-a) translation: glucocorticoids inhibit TNF-a translation by blocking JNK/SAPK. Mol Cell Biol 17, 6274-6282.

Szary J, Missol E, Tarnawski R, Szala S (1997) Selective augmentation of radiation effects by 5-fluorocytosine on murine B16(F10) melanoma cells transfected with cytosine deaminase gene. Cancer Gene Ther 4, 269-272.

Szostak, J.W., Orr-Weaver, T.L., Rothstein, R.J. and Stahl, F.W. (1983) The double-strand-break repair model for recombination. Cell 33, 25-35.

Tahara H, Lotze MT, Robbins PD, Storkus WJ, Zitvogel L (1995) IL-12 gene therapy using direct injection of tumors with genetically engineered autologous fibroblasts. Hum Gene Ther 6, 1607-1624.

Tahara H, Zitvogel L, Storkus WJ, Robbins PD, Lotze MT (1996) Murine models of cancer cytokine gene therapy using interleukin-12. Ann N Y Acad Sci 795, 275-283.

Takahashi JC, Saiki M, Miyatake S, Tani S, Kubo H, Goto K, Aoki T, Takahashi JA, Nagata I, Kikuchi H (1997) Adenovirus-mediated gene transfer of basic fibroblast growth factor induces in vitro angiogenesis. Atherosclerosis 132, 199-205.

Takahashi T, Nau MM, Chiba I, Birrer MJ, Rosenberg RK, Vinocour M, Levitt M, Pass H, Gazdar AF, Minna JD (1989) p53: A frequent target for genetic abnormalities in lung cancer. Science 246: 491-494.

Takehara T, Hayashi N, Yamamoto M, Miyamoto Y, Fusamoto H, Kamada T (1996) In vivo gene transfer and expression in rat stomach by submucosal injection of plasmid DNA. Hum Gene Ther 7, 589-593.

Takeshita S, Isshiki T, Mori H, Tanaka E, Tanaka A, Umetani K, Eto K, Miyazawa Y, Ochiai M, Sato T (1997) Microangiographic assessment of collateral vessel formation following direct gene transfer of vascular endothelial growth factor in rats. Cardiovasc Res 35, 547-552.

Takeshita S, Losordo DW, Kearney M, Rossow ST, and Isner JM (1994b) Time course of recombinant protein secretion after liposome-mediated gene transfer in a rabbit arterial organ culture model. Lab Invest 71, 387-391.

Takeshita S, Zheng LP, Brogi E, Kearney M, Pu L-Q, Bunting S, Ferrara N, Symes JF, and Isner JM (1994a) Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic limb model. J Clin Invest 93, 662-670.

Takle GB, Thierry AR, Flynn SM, Peng B, White L, Devonish W, Galbraith RA, Goldberg AR, George ST (1997) Delivery of oligoribonucleotides to human hepatoma cells using cationic lipid particles conjugated to ferric protoporphyrin IX (heme). Antisense Nucleic Acid Drug Dev 7, 177-185.

Tamayose K, Hirai Y, Shimada T (1996) A new strategy for large-scale preparation of high-titer recombinant adeno-associated virus vectors by using packaging cell lines and sulfonated cellulose column chromatography. Hum Gene Ther 7, 507-513.

Tamura H, Schild L, Enomoto N, Matsui N, Marumo F, Rossier BC (1996) Liddle disease caused by a missense mutation of b subunit of the epithelial sodium channel gene. J Clin Invest 97, 1780-1784.

Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, et al (1995) Identification and expression cloning of a leptin receptor, OB-R. Cell 83, 1263-1271.

Tatulain SA, Hinterdorfer P, Baber G, Tamm LK (1995) Influenza hemagglutinin assumes a tilted conformation during membrane fusion as determined by attenuated total reflection FTIR spectroscopy. EMBO J 14, 5514-5523.

Templeton NS, Lasic DD, Frederik PM, Strey HH, Roberts DD, Pavlakis GN (1997) Improved DNA: liposome complexes for increased systemic delivery and gene expression. Nat Biotechnol 15, 647-652.

Tepper RI, Pattengale PK, Leder P (1989) Murine Interleukin-4 displays potent anti-tumor activity in vitro. Cell 57, 503-512.

Thierry AR and Dritschilo A (1992) Intracellular availability of unmodified, phosphorothioated and liposomally encapsulated oligodeoxynucleotides for antisense activity. Nucleic Acids Res 20, 5691-5698.

Thierry AR, Lunardi-Iskandar Y, Bryant JL, Rabinovitch P, Gallo RC, and Mahan LC (1995) Systemic gene therapy: biodistribution and long-term expression of a transgene in mice. Proc Natl Acad Sci USA 92, 9742-9746.

Thompson L (1992) At age 2, gene therapy enters a growth phase. Science 258, 744-746.

Thompson, E.M., Christians, E., Stinnakre, M.G., and Renard, J.P. (1994) Scaffold attachment regions stimulate HSP70.1 expression in mouse preimplantation embryos but not in differentiated tissues. Mol Cell Biol 14, 4694-4703.

Thut CJ, Chen J-L, Klemm R, and Tjian R (1995) p53 transcriptional activation mediated by coactivators TAFII40 and TAFII60. Science 267, 100-104.

Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, Fiddes JC, and Abraham JA (1991) The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 266, 11947-11954.

Tomita N, Morishita R, Higaki J, Aoki M, Nakamura Y, Mikami H, Fukamizu A, Murakami K, Kaneda Y, Ogihara T (1995) Transient decrease in high blood pressure by in vivo transfer of antisense oligodeoxynucleotides against rat angiotensinogen. Hypertension 26, 131-136.

Tomita N, Morishita R, Higaki J, Tomita S, Aoki M, Ogihara T, Kaneda Y (1996) In vivo gene transfer of insulin gene into neonatal rats by the HVJ-liposome method resulted in sustained transgene expression. Gene Ther 3, 477-482.

Troelstra C, van Gool A, de Wit J, Vermeulen W, Bootsma D and Hoeijmakers JHJ (1992) ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne's syndrome and preferential repair of active genes. Cell 71, 939-953.

Trono D, Feinberg MB, Baltimore D (1989) HIV-1 Gag mutants can dominantly interfere with the replication of the wild-type virus. Cell 59, 113-120

Trouche D, Le Chalony C, Muchardt C, Yaniv M, Kouzarides T (1997) RB and hbrm cooperate to repress the activation functions of E2F1. Proc Natl Acad Sci USA 94, 11268-11273.

Troy CM, Stefanis L, Prochiantz A, Greene LA, and Shelanski ML (1996) The contrasting roles of ICE family proteases and interleukin-1b in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation. Proc Natl Acad Sci USA 93, 5635-5640.

Truant R, Xiao H, Ingles J, Greenblatt J (1993) Direct interaction between the transcriptional activation domain of human p53 and the TATA box-binding protein. J Biol Chem 268: 2284-2287.

Tsurumi Y, Kearney M, Chen D, Silver M, Takeshita S, Yang J, Symes JF, Isner JM (1997) Treatment of acute limb ischemia by intramuscular injection of vascular endothelial growth factor gene. Circulation 96 (9 Suppl):II-II3828.

Tsurumi Y, Takeshita S, Chen D, Kearney M, Rossow ST, Passeri J, Horowitz JR, Symes JF, Isner JM (1996) Direct intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor augments collateral development and tissue perfusion. Circulation 94, 3281-3290.

Tsutsumi-Ishi Y, Tadokoro K, Hanaoka F, Tsuchida N (1995) Response of heat shock element with the human HSP70 promoter to mutated p53 genes. Cell Growth Differ 6, 1-8.

Tucker, P.A., Tsernoglou, D., Tucker, A.D., Coenjaerts, F.E.J., Leenders, H., and van der Vliet, P.C. (1994) Crystal structure of the adenovirus DNA binding protein reveals a hook-on model for cooperative DNA binding. EMBO J 13, 2994-3002.

Tursz T, Cesne AL, Baldeyrou P, Gautier E, Opolon P, Schatz C, Pavirani A, Courtney M, Lamy D, Ragot T, Saulnier P, Andremont A, Monier R, Perricaudet M, Le Chevalier T (1996) Phase I study of a recombinant adenovirus-mediated gene transfer in lung cancer patients. J Natl Cancer Inst 88, 1857-1863.

Uchiyama et al, (1993) Transfection of interleukin 2 gene into human melanoma cells augments cellular immune response. Cancer Res 53, 949-952.

Ueda K, Cardarelli C, Gottesman MM and Pastan I (1987) Expression of a full-length cDNA for the human “MDR1” gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc. Natl. Acad. Sci. USA 84, 3004-3008.

Ueno H, Masuda S, Nishio S, Li JJ, Yamamoto H, Takeshita A (1997a) Adenovirus-mediated transfer of cyclin-dependent kinase inhibitor-p21 suppresses neointimal formation in the balloon-injured rat carotid arteries in vivo. Ann N Y Acad Sci 811, 401-411.

Ueno H, Yamamoto H, Ito S, Li JJ, Takeshita A (1997b) Adenovirus-mediated transfer of a dominant-negative H-ras suppresses neointimal formation in balloon-injured arteries in vivo. Arterioscler Thromb Vasc Biol 17, 898-904.

Ueno NT, Yu D, Hung MC (1997) Chemosensitization of HER-2/neu-overexpressing human breast cancer cells to paclitaxel (Taxol) by adenovirus type 5 E1A. Oncogene 15, 953-960.

Upadhyay S, Li G, Liu H, Chen YQ, Sarkar FH, Kim H-R (1995) bcl-2 suppresses expression of p21WAF1/CIP1 in breast epithelial cells. Cancer Res 55, 4520-4524.

Urashima M, Ogata A, Chauhan D, Vidriales MB, Teoh G, Hoshi Y, Schlossman RL, DeCaprio JA, Anderson KC (1996) Interleukin-6 promotes multiple myeloma cell growth via phosphorylation of retinoblastoma protein. Blood 88, 2219-2227.

Van Belle E, Tio FO, Chen D, Maillard L, Chen D, Kearney M, Isner JM (1997) Passivation of metallic stents after arterial gene transfer of phVEGF165 inhibits thrombus formation and intimal thickening. J Am Coll Cardiol 29, 1371-1379.

Van Beusechem VW, Valerio D (1996) Gene transfer into hematopoietic stem cells of nonhuman primates. Hum Gene Ther 7, 1649-1668.

van den Heuvel SJL, van Laar T, Kast WM, Melief CJM, Zantema A, van der Eb AJ (1990) Association between the cellular p53 and the adenovirus 5 E1B-55kd proteins reduces the oncogenicity of Ad-transformed cells. EMBO J 9, 2621-2629.

van Gent DC, Mizuuchi K, Gellert M (1996) Similarities between initiation of V(D)J recombination and retroviral integration. Science 271, 1592-1594.

Van Heek M, Compton DS, France CF, Tedesco RP, Fawzi AB, Graziano MP, Sybertz EJ, Strader CD, Davis HR Jr (1997) Diet-induced obese mice develop peripheral, but not central, resistance to leptin. J Clin Invest 99, 385-390.

Vanin EF, Cerruti L, Tran N, Grosveld G, Cunningham JM, Jane SM (1997) Development of high-titer retroviral producer cell lines by using Cre-mediated recombination. J Virol 71, 7820-7826.

Vassalli J-D, Saurat J-H (1996) Cuts and scrapes? Plasmin heals! Nature Med 2, 284-285.

Velculescu VE, Zhang L, Vogelstein B, Kinzler KW (1995) Serial analysis of gene expression. Science 270, 484-487.

Venkatesh LK, Arens MQ, Subramanian T, Chinnadurai G (1990) Selective induction of toxicity to human cells expressing human immunodeficiency virus type 1 Tat by a conditionally cytotoxic adenovirus vector. Proc Natl Acad Sci USA 87, 8746-8750.

Veres G, Escaich S, Baker J, Barske C, Kalfoglou C, Ilves H, Kaneshima H, Bohnlein E (1996) Intracellular expression of RNA transcripts complementary to the human immunodeficiency virus type 1 gag gene inhibits viral replication in human CD4⁺ lymphocytes. J Virol 70, 8792-8800.

Verrijzer CP, Kal AJ, and van der Vliet PC (1990) The DNA binding domain (POU domain) of the transcription factor Oct-1 suffices for stimulation of DNA replication. EMBO J. 9, 1883-1888.

Vierboom MP, Nijman HW, Offringa R, van der Voort EI, van Hall T, van den Broek L, Fleuren GJ, Kenemans P, Kast WM, Melief CJ (1997) Tumor eradication by wild-type p53-specific cytotoxic T lymphocytes. J Exp Med 186, 695-704.

Vieweg J, Boczkowski D, Roberson KM, Edwards DW, Philip M, Philip R, Rudoll T, Smith C, Robertson C, and Gilboa E (1995) Efficient gene transfer with adeno-associated virus-based plasmids complexed to cationic liposomes for gene therapy of human prostate cancer. Cancer Res 55, 2366-2372.

Vieweg J, Rosenthal F, Bannerji R, Heston W, Fair W, Gansbacher B, and Gilboa E (1994) Immunotherapy of prostate cancer in the Dunning rat model: Use of cytokine gene modified tumor vaccines. Cancer Res 54: 1760-1765.

Vikkula M, Boon LM, Carraway III KL, Calvert JT, Diamonti AJ, Goumnerov B, Pasyk KA, Marchuk DA, Warman ML, Cantley LC, Mulliken JB, and Olsen BR (1996) Vascular dysmorphogenesis caused by an activating mutation in the receptor tyrosine kinase TIE2. Cell 87, 1181-1190.

Vile RG and Russell SJ (1995) Retroviruses as vectors. Br Med Bull 51, 12-30.

Villarreal FJ, Lee AA, Dillmann WH, Giordano FJ (1996) Adenovirus-mediated overexpression of human transforming growth factor-b1 in rat cardiac fibroblasts, myocytes and smooth muscle cells. Mol Cell Cardiol 28, 735-742.

Vincent AJ, Esandi MC, Avezaat CJ, Vecht CJ, Sillevis Smitt P, van Bekkum DW, Valerio D, Hoogerbrugge PM, Bout A (1997) Preclinical testing of recombinant adenoviral herpes simplex virus-thymidine kinase gene therapy for central nervous system malignancies. Neurosurgery 41, 442-451.

Voeller HJ, Sugars LY, Pretlow T, and Gelmann EP (1994) p53 mutations in human prostate cancer specimens. J Urol 151, 492-495.

Vogelstein B (1990) A deadly inheritance. Nature 348, 681-682.

Vogelstein B and Kinzler K (1992) p53 function and dysfunction. Cell 70: 523-526.

von der Leyden HE, Gibbons GH, Morishita R, Lewis NP, Zhang L., Nakajima M, Kaneda Y, Cooke JP, and Dzau VJ (1995) Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene. Proc Natl Acad Sci USA 92, 1137-1141.

Wachtel SR, Bencsics C, Kang UJ (1997) Role of aromatic L-amino acid decarboxylase for dopamine replacement by genetically modified fibroblasts in a rat model of Parkinson's disease. Neurochem 69, 2055-2063.

Wagner AJ, Kokontis JM, and Hay N (1994) Myc-mediated apoptosis requires wild-type p53 in a mannert independent of cell cycle arrest and ability of p53 to induce p21waf1/cip1. Genes Dev 8, 2817-2830.

Wagner E, Cotten M, Foisner R, Birnstiel ML (1991) Transferrin-polycation-DNA complexes: The effect of polycations on the structure of the complex and DNA delivery to cells. Proc Natl Acad Sci USA 88, 4255-4259.

Wagner E, Plank C, Zatloukal K, Cotten M, and Birnstiel ML (1992a) Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: Toward a synthetic virus-like gene-transfer vehicle. Proc Natl Acad Sci USA 89, 7934-7938.

Wagner E, Zatloukal K, Cotten M, Kirlappos H, Mechtler K, Curiel DT, and Birnstiel ML (1992b) Coupling of adenovirus to tranferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes. Proc Natl Acad Sci USA 89, 6099-6103.

Walsh CE, Nienhuis AW, Samulski RJ, Brown MG, Miller JL, Young NS, and Liu JM (1994) Phenotypic correction of Fanconi anemia in human hematopoietic cells with a recombinant adeno-associated virus vector . J Clin Invest 94, 1440-1448.

Wang B, Ugen KE, Srikantan V, Agadjanyan MG, Dang K, Refaeli Y, Sato AI, Boyer J, Williams WV, Weiner DB (1993) Gene inoculation generates immune responses against human immunodeficiency virus type 1. Proc Natl Acad Sci USA 90, 4156-4160.

Wang C-Y and Huang L (1987) pH-sensitive immunoliposomes mediate target-cell-specific delivery and controlled expression of a foreign gene in mouse. Proc Natl Acad Sci USA 84, 7851-7855.

Wang EH, Friedman PN, Prives C (1989) The murine p53 protein blocks replication of SV40 DNA in vitro by inhibiting the initiation functions of SV40 large T antigen. Cell 57, 379-392.

Wang J, Kim H-H, Yuan X, and Herrin DL (1997) Purification, biochemical characterization and protein-DNA interactions of the I-CreI endonuclease produced in Escherichia coli. Nucleic Acids Res 25, 3767-3776.

Wang JM, Zheng H, Blaivas M, Kurachi K (1997) Persistent systemic production of human factor IX in mice by skeletal myoblast-mediated gene transfer: feasibility of repeat application to obtain therapeutic levels. Blood 90, 1075-1082.

Wang L, Zoppe M, Hackeng TM, Griffin JH, Lee KF, Verma IM (1997) A factor IX-deficient mouse model for hemophilia B gene therapy. Proc Natl Acad Sci USA 94, 11563-11566.

Wang NP, To H, Lee WH, Lee EY (1993) Tumor suppressor activity of RB and p53 genes in human breast carcinoma cells. Oncogene 8, 279-288.

Wang S and Vos J-M (1996) A hybrid herpesvirus infectious vector based on Epstein-Barr virus and Herpes Simplex virus type 1 for gene transfer into human cells in vitro and in vivo. J Virol 70, 8422-8430.

Wang X, Zelenski NG, Yang J, Sakai J, Brown MS, Goldstein JL (1996) Cleavage of sterol regulatory element binding proteins (SREBPs) by CPP32 during apoptosis. EMBO J 15, 1012-1020.

Wang Y and Becker D (1997) Antisense targeting of basic fibroblast growth factor and fibroblast growth factor receptor-1 in human melanomas blocks intratumoral angiogenesis and tumor growth. Nat Med 3, 887-893.

Wang Y, O'Malley BW Jr, Tsai SY, O'Malley BW (1994) A regulatory system for use in gene transfer. Proc Natl Acad Sci USA 91, 8180-8184.

Wang Z-Q, Auer B, Sting L, Berghammer H, Haidacher D, Schweiher M, and Wagner EF (1995) Mice lacking ADPRT and poly(ADP-ribosyl)ation develop normally but are susceptible to skin disease. Genes Dev 9, 509-520.

Ward M, Richardson C, Piuoli P, Smith L, Podda S, Goff S, Hesdorffer C, and Bank A (1994) Transfer and expression of the human multiple drug resistance gene in human CD34⁺ cells. Blood 84, 1408-1414.

Wattiaux R, Jadot M, Warnier-Pirotte MT, Wattiaux-De Coninck S (1997) Cationic lipids destabilize lysosomal membrane in vitro. FEBS Lett 417, 199-202.

Webb A, Cunningham D, Cotter F, Clarke PA, di Stefano F, Ross P, Corbo M, Dziewanowska Z (1997) BCL-2 antisense therapy in patients with non-Hodgkin lymphoma. Lancet 349, 1137-1141.

Webster MK and Donoghue DJ (1998) Constitutive activation of fibroblast growth factor receptors in human developmental syndromes. Gene Ther Mol Biol 1, 365-379.

Weih F, Ryseck R-P, Chen L, and Bravo R (1996) Apoptosis of nur77/N10-transgenic thymocytes involves the Fas/Fas ligand pathway. Proc Natl Acad Sci USA 93, 5533-5538.

Weir L, Chen D, Pastore C, Isner JM, Walsh K (1995) Expression of gax, a growth arrest homeobox gene, is rapidly down-regulated in the rat carotid artery during the proliferative response to balloon injury. J Biol Chem 270, 5457-5461.

Weiss DJ, Liggitt D, Clark JG (1997) In situ histochemical detection of b-galactosidase activity in lung: assessment of X-Gal reagent in distinguishing lacZ gene expression and endogenous b-galactosidase activity. Hum Gene Ther 8, 1545-1554.

Weitzman MD, Fisher KJ, Wilson JM (1996) Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers. J Virol 70, 1845-1854.

Werness BA, Levine AJ, Howley PM (1990) Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248, 76-79.

Westerman KA, Leboulch P (1996) Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination. Proc Natl Acad Sci USA 93, 8971-8976.

White EM, Allis CD, Goldfarb DS, Srivastva A, Weir JW, and Gorovsky MA: Nucleus-specific and temporally restricted localization of proteins in Tetrahymena macronuclei and micronuclei. J Cell Biol 109: 1983-1992, 1989.

White RJ (1998) Control of growth and proliferation by the retinoblastoma protein. Gene Therapy Mol Biol 1, 613-628.

White, E. (1993) Death-defying acts: a meeting review on apoptosis. Genes Dev. 7, 2277-2284.

Whyte, P., Buchkovich, K.J., Horowitz, J.M., Friend, S.H., Raybuck, M., Weinberg, R.A. and Harlow, E. (1988) Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature 334, 124-129.

Wieder R, Wang H, Shirke S, Wang Q, Menzel T, Feirt N, Jakubowski AA, Gabrilove JL (1997) Low level expression of basic FGF upregulates Bcl-2 and delays apoptosis, but high intracellular levels are required to induce transformation in NIH 3T3 cells. Growth Factors 15, 41-60.

Wilcock D and Lane DP (1991) Localization of p53, retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells. Nature 349, 429-431.

Will K and Deppert W (1998) Analysis of mutant p53 for MAR-DNA binding: determining the dominant-oncogenic function of mutant p53. Gene Ther Mol Biol Vol 1, 543-549.

Will K, Warnecke G, Albrechtsen N, Boulikas T, and Deppert W (1998) High affinity MAR/SAR-binding is a common property of murine and human mutant p53. J Cell Biochem In press

Williams SA, Chang L, Buzby JS, Suen Y, Cairo MS (1996) Cationic lipids reduce time and dose of c-myc antisense oligodeoxynucleotides required to specifically inhibit Burkitt's lymphoma cell growth. Leukemia 10, 1980-1989.

Wills KN, Maneval DC, Menzel P, Harris MP, Sutjipto S, Vaillancourt M-T, Huang W-M, Johnson DE, Anderson SC, Wen SF, Bookstein R, Shepard HM, Gregory RJ (1994) Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancer. Hum Gene Ther 5, 1079-1088.

Wilson C, Bellen HJ and Gehring WJ (1990) Position effects on eukaryotic gene expression. Annu Rev Cell Biol 6, 679-714.

Wilson JM (1995) Gene therapy for cystic fibrosis: challenges and future directions. J Clin Invest 96, 2547-2554.

Wilson JM, Danos O, Grossman M, Raulet DH, Mulligan RC (1990) Expression of human adenosine deaminase inmice reconstituted with retrovirus-transduced hematopoietic stem cells. Proc Natl Acad Sci USA 87, 439-443.

Wilson JM, Grossman M, Raper SE, Baker JR, Newton RS, and Thoene JG (1992) Clinical protocol. Ex vivo gene therapy of familial hypercholesterolemia. Hum Gene Ther 3, 179-222.

Wiltrout RH, Gregorio TA, Fenton RG, Longo DL, Ghosh P, Murphy WJ, Komschlies KL (1995) Cellular and molecular studies in the treatment of murine renal cancer. Semin Oncol 22, 9-16.

Witte ON and Baltimore D (1977) Mechanism of formation of pseudotypes between vesicular stomatitis virus and murine leukemia virus. Cell 11, 505-511.

Wolfe JH, Sands MS, Barker JE, Gwynn B, Rowe LB, Vogler CA, and Birkenmeier EH (1992) Reversal of pathology in murine mucopolysaccharidosis type VII by somatic cell gene transfer. Nature 360, 749-753.

Wolff JA, Fisher LJ, Xu L, Jinnah HA, Langlais PJ, Iuvone PM, O'Malley KL, Rosenberg MB, Shimohama S, Friedmann T, Gage FH (1989) Grafting fibroblasts genetically modified to produce L-dopa in a rat model of Parkinson disease. Proc Natl Acad Sci USA 86, 9011-9014.

Wu P, de Fiebre CM, Millard WJ, King MA, Wang S, Bryant SO, Gao YP, Martin EJ, Meyer EM (1996) An AAV promoter-driven neuropeptide Y gene delivery system using Sendai virosomes for neurons and rat brain. Gene Ther 3, 246-253.

Wu X and Levine AJ (1994) p53 and E2F-1 cooperate to mediate apoptosis. Proc Natl Acad Sci USA 91, 3602-3606.

Wyman TB, Nicol F, Zelphati O, Scaria PV, Plank C, Szoka FC Jr (1997) Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry 36, 3008-3017.

Xiao H, Pearson A, Coulombe B, Truant R, Zhang S, Regier JL, Triezenberg SJ, Reinberg D, Flores O, Ingles CJ, Greenblatt J (1994) Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53. Mol Cell Biol 14, 7013-7024.

Xiao X, Xiao W, Li J, Samulski RJ (1997) A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle. J Virol 71, 941-948.

Xing X, Liu V, Xia W, Stephens LC, Huang L, Lopez-Berestein G, Hung MC (1997) Safety studies of the intraperitoneal injection of E1A--liposome complex in mice. Gene Ther 4, 238-243.

Xing Z, Tremblay GM, Sime PJ, Gauldie J (1997) Overexpression of granulocyte-macrophage colony-stimulating factor induces pulmonary granulation tissue formation and fibrosis by induction of transforming growth factor-b1 and myofibroblast accumulation. Am J Pathol 150, 59-66.

Yamamoto R, Murakami K, Taira K, and Kumar PKR (1998) Isolation and characterization of an RNA that binds with high affinity to Tat protein of HIV-1 from a completely random pool of RNA. Gene Ther Mol Biol 1, 451-466.

Yamamoto S, Suzuki S, Hoshino A, Akimoto M, Shimada T (1997) Herpes simplex virus thymidine kinase/ganciclovir-mediated killing of tumor cell induces tumor-specific cytotoxic T cells in mice. Cancer Gene Ther 4, 91-96.

Yamanaka S, Balestra ME, Ferrell LD, Fan J, Arnold KS, Taylor S, Taylor JM, Innerarity TL (1995) Apolipoprotein B mRNA-editing protein induces hepatocellular carcinoma and dysplasia in transgenic animals. Proc Natl Acad Sci USA 92, 8483-8487.

Yang AG, Bai X, Huang XF, Yao C, Chen S (1997) Phenotypic knockout of HIV type 1 chemokine coreceptor CCR-5 by intrakines as potential therapeutic approach for HIV-1 infection. Proc Natl Acad Sci USA 94, 11567-11572.

Yang C, Cirielli C, Capogrossi MC, and Passaniti A (1995) Adenovirus-mediated wild-type p53 expression induces apoptosis and suppresses tumorigenesis of prostatic tumor cells. Cancer Res 55, 4210-4213.

Yang CC, Xiao X, Zhu X, Ansardi DC, Epstein ND, Frey MR, Matera AG, Samulski RJ (1997) Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro. J Virol 71, 9231-9247.

Yang JP and Huang L (1997) Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA. Gene Ther 4, 950-960.

Yang Y, Li Q, Ertl HC, and Wilson JM (1995) Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol 69, 2004-2015.

Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, and Wilson JM (1994a) Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci USA 91, 4407-4411.

Yang Y, Nunes FA, Berencsi K, Gonczol E, Engelhardt JF, Wilson JM (1994b) Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat Genet 7, 362-369.

Yang Y, Vanin EF, Whitt MA, Fornerod M, Zwart R, Schneiderman RD, Grosveld G, Nienhuis AW (1995) Inducible, high-level production of infectious murine leukemia retroviral vector particles pseudotyped with vesicular stomatitis virus G envelope protein. Hum Gene Ther 6, 1203-1213.

Yang Z-Y, Perkins ND, Ohno T, Nabel EG, Nabel GJ (1995) The p21 cyclin-dependent kinase inhibitor suppresses tumorigenicity in vivo. Nature Med 1, 1052-1056.

Yang ZY, Simari RD, Perkins ND, San H, Gordon D, Nabel GJ, Nabel EG (1996) Role of the p21 cyclin-dependent kinase inhibitor in limiting intimal cell proliferation in response to arterial injury. Proc Natl Acad Sci USA 93, 7905-7910.

Yanofsky SD, Baldwin DN, Butler JH, Holden FR, Jacobs JW, Balasubramanian P, Chinn JP, Cwirla SE, Peters-Bhatt E, Whitehorn EA, Tate EH, Akeson A, Bowlin TL, Dower WJ, Barrett RW (1996) High affinity type I interleukin 1 receptor antagonists discovered by screening recombinant peptide libraries. Proc Natl Acad Sci USA 93, 7381-7386.

Yao SN, Smith KJ, Kurachi K (1994) Primary myoblast-mediated gene transfer: persistent expression of human factor IX in mice. Gene Ther 1, 99-107.

Yee JK, Miyanohara A, LaPorte P, Bouic K, Burns JC, Friedmann T (1994) A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes. Proc Natl Acad Sci USA 91, 9564-9568.

Yew NS, Wysokenski DM, Wang KX, Ziegler RJ, Marshall J, McNeilly D, Cherry M, Osburn W, Cheng SH (1997) Optimization of plasmid vectors for high-level expression in lung epithelial cells. Hum Gene Ther 8, 575-584.

Yew PR and Berk AJ (1992) Inhibition of p53 transactivation required for transformation by adenovirus early 1B protein. Nature 357, 82-85.

Ylä-Herttuala S (1996) Gene therapy for cardiovascular diseases. Ann Med 28, 89-93.

Yokoyama Y, Takahashi Y, Morishita S, Hashimoto M, Tamaya T (1997) Introduction of p21(Waf1/Cip1) gene into a carcinoma cell line of the uterine cervix with inactivated p53. Cancer Lett 116, 233-239.

Yonish-Rouach E, Grunwald D, Wilder S, Kimchi A, May E, Lawrence J-J, May P, and Oren M (1993) p53-mediated cell death: relationship to cell cycle control. Mol Cell Biol 13, 1415-1423.

Yoshimura K, Rosenfeld MA, Nakamura H, Scherer EM, Pavirani A, Lecocq J-P, Crystal RG (1992) Expression of the human cystic fibrosis transmembrane conductance regulator gene in the mouse lung after in vivo intratracheal plasmid-mediated gene transfer. Nucleic Acids Res 30, 3233-3240.

Youdim MBH and Riederer P (1997) Understanding Parkinson’s disease. Scientific Am. Jan 1997, 52-59.

Yovandich J, O'Malley Jr B, Sikes M, Ledley FD (1995) Gene transfer to synovial cells by intra-articular administration of plasmid DNA. Hum Gene Ther 6, 603-610.

Yu D, Matin A, Xia W, Sorgi F, Huang L, Hung MC (1995) Liposome-mediated in vivo E1A gene transfer suppressed dissemination of ovarian cancer cells that overexpress HER-2/neu. Oncogene 11, 1383-1388.

Yuan J, Shaham S, Ledoux S, Ellis HM, and Horvitz HR (1993) The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1b-converting enzyme. Cell 75, 641-652.

Yung WK (1994) New approaches to molecular therapy of brain tumors. Curr Opin Neurol 7, 501-505.

Zabner J, Cheng SH, Meeker D, Launspach J, Balfour R, Perricone MA, Morris JE, Marshall J, Fasbender A, Smith AE, Welsh MJ (1997) Comparison of DNA-lipid complexes and DNA alone for gene transfer to cystic fibrosis airway epithelia in vivo. J Clin Invest 100, 1529-1537.

Zacksenhaus E, Gill RM, Phillips RA, Gallie BL (1993) Molecular cloning and characterization of the mouse RB1 promoter. Oncogene 8, 2343-2351.

Zaitsev SV, Haberland A, Otto A, Vorob'ev VI, Haller H, Bottger M (1997) H1 and HMG17 extracted from calf thymus nuclei are efficient DNA carriers in gene transfer. Gene Ther 4, 586-592.

Zakrzewska KE, Cusin I, Sainsbury A, Rohner-Jeanrenaud F, Jeanrenaud B (1997) Glucocorticoids as counterregulatory hormones of leptin: toward an understanding of leptin resistance. Diabetes 46, 717-719.

Zambetti GP and Levine AJ (1993) A comparison of the biological activities of wild-type and mutant p53. FASEB J 7, 855-865.

Zambetti GP, Bargonetti J, Walker K, Prives C, Levine AJ (1992) Wild-type p53 mediates positive regulation of gene expression through a specific DNA sequence element. Genes Dev 6, 1143-1152.

Zardo G, Marenzi S and Caiafa P (1998) Correlation between DNA methylation and poly(ADP-ribosyl)ation processes. Gene Ther Mol Biol 1, 661-679.

Zaretsky JZ, Candotti F, Boerkoel C, Adams EM, Yewdell JW, Blaese RM, Plotz PH (1997) Retroviral transfer of acid a-glucosidase cDNA to enzyme-deficient myoblasts results in phenotypic spread of the genotypic correction by both secretion and fusion. Hum Gene Ther 8, 1555-1563.

Zastawny RL, Salvino R, Chen J, Benchimol S, Ling V (1993) The core promoter region of the P-glycoprotein gene is sufficient to confer differential responsiveness to wild-type and mutant p53. Oncogene 8, 1529-1535.

Zatloukal K, Cotten M, Berger M, Schimdt W, Wagner E, Birnstiel ML (1994) In vivo production of human factor VIII in mice after intrasplenic implantation of primary fibroblasts transfected by receptor-mediated, adenovirus-augmented gene delivery. Proc Natl Acad Sci USA 91, 5148-5152.

Zelphati O, Szoka FC Jr (1997) Intracellular distribution and mechanism of delivery of oligonucleotides mediated by cationic lipids. Pharm Res 13, 1367-1372.

Zennaro MC, Farman N, Bonvalet JP, Lombes M (1997) Tissue-specific expression of a and b messenger ribonucleic acid isoforms of the human mineralocorticoid receptor in normal and pathological states. J Clin Endocrinol Metab 82, 1345-1352.

Zhang H, Yang Y, Horton JL, Samoilova EB, Judge TA, Turka LA, Wilson JM, Chen Y (1997) Amelioration of collagen-induced arthritis by CD95 (Apo-1/Fas)-ligand gene transfer. J Clin Invest 100, 1951-1957.

Zhang JF, Hu C, Geng Y, Blatt LM, Taylor MW (1996) Gene therapy with an adeno-associated virus carrying an interferon gene results in tumor growth suppression and regression. Cancer Gene Ther 3, 31-38.

Zhang LX, Wu M, Han JS (1992) Suppression of audiogenic epileptic seizures by intracerebral injection of a CCK gene vector. Neuroreport 3, 700-702.

Zhang W-W, Alemany R, Wang J, Koch PE, Ordonez NG, Roth JA (1995) Safety evaluation of Ad5CMV-p53 in vitro and in vivo. Hum Gene Ther 6, 155-164.

Zhang WW, Fang X, Mazur W, French BA, Georges RN, Roth JA (1994) High-efficiency gene transfer and high-level expression of wild-type p53 in human lung cancer cells mediated by recombinant adenovirus. Cancer Gene Ther 1, 5-13.

Zhang XY, Ni YS, Saifudeen Z, Asiedu CK, Supakar PC, Ehrlich M (1995) Increasing binding of a transcription factor immediately downstream of the cap site of a cytomegalovirus gene represses expression. Nucleic Acids Res 23, 3026-3033.

Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM (1994) Positional cloning of the mouse obese gene and its human homologue. Nature 372, 425-432.

Zhao JE, Lochumuller H, Nalbantoglu J, Allen C, Prescott S, Massie B, Karpati G (1997) Study of adenovirus-mediated dystrophin minigene transfer to skeletal muscle by combined microscopic display of adenoviral DNA and dystrophin. Hum Gene Ther 8, 1565-1573.

Zhao S-C, Banerjee D, Mineishi S, and Bertino JR (1997) Post-transplant methotrexate administration leads to improved curability of mice bearing a mammary tumor transplanted with marrow transduced with a mutant human dihydrofolate reductase cDNA. Hum Gene Ther 8, 903-909.

Zhonghe, Z., Nickerson, J.A., Krochmalnic, G., and Penman, S. (1987) Alterations in nuclear matrix structure after adenovirus infection. J Virology 61, 1007-1018.

Zhou H, Zeng G, Zhu X, Tang J, Chen G, Huang Q, Peng T, Hu B (1995) Enhanced adeno-associated virus vector expression by adenovirus protein-cationic liposome complex. A novel and high efficient way to introduce foreign DNA into endothelial cells. Chin Med J (Engl) 108, 332-337.

Zhou P, Goldstein S, Devadas K, Tewari D, Notkins AL (1997) Human CD4⁺ cells transfected with IL-16 cDNA are resistant to HIV-1 infection: inhibition of mRNA expression. Nat Med 3, 659-664.

Zhou SZ, Li Q, Stamatoyannopoulos G, Srivastava A (1996) Adeno-associated virus 2-mediated transduction and erythroid cell-specific expression of a human b-globin gene. Gene Ther 3, 223-229.

Zhu C, Bogue MA, Lim D-S, Hasty P, Roth DB (1996) Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell 86, 379-389.

Zhu N, Liggitt D, Liu Y, and Debs R (1993) Systemic gene expression after intravenous DNA delivery into adult mice. Science 261, 209-211.

Zhu NL, Wu L, Liu PX, Gordon EM, Anderson WF, Starnes VA, Hall FL (1997) Downregulation of cyclin G1 expression by retrovirus-mediated antisense gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation. Circulation 96, 628-635.

Zhu Y, Carroll M, Papa FR, Hochstrasser M, and D'Andrea AD (1996) DUB-1, a deubiquitinating enzyme with growth-suppressing activity. Proc Natl Acad Sci USA 93, 3275-3279.

Ziegler A, Jonason AS, Leffell DJ, Simon JA, Sharma HW, Kimmelman J, Remington L, Jacks T, and Brash DE (1994) Sunburn and p53 in the onset of skin cancer. Nature 372, 773-776.

Zou Z, Anisowicz A, Hendrix MJC, Thor A, Neveu M, SHeng S, Rafidi K, Seftor E, Sager R (1994) Maspin, a serpin with tumor-suppressing acivity in human mammary epithelial cells. Science 263, 526-529.

Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D (1997) Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15, 871-875.

Zuidam NJ, Barenholz Y (1997) Electrostatic parameters of cationic liposomes commonly used for gene delivery as determined by 4-heptadecyl-7-hydroxycoumarin. Biochim Biophys Acta 1329, 211-222.

Gene deliv. system	Advantages	Drawbacks
Murine retroviral vectors	Very safe; may achieve high efficiency of transduction; infects only dividing cells; integrates into host DNA.	Loss in expression soon after infection; low efficiency in vivo; up to 8 kb of DNA; high titers required for in vivo gene delivery; immunogenicity.
Recombinant adenoviruses	Infect nondividing cells; rarity of recombination events between adenoviral vectors and the host chromosomes; high efficiency of transduction; adenovirus vectors efficiently escape from the endosome and enter the nucleus; episomaly-replicating virus.	Induction of immune responses that eliminates therapeutic cells; may induce unwanted infections to humans; only up to 7.5 kb of exogenous DNA can be inserted; loss of adenoviral episomes in progeny cells.
AAV	Does not stimulate inflammation or immune reaction; enters nondividing cells and does not replicate; nonpathogenic virus.	Low efficiency of gene transfer; only up to 4.1 and 4.9 kb can be incorporated; wt AAV integrates on chromosome 19 but recombinant AAV integrates at different sites (e.g. chromosome 2); integration may cause inactivation of the transgene by chromatin effects.
HSV-1	Can take up to 30 kb of exogenous DNA; high titer viral stocks; wide range; can infect nonreplicating cells	Infection with HSV-1 is cytotoxic.
Baculovirus	Specificity for hepatocytes; high efficiency of infection	Not applicable in vivo at present.
EBV	Episomaly-replicating virus	wt EBV infects human cells causing mononucleosis.
HIV-1	Transduces non-dividing cells; broad range of tissues and cell types; no inflammation; sustains expression of GFP for 8-22 weeks in muscle and liver after injection to animals	Start up technology, not broadly tested.
Hybrid HSV/EBV	High efficiency of infection (95-99% after intratumoral liver injection)	Not broadly tested.
Cationic lipids	High efficiency of transfection via membrane destabilization (cell membrane and endosomal); destabilize lysosomal membranes and promote release of plasmid in the cytoplasm.	Toxic, not suited for i.v. injection; can interact with negatively charged serum proteins in vivo causing transgene inactivation; gene expression is transient; i.v. injection targets mainly the lung
Stealth liposomes	Non toxic, escape immune surveillance and concentrate into solid tumors by extravasation.	Not taken up by tumor cells but remain in the extracellular space.
Naked plasmid DNA	Suited for intramuscular injection and DNA vaccination; easy to use; no viral antigens.	Low transfection; not widely applicable method; naked plasmid is cleared from blood rapidly.
Gene gun	Easy to use (plasmid-coated gold particles are delivered to tumor cells using helium pressure); rapid, suited for gene transfer to tumor specimens from patients for immunotherapy.	Not broadly tested.

Gene delivered	Human disease	Vector	Method/Goal	Results	Reference
CAT and luciferase	none	DOTMA: DOPE	Expression of reporter genes in mouse tissues after a single intravenous injection.	CMV promoter was most effective; expression in vascular endothelia, extravascular and parenchymal cells present in lung, spleen, and heart; lower expression in all other tissues; persisted for 9 weeks.	Zhu et al, 1993
Luciferase/CMV construct and lacZ	none	Transfectam (DOGS:DOPE)	To show effectiveness in gene transfer after intracranial injection of liposome-plasmid complexes into the newborn mouse.	Transient expression of luciferase in striatal parenchymal cells; lipospermine:DNA charge ratio of 2 or smaller was most effective in vivo.	Schwartz et al, 1995
Luciferase/CMV	none	Cationic liposomes	Distribution of luciferase gene expression among tissues and persistence of expression after systemic injection.	Use of viral origins of replication and T antigen cDNA in vector sustained expression up to 3 months primarily in the lung in mice.	Thierry et al, 1995
lacZ and HSV-tk/IL-2	glioma	AAV	Direct injection of tumors induced from human glioma cells into the brains of nude mice.	30-40% of the cells along the needle track expressed b-galactosidase; administration of GCV to the HSV-tk/IL-2 treated animals for 6 days, resulted in a 35-fold reduction in the mean volume of tumors compared with controls.	Okada et al,. 1996
lacZ and human erythropoietin	none	AAV	Single intramuscular injection into adult BALB/c mice.	Protein expression was detected in myofibers for at least 32 weeks; dose-dependent secretion of erythropoietin and corresponding increases in red blood cell production in mice persisted for up to 40 weeks.	Kessler et al, 1996
lacZ	Retinal degeneration; retinitis pigmentosa	AAV	Subretinal injection of recombinant AAV particles encoding lacZ.	Successful transduction of all layers of the neuroretina as well as the retinal pigment epithelium; the efficiency of transduction of photoreceptors was significantly higher than that achieved with an equivalent adenoviral vector.	Ali et al, 1996
lacZ	Inherited hearing disorders	AAV	To assess the feasibility of introducing genetic material directly into the peripheral auditory system; infusion into the cochlea of guinea pigs.	Thin sections of cochleae showed intense immunohistochemical reactivity in the spiral limbus, spiral ligament, spiral ganglion cells and the organ of Corti but much weaker staining in the contralateral ear.	Lalwani et al, 1996
lacZ	Parkinson’s disease	HSV-1	Stereotactic injection into the midbrain of adult rats.	A 6.8-kb fragment of the rat tyrosine hydroxylase promoter supported a 7- to 20-fold increase in reporter gene expression in catecholaminergic tyrosine hydroxylase-expressing neurons in the substantia nigra.	Song et al, 1997
lacZ	Leptomeningeal cancer	AAV	To test the feasibility of AAV-mediated gene therapy.	Successful transduction of medulloblastoma (DAOY) cells in a nude rat model of leptomeningeal disease.	Rosenfeld et al, 1997
lacZ	vascular disorders	AAV	To develop gene therapies for vascular disorders by gene transfer into isolated segments of normal and balloon-injured rat carotid arteries.	Comparable gene transfer into medial and adventitial cells, with significantly higher efficiency of transduction in injured compared with normal vessels.	Rolling et al, 1997
human placental alkaline phosphatase (AP)	lung disease	AAV	To assess the ability of AAV vectors to transduce airway cells; AP gene was delivered to one lobe of the rabbit lung using a balloon catheter.	AP staining was almost exclusively in the epithelial and smooth muscle cells in the bronchus at the region of balloon placement; staining was in ciliated cells but was also in basal cells and airway smooth muscle cells.	Halbert et al, 1997

Prodrug-activating enzyme	Prodrug	Toxic substance it is converted to
Thymidine kinase from HSV	9-{[2-hydroxy-1-(hydroxymethyl)-ethoxy]methyl}guanine or ganciclovir (GCV)	GCV monophosphate
Cytosine deaminase (CD) from E. coli	5-fluorocytosine (5FC)	5-fluorouracil (5FU)
Purine nucleoside phosphorylase (PNP) from E. coli	6-methylpurine-2’-deoxyriboside (MeP-dR)	6-methylpurine (a very toxic adenine analog)
Purine nucleoside phosphorylase (PNP) from E. coli	Arabinofuranosyl-2-fluoroadenine monophosphate (F-araAMP) commercially known as fludarabine	A very toxic adenine analog
Human deoxycytidine kinase (dCK)	Cytosine arabinoside (ara-C)	A toxic drug inducing lethal strand breaks in DNA
Nitroreductase from E. coli	5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)	A potent dysfunctional alkylating agent which crosslinks DNA

Drug resistance gene	Confers resistance to	Reference
MDR1 (multidrug resistance)	Daunomycin, doxorubicin, taxol	Galski et al, 1989; Podda et al, 1992; Sorrentino et al, 1992 (see below)
Mutant dihydrofolate reductase	Methotrexate (MTX)	Williams et al, 1987; Corey et al, 1990; Li et al, 1994; Zhao et al, 1997
Glutathione transferase	DNA alkylating agents	reviewed by Maze et al, 1997
O⁶-methyl guanine transferase	Nitrosoureas	Allay et al, 1995
Cytidine deaminase	Cytosine arabinoside (Ara-C)	Momparler et al, 1996
Aldehyde dehydrogenase	Cyclophosphamide	reviewed by Koc et al, 1996

Gene target	Human disease	Method	Animal model, objective, and method	Results	Reference
ADA	SCID (severe combined immunodeficiency)	Retrovirus	Immunodeficiient mice were injected with peripheral blood lymphocytes from ADA- patients transduced with a retroviral vector for human ADA	Restoration of immune functions (presence of human immunoglobulin and antigen-specific T cells)	Ferrari et al, 1991
bcl-2	prostate cancer		bcl-2 expressing LNCaP human prostate cancer cells are rendered highly resistant to apoptotic stimuli	LNCaP-bcl-2 cells induced earlier, larger, and hormone-refractory prostate tumors in nude mice	Rafo et al, 1995
Factor IX	hemophilia B		Injection of transduced primary myoblasts into the muscle	Factor IX was being synthesized and delivered to the circulation for over 6 months	Dai et al, 1992
Factor IX	hemophilia B	retr	Transplantation of retrovirus-transduced keratinocytes	Human factor IX was detected in the bloodstream of nude mice in small quantities for one week	Gerrard et al, 1993,
Factor IX	hemophilia B	retrovirus	Mouse primary myoblasts were infected with retrovirus expressing the canine factor IX under control of mouse muscle creatine kinase and human CMV promoter; myoblasts were injected into the hindlegs of recipient mice; secreted canine factor IX was monitored in the plasma	Sustained expression of factor IX for over six months without any apparent adverse effects on the recipient mice; however, the levels of the factor IX protein secreted into the plasma (10 ng/ml for 107 injected cells) were not sufficient to be of therapeutic value; 100 times higher amounts of factor IX were needed	Dai et al, 1992; Yao et al, 1994
Factor VIII	Hemophilia A	transferrin	Transfection of fibroblasts and myoblasts with B-domain-deleted factor VIII gene followed by implantation into mice	Therapeutic levels of factor VIII in the blood of the animals for 24 hours	Zatloukal et al, 1994
Factor VIII	Hemophilia A	Retrovirus	Mouse primary fibroblasts infected with a recombinant retrovirus containing factor VIII gene deleted at the B domain	Therapeutic levels of factor VIII in blood of animals for 1 week after surgical implantation into the peritoneal cavity in SCID mice of 15 million cells in the form of neo-organs	Dwarki et al, 1995
Growth hormone (human)	none	mice electroporation	Ex vivo modified C2C12 cells with the hGH gene under control of the inducible UAS promoter and a synthetic hybrid steroid receptor (TAXI), activating transcription from the inducible promoter after treatment with the synthetic nontoxic drug inducer RU486; transplanted in mouse muscle	This model allows up to 100-fold induction of the hGH gene and can be finely tuned to lower levels of induction	Delort and Capecchi, 1996
Growth hormone (human, hGH)	general	retr	Injection of genetically engineered myoblasts into mouse muscle	hGH could be detected in serum for 3 months; myoblasts were fused into preexisting multinucleated myofibers that were vascularized and innervated	Dhawan et al, 1991
HSV-tk	glioma	Retr	To directly transfer HSV TK gene and kill transduced proliferating brain tumor cells with ganciclovir without affecting nondividing normal cells	Murine fibroblasts transduced ex vivo with HSV TK retroviral vectors caused complete regression of gliomas in rat brain after intratumor injection	Culver et al, 1992
HSV-tk	pancreatic cancer	retrovirus	BXPC3 primary human pancreatic adenocarcinoma cells were transduced with retroviral vector carrying the HSV-tk gene driven by the CEA promoter; engrafted subcutaneously into nude mice eliciting pancreatic tumors	Animals treated with 0.1 mg/Kg ganciclovir exhibited a significant reduction in tumor growth	DiMaio et al, 1994
HSV-tk	proliferative vitreoretinopathy (PVR)	retrovirus-transduced rabbit dermal fibroblasts	Traction retinal detachment results from proliferation of retinal pigment cells in the vitreous cavity of the eye; PVR may ensue after retinal surgery or trauma and can be induced in rabbit models by surgical vitrectomy.	Significant inhibition of PVR (killing of proliferating cells in the retina) was observed in rabbit PVR models after injection into the vitreous cavity of rabbit dermal fibroblasts transduced in vitro; all eyes received 0.2 mg GCV on the following day and on day 4;	Kimura et al, 1996
IL-1 -receptor antagonist protein gene	Rheumatoid arthritis (RA)	Retr	Synovial cells were surgically removed from joints of animals with experimental arthritis, cultured, transduced with the IL-1 -receptor antagonist protein gene and reimplanted into the respective donors by intraarticular injection	Improvement in RA symptoms	Bandara et al, 1993
IL-1 receptor antagonist (IL-1Ra)	Rheumatoid arthritis (RA)	Retr	Degradation of cartilage in RA is stimulated by IL-1; to inhibit IL-1; RA synovial fibroblasts transfected with the IL-1Ra gene were coimplanted with normal human cartilage in SCID mice	IL-1Ra expression protected the cartilage from chondrocyte-mediated degradation.	Otani et al, 1996; Makarov et al, 1996; Muller-Ladner et al, 1997b
IL-2; GM-CSF	prostate cancer		Dunning rat R3327-MatLyLu prostate tumor model (an anplastic androgen-dependent, nonimmunogenic tumor that metastasizes to the lymph nodes and the lung); cytokine (IL-2)-secreting human tumor cell preparations (tumor vaccines) were used for the treatment of advanced human prostate cancer in rats	All animals with subcutaneously established tumors were cured; the cancer vaccine induced immunological memory that protected the animals from subsequent tumor challenge; GM-CSF was less effective than IL-2.	Vieweg et al, 1994
LDL receptor	Familial hypercholesterolemia (FH)	Retr	Watanabe heritable hyperlipidemic rabbit (deficient in both alleles of LDL receptor gene); establish hepatocyte culture from animal liver; transduce with LDL receptor gene responsible for LDL internalization into hepatocytes to reduce blood serum cholesterol; transplant hepatocytes into the animal	30-40% decrease in serum cholesterol that persisted for 4 months	Chowdhury et al, 1991
Nerve Growth Factor (NGF)	Alzheimer's disease	rat and primate	Delivery of NGF by ex vivo-modified allogeneic cells surrounded by a semipermeable membrane and implanted intrathecally	Release of NGF by the implant which is not subject to immunologic rejection due to the membrane	Kordower et al, 1994
XPD (ERCC2)	xeroderma pigmentosum (XP)	Retr	To transduce ex vivo human keratinocytes and produce skin grafts on immunodeficient mice; use it on XP patients as reconstructive surgery to alleviate cancers in UV-exposed areas	The retroviral vector carrying the XPD gene and neoR under control of SV40 enhancer fully complemented the DNA repair deficiency of primary skin fibroblasts	Gözükara et al, 1994; Carreau et al, 1995
TH (Tyrosine hydroxylase)	Parkinson's disease (PD)	Retrovirus	Rat fibroblasts transduced with tyrosine hydroxylase (TH) produced and released L-dopa to the culture medium;	When grafted to the striatum of Fischer rats with a prior 6-hydroxydopamine lesion, primary fibroblasts containing the TH transgene survived for 10 weeks, continued to express the transgene, and reduced rotational asymmetry.	Wolff et al, 1989
TH	Parkinson disease (PD)		Overexpress TH that converts tyrosine to L-DOPA to alleviate degeneration of dopaminergic nigrostriatal neurons (DNN) in PD rat models; unilateral destruction of DNN in animals with 6-hydroxydopamine and administration of apomorphine caused PD rats to turn contralaterally (7 or more rotations/min).	Implantation of transgenic immortalized fibroblasts and myoblasts intracerebrally improved rotational behavior	Fischer et al, 1991
TH	Parkinson's disease (PD)	HSV	Infection of 6-hydroxydopamine-lesioned rats, used as a model of PD, with a defective herpes simplex virus type 1 vector expressing TH	Conversion of endogenous striatal cells into L-dopa-producing cells	During et al, 1994
TH	Parkinson's disease (PD)	Lipofectin	To alleviate the symptoms of PD in TH-deficient rats (PD animal models; perform colateral rotations at 15 rounds/min upon administration of apomorphine)	Primary muscle cells were transduced with TH cDNA under control of CMV promoter; 10 million cells were injected into brains of TH-deficient rats; this resulted in 75% decrease in the number of rotations/min for more than 6 months	Jiao et al, 1993

Gene target or delivered	Human disease	Method	Animal model, objective, and method	Results	Reference
HSV TK and ganciclovir	hepatocellular carcinoma	AAV mice	To preferentially kill hepatocellular carcinoma cells by the suicadal gene HSV TK (driven by the a-fetal protein (AFP) enhancer and albumin promoter) with ganciclovir	Selective killing of AFP-positive cells in culture; transgenic mice were established by injection of AAV ITRs, neoR, and HSV TK genes as a linear DNA fragment; HSV TK was expressed predominantly in adult liver.	Su et al, 1996
Prostaglandin G/H synthase	acute lung injury	Cat lipid rabbit	Rabbits intravenously transfected with the PGH synthase gene	Increased plasma levels of prostacyclin and PGE2; protection of lungs in rabbits against endotoxin-induced inflammation, pulmonary edema, release of thromboxane B2, and pulmonary hypertension	Conary et al 1994
Ornithine transcarbamylase (OTC)	OTC-deficiency	Adenovirus	iv injection of recombinant adenovirus to spf-ash mice (OTC-deficient)	Correction of enzyme deficiency in OTC-deficient mice for over 1 year.	Stratford-Perricaudet et al, 1990
a1-antitrypsin	a1-antitrypsin-deficiency in lung	Adenovirus	The adenovirus major late promoter was linked to a human a1-antitrypsin gene for its transfer to lung epithelia of cotton rat respiratory pathway	Both in vitro and in vivo infections have shown production and secretion of a1-antitrypsin by the lung cells for over 1 week	Rosenfeld et al, 1991
a1-antitrypsin	a1-antitrypsin-deficiency in liver	Cat lipid mice	Protect connective tissue from the lytic action of the leukocyte neutrophil elastase; plasmid was encapsulated into negatively-charged liposomes containing phoshpatidylcholine	Small liposomes were much more effective in delivering the a1-antitrypsin gene to mouse hepatocytes in vivo.	Aliño et al, 1996
a1-antitrypsin (AT, human)	acute and chronic lung diseases.	Cat lipid	Aerosol and intravenous transfection to lungs of rabbits	Human a1AT mRNA and protein were detected for at least 7 days; immunohistochemical staining showed a1AT protein in the pulmonary endothelium following intravenous administration, in alveolar epithelial cells following aerosol administration, and in the airway epithelium by either route	Canonico et al, 1994
CFTR	Cystic fibrosis (CF)	Adenovirus	To alleviate the symptoms of CF	Expression of CFTR after intratracheal instillation into lungs of cotton rats; expression between days 2-10	Rosenfeld et al, 1992
CFTR (cystic fibrosis transmembrane conductance regulator)	Cystic fibrosis (CF)	Cat lipid	To express the normal CFTR gene in lungs of Edinburgh insertional mutant mouse (cf/cf) after delivering CFTR cDNA-liposome complexes into the airways by nebulization.	Full restoration of cAMP related chloride responses in some animals; human CFTR cDNA expression in the same tissues	Alton et al, 1993
CFTR	Cystic fibrosis (CF)	Lipofectin	To express the human CFTR gene in lungs in CFTR-deficient transgenic mice by tracheal instillation of lipofectin-plasmid	Successful transfer of the CFTR gene to epithelia and to alveoli deep in the lung leading to correction of the ion conductance defects found in the trachea of transgenic mice	Hyde et al, 1993.
CFTR	CF	Lipofectin	Transduction of airway epithelial cells in normal mice by intratracheal instillation of a plasmid carrying the CFTR gene under control of the Rous sarcoma virus promoter	Airway epithelial cells were the major target and site of expression of CFTR	Yoshimura et al, 1992
CFTR cDNA (human)	Cystic fibrosis (CF)	AAV	Intratracheal instillation into neonatal New Zealand white rabbits	Epithelial expression of the human CFTR fusion protein was detected using antisera to both the human CFTR R domain and the amino-terminal epitope at up to 6 weeks after vector inoculation, a time coinciding with the completion of the alveolar phase of lagomorph lung development	Rubenstein et al, 1997
p53	lung cancer	retrovirus	Lung tumors were elicited in nu/nu mice after intratracheal inoculation with human lung cancer H226Br cells whose p53 gene has a homozygous mutation at codon 254	Intratracheal injection of a recombinant retrovirus containing the wt p53 gene was shown to inhibit the growth of the tumor	Fujiwara et al, 1994
MHC class I HLA-B7 heavy chain gene plus b2-microglobulin		Cat lipid:DNA 1:5 mice	To give a complete Class I molecule with heavy and light chains; MHC class I HLA-B7 heavy chain gene has an internal ribosome entry site; the b2-microglobulin was driven by RSV promoter	Experiments in mice showed rapid (1 day) destruction of the plasmid in tissues; femtogram amounts only in muscle at 6 months postinjection	Lew et al, 1995
CCK (cholecystokinin)	congenital audiogenic epileptic seizures (AS)	Lipofectin (DOTMA:DOPE)	To suppress audiogenic epileptic seizures by cholecystokinin octapeptide (CCK-8) injected intracerebroventricularly (i.c.v.)	AS in rats was markedly reduced between day 3 and 4.	Zhang et al, 1992
hirudin	restenosis; arterial injury	adenovirus	Virus-injured rat carotid arteries; hirudin (from medicinal leech) is a potent protein inhibitor of thrombin; thrombin converts fibrinogen to fibrin and also stimulates smooth muscle cell proliferation during neointima formation in the arterial walls	35% reduction in neointima formation in hirudin cDNA-transduced arterial wall cells in vivo	Rade et al, 1996
HSV-tk	arterial injury; restenosis	Adenovirus	To kill preferentially the smooth muscle cells and reduce neointima formation in the arterial wall	47% reduction in I/M area ratio following local delivery of HSV-tk and systemic ganciclovir therapy	Ohno et al, 1994; Guzman et al, 1994
RB	atherosclerosis, restenosis, arterial injury	Adenovirus	To inhibit vascular smooth muscle cell (VSMC) proliferation after arterial injury	42% reduction in I/M area ratio	Chang et al, 1995a
p21	atherosclerosis, restenosis, arterial injury	Adenovirus	To inhibit vascular smooth muscle cell (VSMC) proliferation after arterial injury; p21 protein functions both by inhibiting cyclin dependent kinases (CDKs) required for the initiation of S phase and by binding to and inhibiting PCNA	Over-expression of human p21 inhibited growth factor-stimulated VSMC proliferation and neointima formation in the rat carotid artery model of balloon angioplasty in vitro by arresting VSMCs in the G1 phase of the cell cycle	Chang et al, 1995b
ras	arterial injury		Transfer of ras transdominant negative mutants to rats in which the common carotid artery was subjected to balloon injury	Reduced neointimal formation	Indolfi et al, 1995
TGF-b	arterial injury		TGF-b accelerates wound healing and inhibits epithelial and smooth cell proliferation; loss of functional TGF-b receptors in cancer cells	Inhibition in epithelial and smooth cell proliferation	Grainger et al, 1995
TGF-b1	Rheumatoid arthritis (RA)	Retr	Mice with induced arthritis	Effective in lowering inflammation of joints with already established arthritis and inhibiting the spreading of the disease to other joints in mice	Chernajovsky et al, 1997
Gene target or delivered	Human disease	Method	Goal/rationale	Results	Reference
LDL receptor	Familial hypercholesterolemia (FH)	Adeno	Infusion of adenovirus human LDL-R cDNA into the portal vein of rabbits deficient in LDL receptor	Human LDL receptor protein was produced in the majority of hepatocytes that exceeded the levels found in human liver by at least 10-fold	Kozarsky et al, 1994
Very low density lipoprotein receptor (VLDL-R)	Familial hypercholesterolemia (FH)	Adeno	LDL-R knockout mice	A single intravenous injection resulted in reduction in total cholesterol by approximately 50% at days 4 and 9; marked reduction in the intermediate density lipoprotein/low density lipoprotein (IDL/LDL)	Kobayashi et al, 1996; Kozarsky et al, 1996
HSV TK+ganciclovir (GCV)	Gliomas	Retr	Treatment of rats with cerebral glioma; intratumoral stereotaxic injection of murine fibroblasts (G418-selected) producing a retroviral vector with the HSV TK gene	Tretament with GCV 5 days postinjection resulted in complete regression of gliomas	Culver et al, 1992
HSV TK plus ganciclovir	prostate cancer	Adenovirus mouse	Ganciclovir is converted by HSV TK into its triphosphate form which is then incorporated into the DNA of replicating mammalian cells leading to inhibition in DNA replication and cell death; it is only viral TK, not the mammalian enzyme, that can use efficiently phosphorylated ganciclovir as a substrate	Subcutaneous tumors induced by injection of RM-1 (mouse prostate cancer) cells followed by injection of HSV TK and treatment with ganciclovir for 6 days showed reduction in tumor volume (16% of control) and higher apoptotic index in tumor cells	Eastham et al, 1996
HSV TK plus ganciclovir	adenocarcinoma		Carcinoembryonic antigen-producing human lung cancer cells	Cell type-specific expression of herpes simplex virus thymidine kinase gene	Osaki et al, 1994
TH	PD	Cat lipos rat	Overexpress TH to alleviate degeneration of dopaminergic nigrostriatal neurons (DNN) in PD rat models	Direct injection of lipofectin-TH expression plasmid on nigra-lesioned side generated L-DOPA locally and decreased contralateral rotations.	Cao et al, 1995
rat glial cell line-derived neurotrophic factor (rGDNF),	PD	AAV	To protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model of Parkinson's disease (back-labeled fluorogold-positive neurons in the substantia nigra)	94% cell protection; 85% of tyrosine hydroxylase-positive cells	Mandel et al, 1997

Factor IX	Hemophilia B	Adenovirus mice	Correction of FIX defect by injection of recombinant adenovirus hosting the canine FIX gene into hind leg muscle of mice	Establishment of factor IX levels in the blood in nude mice for 300 days; only for 10 days in normal mice; transduced cells were removed by cell-mediated as well as humoral immune responses; cyclophosphamide or cyclosporin A immunosuppression maintained FIX protein for 5 months	Dai et al, 1995
Factor IX	Hemophilia B	Adenovirus mice in vivo	Correction of the factor IX gene	Therapeutic plasma levels of factor IX in mice	Smith et al, 1993
Factor IX	Hemophilia B	Retrovirus dogs in vivo	Expression of factor IX gene in liver in hemophilia B dogs	1% of hepatocytes were transduced with a retroviral vector carrying the canine factor IX gene injected into the portal vein (2/3 hepatectomy was required); low therapeutic levels of factor IX	Kay et al, 1993
Factor IX	Hemophilia B	Adenovirus dogs in vivo	Establishment of the blood serum factor IX levels in hemophilic B dogs by infection with recombinant adenovirus vectors delivering the Factor IX gene and treatment with cyclosporin A	Treatment with cyclosporin A suppressed T cell activation; T cells attack the adenovirus-transduced cells eliminating them from the body; Cycl A tretment led to prolonged Factor IX levels in the blood of hemophilic dogs	Kay et al, 1994; Fang et al, 1995
Factor IX	Hemophilia B	AAV		Successful transduction of the mouse liver in vivo after a single administration; persistent, curative concentrations of functional human factor IX can be achieved	Snyder et al, 1997
Gene target or delivered	Human disease	Method	Goal/rationale	Results	Reference
Factor IX	Hemophilia B	AAV	Intramuscular injection into hindlimb muscles of C57BL/6 mice and Rag 1 mice	Presence of hF.IX protein by immunofluorescence staining of muscles harvested 3 months after injection in both strains of mice; no plasma FIX in immunocompetent mice; Rag 1 mice which lack functional B and T cells, displayed therapeutic levels (200-350 ng/ml) of F. IX in the plasma in addition to F.IX in muscle cells	Herzog et al, 1997
Factor X	Hemophilia B	Retr	Delivery to rat hepatocytes in vivo during liver regeneration; under control of a1-antitrypsin promoter	10% to 43% of normal human factor X levels in 4 rats; expression remained stable for more than 10 months in two rats	Le et al, 1997
p53	breast carcinoma MDA-MB-435 cells	DOTMA:DOPE	Nude mice inoculated with breast carcinoma cells (have mutated p53)	Iv injection of p53 gene under control of b-actin promoter and intron every 10-12 days resulted in more than 60% reduction in tumor volume	Lesoon-Wood et al, 1995
Cdc2 kinase and PCNA	Restenosis	liposome-Sendai virus	Delivery to rat carotids after balloon injury; inhibition of Cdc2 kinase and PCNA with antisense oligonucleotides using PS:PC:Chol liposomes	Whereas antisense cdc2 kinase or PCNA alone failed to have an effect, combination of the two antisense oligos significantly reduced neointima formation and smooth muscle cell proliferation after balloon injury	Morishita et al, 1993
vascular endothelial growth factor (VEGF)	restenosis	naked plasmid	VEGF promotes endothelial cell proliferation to accelerate re-endothelialization of the artery reducing intimal thickening	VEGF gene expression using ELISA or RT-PCR was detected for 3-14 days after a single transfer using a hydrogel/polymer-coated ballon angioplasty catheter to induce simultaneous injury and delivery of plasmid to the femoral artery in rabbits.	Isner et al, 1996
Kallikrein	hypertension	naked plasmid	Tissue kallikrein is a serine proteinase cleaving the kininogen to produce the vasoactive kinin peptide; kinin causes smooth muscle contraction and relaxation, increase in vascular permeability, and vasodilatation	Significant reduction in blood pressure in spontaneously hypersensitive rats after a single delivery of naked DNA to portal vein which lasted for 5-6 weeks.	Chao et al, 1996
Kallikrein	hypertension	Adeno		Sustained delay in the increase in blood pressure from day 2 to day 41 post injection (iv) into spontaneously hypertensive rats; human tissue kallikrein mRNA was detected in the liver, kidney, spleen, adrenal gland, and aorta.	Jin et al, 1997
Tissue kallikrein-binding protein (HKBP) or kallistatin	hypertension	Adeno	Kallistatin, a serine proteinase inhibitor, may function as a vasodilator in vivo	Delivery of the human kallistatin cDNA/CMV by portal vein injection resulted in a significant reduction of blood pressure of hypertensive rats for 4 weeks.	Chen et al, 1997
Human endothelial NO synthase (eNOS) gene	hypertension		Blood pressure is controled by the endothelium-derived nitric oxide (NO) in peripheral vessels	Significant reduction of systemic blood pressure for 5 to 6 weeks.	Lin et al, 1997
Antisense oligonucleotides to AT1-receptor mRNA and to angiotensinogen mRNA	hypertension	Liposomes	Angiotensinogen, produces angiotensin I in the liver (component of the renin-angiotensin system); mutations in the angiotensinogen (AT) gene are associated with hypertension	Antisense oligonucleotides delivered to rat liver via the portal vein diminished the expression of hepatic angiotensinogen mRNA and reduced blood pressure.	Tomita et al, 1995; Phillips, 1997; Phillips et al, 1997
Endothelial basic FGF (bFGF)	hypertension		Subphysiological amounts in blood vessels of spontaneously hypertensive rats	Restored the physiological levels levels of bFGF in the vascular wall and corrected hypertension	Cuevas et al, 1996
Atrial natriuretic peptide (ANP) gene	hypertension		Chronic infusion of ANP causes natriuresis, diuresis, and hypotension	Significant reduction of systemic blood pressure in young hypertensive rats (4 weeks old); the effect continued for 7 weeks.	Lin et al, 1995
IL-2	prostate cancer	liposomes	Direct transfer of the IL-2 gene under control of the CMV promoter with or without the AAV inverted terminal repeats	Plasmid DNA containing the AAV inverted terminal repeats showed 3-10 fold higher levels of gene transfer and IL-2 expression compared with constructs lacking the AAV sequences.	Vieweg et al, 1995
mouse leptin cDNA	obesity	Adeno	ob/ob mouse (which is genetically deficient in leptin and exhibits both an obese and a mild non-insulin-dependent diabetic phenotype)	Dramatic reductions in both food intake and body weight, as well as in normalization of serum insulin levels and glucose tolerance	Muzzin et al, 1996
rat leptin cDNA/CMV	obesity	Adeno	Wistar rats infused with 8 ng/ml adeno/leptin cDNA for 28 days	Animals became hyperleptinemic; 30-50% reduction in food intake; gained only 22 g over the experimental period versus 115-132 gained by control animals	Chen et al, 1996
VEGF₁₆₅	cancer (vascularization)	calcium phosphate	Expression of VEGF₁₆₅ in rat C6 glioma cells and subcutaneous injection of the transduced cells in athymic mice.	Tumors from cells expressing VEGF grew slower than tumors developed from nontransduced C6 cells, were highly vascularized, and contained varying degrees of necrosis and eosinophilic infiltrate	Saleh, 1996
cGMP phosphodiesterase-b (PDE-b) gene	retinal degeneration	AAV	To treat retinal degeneration due to recessive mutation in the endogenous gene	Intraocular injection of AAV-PDE-b cDNA increased retinal expression of immunoreactive PDE protein; treated eyes showed increased numbers of photoreceptors and a two-fold increase in sensitivity to light	Jomary et al, 1997

Gene or antisense	Reference

VEGF gene transfer	Isner et al, 1996a; Van Belle et al, 1997; Laitinen et al, 1997a,b
HSV-tk gene /GCV	Guzman et al, 1994; Ohno et al, 1994
Cytosine deaminase (CD) gene /5-fluorocytosine	Harrell et al, 1997
RB gene	Chang et al, 1995a; Smith et al, 1997
p21 gene	Chang et al, 1995b
Hirudin gene	Rade et al, 1996
Dominant-negative mutated form of c-H-ras gene	Indolfi et al, 1995; Ueno H et al, 1997b
TGFb gene	Grainger et al, 1995
Nitric oxide synthase gene	von der Leyen et al, 1995
gax gene	Weir et al, 1995; Maillard and Walsh, 1996; Smith et al, 1997
Protein kinase Cd gene (by suppressing G1 cyclin expression)	Fukumoto et al, 1997
Bone morphogenetic protein-2 (BMP-2) gene	Nakaoka et al, 1997
b-interferon gene	Stephan et al, 1997
Antisense cdk2 oligonucleotides	Morishita et al, 1994
Antisense oligodeoxynucleotides to c-myc	Bennettet al, 1994
Antisense oligonucleotides to PCNA	Simons et al, 1994
Antisense cyclin G1	Zhu et al, 1997

Company	Drug/Gene type	Goal/Disease	Status of drug development (end of 1997)
Calydon (San Francisco, CA)	Prostate cancer gene therapy		Preclinical trials
Canji/Schering-Plough (San Diego, CA)	p53 to restore p53-mediated tumor suppression or apoptosis	Colon with hepatic metastases(#131); prostate (#148)	Phase I
Canji/Schering-Plough (San Diego, CA)	RB to restore RB-mediated cell cycle arrest	Bladder(#140), non-small cell lung cancer (#156), lung, ovarian, and liver cancers	Early clinical trials
Cell Genesys, Inc	CC49-Zeta T cell receptor	Colorectal carcinomas expressing the tumor-associated antigen, TAG-72 (#110)	Phase I /II
Cell Genesys, Inc	CD4-zeta Chimeric Receptor	HIV (#85, 116)	Phase I-II
Chiron Corporation (Emerville & San Diego CA)	HIV-1IIIB envelope protein (#108)	HIV	Phase I
IDUN Pharmaceutical (San Diego, CA)	bcl-2 inhibition		Preclinical trials
Incyte Pharmaceuticals (Palo Alto, CA)	Gene sequence databases		License access to database for drug discovery to different companies
Introgen Therapeutics (Austin and Houston, TX)	p53 gene therapies to restore p53-mediated tumor suppression or apoptosis	Lung, head and neck cancers, prostate cancer (#154)	Phase I-II
Gene Medicine, Inc. (The Woodlands, Texas)	a1-Antitrypsin cDNA/cationic lipids	a1-antitrypsin deficiency (#194)	Phase I
Gene Medicine, Inc.	Human insulin-like growth factor-1(hIGF-1)	Cubital Tunnel syndrome (#164)	Phase I
Genetic Therapy, Inc. /Novartis (Gaithersburg, MD)	HSV-tk	brain tumors (#17), recurrent pediatric brain tumors (#45), recurrent glioblastoma (#32, 97, 99), astrocytoma (#42, 68), leptomeningeal carcinomatosis (#49), multiple myeloma (#75)	Phase I
Genetic Therapy, Inc. /Novartis	CFTR/adenovirus vector	Cystic fibrosis (#121)	Phase I
Genetic Therapy, Inc. /Novartis	Glucocerebrosidase cDNA	Gaucher disease (#39)	Phase I
Genset		Isolation of regulatory regions
Genta (La Jolla, CA)	bcl-2 inhibition with antisense	cancer	Early clinical trials
Genzyme Corporation (Framingham, MA)	CFTR/adenovirus vector/cationic lipids	Cystic fibrosis (#120, 128, 129, 203)	Phase I
GenVec, Inc.	VEGF₁₂₁ cDNA to the ischemic myocardium	Coronary artery disease (#157)	Phase I
Glaxo Wellcome Inc.	CFTR/Cationic Liposome Complex	Cystic fibrosis	Phase I
LXR (Richmond, CA)	Antiapoptotics		Advanced clinical trials
Mitotix (Cambridge, MA)	Cell cycle inhibitors		Preclinical trials
Myriad Genetics (Salt Lake City, UT)	Diagnostic genetic testing	BRAC analysis kit for breast cancer	In the market
Onyx (Richmond, CA)	p53 gene therapies killing cells that lack p53		Early clinical trials
Ribozyme Pharmaceuticals, Inc (Boulder, CO)	Tat and Rev Hammerhead Ribozyme	HIV replication inhibition (#117)	Phase II
Ribozyme Pharmaceuticals, Inc (Boulder, CO)	Growth factor inhibition		Preclinical trials
Rhône-Poulenc Rorer Gencell (Vitry-sur-Seine, France & Santa Clara, California)	p53	Head and neck squamous cell carcinoma (#152)	Phase II
SEQUUS Pharmaceuticals (Menlo Park, CA)	Doxorubicin encapsulated into “stealth” liposomes	Kaposis sarcoma	In the market (Doxil); clinical trials for other cancers
SUGEN (Redwood City, CA)	Tyrosine kinase inhibitor to inhibit PDGF receptor	Gliomas	Clinical trials
Targeted Genetics Corporation	HSV-tk	HIV (#81)	Phase I/II
Targeted Genetics Corporation	E1A/ DC-Chol-DOPE	Metastatic breast or ovarian cancer, metastatic solid tumors that overexpress HER-2 /neu	Phase I
Targeted Genetics Corporation	CFTR/AAV	Cystic fibrosis	Phase I
Vical, Inc.	Tumor idiotype	Non-Hodgkin’s B-cell lymphoma (#161)	Phase I/II
Vical, Inc. (San Diego, California)	b-2 Microglobulin cDNA/cationic lipids	Immunotherapy of advanced colorectal carcinoma, renal cancer (#195), melanoma (#196), metastatic malignancies (#201)	Phase I/II
Vical, Inc.	Interleukin-2 cDNA	Lymphoma (#198), solid tumor immunotherapy (#211)	Phase I
Vical, Inc.	HLA B7 cDNA	Renal cancer immunotherapy	Phase I

RETROVIRAL GENE DELIVERY
Disease	Protocol title	Procedures	Principal investigator
1. Gene Marking /Cancer	The Treatment of Patients with Advanced Cancer Using Cyclophosphamide, Interleukin-2 and Tumor Infiltrating Lymphocytes.	In Vitro /Tumor Infiltrating Lymphocytes /Retrovirus /Neomycin Phosphotransferase cDNA /Intravenous	Rosenberg, Steven A
2. Gene Therapy /Phase I /Monogenic Disease /Severe Combined Immune Deficiency due to Adenosine Deaminase Deficiency	Treatment of Severe Combined Immune Deficiency (SCID) due to Adenosine Deaminase (ADA) Deficiency with Autologous Lymphocytes Transduced with the Human ADA Gene: An Experimental Study.	In Vitro /Autologous Peripheral Blood Cells /CD34⁺ Autologous Peripheral Blood Cells /Cord Blood /Placenta Cells /Retrovirus /Adenosine Deaminase cDNA /Neomycin Phosphotransferase cDNA /Intravenous	Blaese, R. Michael
3. Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy	Gene Therapy of Patients with Advanced Cancer Using Tumor Infiltrating Lymphocytes Transduced with the Gene Coding for Tumor Necrosis Factor.	In Vitro /Tumor Infiltrating Lymphocytes /Retrovirus /Cytokine /Tumor Necrosis Factor cDNA /Neomycin Phosphotransferase cDNA /Intravenous	Rosenberg, Steven A
4. Gene Marking /Cancer /Acute Myelogenous Leukemia	Autologous Bone Marrow Transplant for Children with Acute Myelogenous Leukemia in First Complete Remission: Use of Marker Genes to Investigate the Biology of Marrow Reconstitution and the Mechanism of Relapse.	In Vitro /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant	Brenner, Malcolm K.
5. Gene Marking /Cancer /Neuroblastoma	A Phase I /II Trial of High Dose Carboplatin and Etoposide with Autologous Marrow Support for Treatment of Stage D Neuroblastoma in First Remission: Use of Marker Genes to Investigate the Biology of Marrow Reconstitution and the Mechanism of Relapse.	In Vitro /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant	Brenner, Malcolm K.
6. Gene Marking /Cancer /Neuroblastoma	A Phase II Trial of High-Dose Carboplatin and Etoposide with Autologous Marrow Support for Treatment of Relapse /Refractory Neuroblastoma Without Apparent Bone Marrow Involvement.	In Vitro /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant	Brenner, Malcolm K
7. Gene Marking /Cancer /Chronic Myelogenous Leukemia	Autologous Bone Marrow Transplantation for Chronic Myelogenous Leukemia in which Retroviral Markers are Used to Discriminate between Relapse which Arises from Systemic Disease Remaining after PreparativeTherapy Versus Relapse due to Residual Leukemic Cells in Autologous Marrow: A Pilot Trial.	In Vitro /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant	Deisseroth, Albert B
8. Gene Marking /Acute Hepatic Failure	Hepatocellular Transplantation in Acute Hepatic Failure and Targeting Genetic Markers to Hepatic Cells.	In Vitro /Autologous Hepatocytes /Retrovirus /Neomycin Phosphotransferase cDNA /Intrahepatic	Ledley, Fred D.
9. Gene Marking /Cancer /Melanoma	The Administration of Interleukin-2 and Tumor Infiltrating Lymphocytes to Patients with Melanoma.	In Vitro /Tumor Infiltrating Lymphocytes /Retrovirus /Neomycin Phosphotransferase cDNA /Intravenous	Lotze, Michael T
10. Gene Therapy /Phase I /Cancer /Melanoma /Renal Cell /Colon /Breast /Immunotherapy	Immunization of Cancer Patients Using Autologous Cancer Cells Modified by Insertion of the Gene for Tumor Necrosis Factor (TNF).	In Vitro /Autologous Tumor Cells /Lethally Irradiated /Retrovirus /Cytokine /Tumor Necrosis Factor cDNA /Neomycin Phosphotransferase cDNA /Subcutaneous Injection	Rosenberg, Steven A
11. Gene Therapy /Phase I /Cancer /Melanoma /Renal Cell /Colon /Immunotherapy	Immunization of Cancer Patients Using Autologous Cancer Cells Modified by Insertion of the Gene for Interleukin-2 (IL-2).	In Vitro /Autologous Tumor Cells /Lethally Irradiated /Retrovirus /Cytokine /Interleukin-2 cDNA /Subcutaneous Injection	Rosenberg, Steven A
12. Gene Marking /Cancer /Acute Myelogenous Leukemia /Acute Lymphocytic Leukemia	Retroviral-Mediated Gene Transfer of Bone Marrow Cells during Autologous Bone Marrow Transplantation for Acute Leukemia.	In Vitro /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant	Cornetta, Kenneth
13. Gene Marking /Cancer /Melanoma /Renal Cell	The Treatment of Patients with Metastatic Melanoma and Renal Cell Cancer Using In Vitro Expanded and Genetically-Engineered (Neomycin Phosphotransferase) Bulk, CD8⁺ and /or CD4⁺ Tumor Infiltrating Lymphocytes and Bulk, CD8⁺ and /or CD4⁺ Peripheral Blood Leukocytes in Combination with Recombinant Interleukin-2 Alone, or with Recombinant Interleukin-2 and Recombinant a Interferon.	In Vitro /CD4⁺ Autologous Peripheral Blood Lymphocytes /CD8⁺ Autologous Peripheral Blood Lymphocytes /CD4⁺ Autologous Tumor Infiltrating Lymphocytes /CD8⁺ Autologous Tumor Infiltrating Lymphocytes /Retrovirus /Neomycin Phosphotransferase cDNA /Intravenous	Economou, James S. and Belldegrun, Arie
14. Gene Therapy /Phase I /Cancer /Ovarian /Pro-Drug	Gene Transfer for the Treatment of Cancer.	In Vitro /Allogeneic Tumor Cells /Lethally Irradiated /PA317 /Retrovirus /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intraperitoneal Administration	Freeman, Scott M
15. Gene Therapy /Infectious Disease /Human Immunodeficiency Virus	Phase I Study to Evaluate the Safety of Cellular Adoptive Immunotherapy Using Genetically Modified CD8⁺ HIV-Specific T Cells in HIV Seropositive Individuals.	In Vitro /CD8⁺ Allogeneic Cytotoxic T Lymphocytes /CD8⁺ Syngeneic Cytotoxic T Lymphocytes /Retrovirus /Hygromycin Phosphotransferase /Herpes Simplex Virus Thymidine Kinase cDNA /Intravenous	Greenberg, Philip D. and Riddell, Stanley
16. Gene Therapy /Phase I /Cancer /Relapsed-Refractory Neuroblastoma /Immunotherapy	Phase I Study of Cytokine-Gene Modified Autologous Neuroblastoma Cells for Treatment of Relapsed /Refractory Neuroblastoma.	In Vitro /Autologous Neuroblastoma Cells /Allogeneic Partially HLA-Matched /Retrovirus /Cytokine /Interleukin-2 cDNA /Subcutaneous Injection	Brenner, Malcolm K.
17. Gene Therapy /Phase I /Cancer /Brain /Pro-Drug	Gene Therapy for the Treatment of Brain Tumors Using Intra-Tumoral Transduction with the Thymidine Kinase Gene and Intravenous Ganciclovir. Sponsor: Genetic Therapy, Inc. /Novartis	In Vivo /Autologous Tumor Cells /PA317 /Retrovirus /Herpes Simplex Virus Thymidine Kinase cDNA /Ganciclovir /Intratumoral /Stereotactic Injection	Oldfield, Edward
18. Gene Marking /Cancer /Chronic Myelogenous Leukemia	Use of Two Retroviral Markers to Test Relative Contribution of Marrow and Peripheral Blood Autologous Cells to Recovery After Preparative Therapy.	In Vitro /Autologous Bone Marrow Cells /Autologous Peripheral Blood Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant	Deisseroth, Albert B
19. Gene Therapy /Phase I /Cancer /Melanoma /Immunotherapy	Immunization with HLA-A2 matched Allogeneic Melanoma Cells that Secrete Interleukin-2 in Patients with Metastatic Melanoma.	In Vitro /Allogeneic Partially HLA-Matched /Retrovirus /Cytokine /Interleukin-2 cDNA /Subcutaneous Injection	Gansbacher, Bernd
20. Gene Therapy /Phase I /Cancer /Renal Cell /Immunotherapy	Immunization with Interleukin-2 Secreting Allogeneic HLA-A2 Matched Renal Cell Carcinoma Cells in Patients with Advanced Renal Cell Carcinoma.	In Vitro /Allogeneic Partially HLA-Matched /Retrovirus /Cytokine /Interleukin-2 cDNA /Subcutaneous Injection	Gansbacher, Bernd
21. Gene Marking /Cancer /Multiple Myeloma	Retroviral-Mediated Gene Transfer of Bone Marrow and Peripheral Blood Stem Cells During Autologous Bone Marrow Transplantation for Multiple Myeloma.	In Vitro /CD34⁺ Autologous Peripheral Blood Cells /Intravenous /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant	Dunbar, Cynthia
22. Gene Marking /Cancer /Breast	Retroviral-Mediated Gene Transfer of Bone Marrow and Peripheral Blood Stem Cells During Autologous Bone Marrow Transplantation for Metastatic Breast Cancer.	In Vitro /CD34⁺ Autologous Peripheral Blood Cells /Intravenous /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant	Dunbar, Cynthia
23. Gene Marking /Cancer /Chronic Myelogenous Leukemia	Retroviral-Mediated Gene Transfer of Bone Marrow and Peripheral Blood Stem Cells During Autologous Bone Marrow Transplantation for Chronic Myelogenous Leukemia.	In Vitro /CD34⁺ Autologous Peripheral Blood Cells /Intravenous /Autologous Bone Marrow Cells /Retrovirus /Neomycin Phosphotransferase cDNA /Bone Marrow Transplant	Dunbar, Cynthia
24. Gene Marking /Infectious Disease /Human Immunodeficiency Virus	A Study of the Safety and Survival of the Adoptive Transfer of Genetically Marked Syngeneic Lymphocytes in HIV Infected Identical Twins.	In Vitro /Syngeneic Peripheral Blood Lymphocytes /Retrovirus /Neomycin Phosphotransferase cDNA /Intravenous	Walker, Robert E
25. Gene Marking /Cancer	Study on Contribution of Genetically Marked Peripheral Blood Repopulating Cells to Hematopoietic Reconstitution after Transplantation.