Status of gene therapy in 1997: molecular mechanisms, disease targets, and clinical applications

I. Introduction

Monumental progress in several fields including DNA replication, transcription factors and gene expression, repair, recombination, signal transduction, oncogenes and tumor suppressor genes, genome mapping and sequencing, and on the molecular basis of human disease are providing the foundation of a new era of biomedical research aimed at introducing therapeutically important genes into somatic cells of patients. The main targets of gene therapy are to repair or replace mutated genes, regulate gene expression and signal transduction, manipulate the immune system, or target malignant and other cells for destruction (reviewed by Anderson, 1992; Nowak, 1995; Boulikas, 1996a,b; Culver, 1996; Ross et al, 1996).

Two main approaches have been pursued for gene transfer to somatic cells (i) direct gene delivery using murine retroviruses, adenoviruses, adeno-associated virus, HSV, EBV, liposomes, polymers, or direct plasmid injection (gene therapy in vivo); and (ii) ex vivo gene therapy involving removal of syngeneic cells from a specific organ or tumor of an individual, genetic correction of the defect in cell culture (ADA deficiency, LDL-R for FH) or transfer of a different gene (IL-2 to tumor infiltrating lymphocytes to potentiate the cytotoxicity to tumors, cytokine genes to tumor cells from a patient for cancer immunotherapy, multidrug resistance gene transfer to render bone marrow cells resistant to certain antineoplastic drugs), followed by reimplantation of the cells. The reimplanted cells produce the therapeutic protein.

Several key factors or steps appear to be involved for the effective gene transfer to somatic cells in a patient or animal model: (i) the type of vehicle used for gene delivery (liposomes, adenoviruses, retroviruses, AAV, HSV, EBV, polymer, naked plasmid) which will determine not only the half-life in circulation, the biodistribution in tissues, and efficacy of delivery but also the route through the cell membrane and fate of the transgene in the nucleus; (ii) interaction of the gene-vehicle system with components in the serum or body fluids (plasma proteins, macrophages, immune response cells); (iii) targeting to the cell type, organ, or tumor, and binding to the cell surface; (iv) port and mode of entrance to the cell (poration through the cell membrane, receptor-mediated endocytosis), (v) release from cytoplasmic compartments (endosomes, lysosomes), (vi) transport across the nuclear envelope (nuclear import); (vii) type and potency of regulatory elements for driving the expression of the transferred gene in a particular cell type including DNA sequences that determine integration versus maintenance of a plasmid or recombinant virus/retrovirus as an extrachromosomal element; (viii) expression (transcription) of the transgene producing heterogeneous nuclear RNA (HnRNA) which is then (ix) spliced and processed in the nucleus to mature mRNA and is (x) exported to the cytoplasm to be (xi) translated into protein. Additional steps may include posttranslational modification of the protein and addition of a signal peptide (at the gene level) for secretion.

All steps can be experimentally manipulated and improvements in each one can enormously enhance the level of expression and therapeutic index of a gene therapy approach. It has been proposed that the plasmid vector is unable to translocate to the nucleus unless complexed in the cytoplasm with nuclear proteins possessing nuclear localization signals (NLSs). NLSs are short karyophilic peptides on proteins destined to function in the nucleus used for binding to specific transporter molecules in the cytoplasm, mediating their passage through the pore complexes to the nucleus (see Boulikas, 1998, this volume). NLS are present on histones, transcription factors, nuclear enzymes, and a number of other nuclear proteins; nascent chains of DNA-binding polypeptides could bind to the supercoiled plasmid in the cytoplasm mediating its translocation to the nucleus.

During delivery of foreign DNA in vivo vehicles may be attacked by macrophages, lymphocytes, or other components of the immune system and the vast majority will be cleared from blood, intracellular, or other body fluids before it is given the chance to reach the membrane of the cell target; the half-life of naked plasmids injected intravenously into animals is about 5 min (Lew et al, 1995). Cationic lipids, other than being very toxic, mediate efficient gene delivery passing through biological membranes; those lipid-DNA complexes surviving the immediate neutralization by serum proteins in the blood can reach the lung, heart and other tissues after vein or artery injection with one heart beat and transform endothelial vascular cells (reviewed by Boulikas, 1996d).

A variety of viral vectors have been developed to exploit the characteristic properties of each group to maintain persistence and viral gene expression in infected cells. Retroviral vectors and AAV integrate into target chromosomes and the transgene they carry can be inactivated from position effects from chromatin surroundings. Vectors with persistence/integration functions may not result in high levels of gene delivery in vivo.

Adenoviruses and retroviruses which are of the most frequently used vehicles for gene transfer can accommodate up to 7kb of total foreign DNA into their genome because of packaging limitations. This precludes their use for the transfer of large genomic regions. Transfer of intact yeast artificial chromosome (YAC) into transgenic mice will enable the analysis of large genes or multigenic loci such as human b-globin locus (reviewed by Peterson et al, 1997).

A small portion of plasmid molecules crossing the cell membrane will escape degradation from nucleases in the lysosomes and become released to the cytoplasm; even a smaller portion of these molecules will enter nuclei; finally, after successfully reaching the nucleus, plasmids with therapeutic genes are usually degraded by nuclear enzymes and transgene expression is permanently lost after about 2-7 days from animal tissues following successful gene delivery. During the peak of transgene expression (usually 7-48 h from injection) the transgene transcript can follow the normal fate of other nuclear transcripts when proper polyadenylation signals are provided; its processed mRNA will be exported to the cytoplasm and translated into the therapeutic protein.

The choice of the appropriate delivery system for successful somatic gene transfer demands understanding of the drawbacks and advantages of each delivery system, such as limitations in the total length of the DNA that can be introduced, including the cDNA of the therapeutically important gene and control elements. Understanding the pathophysiology of the disease and the cell targets can give clues on the way of introducing the gene (i.v., i.p., intratumoral, s.c. injection) or direct the gene therapist to designing methods to target and secrete a therapeutic protein from a tissue which is not the normal site of production of a therapeutic protein. The type of control elements required for the anticipated tissue-specific expression of the construct, the presence of viral or other origins of replication as well as of the cDNA encoding the viral replication initiator protein for an episomal replication of the transgene, sequences that prompt integration and others that insulate the gene from the chromatin surroundings at the integration site, are also important for successful gene transfer.

Cancer gene therapy and immunotherapy has been the first priority of human gene therapy protocols. New gene targets are being defined and new clinical protocols are being proposed and approved. Effective eradication of a great variety of tumors with drugs which inhibit angiogenesis has been extraordinarily successful on animal models and the method moves fast to clinical trials; transfer of anti-angiogenesis genes will be the next step. A number of anticancer genes are being tested in preclinical or clinical cancer trials including p53, RB, BRCA1, E1A, bcl-2, MDR-1, HER2, p21, p16, bax, bcl-xs, E2F, antisense IGF-I, antisense c-fos, antisense c-myc, antisense K-ras and the cytokine genes GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-g, and TNF-a. A promising approach is transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene (suicide gene) and systemic treatment with the prodrug ganciclovir which is converted by HSV-tk into a toxic drug killing dividing cells. Theoretically, expression of therapeutic genes preferentially in cancer cells could be achieved by regulatory elements from tumor-specific genes such as carcinoembryonic antigen.

The first gene therapy products are expected to receive FDA approval by the year 2000; the market for gene therapy products is expected to exceed $45 billion by 2010.

This article reviews the molecular mechanisms and recent developments for the gene therapy of cancer, HIV, ADA deficiency, Parkinson's disease, lysosomal storage disease, hemophilia A and B, a1-antitrypsin deficiency, cystic fibrosis, rheumatoid arthritis, hypertension, familial hypercholesterolemia, atherosclerosis/restenosis, wound healing, and obesity including the treatment of cancer and heart diseases with angiogenesis inhibitors and gene transfer to the arterial wall. It is my intention to give a general overview rather to exhaust the field.

DIVISION ONE: GENE DELIVERY SYSTEMS AND GENE EXPRESSION

II. Gene delivery using retroviruses

A. Recombinant murine retroviruses

The recombinant Moloney murine leukemia virus (Mo-MLV or MLV) has been extensively used for gene transfer. Retroviral vectors derived from Mo-MLV promote the efficient transfer of genes into a variety of cell types from many animal species; up to 8 kb of foreign DNA can be packaged in a retroviral vector. Recombinant retroviruses have been the most frequently used and promising vehicles for the delivery of therapeutic genes in human gene therapy protocols (Appendix 1). Retroviral vectors cause no detectable harm as they enter their target cells; the retroviral nucleic acid becomes integrated into chromosomal DNA, ensuring its long-term persistence and stable transmission to all future progeny of the transduced cell.

The life cycle of the retrovirus is well understood and can be effectively manipulated to generate vectors that can be efficiently and safely packaged. An important contribution to their utility has been the development of retrovirus packaging cells, which allow the production of retroviral vectors in the absence of replication-competent virus.

Recombinant retroviruses stably integrate into the DNA of actively dividing cells, requiring host DNA synthesis for this process (Miller et al, 1990). Although this is a disadvantage for targeting cells at G0, such as the totipotent bone marrow stem cells, it is a great advantage for targeting tumor cells in an organ without affecting the normal cells in the surroundings. This approach has been used to kill gliomas in rat brain tumors by injection of murine fibroblasts stably transduced with a retroviral vector expressing the HSV-tk gene (Culver et al, 1992; see below).

B. Retrovirus packaging cell lines

The use of retroviral vectors in human gene therapy requires a packaging cell line which is incapable of producing replication-competent virus and which produces high titers of replication-deficient vector virus. The packaging cell lines have been stably transduced with viral genes and produce constantly viral proteins needed by viruses to package their genome. Wild-type virus can be produced through recombinational events between the helper virus and a retroviral vector. Methods are also available for generating cell lines which secrete a broad host range retrovirus vectors in the absence of helper virus.

Retrovirus packaging cell lines containing the gag-pol genes from spleen necrosis virus and the env gene from spleen necrosis virus or from amphotropic murine leukemia virus on a separate vector have been used; retrovirus vectors were produced from these helper cell lines without any genetic interactions between the vectors and sequences in the helper cells (Dougherty et al, 1989). An ecotropic packaging cell line and an amphotropic packaging cell line, in which the viral gag and pol genes were on one plasmid and the viral env gene were on another plasmid have been constructed; both plasmids contained deletions of the packaging sequence and the 3' LTR; when the fragmented helper virus genomes were introduced into 3T3 cells they produced titers of retrovirus which were comparable to the titers produced from packaging cells containing the helper virus genome on a single plasmid (Markowitz et al, 1990).

The pBabe retroviral vector constructs which transmit inserted genes at high titers and express them from the Mo-MLV LTR have been designed with one of four different dominantly acting selectable markers, allowing the growth of infected mammalian cells in the presence of G418, hygromycin B, bleomycin/phleomycin or puromycin, respectively. The packaging cell line, omega E, generated with separate gag/pol and ecotropic env expression constructs, was designed in conjunction with the pBabe vectors to reduce the risk of generation of wild type Mo-MLV via homologous recombination events (Morgenstern and Land, 1990).

C. Pseudotyped retroviral vectors

The traditional retroviral vector enters the target cell by binding of a viral envelope glycoprotein to a cell membrane viral receptor. Coinfection of cells with a retrovirus and VSV (vesicular stomatitis virus) produces progeny virions containing the genome of one virus encapsidated by the envelope protein of the other (pseudotypes of viruses); this led to the development of pseudotyped retroviral vectors where the Moloney murine leukemia env gene product is replaced by the VSV-G protein able to interact with other membrane-bound receptors as well as with some components of the lipid bilayer (phosphatidylserine); because of the ubiquitous distribution of these membrane components pseudotyped particles display a very broad host range (Friedmann and Yee, 1995). Use of pseudotyped vectors has been a significant advancement for retroviral gene transfer.

Pseudotypes of VSV and Mo-MLV, are released preferentially at early times after infection of MuLV-producing cells with VSV; at later times, after synthesis of M-MLV proteins has been inhibited by the VSV infection, neither Mo-MLV virions nor the VSV (Mo-MLV) pseudotypes are made. There appears to be a stringent requirement for recognition of the viral core by homologous envelope components for the production of VSV (M-MLV) pseudotypes (Witte and Baltimore, 1977).

The finding that the G protein of vesicular stomatitis virus (VSV) can serve as the exclusive envelope protein component for one specific retroviral vector that expresses VSV G protein was extended to a general transient transfection scheme for producing very high-titer VSV G-enveloped pseudotypes from any Moloney murine leukemia-based retroviral vector (Yee et al, 1994). Pseudotyping of MuLV particles with VSV-G expressed transiently in cells producing MLV Gag and Pol proteins, has yielded vector preparations with a broader host range that could be concentrated by ultracentrifugation. For example, this technology allowed for efficient concentration of vector by ultracentrifugation to titers > 10⁹ colony-forming units/ml and offers hope for potential use for gene transfer in vivo. Furthermore, these vectors could infect cells, such as hamster and fish cell lines, that are ordinarily resistant to infection with vectors containing the retroviral envelope protein (Burns et al, 1993).

A human 293-derived retroviral packaging cell line was generated by Ory et al (1996) capable of producing high titers of recombinant Mo-MLV particles that have incorporated the VSV-G protein. This new packaging cell line may be used for direct in vivo gene transfer using retroviral vectors because the retroviral/VSV-G pseudotypes generated with these cells were significantly more resistant to human complement than commonly used amphotropic vectors.

A human immunodeficiency virus type 1 (HIV-1)-based retroviral vector containing the firefly luciferase reporter gene could be pseudotyped with a broad-host-range VSV envelope glycoprotein G; higher-efficiency gene transfer into CD34⁺ cells was achieved with a VSV-G-pseudotyped HIV-1 vector than with a vector packaged in an amphotropic envelope (Akkina et al, 1996).

Because the VSV-G protein is toxic to cells when constitutively expressed, Yang et al (1995) have used steroid-inducible and tetracycline-modulated promoter systems to derive stable producer cell lines capable of substantial production of VSV-G pseudotyped MLV particles. Similarly, the toxic G protein of VSV could be induced in a cell line by the removal of tetracycline and the addition of estrogen; this cell line was transduced with a modified tTA transactivator gene engineered with the ligand-binding domain of the estrogen receptor to the carboxy terminus of the tTA transactivator; a single retroviral vector could transduce both the transactivator gene and the VSV-G protein gene controlled by the tTA-inducible promoter into mammalian cells (Iida et al, 1996). The tetracycline-inducible system was modified by fusing the ligand binding domain of the estrogen receptor to the carboxy terminus of a tetracycline-regulated transactivator to regulate VSV-G expression in a tetracycline-dependent manner that could be modulated by b-estradiol in stable packaging cell lines (Chen et al, 1996).

D. Limitations and advancements using retroviral vectors

Before the in vivo gene therapy with retroviruses becomes a successful reality a number of problems must be overcome. Despite the extensive use of retroviral vectors in gene therapy, there are still problems to be solved and there is an ultimate need for the development of new, improved retroviral vectors and packaging systems to fuel further advances in the field of human gene therapy. The principle limitation of retroviruses has been poor gene expression in vivo which has been overcome through the use of tissue-specific promoters. Use of internal ribosome entry sites from picornaviruses in retroviral vectors has provided stable expression of multiple gene enhancers (reviewed by Naviaux and Verma, 1992; Boris-Lawrie and Temin, 1993).

Little is known about the factors that influence the efficiency of retroviral infection in vivo. Many commonly used experimental animal strains, such as mice, harbor endogenous C-type proviruses, some of which are expressed and have circulating antibodies against the viral envelope glycoproteins that cross-react with the Mo-MLV; the efficiency of retrovirus-mediated transfection in vivo using a variety of mouse strains was affected by humoral immune competence and interference between endogenous MLVs and exogenous recombinant Mo-MLV (Fassati et al, 1995).

One of the drawbacks of retroviruses for their exploitation in gene therapy has been the low viral titers obtained, too low to achieve therapeutic levels of gene expression; methods for the efficient concentration from large volumes of supernatant and purification of amphotropic retrovirus particles have been developed in several laboratories. For example, Bowles et al (1996) have used concentration and further purification of virus particles by sucrose banding ultracentrifugation; animal studies have shown that viral transduction increased proportionally with titer of the retrovirus.

Transduced cells producing retrovirus are tissue-incompatible and are, therefore, expected to be attacked by the immune system; this will lead to the elimination of therapeutic cells from the body, a phenomenon markedly associated also with adenoviral gene transfer. A privileged exception are brain tumor cells expressing recombinant retrovirus which persist without immunologic rejection (Culver et al, 1992).

Sodium butyrate treatment of murine retrovirus packaging cells producing a CFTR vector increased the production of the retrovirus vector between 40- and 1,000-fold (Olsen and Sechelski, 1995).

The Cre/LoxP recombinase strategy (see below) has been used to generate retroviral vectors that have the ability to excise themselves after inserting a gene into the genome, thereby avoiding problems encountered with conventional retrovirus vectors, such as recombination with helper viruses or transcriptional repression of transduced genes (Russ et al, 1996). Retroviral vectors with the Cre/LoxP technology have also been used to deliver the GM-CSF gene to K562 cell culture (Fernex et al, 1997), for the development of retroviral suicide vectors for gene therapy using the HSV-tk gene (Bergemann et al, 1995), and for the production of a high-titer producer cell line containing a single LoxP site flanked by the viral LTRs (Vanin et al, 1997).

Because retrovirus vectors are integrated into the genome, transcriptional repression of transduced genes will often take place from position effects exerted from neighboring chromatin domains; two matrix-attached regions (MARs), one at either flank of the transgene, are proposed here to insulating the gene in the retrovirus vector from chromatin effects at the integration site by creating an independent realm of chromatin structure harboring the transgene. MAR insulators have been used and can enhance up to 2,000-fold the expression of genes in transgenic animals and plants (McKnight et al, 1992; Breyne et al, 1992; Allen et al, 1993; Brooks et al, 1994; Thompson et al, 1994; Forrester et al, 1994).

E. Targeting of retrovirus to specific cell types

A number of approaches have been directed to develop retroviral vectors that are able to target particular cell types; also efforts focus toward retroviral vectors that incorporate nonretroviral features and are tailored to desired needs for specific uses (reviewed by Vile and Russell, 1995; Gunzburg and Salmons, 1996).

Ideally, therapeutic genes should be delivered only to the relevant cell type and/or expressed in this cell type. Viral and nonviral vectors can be targeted through ligand-receptor interactions. Retroviral targeting through protease-substrate interactions has also been described; epidermal growth factor (EGF) was fused to a retroviral envelope glycoprotein via a cleavable linker comprising a factor Xa protease recognition signal. Vector particles displaying the cleavable EGF domain could bind to EGF receptors on human cells but did not transfer their genes until they were cleaved by factor Xa protease (Nilson et al, 1996).

A retroviral vector that infects human cells specifically through recognition of the low density lipoprotein receptor has been described by adding onto the ecotropic envelope protein of M-MLV a single-chain variable fragment derived from a monoclonal antibody recognizing the human LDL-R; the chimeric envelope protein was used to construct a packaging cell line producing a retroviral vector capable of transfer of the lacZ gene to human cells expressing LDL-R (Somia et al, 1995).

F. Other retroviruses

Viruses that contain RNA as their genetic material may be either negative- or positive-strand RNA viruses. The very large group of negative-strand RNA viruses includes some of the most serious and notorious pathogens subdivided into those with segmented RNA (influenza viruses, comprising eight separate segments of RNA and bunyaviruses containing three segments of single-stranded RNA, the large, L, the medium, M, and the small, S) and those with nonsegmented RNA (VSV, rabies, measles, Sendai, respiratory syncytial virus, Ebola viruses). Positive-strand RNA viruses include poliovirus.

Cloned positive-strand poliovirus cDNA is infectious but neither isolated genome nor antigenome RNA of negative-strand viruses is infectious; this is because the negative-strand viral RNA is assembled with viral nucleoprotein into an RNP complex that becomes the template for the viral RNA-dependent RNA polymerase. Helper influenza virus-dependent procedures have been developed in which an influenza virus-like RNA molecule, containing a reporter gene, was mixed with disrupted virion core proteins to reconstitute RNP complexes in vitro which were then transfected into influenza virus-transfected cells. Recombinant nucleocapsid and polymerase proteins for the unsegmented RNA viruses have also been used to produce infectious virus without help from an homologous virus using full-length cDNA clones of intracellularly transcribed antigenomes (rabies, VSV, measles, Sendai) (see Bridgen and Elliott, 1996 and the references cited therein).

Plasmids containing full-length cDNA copies of the three RNA genome molecules of Bunyamwera bunyavirus and a negative-sense copy of the GFP gene, flanked by T7 promoter and hepatitis delta virus ribozyme sequences, were used to produce infectious virus particles without helper virus; these plasmids were used to transfect HeLa cells which expressed T7 RNA polymerase and recombinant Bunyamwera bunyavirus proteins by previous transfection with the appropriate plasmids; 24 h after infection about 1 in 1,000 HeLa cells displayed fluorescence indicative of transcription and replication of the reporter RNA (Bridgen and Elliott, 1996).

III. Adenoviral gene delivery

A. Adenovirus replication, transcription, and attachment to the nuclear matrix

Before understanding the principle of adenoviral gene transfer, it is essential to comprehend the molecular events which are involved in the life cycle of the adenovirus. Adenoviruses posses a well-defined origin of replication which is stimulated by transcription factors NFI and NFIII (Hay, 1985; Pruijn et al, 1986). The transcription factor NF-I (also called CTF, CCAAT box-binding protein, or C/EBP) stimulates replication of adenovirus DNA in vitro (Pruijn et al, 1986; Jones et al, 1987; Santoro et al, 1988; Coenjaerts et al, 1991) by establishing cooperative interactions with Ad-DBP (Adenovirus DNA-binding protein) (Cleat and Hay, 1989). The transcription factor NFIII (also called Oct-1 or OTF-1), involved in the regulation of the histone H2B and immunoglobulin genes, can stimulate initiation of adenovirus DNA replication in vitro (O'Neil et al., 1988; Mul et al, 1990; Verrijzer et al, 1990; Coenjaerts et al, 1991).

The adenovirus 5 protein Ad-DBP is a single-stranded DNA binding protein product of the viral E2A absolutely required for chain elongation during Ad5 DNA replication; other than facilitating unwinding of the DNA, Ad-DBP might also protect single-stranded DNA at the replication fork from nuclease attack, increase the rate of processivity of the viral DNA polymerase, and increase binding of NFI of the core origin of Ad5 (Cleat and Hay 1989). This protein has a size of 529 amino acids, is phosphorylated and apart from its role in DNA replication is also involved in transcription, recombination, transformation, and virus assembly (see Tucker et al 1994). Crystal structure at 2.6 A resolution of Ad-DBP shows that a 17 aa C-terminal domain hooks onto a second Ad-DBP molecule thus promoting its cooperativity during DNA binding; Ad DBP was proposed to act by forming a core around which single-stranded DNA winds (Tucker et al, 1994).

Adenoviruses replicate episomally; they need to attach to the nuclear matrix of the host cell for their replication. Two adenoviral proteins have been found attached to the nuclear matrix and presumably mediating the anchorage of the adenovirus: (i) the E1a protein (11 kDa), a transcription and replication factor sufficient to immortalize primary rodent cells, which was crosslinked to matrix proteins with oxidation with o-phenanthroline/Cu²⁺ (Chatterjee and Flint, 1986) and (ii) the adenovirus terminal protein (55 kDa) which is covalently attached to the 5' end of Ad DNA and initiates DNA replication; the adenovirus terminal protein mediated adenovirus anchorage to nuclear matrix was resistant to 1M guanidine extraction (Bodnar et al, 1989; Schaack et al, 1990; Fredman and Engler, 1993).

Three types of internal matrix structures were recognized in HeLa cells infected with adenovirus 2; an amorphously dense region; granular regions representing virus capsid assembly structures; and filaments connecting these regions to one another and to the peripheral lamina (Zhonghe et al, 1987); the perinuclear matrix was also rearranged after adenovirus infection.

Electron micrographs of thin sections through nuclei of adenovirus-infected HeLa cells showed that the ³H-deoxyuridine grains were located at the periphery as well as in the interior of nuclei. Simultaneous visualization of adenovirus transcription and replication showed that the two processes occurred in adjacent, yet distinct, foci throughout the interior and periphery of nuclei presumably in association with the nuclear matrix; DNA molecules were found to be displaced from the replication foci and to become spread in the surrounding nucleoplasm serving as templates for transcription (Pombo et al, 1994).

Adenovirus infection provokes dramatic rearrangements to the nuclear matrix. A reorganization in both internal and peripheral NM was also observed in HeLa cells after infection with adenovirus 2 giving structures able to support the increased replication demands and capsid assembly of the virus (Zhonghe et al, 1987). Splicing of adenoviral HnRNA takes place on the nuclear matrix. All adenovirus 2 polyadenylated RNAs could be UV crosslinked to two host HnRNP proteins that are involved in the association of HnRNA to the matrix (Mariman et al, 1982).

Adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur; although the rAAV genome was faintly detectable in a perinuclear distribution after successfully entering the cell, AAV was mobilized to the adenovirus replication centers when the cell was infected with adenovirus; thus AAV colocalizes with the adenovirus replication centers (Weitzman et al, 1996).

B. Adenovirus E1A and E1B proteins in apoptosis and control of the host cell cycle

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. In response, many cell types commit suicide after viral infection to protect the organism from further infection. Striking back, viruses have evolved mechanisms to prevent infected cells from perishing using mechanisms that inhibit apoptosis of the host cell; adenoviruses synthesize the 19 kDa E1B protein which has a domain similar to that of the cellular protein Bcl-2, the apoptosis inhibitor (Sarnow et al, 1982; van den Heuvel et al., 1990). p53 can be complexed with adenovirus E1B (Sarnow et al, 1982; van den Heuvel et al., 1990).

Expression of the adenovirus E1A protein stimulates host DNA synthesis and induces apoptosis; on the contrary E1B 19 kDa associates with Bax protein and inhibits apoptosis (Figure 1). The E1A oncogene of adenovirus exerts its effect via p53 protein (Debbas and White, 1993; White, 1993). Indeed, expression of E1A increases the half-life of p53 resulting in accumulation of p53 molecules in adenovirus-infected cells leading to apoptosis. Although induction of host DNA synthesis by E1A provides a suitable environment for virus replication, the induction of apoptosis by the same protein impairs virus production since virus-infected cells are eliminated (see Han et al, 1996 for references). p53-deficient cells are transformed by E1A because of absence of the pathway for induction of apoptosis by p53 (Lowe et al, 1994).

E1A represses HER-2/neu transcription and functions as a tumor suppressor gene in HER-2/neu-overexpressing cancer cells. Transfer the E1A gene into cancer cells that overexpress HER-2/neu is an interesting aspect of gene therapy (see E1A in gene therapy; Yu et al, 1995; Chang et al, 1996; Ueno NT et al, 1997; Rodriguez et al, 1997; Xing et al, 1997).

The E1B oncogene products inhibit apoptosis induced by E1A expression thus preventing premature death of host cells during adenovirus infection. This gives an advantage to virus for its proliferation and E1B proteins (19 kDa and 55 kDa) are necessary for transformation of primary rodent cells by E1A. E1A alone is unable to transform primary rodent cells (White, 1993).

The E1B 19K protein of adenovirus is the putative viral homolog of the cellular Bcl-2 protein; using the yeast two-hybrid system for the identification of proteins interacting with E1B, Han and coworkers (1996) have identified Bax as one of the seven 19k-interacting clones. The 50-78 amino acid domain of Bax contains a conserved region homologous to Bcl-2 which is able to interact specifically with either Bcl-2 or E1B. In p53 mutant cells expression of Bax induced apoptosis; inhibition of apoptosis by Bcl-2 may proceed via its ability to bind the death-promoting Bax protein (Han et al, 1996). The bax gene is upregulated by p53.

Expression of p53 and of adenovirus E1A induce apoptosis (Debbas and White, 1993; Lowe and Rudley, 1993). 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). 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). 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.

The transcription factor E2F was originally identified as an activator of the adenovirus E2 gene and is implicated in the regulation of DNA replication (Shirodkar et al., 1992). Following infection of cells with adenovirus, the DNA binding activity of E2F increases and as a consequence transcription of the E2 gene of adenovirus increases (Kovesdi et al., 1987). These changes in E2F are mediated by E1A protein of adenovirus. RB forms specific complexes with E2F keeping E2F in a form unable to upregulate its target regulatory sequences. E2F can form specific complexes also with cyclin A during S-phase in NIH 3T3 cells (Mudryj et al., 1991). Both types of complexes, E2F-RB and E2F-cyclin A, can be dissociated by the adenovirus E1A protein (Chellappan et al., 1991; Bagchi et al., 1990; reviewed by White, 1998 this volume) but also by phosphorylation of RB at G1/S causing release of E2F and stimulation in transcription of genes required for DNA replication (myc, DHFR). These events contribute to the uncontrolled proliferation of adenovirus-transformed cells (Mudryj et al., 1990, 1991). Release of E2F from RB induced by E1A is critical for transformation of cells by E1A (for references see Hiebert et al, 1995).

C. Strategies of adenoviruses to enter the cell

In order to enter the host cell the adenovirus first attaches with a high affinity to a cell surface receptor, whose nature still remains elusive, using the head domains of the protruding viral fibers; the fibronectin-binding integrin on the cell surface then associates with the penton base protein on the adenovirus triggering endocytosis of the virus particle via coated pits and coated vesicles (Svensson and Persson, 1984; Greber et al, 1996). The third step in adenovirus entry into the host cell includes

AAV-mediated delivery of the lacZ gene by direct injection to brain tumors which were induced from human glioma cells in nude mice showed that 30-40% of the cells along the needle track expressed b-galactosidase; subsequent delivery of the HSV-tk/IL-2 genes to these tumors with AAV and administration of GCV to the animals for 6 days resulted in a 35-fold reduction in the mean volume of tumors compared with controls by a significant contribution from the bystander effect (Okada et al, 1996).

A phase I clinical trial for CF is being conducted at Johns Hopkins Hospital using AAV (see Kearns et al, 1996, and protocols #165, 166 in Appendix 1).

V. Herpes Simplex Virus-1 (HSV-1) and miniviral vectors

HSV-1 has a capacity of inserting up to 30 kb of exogenous DNA which is a clear advantage over the adenovirus (up to 7.5 kb of exogenous DNA). High titer viral stocks can be prepared from HSV-1. HSV-1 also displays a wide range of host cells and can infect nonreplicating cells such as neuron cells in which the vectors can be maintained indefinitely in a latent state. However, infection with HSV-1 is cytotoxic to cells because of residual viral proteins produced by the virus. Strategies to circumvent this drawback led to the development of viral vectors with a very large capacity for insertion (almost as large as the size of the virus) which depend on defective helper virus for replication and packaging into infectious virions (see below). A miniviral vector can combine the advantage of cloning the gene in bacterial plasmids, the high efficiency of virus-mediated gene transfer, and the possibility to transfer large genomic DNA fragments including far upstream, downstream and intronic regulatory elements.

The HSV-1 genome is a 152 kb double-stranded DNA containing three origins of replication and encoding at least 72 unique proteins; it consists of a unique long segment replicated from oriL and two repeats flanking the unique segment each replicated from oriS. Spaete and Frenkel (1982) have constructed plasmids containing the lytic viral origin of replication, foreign DNA inserts, and the terminal packaging signal sequences; in the presence of a wild-type helper virus such an amplicon was amplified into multimeric tandemly-repeated forms of the original vector by rolling-circle replication and was packaged into infectious HSV virions (Spaete and Frenkel, 1982). However, the helper virus caused death of the infected cells due to lytic replication and this system is not amenable to gene therapy.

To circumvent this bottleneck two strategies have been developed leading to replication-defective helper HSV: (i) a temperature-sensitive system permitted production of virion stocks at 31^o C whereas infection of cells at 37^o C caused inactivation of the helper virus which was incapable of entering the lytic cycle and allowed delivery of the miniviral vector to the target cell without causing its death. (ii) In a different system, the immediately early gene IE3 was deleted from the helper virus; IE3 encodes for a protein (ICP4) essential for early and late viral gene expression and replication; the helper cell line used for packaging had a genomic insertion of the IE3 gene of HSV which was functionally expressed allowing for complementation and for lytic infection using the IE3-defective HSV virus (DeLuca and Schaffer, 1987; Geller and Freese, 1990).

Two types of viral vectors have been used for gene transfer to cancer cells: replication-incompetent vectors expressing a gene product that leads to the destruction of the tumor or replication-competent vectors that are inherently cytotoxic to the tumor cells. In order to combine the two modes of action Miyatake et al (1997) used a defective HSV vector that consisted of a defective particle, containing tandem repeats of the HSV-tk gene, and a replication-competent, non-neurovirulent HSV mutant as a helper virus. When glioma GL261 cells were infected with the tk-defective vector/helper virus the HSV-TK activity was significantly higher than that in helper virus-infected cells which contained a single copy of HSV-tk; subcutaneous injection of these cells to C57BL/6 mice inducing gliomas led to a significant decrease in tumor size after GCV treatment.

An HSV-1 vector containing a 6.8-kb fragment of the rat tyrosine hydroxylase promoter (pTHlac) supported a seven- to 20-fold increase in reporter gene expression in catecholaminergic cell lines compared to noncatecho-laminergic cell lines. Furthermore, 4 days after stereotactic injection into the midbrain of adult rats and for a duration of 6 weeks, pTHlac supported a 10-fold targeting of b-galactosidase expression to tyrosine hydroxylase-expressing neurons in the substantia nigra pars compacta compared with pHSVlac; this long term expression was significant compared to that from pHSVlac which decreased approximately 30-fold between 4 days and 6 weeks after gene transfer (Song et al, 1997); this study also shows the importance of large control regions in the order of 7 kb in sustaining cell type-correct gene expression, something feasible with HSV and liposomes but nor with recombinant retrovirus, adenovirus, or AAV.

VI. HIV vectors for gene transfer

Recent studies have succeeded in exploiting the deadly HIV-1 virus, after crippling some functions, as a gene delivery vehicle. An advantage of HIV vectors has been the broad range of tissues and cell types they can transduce, a property granted because lentiviral vectors are pseudotyped with vesicular stomatitis virus G glycoprotein. Human lentiviral (HIV)-based vectors can transduce non-dividing cells in vitro and deliver genes in vivo; expression of transgenes in the brain has been detected for more than six months. HIV vectors have been also used to introduce genes directly into liver and muscle; 3-4% of the total liver tissue was transduced by a single injection of 1-3 x 10⁷infectious units (I.U.) of recombinant HIV with no inflammation or recruitment of lymphocytes at the site of injection. Whereas expression of green fluorescent protein (GFP), used as a surrogate for therapeutic protein, was observed for more than 22 weeks in the liver and for over 8 weeks in the muscle using lentiviral vectors, little or no GFP could be detected in liver or muscle transduced with the Moloney murine leukemia virus (Mo-MLV), a prototypic retroviral vector (Kafri et al, 1997).

The development of a stable noninfectious HIV-1 packaging cell line capable of generating high-titer HIV-1 vectors is another important step towards use of HIV vectors in gene therapy (Corbeau et al, 1996). A hybrid murine leukemia virus-based vector containing U3 and R sequences from HIV-1 in place of the MLV U3 and R regions gave single transcriptional unit retroviral vectors under the control of Tat; this vector has advantages for anti-HIV gene therapy (Cannon et al, 1996).

Although replication-incompetent HIV vectors displayed a strict CD4⁺ T cell tropism for gene transfer, a feature important for AIDS therapy, it was thought to preclude HIV-based vectors for other gene transfer applications; a two-step gene transfer system, however, was developed to expand the host range of the HIV vector: in the first step, the CD4 gene was introduced into target cells using a replication-defective adenoviral vector; in the second step the CD4-transfected cells were incubated with HIV vectors which resulted in stable integration and HIV-mediated gene transfer (Miyake et al, 1996).

An HIV multiply attenuated vector in which the virulence genes env, vif, vpr, vpu, and nef were deleted was able to deliver genes in vivo into adult neurons (Zufferey et al, 1997).

HIV-mediated gene transfer was used to transfer the GFP gene under control of CMV to retinal cells by injection into the subretinal space of eyes in rats; the GFP gene was efficiently expressed in both photoreceptor cells and retinal pigment epithelium; predominant expression in photoreceptor cells was achieved using the rhodopsin promoter. The transduction efficiency was high and photoreceptor cells in >80% of the area of whole retina were expressing GFP (Miyoshi et al, 1997).

VII. Epstein-Barr virus (EBV) and baculovirus vectors

EBV is an episomaly-replicating virus in synchrony with the cell cycle. EBV infects human cells causing mononucleosis; the presence of the unique latent origin of replication (oriP) in EBV allows for episomal replication of the virus in human cells without entering the lytic cycle. The presence of oriP and of the replication initiator protein EBNA1 cDNA on a vector allows episomal replication in human cells; in addition, plasmids containing only oriP can replicate episomally into cell lines expressing EBNA-1 (Sun et al, 1994; Banerjee et al, 1995).

A hybrid HSV-1/EBV vector has been developed by Wang and Vos (1996), which combines (i) the HSV-1 lytic oriS; (ii) an HSV-1 packaging sequence which allows replication and packaging in the presence of defective helper virus carrying a deletion in the IE3 gene in the E5 cell line expressing the IE3 gene; (iii) the latent oriP of EBV and (iv) the EBNA-1 cDNA allow episomal replication of the infectious vector in the E5 cell line so that viral stocks of high titer can be made. Infection of tumor-derived fibroblast and epithelial cell lines in culture and local injection of human liver tumors in nude mice was used to demonstrate 95-99% efficiency of infection and transfer of the reporter b-galactosidase gene.

Genetically modified baculoviruses (Autographa californica nuclear polyhedrosis virus) were used to efficiently deliver genes into cultured hepatocytes of different origin; delivery into human hepatocytes with baculovirus vectors approached 100% efficiency in cell culture and expression levels were high when mammalian promoters were chosen. A number of drawbacks preclude their direct application in vivo; nevertheless gene transfer was feasible in ex vivo perfused human liver tissue (Sandig et al, 1996; Hofmann et al, 1998 this volume).

VIII. Liposomal gene delivery

Abbreviations:

DC-CHOL: 3b [N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol

DDAB: dimethyldioctadecyl ammonium bromide

DMRIE: N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammonium bromide

DMTAP: 1,2-dimyristoyl-3-trimethylammonium propane

DOGS: Dioctadecylamidoglycylspermine (Transfectam, Promega)

DOPE: dioleyl phosphatidylethanolamine

DOSPA: 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl -1-propanaminium trifluoroacetate

DOTAP: N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride

DOTMA: N-[1-(2,3-dioleyloxy) propyl]-n,n,n-trimethylammonium chloride

DPTAP: 1,2- dipalmitoyl-3-trimethylammonium propane

DSTAP: 1,2-disteroyl-3-trimethylammonium propane

Lipofectin: DOTMA:DOPE 1:1 (GIBCO BRL)

A. Immune responses and toxicity of cationic lipid-DNA complexes

Cationic lipids have been widely used for gene transfer; a number of clinical trials (34 out of 220 total RAC-approved protocols as of December 1997) use cationic lipids (see Table 4 in Martin and Boulikas, 1998, this volume, pages 203-206). Although many cell culture studies have been documented, systemic delivery of genes with cationic lipids in vivo has been very limited. All clinical protocols use subcutaneous, intradermal, intratumoral, and intracranial injection as well as intranasal, intrapleural, or aerosol administration but not i.v. delivery because of the toxicity of the cationic lipids and DOPE (see Table 4 in Martin and Boulikas, 1998, this volume, pages 203-206).

Liposomes formulated from DOPE and cationic lipids based on diacyltrimethylammonium propane (dioleoyl-, dimyristoyl-, dipalmitoyl-, disteroyl-trimethylammonium propane or DOTAP, DMTAP, DPTAP, DSTAP, respectively) or DDAB were highly toxic when incubated in vitro with phagocytic cells (macrophages and U937 cells), but not towards non-phagocytic T lymphocytes; the rank order of toxicity was DOPE/DDAB > DOPE/DOTAP > DOPE/DMTAP > DOPE/DPTAP > DOPE/DSTAP; the toxicity was determined from the effect of the cationic liposomes on the synthesis of nitric oxide (NO) and TNF-a produced by activated macrophages (Filion and Phillips, 1997).

Another factor to be considered before i.v. injections are undertaken is that negatively charged serum proteins can interact and cause inactivation of cationic liposomes (Yang and Huang, 1997). Condensing agents used for plasmid delivery including polylysine, transferrin-polylysine, a fifth-generation poly(amidoamine) (PAMAM) dendrimer, poly(ethyleneimine), and several cationic lipids (DOTAP, DC-Chol/DOPE, DOGS/DOPE, and DOTMA/DOPE) were found to activate the complement system to varying extents. Strong complement activation was seen with long-chain polylysines, the dendrimer, poly(ethyleneimine), and DOGS; complement activation was considerably reduced by modifying the surface of preformed DNA complexes with polyethyleneglycol (Plank et al, 1996).

B. Mechanism of liposome entry to cells

Cationic lipids increase the transfection efficiency by destabilizing the biological membranes including plasma, endosomal, and lysosomal membranes; indeed, incubation of isolated lysosomes with low concentrations of DOTAP caused a striking increase in free activity of b-galactosidase, and even a release of the enzyme into the medium demonstrating that lysosomal membrane is deeply destabilized by the lipid; the mechanism of destabilization was thought to involve an interaction between cationic liposomes and anionic lipids of the lysosomal membrane, allowing a fusion between the lipid bilayers; the process was less pronounced at pH 5 than at pH 7.4 and anionic amphipathic lipids were able to prevent partially this membrane destabilization (Wattiaux et al, 1997).

In contrast to DOTAP and DMRIE which were 100% charged at pH 7.4, DC-CHOL was only about 50% charged as monitored by a pH-sensitive fluorophore; this difference decreases the charge on the external surfaces of the liposomes and was proposed to promote an easier dissociation of bilayers containing DC-CHOL from the plasmid DNA and an increase in release of the DNA-lipid complex into the cytosol from the endosomes (Zuidam and Barenholz, 1997).

C. Tissue targets using cationic liposomes in vivo

Although cationic lipids have been used widely for the delivery of genes very few studies have used systemic i.v. injection of cationic liposome-plasmid complexes because of the toxicity of the lipid component and certainly in animal models, not humans. Administration by i.v. injection of two types of cationic lipids of similar structure, DOTMA and DOTAP, has shown that the transfection efficiency was determined mainly by the structure of the cationic lipid and the ratio of cationic lipid to DNA; the luciferase and GFP gene expression in different organs was transient, with a peak level between 4 and 24 hr, dropping to less than 1% of the peak level by day 4 (Song et al, 1997).

Figure 5 shows the effect of cationic lipid:DNA ratio on transfection efficiency after i.v. tail injection. Luciferase activity was detected in all organs examined with the highest level in lung. In the absence of neutral lipid both DOTMA and DOTAP promoted a linear increase in luciferase activity in the lung with increasing lipid:DNA from 12:1 to 36:1 nmol lipid: mg of DNA. DOTMA was 10 times more efficient than DOTAP (10⁶ versus 10⁷ relative luciferase units (RLU) per mg protein. Cholesterol (Chol) mixed with DOTMA (1:1 molar ratio) decreased the level of gene expression in the lung whereas cholesterol did not affect the transfection efficiency of DOTAP liposomes. Inclusion of DOPE into either DOTAP or DOTMA liposomes significantly decreased the transfection efficiency by 100-fold in the lung.

When a group of four cationic lipids with identical head group but of different fatty acyl chains were tested for their transfection efficiencies (Figure 6); these included DOTAP, DMTAP, DPTAP, and DSTAP. The C₁₄ acyl chain-lipid DMTAP had a similar transfection efficiency as DOTAP which has 18 carbon atoms in the acyl chain and one double bond (C₁₈D₉); on the contrary, the transfection efficiencies of DPTAP (C₁₆) was 10-100 fold lower and that of DSTAP (C₁₈) was 100 to 1000 fold lower.

Confocal microscopy of lung tissue after injection of 25 mg pCMV-GFP plasmid DNA complexed with DOTMA liposomes to mice (Figure 7) has shown that the type of cells that express the transgene are the endothelial cells that have typical characteristics of neighboring multiple air-sac structures (Figure 7D).

A number of different organs in vivo can be targeted after liposomal delivery of genes or oligonucleotides. Intravenous injection of cationic liposome-plasmid complexes by tail vein in mice targeted mainly the lung and to a smaller extend the liver, spleen, heart, kidney and other organs (Zhu et al, 1993). Intraperitoneal injection of a plasmid-liposome complex expressing antisense K-ras RNA in nude mice inoculated i.p. with AsPC-1 pancreatic cancer cells harboring K-ras point mutations and PCR analysis indicated that the injected DNA was delivered to various organs except brain (Aoki et al, 1995).

A number of factors for DOTAP:cholesterol/DNA complex preparation including the DNA:liposome ratio, mild sonication, heating, and extrusion were found to be crucial for improved systemic delivery; maximal gene expression was obtained when a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size were used. Cryo-electron microscopy showed that the DNA was condensed on the interior of invaginated liposomes between two lipid bilayers in these formulations, a factor that was thought to be responsible for the high transfection efficiency in vivo and for the broad tissue distribution (Templeton et al, 1997).

Steps to improve for successful liposome-mediated gene delivery to somatic cells include persistence of the plasmid in blood circulation, port of entry and transport across the cell membrane, release from endosomal compartments into the cytoplasm, nuclear import by docking through the pore complexes of the nuclear envelope, expression driven by the appropriate promoter/enhancer control elements, and persistence of the plasmid in the nucleus for long periods. A number of strategies for liposomal delivery and for enhancing the efficiency of uptake by the cells and release from endosomal compartments of plasmid or oligonucleotide DNA are reviewed in the following article (Martin and Boulikas, 1998).

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

by T lymphocytes thus eliminating the therapeutic effect after reimplantation (e.g. Yang et al, 1994). It was thought that this antigenicity arises from the adenoviral proteins expressed in transduced cells; however, recent data have demonstrated that antigenicity could also arise from the expression of the therapeutic recombinant protein (see above).

Ex vivo approaches have concentrated on correction of mutated genes involved in purine metabolism including adenosine deaminase (ADA) deficiency in severe combined immunodeficiency (SCID) patients, PNP (purine nucleoside phosphorylase) deficiency, and the therapy of Lesh-Nyhan syndrome caused by a deficiency in hypoxanthine-guanine phosphoribosyltransferase (HG-PRT). The first human trial to be approved for ex vivo gene therapy was for the treatment of ADA deficiency which began in 1990 (Karlsson, 1991; Ferrari et al, 1991). Ex vivo studies include transfer of factor IX gene in skin fibroblasts from hemophilia B patients in China followed by subcutaneous injection of the cells to the patient (Wilson et al, 1992; reviewed by Anderson, 1992). 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 vector was integrated and the ADA gene was expressed for long periods (Blaese et al, 1995; Bordignon et al, 1995).

A clinical protocol for the therapy of amyotrophic lateral sclerosis uses a semipermeable membrane to enclose the ex vivo modified xenogenic BKH cells; the membrane is implanted intrathecally to provide human ciliary neurotrophic factor (Deglon et al, 1996; Pochon et al, 1996). An ex vivo clinical trial on humans, homozygous for mutations in the LDL receptor gene, is performed using cultured hepatocytes from the patient which are transduced ex vivo with the LDL receptor gene and transplanted by infusion into the portal vein of the patient (Wilson et al, 1992; Grossman et al, 1994).

Cancer immunotherapy uses transfer of cytokine genes (IL-2, IL-7, IFN-g, GM-CSF) to autologous (cancer patient’s) cells followed by immunization of the patient to elicit activation of tumour-specific T lymphocytes capable of rejecting tumour cells from the patient, especially on immunoresponsive malignancies such as melanomas, colorectal carcinomas, and renal cell carcinomas (Uchiyama et al, 1993; Chang et al, 1996; Finke et al, 1997; Das Gupta et al, 1997; Mahvi et al, 1997).

Table 5. Ex vivo studies on animal models

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

Table 6. In vivo somatic gene transfer strategies to animal models

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

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)

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