Gene therapy for vascular diseases

adenoviral vectors to a patient on a gene therapy clinical

I. Introduction

trial for ornithine transcarbamylase (OTC) deficiency asGene therapeutics have been proposed as a potential well as the evolution of leukaemia in severe combinednovel therapy for a host of diverse disease that encompass immunodeficiency (SCID) patients involving retroviralacquired conditions such as cancer, cardiovascular disease vectors (Cavazzana-C et al, 2000; Somia et al, 2000; Foxand arthritis as well as monogenic diseases through gene 2003) have highlighted safety issues relating to genereplacement strategies. In theory the concept has seemed delivery vectors. In vascular diseases, successful generelatively simply; in practice, however, gene therapy is therapy will require the following:extremely complex, both technically and clinically. It Identification of the optimal transgene cassette.

requires a multifaceted approach involving identification Expression systems vary considerably for different geneof suitable therapeutic gene(s), identification of a suitable therapy applications. Traditionally strong viral promotersgene delivery vehicle together with the availability of have been used to provide maximal levels of expression insatisfactory pre-clinical models in which to evaluate the a multitude of recipient cell types. However, it ispotential benefit of the gene therapeutic approach, becoming increasing important to supply expressionparticularly against alternative pharmacological therapies, selectively to individual cell types or in a regulatedif available. The issue of long-term safety of gene therapy manner through inducible promoters (such as tetracyclinapproaches is still unclear. To date, major progress at the system (Gossen et al, 1992; Vigna et al, 2002) thusclinical level has been made in defined areas, particular circumventing potentially deleterious effects of transgenecancer, cystic fibrosis, haemophilia and some vascular expression in non-target cell types. Additionally, viraldiseases. These advances have not been without major promoters, particularly the cytomegalovirus immediatedrawbacks. Tragic events involving high dose delivery of

early promoter (CMV IEP) is prone to host-mediated silencing in vivo (De Geest et al, 2000) leading to a shut down in transgene expression, an effect not observed with cell-specific promoters. Further optimisation of expression cassettes can be made through incorporation of introns and enhancers to elevate promoter activity as well as posttranscriptional modifications including the Woodchuck post-transcriptional regulatory element (WPRE) which is thought to act through promoting mRNA stability (Loeb et al, 1999; Zufferey et al, 1999).

Optimisation and evaluation of the gene delivery vehicle. At present the repertoire of gene delivery vectors available for human gene therapy is limited. Traditionally, non-viral vectors such as naked DNA and liposome DNA complexes provide low efficiency gene transfer and are restricted to the delivery of highly potent biological agents, such as angiogenic gene therapy (see below). Improvements in the efficiency of non-viral vectors, such as inclusion of targeting peptides into DNA liposome complexes (Hart et al, 1997; Parkes et al, 2002) have been realised but are still someway from the efficiency of viral vectors. Certain viruses, by virtue of evolution, infect human cells with high efficiency resulting in high potency gene transfer and overexpression of candidate therapeutic genes. For gene delivery to vascular tissues the current armoury of viral vectors includes adenoviruses (Ad), adeno-associated viruses (AAV), lentiviruses and Semliki forest viruses.

Efficient modalities for gene delivery to the target site. Certain vascular diseases, such as vein grafting are optimal for gene therapyapy since the target tissue (i.e. the vein to be grafted) is harvested and is available ex vivo for gene delivery prior to grafting within a clinically relevant time window (approximately 30 minutes). This enables delivery of genes in a safe and efficient manner (Baker et al, 1997; Tamirisa et al, 2002). Due to the short time frame, however, efficient vectors are required. Adenoviral vectors have proven particularly suited for this application (Channon et al, 1997; George et al, 2000; Tamirisa et al, 2002). Conversely, gene delivery to blood vessels in vivo requires the use of devices to allow localised in vivo gene delivery. Specific catheter systems have been developed and utilised with high efficiency for post-angioplasty and in-stent restenosis in a variety of animal species and blood vessels (French et al, 1994; Klugherz et al, 2000, 2002). Additionally, local delivery technology has been applied for gene therapyapy aimed at the myocardium. Different applications, such as atherosclerosis or hypertension require alternate delivery systems and often rely on intravenous vehicle administration.

Together, a combined approach to optimise the gene expression system, the delivery vehicle and the route of delivery are required for successful gene therapy. A number of key areas within vascular diseases have successfully exploited this and advanced to clinical trials while other areas have been severely limited due to deficiencies in one or more of the above requirements. Here, we discuss a number of these applications.

There is no doubt that gene therapy may offer advantages above traditional pharmacological therapies in certain respects. Delivery of gene can be achieved locally in the vasculature thereby increasing the selectivity and, potentially, the safety. This would be particularly important when the therapy may have an adverse effect if contact to non-target tissue in vivo occurred. Since many of the strategies that have been designed to be effective in vascular disease may be deleterious if exposed to nontarget tissue, this advantage becomes very important. For example, in development of gene therapy for vein graft failure (see later) pro-apoptotic genes are highly effective but clearly their expression in other tissues such as the liver, may be detrimental. Likewise, in restenosis postangioplasty (cytotoxic or cytostatic strategies) and angiogenesis gene therapy can be delivered locally and is a pre-requisite for clinical translation. A second (and equally important) advantage of gene therapy might be the requirement for only a single administration compared to the requirement for multiple administrations of conventional drugs, often daily for the lifetime of the patient. Again, this depends largely on the application and is to date unproven. Evidence suggests that beneficial effects of gene therapy for hypertension, vein grafting and restenosis can be elicited in the long term from single administrations (see later). This provides ample preclinical evidence to support these concepts.

In the following review, we discuss gene therapy for some vascular diseases and its progression in different experimental and clinical applications.

II. Local gene delivery to the vessel wall

It has been known for over a decade that gene delivery to the vessel wall can result in alterations in cell behaviour (Nabel et al, 1993 a, b, c) thereby initiating a plethora of studies that have evaluated and optimised gene delivery to the vessel wall. Although the first studies revealed that non-viral gene delivery could lead to phenotypic modulation of cell behaviour, it soon became clear that adenoviral vectors provided the most efficient means to achieve high-level gene delivery to the vessel wall in vivo (Lemarchand et al, 1993; French et al, 1994). Pioneering studies by Lemerchand and colleagues (1993) and French et al, (1994) showed that local exposure of high titre adenoviral vectors to normal and diseased blood vessels in vivo led to high-level transduction, in sheep and rabbit models, respectively. Catheter systems were rapidly developed and optimised for gene delivery postangioplasty resulting in transgene expression throughout the vessel wall in a geographical localisation defined by the mode of vector delivery by the catheter utilised. This initiated a host of studies and led to the use of adenoviral vectors as the most commonly used modality through which to deliver genes to the vessel wall in vivo. However, this is not without limitations since adenoviral-mediated gene delivery was found to evoke an inflammatory response in the vessel wall leading to toxicity and endothelial cell activation (Newman et al, 1995). Furthermore, the use of these first-generation Ad vectors only resulted in transient gene expression lasting 7-14 days. Unlike other tissues, second generation vectors (that contained modifications of the Ad genome to reduce expression of Ad-related genes) did not lead to sustained transgene expression in the vessel wall in vivo (Engelhardt et al, 1994; Wen et al, 2000). Other vector systems have recently been tested including improved non-viral systems such as peptide-targeted DNA/liposome complexes (Hart et al, 1997; Parkes et al, 2002), HVJ-modified liposomes (Morishita et al, 1995; Von Der Leyen et al, 1995; Dzau et al, 1996) and ultrasound-enhanced systems (Lawrie et al, 1999; Taniyama et al, 2002). Likewise, other viral vectors (including adeno-associated viruses (Maeda et al, 1997; Richter et al, 2000), Semliki-forest viruses (Lundstrom et al, 2001) and lentiviruses (Dishart et al, 2003) have been utilised. Modified viral systems in particular provide opportunities to modify the longevity of transgene expression as well as the principle cell type transduced. As an example, adeno-associated viruses (AAV) transduce smooth muscle cells in the vessel wall, even in the presence of an intact endothelial layer (Richter et al, 2000). This is in direct comparison to Ad-mediate delivery since endothelial transduction is high when an intact endothelium is present and represents a barrier to transduction (Lemarchand et al, 1993). This finding may in part be due to different physical sizes of Ad and AAV and due to different vector tropisms of each, which is dictated by host expression of viral receptors and coreceptors (Wickham et al, 1993; Bergelson et al, 1997; Tomko et al, 1997; Summerford et al, 1998; Qing et al, 1999; Summerford et al, 1999; Dishart et al, 2003). Hence, these systems have provided researchers with a diverse range of vectors through which to evaluate the phenotypic effects of overexpression of candidate therapeutic genes in the vessel wall in vivo.

III. Gene therapy and vein graft failure

The failure of vein bypass grafts in the coronary or lower extremity circulation is a common clinical occurrence that incurs significant morbidity and mortality. Despite the very common use of saphenous vein grafts to treat coronary and lower extremity occlusions the failure rate is extremely high, approximately 50% and 70% of vein grafts fail within 5-10 years after surgery, respectively (Angelini 1992; Conte et al, 2001). To date, pharmacological approaches to prolong vein graft patency have produced very limited results. Consequently, genetic approaches to modulate bypass grafts are actively being studied both in vitro and in vivo and are progressing to clinical trials. Vein grafts are uniquely amenable to intraoperative genetic modification because of the ability to manipulate the tissue ex vivo with controlled conditions. We will describe how both gene overexpression and gene blockade strategies have been tested, and how the latter is now in clinical trials (see also Figure 1 for schematic summary of gene therapy strategies).

Figure 2: Adenoviral-mediated gene transfer of TIMP-3 reduced intimal thickening in vein grafts. Transverse sections stained for αsmooth muscle cell actin illustrate that intimal thickening was dramatically reduced in porcine arterio-venous vein grafts at one month by Ad-mediated over-expression of TIMP-3 compared to controls (AdlacZ). White dotted line indicates the intimal/medial boundary.

IV. Gene therapy and restenosis

Treatment of symptomatic coronary artery atherosclerotic plaques by angioplasty leads to vascular responses including intimal thickening and constrictive remodelling causing restenosis in approximately 30% of initially successfully treated patients. Although stents prevent constrictive vascular remodelling, they induce vascular injury eventually leading to intimal thickening and thereby restenosis. Gene therapy has been perceived as attractive to treat restenosis as it can be delivered locally and appears to be able to treat excessive vascular cell proliferation.

To date, a number of small (rat, mice) or large size animal modes (rabbit, pig) have been used to evaluate the potential of many gene therapy approaches for restenosis. The gene therapy strategies for treatment of restenosis are summarized below and also in Figure 1. However, despite the successful use of gene therapy to treat animal restenosis by various approaches, application of gene therapy to prevent restenosis in man has only been carried out using a re-endothelialization strategy with VEGF. Before further clinical trials are initiated a better understanding of vascular biology, gene expression, vector design, and catheter-tissue interactions is required. It must also be mentioned that the efficacy of sirolimus (rapamycin) for the treatment of in-stent restenosis (Serruys et al, 2002; Sousa et al, 2003) has reduced the impetus for designing gene therapy for in-stent restenosis.

VI. Therapeutic angiogenesis

Therapeutic angiogenesis represents a novel strategy for the treatment of vascular insufficiency. It is based on supplementation with angiogenic growth factors to enhance native angiogenesis in critical myocardial or peripheral ischaemia. Angiogenic growth factors have been delivered both as protein and by way of gene transfer and have demonstrated positive results (Yla-Herttuala et al, 2003). The recent insights in the molecular basis of angiogenesis have resulted in great interest in the gene therapy field. However, because of the rapid evolution and enthusiasm in the field, angiogenic molecules have been tested without a complete understanding of their mechanism of action. Among the angiogenic growth factors used in pre-clinical studies, VEGF165 and VEGF121, FGF1, FGF2 and hepatocyte growth factor (HGF) have all shown significant improvement of native angiogenic response to ischemia, resulting in accelerated rate of perfusion, (see reviews by Hammond et al, 2001) (Emanueli et al, 2001; Manninen et al, 2002). Besides growth factors a number of other substances have been investigated, such as human tissue kallikrein (Emanueli et al, 2001), angiopoietin (Shyu et al, 1998), leptin (Bouloumie et al, 1998) and thrombopoietin (Brizi et al, 1999).

Although difficulties have been encountered in the field of gene therapy, great progress has been made in the field of pro-angiogenic gene therapy. It has been suggested that this is because the long-term gene expression is not required for therapeutic vascular growth and the current gene therapy vectors induce at least some physiological improvement (Yla-Herttuala et al, 2003). Over 23 clinical trials have been initiated; approximately half are for peripheral disease and the other half for coronary heart disease. The first set of clinical trials involved pioneering attempts to overexpress VEGF165 with naked DNA (Isner et al, 1996; Baumgartner et al, 1998; Losordo et al, 1998) and adenoviruses (Rosengart et al, 1999). The second phase of trials were small, uncontrolled trials using naked DNA and adenoviruses to overexpress VEGF165 and VEGF121; many of these had positive results (Symes et al, 1999; Laitinen et al, 2000; Rajagopalan et al, 2001). Only recently, the third set of clinical trials has begun to test the potential of this gene therapy fully. These randomised, controlled and blinded trials have involved larger numbers of patients and defined primary and secondary endpoints (Grines et al, 2002; Makinen et al, 2002; Stewart et al, 2002; Hedman et al, 2003; Rajagopalan et al, 2003). Several of these have been judged positive according to primary and secondary endpoints but it has been suggested that this may not be transferable to a clear-cut clinical benefit (Yla-Herttuala et al, 2003).

Critically ischaemic lower limbs from diabetes that are not suitable candidates for surgical endovascular approaches may be amenable to gene therapy for therapeutic angiogenesis. Diabetes impairs endogenous neovascularization of ischaemic tissues due to a reduced expression of VEGF (Rivard et al, 1999) and HGF (Taniyama et al, 2001). Consequently Ad-mediated overexpression of VEGF and plasmid HGF restored neovascularization in mouse and rat models of diabetes, respectively (Rivard et al, 1999; Taniyama et al, 2001). Enhanced angiogenesis by such strategies also improves neuropathy both when growth factors including VEGF, are given alone (Rissanen et al, 2001) or in conjunction with the prostacyclin synthase gene (Koike et al, 2003). Furthermore, a small clinical trial which included 6 diabetic patients with critical leg ischaemia, observed neurologic improvement and therapeutic angiogenesis after plasmid injections of VEGF165 in the muscles of the ischaemic limb (Simovic et al, 2001). Inhibition of angiogenesis may also have therapeutic potential for the treatment of retinopathy, since lentiviral delivery of angiostatin inhibited neovascularization in a murine proliferative retinopathy model (Igarashi et al, 2003).

Although, this strategy has made great progress in the last decade there are still some unresolved issues. For example is administration of a single angiogenic molecule sufficient? Will administration of VEGF lead to toxic effects such as oedema? Will an angiogenic factor be suitable for myocardial and peripheral angiogenesis? Since the same adenoviral VEGF121 gave positive effects in the myocardium (Stewart et al, 2002) but failed in peripheral vascular disease (Rajagopalan et al, 2003), will VEGF be proven clinically benefial? Some caution has been cast on the potential of VEGF gene therapy by the observation that VEGF enhances atherosclerotic plaque progression in both mice and rabbits (Celletti et al, 2001). Are other VEGF homologues safer options? Increased lymphogenesis and reduced oedema is observed with VEGFC and VEGFD (Yla-Herttuala et al, 2003).

VII. Future directions

Recent advances through preclinical studies have raised the profile of gene therapy in some vascular diseases, particularly with respect to angiogenic gene therapy in the myocardium and peripheral vasculature as well as in vein graft disease. These studies, presently in phase II, highlight the potential of the technology for relieving symptoms of human vascular diseases.

Despite the lack of dramatic cures, a decade of clinical trials has provided important news about the strengths and weaknesses of current vectors. Both adenoviruses and liposomal vectors have been shown to be able to transduce transgenes in patients with a variety of disorders. From this work, it is now extremely clear that the expression is temporary and is associated with an inflammatory response. However, there are some important points to consider. First, with respect to myocardial and peripheral vascular gene transfer clinical trials, these have been performed with single pro-angiogenic genes with gene delivery using sub-optimal vector systems (e.g. naked DNA/adenoviral vectors). With respect to the former, angiogenic gene therapy may be significantly more therapeutic with respect to collateral vessel formation with a combination of therapeutic genes rather than single gene therapy strategies. With recent advances in adenoviral vector technology [e.g. using "gutted" adenoviral vectors (Kochanek et al, 1996; Parks et al, 1996)] the cloning capacity required for such studies is now available. Equally, the gutted adenoviral vector systems are less immunogenic in vivo and would allow longer term overexpression of transgenes that in turn may promote sustained angiogenic effects. It is known that vascular cell uptake by these vectors (all based on serotype 5 adenoviruses) is extremely poor in comparison to other cells, such as hepatocytes in the liver (Nicklin et al, 2001). Indeed, pre-clinical experiments have shown that local delivery of adenoviruses serotype 5 vectors to the vasculature leads to virion dissemination, not only to the liver but also to testes and other organs posing additional safety concerns (Hiltunen et al, 2000; Baker, 2002).

Given the limited ability of liposomes and adenoviruses to enable long-term gene expression, and given the poor in vivo performance of retroviruses, the AAV vectors are being developed. This virus is smaller than the adenovirus and has a relatively low-capacity size. However, it allows for long-term gene expression (ie, months to years) with only minimal induction of inflammation or antiviral immune responses. A better understanding of the life cycle of this virus, along with improved production techniques, has allowed investigators to conduct clinical trials with AAV in diseases such as hemophilia and cystic fibrosis (see http://www.wiley.co. uk/wileychi/genmed/clinical/). Preclinical data in mice injected intramuscularly with an AAV-human alpha-1antitrypsin (1AT) vector are encouraging (Xiao et al, 1998; Phillips et al, 2002).

To date, the major problem in gene therapy remains the relative inefficiency of current vectors. Currently, this inefficiency, coupled with a relatively poor specificity of most vectors, requires the delivery of large doses of vector. This is both expensive and more likely to lead to side effects. Pathophysiological questions still remain about which and how many cells need to be transduced to obtain a clinical response. One new and very exciting area of gene therapy that has not yet reached clinical trials is the "gene correction" (Gamper et al, 2000; Metz et al, 2002). It is possible to design oligonucleotides that bind to areas of single-nucleotide changes that are associated with abnormal functions and to catalyze corrections of the nucleotide errors. This concept clearly has been demonstrated to work in cell cultures and in animal models, although the efficiency is still quite low. With the development of better oligonucleotides and improved delivery methods, this approach will likely be tested first in diseases such as hemophilia and 1AT.

When it is considered that angiogenic gene therapy should be highly localised due to potential side effects [including potentiation of atherosclerosis (Celletti et al, 2001) and development of cancer (Lee et al, 2000)] other vector systems should now be considered. The choice of potential new vectors is broad and must be considered with caution and evaluated based on current knowledge of existing systems (de Nigris et al, 2003). Additional evidence now suggests that the vast majority of AAV genomes remain in a non-integrative capacity within infected cells (Nakai et al, 2001; Schnepp et al, 2003) further supporting the safety of this vector system. Of equal potential are adenoviral vectors originating from different serotypes. Previous pre-clinical data support of the notion that novel vector systems can be isolated for the capacity to efficiently infect an individual tissue type (Havenga et al, 2001, 2002). For example, adenoviruses based on serotype 16 have a high propensity to transduce both endothelial cells and smooth muscle cells than serotype 5 vectors (Havenga et al, 2001). Again, like AAV-2, this may provide a system through which to optimise gene delivery for defined gene therapeutic applications. The use of cell selective promoters (tissue-specific expression) to drive transgene expression will add a further level of selectivity to such systems. The combined use of vectors and immuno-suppressors may be also reasonable.

Gene therapy remains the key link between advances in genetics and genomics and the translation of this knowledge into useful outcomes for patients. Although progress has been slower than hoped for, clear advances are being made; gene therapy will probably find a number of key therapeutic niches. Together, these modifications will enhance the utility and safety of gene therapy as transition from pre-clinical to clinical gene therapy proceeds for the vascular system and its diseases.

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