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Current status of catheter- and stent-based gene therapy

F. Sharif, K. Daly, J. Crowley, T. O'Brien
DOI: http://dx.doi.org/10.1016/j.cardiores.2004.07.003 208-216 First published online: 1 November 2004

Abstract

Significant progress has been made in the field of cardiovascular gene therapy over the past decade. Animal models of human disease have helped in identifying potential therapeutic genes and have also assisted in the evaluation of an ideal vector. A number of percutaneous catheter systems have been used in animal models with limited success. Stents represent an attractive alternative for localized gene delivery, as they provide a platform for prolonged gene elution and efficient transduction of opposed arterial walls. This gene delivery strategy has the potential to decrease the systemic spread of the viral vectors and hence a reduced host immune response. Both synthetic and naturally occurring stent coatings have shown potential to allow prolonged gene elution with no significant adverse reaction. However, further animal studies are required to evaluate the compatibility of stent coatings, vector solutions, and the arterial wall as well as assessment of the feasibility of this approach to achieve the full potential of gene therapy.

Keywords
  • Atherosclerosis
  • Stents
  • Catheters
  • Gene therapy
  • Re-stenosis

Coronary artery disease is the leading cause of mortality and morbidity in the Western population. While atherosclerosis is a systemic disease, focal manifestations may lead to flow limiting lesions in critical areas of the vasculature. Treatment options for these lesions include arterial bypass and/or mechanical intervention by angioplasty, atherectomy and stenting (percutaneous coronary intervention). At present, more than 1 million PCIs are performed worldwide each year.

Stenting has become popular because it provides favorable acute results after angioplasty, as well as improving the safety of the procedure and long-term clinical outcomes as compared to angioplasty alone in native coronary and graft diseases [1]. Polymer-coated stents have already entered into clinical practice as delivery devices for the timed release of small molecules such as anticoagulants, corticosteroids and antimitotic agents [2]. Despite the ever increasing use of coronary stenting along with periprocedural and long-term administration of antiplatelet agents, post procedural restenosis remains a significant problem (10–15% in high risk patients) [3] (Table 1). This drawback of arterial stenting is due to the fact that PTCA/stenting addresses the structural consequences, rather than biological basis of atherosclerosis. It also causes a local vascular injury which is common to all vasculoproliferative disorders including atherosclerosis, in-stent restenosis and vein graft failure. In addition to mechanical intervention, hypertension and hyperlipidaemia also cause vascular injury. This leads to endothelial disruption and platelet activation followed by mural thrombus formation [4,5]. Cytokines and growth factors are released by activated neutrophils and macrophages [6]. These growth factors cause smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) formation. The thrombus formation, constrictive remodeling, neointimal proliferation, and matrix production are the primary contributors to the development of restenosis.

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Table 1

High risk factors for restenosis

Cinical
(1) Diabetes
(2) Unstable angina
(3) Variant angina
(4) Smoking
(5) Dialysis
(6) Hypercholesteremia
Anatomical
(1) Long lesion (>20 mm)
(2) Multivessel
(3) Multilesion
(4) Chronic total occlusion
(5) Vein graft lesion
(6) Ostial lesion
(7) Angulation (>45°)
Procedural
(1) Pressure gradient (>20 mm)
(2) Residual stenosis (>30%)

Vascular gene therapy, like most gene therapy protocols, requires the combination of a therapeutic gene product, an appropriate vector for introduction of these genes into the cells and a device for mechanical delivery of vectors to the target cells. While the former two have received considerable attention, the latter has been relatively neglected. Animal models of cardiovascular diseases are used to assess the feasibility of vascular gene therapy and provide an important step in the development of gene therapy approaches, linking gene discovery, characterization, and clinical experiments. Difficulties with the development of an effective percutaneous gene delivery system to the diseased site in animal models have proven to be a major hurdle to the advancement of vascular gene therapy.

The aim of this article is to review the delivery methods for vascular gene therapy and to summarize the currently available therapeutic genes and vectors for vascular gene therapy. We will also review the potentially treatable vascular targets with stent-based gene therapy.

1 Catheter delivery systems

Percutaneous gene transfer requires the delivery of vector-containing solutions at sufficient concentrations and for adequate periods of time for optimal transfer into the target cells. Since the first successful in vivo gene transfer in 1989 [7], several balloon catheters have been used for vector delivery. However, no catheter system has yet achieved reliable, reproducible and highly efficient percutaneous gene delivery to the blood vessels in animal models.

The main types of catheters used for gene transfer in animal models are next reviewed.

1.1 Double balloon catheters

Double balloon catheters were the first catheters used for vascular gene delivery [8,9]. This catheter includes two inflatable balloons separated by a potential space into which the solution containing a gene transfer reagent can be infused through a separate lumen. After infusion, the vector-containing solution remains in contact with the vessel wall between the proximal and distal occluding balloon. The solution can be withdrawn as required, provided that the run-off through side branches is minimal. The efficiency of gene delivery using this system is directly proportional to the pressure employed [8]. The potential of these catheters for in vivo gene delivery has also been determined in animal models. Efficient transgene expression (30%) up to 4 weeks post transduction has been confirmed with the use of these catheters following adenoviral vector-mediated gene transfer in sheep carotid and femoral arteries [9]. The major drawback of these catheters is the requirement for total occlusion of the blood vessel for prolonged durations in order to deliver the vector with potential for inducing ischemia.

1.2 Porous and microporous catheters

To use a porous balloon catheter for gene therapy, a gene transfer solution is injected into the lumen of a single balloon [10]. This single balloon contains multiple and variable microscopic perforations. On injection the vector solution expands the balloon in close apposition to the vessel wall, then exits from the balloon through the pores and enters the vessel wall under relatively high pressure. Perforated balloon catheters were used to inject retroviral vectors expressing β-galactosidase into rabbit aortas [11]. The viral particles were injected within 1 min. PCR-based protocols to amplify vector-related sequences showed less than a hundred transduced cells in the aortic tissue 5–14 days after injection. Thus, this method is associated with a low efficiency of gene delivery.

1.3 Hydrogel catheters

Gel-coated balloon catheters have also been used for gene delivery. Plasmid DNA and adenoviral vectors have been incorporated into the gel coatings [12]. Expansion of the catheter in vivo has resulted in recombinant gene expression in vascular cells at the site of balloon expansion. This catheter has been used to deliver genes to induce therapeutic angiogenesis. The plasmid DNA encoding three principle human VEGF isoforms (pHVEGF121,165,189) were applied to the hydrogel polymer coating of an angioplasty balloon and delivered percutaneously to the iliac arteries of rabbits [13] resulting in augmented collateral vessel development. In another study, accelerated re-endothelialization with inhibition of neointimal thickening, reduction in thrombogenicity, and restoration of endothelium-dependant vasomotor reactivity were seen with the use of hydrogel catheter-mediated site-specific arterial gene transfer of pHVEGF165 [14].

1.4 Dispatch catheters

Dispatch catheter systems deploy a spiral coil that permits distal blood flow through a central lumen while allowing simultaneous infusion of a vector-containing solution into the closed compartment between the artery wall and catheter. Dispatch catheters have been used successfully for gene delivery in animal models. In a study by Numaguchi et al. [15], these catheters were used for percutaneous delivery of prostacyclin synthase (PGIS) gene and LacZ marker gene, via a plasmid to the balloon injured iliac arteries of rabbits. Stents were deployed after the balloon injury and this was followed by PGIS gene (plasmid 200 μg) delivery by lipotransfection method via the dispatch catheter. This resulted in 4.8% transduction of cells with accelerated re-endothelialization and reduced neointimal formation. However, prolonged contact time (60 min) between plasmid/lipofectamine solution and the stented arterial wall was required to achieve the above results. In another study, Tahlil et al. [16] also reported efficient arterial gene delivery to endothelial and superficial smooth muscle cells. They used an adenoviral vector-carrying LacZ to achieve transduction of 16±8% of endothelial cells in the normal iliac arteries of rabbits. Again, the duration of gene delivery by balloon inflation was 60 min with the use of these catheters. Transgene expression was also detected in the liver of rabbits during this study. The prolonged incubation period, required for adequate gene transfer, in both these studies poses a risk for tissue ischemia especially in the coronary vasculature. Dispatch multichamber catheters have the potential to permit local delivery to the vasculature, as well as allowing distal blood flow by a separate lumen. This however poses an increased potential risk of systemic dissemination of viral vectors. Further animal studies are required to assess the safety of these catheters in the coronary vasculature, as a tool for gene delivery.

1.5 Infiltrator catheters (direct)

The infiltrator angioplasty catheter was designed to permit injection of solutions directly into the arterial wall. It consists of three longitudinal polyurethane pads attached to the balloon at 2, 6, and 10 o'clock, with three linear arrays of microminiaturized injection needles positioned on the pads. Successful gene transfer has been reported with this catheter. The safety of this catheter was tested in pig models. The infiltration procedure did not damage the vessel angiographically or histologically. Rhodamine tracer was evenly distributed through the whole width of the vessel wall 10 min after infiltration. The medial layer revealed mild local thickening without luminal narrowing [17]. Several preclinical studies have established evidence that infiltrator catheters can be used for gene transfer to the vasculature [18,19]. Wang et al. [18] demonstrated successful gene transfer using recombinant adenovirus, encoding inducible nitric oxide synthase in porcine coronary arteries. Stents were deployed post gene transfer. Follow up at 28 days showed significant reduction of neointimal formation in the iNOS-treated group. Again, the limited amount of data on the safety and feasibility of this catheter has curtailed its clinical application.

2 Summary of catheter-based gene delivery to the blood vessel wall

Although catheter-based gene delivery is simple to use and allows vector localization several factors have hampered its efficiency as a tool for vector delivery to the blood vessel wall (Table 2). Most catheters require prolonged total vascular occlusion for efficient gene delivery to the vasculature increasing the risk of myocardial ischemia. They also cause localized vascular injury with increased inflammatory response and neointimal proliferation. The amount of gene vector that can be transferred is limited because of obligatory dilution of the gene in vector solutions and numerous side branches of the coronary arteries which permit run-off of the infused volume. These shortcomings have curtailed their advancement in the field of vascular gene therapy.

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Table 2

Catheter gene delivery systems in animal models

CatheterAdvantagesDisadvantages
Double BalloonSimple to useNot useful in coronary vasculature because of frequent side branches
Allows vector localizationRequires total occlusion of the target vessel
Minimal disruption of blood vessel because of low pressure balloon systemTrauma to endothelium seems necessary for gene delivery to vascular media
No systemic release of vector
Prolonged contact between gene transfer agent and vessel wall
PorousAbility to infuse gene transfer agent into deeper layer of the vessel wallDamage to the vessel wall by jet-like propulsion of the vector
Potential to minimize vessel occlusion timeNot extensively used in animal models for gene delivery
Lack of dependence on ligation of branch vessels at site of delivery
Ability to combine angioplasty and in vivo gene transfer
HydrogelUsed extensively for percutaneous gene deliveryObligatory dilution of the gene transfer vector in the gel
Enhanced gene transfer to the target cellsLimited amount of vector can be incorporated into the gel
Dependence of gene delivery on balloon inflation causes vessel occlusion
DispatchAllows distal blood flow while allowing simultaneous infusion of vectorLimited data available regarding use in trials
Systemic dissemination of viral vectors
Infiltrator CatheterSolutions injected directly into tissueRisk of vessel injury with increased inflammation
Successful gene delivery into deeper tissue possibleMore preclinical data required
No distal ischemia

3 Stents

Endovascular stents represent an ideal platform for localized vascular gene delivery for vascular disorders because of their permanent scaffolding structure. The increasing popularity of stenting as a primary treatment modality for occlusive vascular disease has led to stent advancement in terms of design, allowing for their employment as drug delivery systems. Stents now represent drug reservoirs allowing localized, prolonged release of a therapeutic agent to the blood vessel. The greatest challenge with this delivery system lies in achieving a compatible relationship between the stent, coating matrix, and vessel wall.

The coating is a crucial element to the design of a gene eluting stent. Coatings provide an inert barrier between the metallic stent and the circulating blood. As a result of the long residence times of coatings on the stents, attention has been focused on using them as reservoirs for prolonged local drug administration. Most coatings under investigation are biocompatible and are thought to be less thrombogenic and inflammatory as compared to bare metallic stents, with the potential to reduce neointimal proliferation. While there is much known about stent coatings for drug elution, less is known about the use of these substances for gene elution.

Van der Giessen et al. [20] reported significant inflammatory and neointimal proliferative responses to eight different polymers. One of the limitations of this study was that the polymer was applied in a nonuniform manner with a comparatively thick layer (75–125 μm) which may have predisposed to these responses. More recent studies using polymers as stent coatings in animal models have shown a favourable effect on stent thrombosis with no increased evidence of inflammation [21–26]. These include both synthetic (polyurethane, polylactic–polyglycolic acid (PLGA)) and naturally occurring polymers (collagen and phosphorylcholine). These latter coatings may be preferable for use in the context of gene elution but all studies should evaluate the potential for polymer induced vascular injury.

The elution of the desired gene from the stent coatings and transfer to the cells of arterial wall has been an area of intensive research over the past few years. A number of animal model studies have shown successful gene transfer from the polymeric matrix after stent deployment. Yun-wei-ye et al. [21] first showed significant expression of the LacZ reporter gene in vascular tissue after surgical implantation of Adenovirus-expressing βGal-coated stents in the carotid arteries of rabbits. Klugherz et al. [22] demonstrated that plasmid DNA can be formulated into a DNA delivery stent, using a PLGA coating. They achieved in vivo transfection efficiency of 1.14±0.08%. In another study, the same group reported in vivo pig arterial wall cell transduction efficiency of 5.9±1.1% with a marker gene using adenovirus [23]. They tethered the virus onto the collagen coating with an antiviral monoclonal antibody. This resulted in highly localized gene delivery as compared to nonspecific antibody plus adenovirus control. Perlstein et al. [24] demonstrated enhanced transfection of cells with denatured collagen–PLGA-coated stents containing plasmid DNA. They achieved in vivo transduction efficiency of 10.4±1.23% of neointimal cells. This study was comparable to Klugerz et al.'s study with regard to vector, DNA loading, PLGA component, stents, animal strain, duration of treatment, and operating team. The much higher magnitude of transduction achieved with Perlstein's study (1.1% vs. 10.4%) may be attributable to the use of denatured collagen as a coating which is an αvβ3 integrin ligand targeting specific receptors on smooth muscle cells. This receptor–ligand interaction may explain the higher transfection achieved with the use of this coating.

Takahashi et al. [25] characterized gene delivery properties of a biostable coating (polyurethane) expressing multiple marker genes in the vessel wall. They reported a dose–response relation for transgene expression and also demonstrated the ability of this polymer to deliver transgenes to inflammatory cells at the site of stent implantation. This characteristic has the potential to modulate the activity of immune cells by delivering immunoregulatory genes to reduce inflammation. Wu et al. [26] also demonstrated overexpression of a therapeutic gene, TIMP 3, via stent-based adenoviral delivery, using a high molecular mass phosphorylcholine coating in the porcine coronary arteries. This resulted in increased transduction of cells with TIMP 3 which was associated with significant reduction in neointimal hyperplasia at 28 days in the Ad TIMP 3 group (0.34±017 mm) as compared to the uncoated control group (0.81±0.32 mm). The naturally occurring phosphorylcholine polymer used in this study behaves as an intact tissue element causing improved haemocompatability without causing excessive tissue reaction. The newer positively charged phosphorylcholine coating has the potential to carry and deliver higher molecular weight therapeutic agents. In most of these studies, the bioactive agent (vector and genes) was attached to the stent surface by dipping or spraying the stent.

In conclusion, both synthetic and naturally occurring biopolymers when applied uniformly can lead to site-specific gene expression without excessive inflammation. The ideal coating must have excellent binding properties and targeted release kinetics as well as having sufficient vector capacity to attain an appropriate level of transduction. The task of incorporating naked DNA or viral vector-containing DNA into a polymeric matrix under conditions that would facilitate higher cellular uptake and transgene expression represents a significant challenge.

4 Vector systems

The eluting bioactive agent in the case of gene therapy is a suitable vector carrying a potential therapeutic gene. A multitude of vector systems have been evaluated for the introduction of genes into the vascular tissues. The vascular structure provides several layers of cellular and extracellular components that limit gene delivery, therefore gene transfer requires potent and cell-specific vectors. Vectors for gene transfer include nonviral (plasmid/liposome) and viral vectors which will be reviewed here.

4.1 Plasmids

Naked DNA is the simplest gene delivery vector, consisting of DNA molecule containing the recombinant gene and adjoining DNA sequences that permit its replication as a plasmid in bacterial hosts. Several early studies of direct in vivo vascular gene delivery used plasmid DNA [7,11,27], in complex with liposome-based carriers. The level of transduction achieved with these vectors was low (0.1–1%), even with modification of lipid composition (5% transduction of target cells). However, plasmid-based gene transfer has achieved satisfactory biological effects when used to mediate in vivo expression of potent (VEGF) secreted molecules [14,28]. Inefficient transduction using plasmid-mediated gene delivery may be adequate when a potent secretory transgene product with effects on surrounding cells is used. This may particularly be true when the plasmid is eluted off a stent over a period of time.

Advances in nonviral vector-based gene transfer could have significant advantages, especially because of their enhanced safety profile as compared to viral vectors.

4.2 Retroviruses

Retroviruses were among the first vectors used for in vivo vascular gene transfer [29]. Recombinant retroviruses are RNA viruses that are capable of integrating the transgene into the target genome. This leads to the desirable effect of long-term gene expression. However, random insertion of the virus into the target genome has the potential to trigger neoplastic proliferation by enhancing the activity of proto-oncogenes [30]. Retroviral vectors were associated with low efficiency gene transfer because of two significant shortcomings: firstly, their requirement for target cell division in order to accomplish gene transfer [31] and, secondly, their inability to attain very high titers, required for vascular gene delivery. These shortcomings have been compounded by the fact that vascular endothelial and smooth muscle cells have low mitotic rates, even in diseased states [32]. The exciting application of pseudo typing [33] and the development of novel lentiviral vectors that can mediate gene delivery to nondividing cells [34] has overcome these drawbacks. The stabilization of the vector during the concentration process has facilitated the generation of highly concentrated stocks. This is expected to permit efficient gene transfer in quiescent cells of the vascular system. In fact, successful transfer and expression of marker and therapeutic genes in endothelial cells have been reported in some studies [35,36]. In one of these studies, Totsugawa et al. [35] demonstrated in vitro 70% transfection of human umbilical vein endothelial cells (HUVEC) by a vesicular stomatitis virus G-protein (VSV-G)-pseudotyped lentiviral vector expressing the NLS/LacZ gene. This vector has also been shown to transduce human saphenous vein endothelial and smooth muscle cells in a study by Dischart et al. [36]. In this study, overexpression of a therapeutic gene (TIMP-3) was achieved, with concomitant blockade of smooth muscle migration and induction of cell death. However, lentiviral vectors have not been used for in vivo gene delivery to the blood vessel wall.

4.3 Adenoviruses

Adenoviral vectors have become increasingly popular in vascular gene therapy. They have emerged as the vector of choice for many in vivo applications of gene therapy. Over the past decade, an enormous amount of data on adenovirus vector has become available from numerous experimental studies [37–40]. The advantages of adenovirus as a gene transfer vector are its ability to be concentrated to very high titers, its broad host range, its high infectivity in vitro and in vivo, its exhibition of tropism for most human cells, and, finally, its ability to infect quiescent as well as dividing cells [41]. Adenoviral vectors have been shown to achieve transgene expression in vivo in up to 30% of target vascular cells [38,39].

The disadvantages of adenoviral vectors include the short duration of transgene expression of genes secondary to both immune [42,43] and inflammatory [44] responses to adenovirus transduced cells. In many individuals, it leads to the production of neutralizing antibodies [43] and memory T cells [45], directed at adenoviral proteins. Transient immunosuppresion has shown some promise in permitting gene delivery to animals with a preexisting immunity to the virus [46].

Current research into adenovirus vectors is focusing on strategies to reduce host immune response to attain long-term persistent transgene expression. A new generation of adenovirus vectors is being developed that involves complete elimination of all adenoviral coding sequences, retaining only cis-acting elements essential for adenoviral replication and packaging [47,48]. These highly ablated viral vectors hold great potential for the clinical application of vascular gene therapies.

4.4 Adeno-associated viruses

AAV has emerged recently as an attractive candidate for vascular gene therapy. It has several unique features. Firstly, AAV is a nonpathogenic virus that can be concentrated to high titers. Secondly, it can mediate efficient gene delivery to post mitotic cells. Thirdly, AAV vectors are capable of long-term expression because of their stable integration into the host cell genome in nature. And finally, these vectors lack viral sequences encoding viral proteins, resulting in far less immunogenic potential. In fact when AAV vector was injected into the same muscle as an adenoviral vector, expression from AAV transduced cells was stable and prolonged as compared to adenovirus transduced cells [49]. AAV vector has been shown to mediate in vivo gene delivery to vascular cells in several reports [50,51]. However, results have been inconsistent. Earlier work by our group has shown that the pattern of transgene expression in the vascular cells is affected by the serotype of AAV, with AAV-2 incapable of endothelial cell transduction and AAV 1 and 5 showing expression in vascular endothelial and smooth muscle cells both in vitro and in vivo [52]. More extensive investigations are required to determine the role of AAV vectors in cardiovascular gene therapy.

Advances in vector targeting strategies have been rapid especially using adenovirus and, more recently, AAV-based vectors. Targeting may be achieved by modification of the viral capsid or by the use of tissue-specific promoters. Work et al. [53] demonstrated that incorporation of EYH peptide into adenovirus and AAV capsids resulted in a significant and selective enhancement in transduction of smooth muscle cells in vitro. White et al. [54] also demonstrated that peptide-modified AAV when given intravenously produced enhanced uptake of virions in vena caval endothelial cells with selective gene expression. These genetically modified viruses with vascular cell tropism hold the potential for enhanced transduction especially when delivered to a diseased site via stent. To our knowledge, however, they have not been studied in the setting of stent-based gene delivery.

In summary, both gene transfer vectors, viral and nonviral, have been used extensively to deliver recombinant genes to vascular cells. An ideal vector is characterized by its high efficiency, cell specificity, low toxicity, unlimited insert size, prolonged expression, and lack of immunogenicity. At present, no single vector has all of these characteristics.

5 Cardiovascular disease targets for stent-based gene therapy

5.1 Restenosis

The overexpression of several gene products has demonstrated their ability to reduce restenosis. The pathogenesis of restenosis is likely to be multifactorial. One key element is increased vascular smooth muscle cell migration and proliferation. Normally vascular smooth muscle cells are quiescent cells in the G0 phase of the cell cycle. Following vascular injury, vascular smooth muscle cells are stimulated by growth factors and cytokines to divide and enter the G1 phase of the cell cycle. Major approaches to inhibiting neointimal formation include a large number of cytostatic and cytotoxic strategies [55,56].

Overexpression of a constitutively active mutant form of Rb and CKI protein p21 [56] has resulted in inhibition of vascular smooth cell in vitro and in vivo (cytostatic strategy). The cytotoxic approach involves inhibition of DNA synthesis in the S phase of the cell cycle. Several studies have suggested that using the herpes simplex virus thymidine kinase gene (HSV-tk) and the nucleoside analogue ganciclovir prevent restenosis, in atherosclerotic blood vessels [37,57]. HSV-tk encodes the enzyme thymidine kinase that phosphorylates ganciclovir to a toxic form. Incorporation of phophorylated ganciclovir into replicating DNA in dividing cells results in cell death.

Another approach to the treatment of neointimal formation includes the expression of nitric oxide synthase. This enzyme increases production of nitric oxide, which is a pleotropic agent acting as a potent vasodilator with antithrombotic and antiproliferative properties. Our group has shown that adenovirus-mediated transfer of genes for recombinant endothelial nitric-oxide synthase to the adventitia of rabbit carotid arteries alters vascular reactivity [58,59]. Adenoviral-mediated gene transfer of eNOS has also been shown to significantly reduce the intimal to medial ratio at 14 days compared with vehicle and virus controls in incubated human saphenous veins [60]. Preliminary results suggest that in vivo delivery of eNOS to canine saphenous vein grafts results in outward remodeling of the grafts [61]. Transfection of eNOS with HVJ/liposome vectors into the balloon injured arteries of rats was associated with local NO generation and a reduction in intimal hyperplasia [62]. This observation has been confirmed after transfection with adenoviral vectors encoding eNOS, showing up to 50% reduction in neointimal formation in porcine coronary balloon injury model [39].

Another approach to preventing neointimal hyperplasia is to accelerate re-endothelialization, with the expression of genes promoting endothelial growth and recovery. This, in turn, limits smooth muscle cells proliferation and migration after vascular injury. Some earlier preclinical studies demonstrated that VEGF expression was associated with accelerated endothelial coverage [14] with reduction in medial proliferation [28]. However, this beneficial effect of VEGF on restenosis was not seen in recent studies. Swanson et al. [63] reported no accelerated re-endothelialization or reduction in restenosis following deployment of VEGF-eluting stent in rabbit iliac artery model. This was confirmed in a clinical study by Hedman et al. [64] where no difference in clinical restenosis was observed at 6 months following VEGF delivery to the arterial wall post angioplasty.

In the era of drug eluting stents (DES), the question arises: do we need gene-based approaches for vascular diseases? Rapamycin and paclitaxel have been shown to reduce the incidence of restenosis after primary angioplasty by targeting the proliferating VSMC. This antiproliferative approach has the potential to impair endothelial cell proliferation resulting in delayed recovery of the endothelial cell layer [65]. This apparent inhibition of re-endothelialization can manifest as late thrombosis and increased severity of the intimal disease. The other potential problems with drug eluting stents are the possibility of intrinsic drug resistance and late development of aneurysm. Furthermore, long-term experience with DES in coronary arteries and use in long peripheral vascular lesions are awaited. Stent-based delivery of genes with pleotrophic effects, which inhibit smooth muscle cell proliferation and enhance re-endothelialization, is worthy of study.

5.2 Thrombosis

The role of antithrombotic gene therapy in interventional cardiology is not well defined at present. However, the targets for antithrombotic gene therapy (i.e. platelet aggregation and fibrin clot formation) as well as the candidate genes that encode the key proteins (urokinase, t-PA, cyclo oxygenase, and hirudin) are well characterized in animal models [40,66].

Thrombosis formation after coronary stenting has been reduced to less than 1% of patients because of the technical advances in stent deployment and the aggressive treatment with antiplatelet agents (aspirin, clopidogrel, heparin, abciximab) [67]. This reduced rate of thrombosis has dampened the clinical application of antithrombotic gene therapy. However, the effective advantage of antithrombotic gene therapy post angioplasty and stenting lies in its ability to deliver site-specific gene delivery, thereby reducing systemic bleeding complications from intravenous antiplatelet agents. Also, the fact that certain antithrombotic agents have been shown to reduce neointimal formation in several animal studies provides a rationale for exploring this therapeutic avenue [68].

5.3 Vascular graft disease

Vascular graft failure represents a significant clinical problem and atherosclerosis is the primary pathophysiological event. Revascularization of vein grafts with stenting is associated with a higher rate of restenosis when compared to native arterial coronary vessels. Effective stent-based gene therapy of symptomatic vascular graft stenosis or restenosis is an area of great potential and interest to the interventional cardiologist. Several studies in animal models have demonstrated direct gene transfer to diseased vascular grafts [60,69,70]. Cell-based gene transfer using a stent as a platform have shown to provide site-specific gene expression in grafts [70]. Autologous porcine smooth muscle cells were transduced with plasmid encoding GFP and seeded on to the mesh-stent which was then deployed in the porcine coronary artery. Stable in vivo gene expression was demonstrated up to 4weeks after implantation of the stent. This study implies that stents can be used for long-term gene delivery and represents a desirable therapeutic option.

In summary, stent-based gene delivery for vascular graft stenosis and restenosis has enormous clinical application but extensive preclinical and clinical studies are required to achieve this application.

5.4 Chronic vascular occlusion

The primary objective of percutaneous coronary intervention (PCI) is to alleviate an obstruction and to restore blood flow to the myocardium. This however may not be possible in patients with chronic total vascular occlusion or heavily calcified coronary lesions. The concept of using gene therapy to promote angiogenesis to restore myocardial blood flow that bypasses the stenotic segment has gained considerable attention. Successful angiogenesis has been achieved in ischemic and non-ischemic models from delivery of angiogenic cytokines (VEGF/FGF) by plasmid or adenoviral vectors [71,72]. The therapeutic angiogenic response in myocardium can be achieved by delivering these genes and/or proteins by direct intracoronary or intramyocardial injection. Safety of these angiogenic cytokines to achieve focal angiogenesis without systemic effects appears well established in animal models. In humans, the major safety concern with these growth factors is the potential acceleration of a neoplastic disease or retinopathy. Also, the mitogenic effects of these proteins may accelerate atherosclerotic lesion expansion or stimulate intraplaque angiogenesis and hemorrhage [73,74]. None of these concerns have been observed in practice. Clinical studies with these angiogenic cytokines did not show any exacerbation of ocular pathology when used in patients with diabetic retinopathy. An alternative approach to intravascular or direct injection might be to use a gene eluting stent upstream of the chronic total occlusion.

6 Conclusion

Cardiovascular gene therapy has made a tremendous amount of progress in the last decade. An enormous amount of research has been conducted in this area to find the appropriate therapeutic gene product, to define the ideal vector, and to identify the most effective delivery strategies.

The development of animal models for human cardiovascular diseases has been instrumental and rewarding in achieving these goals and also in defining the biological efficacy of gene therapy approaches. Several genes have been confirmed to have therapeutic effects in cardiovascular diseases. The advent of adenovirus and adeno-associated virus vectors has helped improve the problem of efficiency. Site-specific stent-based delivery of genes for cardiovascular diseases holds great promise.

Nevertheless, significant technical hurdles remain to clinical translation of stent-based gene delivery. To overcome these challenges, more effort is required to increase the incorporation of viral and nonviral vectors in the coating matrix of stents with standard release kinetics and without potential inflammatory effects. Also, the improvement of vector systems that allow cell-specific improved transfection without toxicity remains elusive.

Cardiovascular gene therapy has come a long way and carries the potential to provide a lasting biological solution for many cardiovascular diseases. However, significant challenges remain in vector development, gene incorporation, and percutaneous deployment in appropriate preclinical models.

Footnotes

  • Time for primary review 18 days

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View Abstract