Journal of Molecular and Cellular Cardiology 42 (2007) 461–468www.elsevier.com/locate/yjmcc

Review article

Gene therapy for restenosis: Biological solution to a biological problem

Raj Kishore, Douglas W. Losordo ⁎

Division of Cardiovascular Research, Caritas St. Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, MA 02135, USA

Received 19 October 2006; accepted 16 November 2006Available online 11 January 2007

Abstract

Coronary artery disease remains a significant health threat afflicting millions of individuals worldwide. Despite the development of a variety oftechnologies and catheter based interventions, post-procedure restenosis is still a significant concern. Gene therapy has emerged as a promisingapproach aimed at modification of cellular processes that give rise to restenosis. When juxtaposed alongside the failure of traditionalpharmacotherapeutics to eliminate restenosis, gene therapy has engendered great expectations for cubing coronary restenosis. In this review wehave discussed an overview of gene therapy approaches that hve been utilized to reduce restenosis in preclinical and clinical studies, current statusof anti-restenosis gene therapy and perspectives on its future application. For brevity, we have limited our discussion on anti-restenosis genetherapy to the introduction of a nucleic acid to the cell, tissue, organ or organism in order to give rise to the expression of a protein, the function ofwhich will confer therapeutic effect. For the purpose of this review, we have focused ou discussion on two relevant anti-restenosis strategies, anti-proliferative and pro-endothelialization.© 2006 Elsevier Inc. All rights reserved.

Keywords: Restenosis; Gene therapy; Proliferation; Re-endothelialization

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4612. Anti-proliferative gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462

2.1. Cytotoxic gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4622.2. Cytostatic gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4632.3. Anti-migrational gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4632.4. Pleiotropic gene transfer strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463

3. Pro-reendothelialization gene therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4644. Conclusion—a promise of clinical trial? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465

1. Introduction

Diseases desperate grownBy desperate application are reliev’d,Or not at all: Shakespeare (in Hamlet)

The health threat posed by coronary artery disease (CAD),which currently affects more than 13 million Americans, has

⁎ Corresponding author.E-mail address: douglas.losordo@tufts.edu (D.W. Losordo).

0022-2828/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.yjmcc.2006.11.012

inspired the development of a variety of technologies andtechniques for coronary revascularization. With the developmentand subsequent evolution of catheter-based interventions, there hasbeen a gradual progressive decline in the frequency of surgicalreferral. The advent of drug-eluting stents, for example, sirolimusand paclitaxel eluting stents, have further reduced the incidence ofpost-procedure restenosis [1–3]. Although this seems an impres-sive achievement, it should be noted that up to 13.3% of thepatients with drug-eluting stents require additional revasculariza-tion (either of the target vessel or another coronary vessel) withinthe first year after the stent was inserted. Additionally, the extents

462 R. Kishore, D.W. Losordo / Journal of Molecular and Cellular Cardiology 42 (2007) 461–468

to which the results of the drug-eluting stent trials can begeneralized to the CAD population are somewhat limited. Forexample in SIRIUS trial, patients were excluded if they had recentor evolving myocardial infarction, left main coronary arterydisease, ostial lesions, severely calcified or thrombotic lesions, orejection fractions below 25%. As a result, patients with moresevere CAD were largely excluded. Given recent statisticsshowing a startling increase in the prevalence of obesity in theUnited States, it is likely that chronic total occlusions andbifurcation stenoses will become even more prevalent in the nearfuture, further shrinking the population in which drug-elutingstents can be used with confidence. Moreover, despite thesignificant advances in drug-eluting stent technology, catheter-based interventions in general have not proven to be well suited topatients with a large atherosclerotic burden and patients withdiffused disease, especially those with small vessels, stillexperience a high incidence of restenosis. New technology mayimprove stenting outcomes for these challenging groups, but theyleave room for complimentary therapies that would furtherdecrease restenosis or eliminate it entirely so that coronaryinterventions become cure for symptomatic stenoses rather thanmerely a temporizing procedure.

Against this background, gene therapy has emerged as apromising approach aimed at modification of cellularprocesses that give rise to restenosis. The attractiveness ofgene therapy is based on several widely held perceptions.First, gene therapy appears capable of delivering therapeuticagents specifically to the location of the disease, at a precisesite in the arterial wall, thus maximizing the therapeutic effectswith minimal side effects. Maximal therapeutic efficacy mightbe achieved with minimal systemic side effects. Second, genetherapy proposes a ‘biological solution’ to an essentially‘biological problem’: regrowth of intimal mass or artery wallremodeling. Because restenosis is fundamentally the manifes-tation of a failed mechanical solution to a biological problem,a biological approach is intuitively attractive. Third, certaingene therapy approaches appear capable of precisely treatingexcessive vascular cell proliferation, potentially a keycomponent of the pathophysiology of restenosis. Fourth,gene therapy approaches have appeared imminently applicableto large populations. Finally, since many critical changes ingene expression within the arterial wall are confined to thefirst 14 days, hence short-term transgene expression may betherapeutically effective. All these perceptions are based onsolid experimental data produced either in vitro or inexperimental animals [4]. When considered together, andparticularly when juxtaposed alongside the failure of tradi-tional pharmacotherapeutics to eliminate restenosis, theseperceptions have engendered great expectations concerningthe current potential of gene therapy for coronary restenosis.For brevity, we will limit our discussion on anti-restenosisgene therapy approaches to the introduction of a nucleic acidinto a cell, tissue, organ or organism in order to give rise to theexpression of a protein, the function of which will confertherapeutic effect. This definition of gene therapy, therefore,excludes the use of nucleic acids as antisense oligonucleotides,ribozymes, decoys or DNAzymes from this mini-review.

These areas of nucleic acid therapy have been reviewedelsewhere [5–7].

A complete understanding of the pathophysiology of rest-enosis is essential for evaluating gene therapy approaches.Unfortunately, both the biological stimuli that initiate restenosisand the molecular and cellular mechanisms by which it occursare incompletely characterized. However, it is generally agreedthat three distinct processes are involved in the pathogenesis ofrestenosis: vessel recoil, neointimal proliferation, and earlythrombus formation. The relative contribution of each of thesedepends on the type of injury. Coronary stenting virtuallyeliminates vessel recoil, and restenosis is largely the result ofneointimal proliferation. When an artery is injured, depositionof platelets, leukocyte infiltration, proliferation of smoothmuscle cells (SMC), deposition of extracellular matrix, andreendothelialization occur. Growth factors and cytokinesreleased by these leukocytes and platelets stimulate themigration, growth, and multiplication of smooth muscle cellsas well as affect the process of reendothelialization [8,9]. For thepurpose of this review, we will focus our discussions on tworelevant anti-restenosis strategies: (a) anti-proliferative and (b)pro-reendothelialization approaches.

2. Anti-proliferative gene therapy

The complexity of the vascular response to injury, involvingthe integration of SMC/myofibroblast proliferation, migrationand extracellular matrix (ECM) deposition, has naturally givenrise to a wide range of potential targets for therapeuticintervention. SMC proliferation is recognized as a prominentfeature of lesion formation in all the animal models of vascularinjury [10–12], and a host of regulatory steps are involved in theproliferative process. As a consequence, gene transfer to inhibitSMC proliferation has provided a large proportion of candidatetherapeutic genes that have been investigated so far in thesetting of gene transfer after arterial injury. The geneticmanipulation approaches have largely targeted vascular musclecell proliferation by the overexpression of anti-proliferativegenes, introduction of cytotoxic genes, and the inhibition of cellcycle genes. For the convenience of discussion anti-prolifera-tive gene therapy may be categorized under the followingsubgroups.

2.1. Cytotoxic gene transfer

Following the first reports of successful gene transfer tovascular cells [13], follow-up experiments in which herpessimplex virus thymidine kinase (HSV-tk) was delivered toinjured arteries, porcine iliac [14], or rat carotid [15], using anadenoviral vector, were published 5 years later. This approach isexemplified by the use of the cDNA for HSV-tk, which convertsthe inert nucleoside analogue ganciclovir into phosphorylatedtoxic form in transduced cells. Incorporation into host DNAinduces chain termination and cell death, thereby permittingselective elimination of dividing cells. A reduction in theneointima by 86% in the porcine iliac artery model [14] and46% in the rodent carotid model [15] was noted, upon

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adenovirus-mediated HSV-tk gene transfer. In contrast, retrovi-rus-mediated transfer of HSV-tk into injured vessels had nodiscernible effect upon neointimal area or intima:media (I:M)ratio [16]. In a separate study, adenovirus-mediated transfer ofHSV-tk into pig coronaries followed by daily injections ofganciclovir resulted in a 30% reduction in neointima formationat 28 days [17,18]. Studies of the effects of gene transfer afterballoon injury in non-diseased animal vessels are not represen-tative of human arterial disease, which is commonly theconsequence of atherosclerosis. Thus, evaluation of the injuryprocess in atherosclerotic animal vessels is important and canyield pertinent information that may be more directlycomparable to clinical scenarios. Using an atheroscleroticrabbit model, investigators delivered an adenoviral vectorcarrying the HSV-tk gene to injured vessels and demonstrateda 42% reduction in the I:M ratio at 4 weeks after injury [19].Similarly, another study reported a 35% to 49% reduction in theintimal area at 3 weeks but only a 21% reduction in the I:M ratioin an atherosclerotic rabbit model of arterial injury [20].Similarly, gene transfer of another cytotoxic gene product,cytosine deaminase, which converts 5-fluorocytosine to apowerful anti-metabolite 5-fluorouracil, has also been proveneffective at reducing intimal hyperplasia in a rabbit model ofvascular injury [21]. Other cytotoxic gene strategies that havebeen investigated include transfer of Fas ligand (FasL) and amutant cyclin G1. Fas, a death receptor belonging to TNFreceptor family, is expressed abundantly in vascular SMC andupon binding to FasL leads to apoptosis. Studies evaluatingFasL expression in the vessel wall have demonstratedsignificant reductions in neointima formation [22,23]. Retrovi-rus-mediated transfer of cyclin G1, an inducible cell cyclecontrol element, has also been shown to inhibit SMCproliferation in the injured rat carotid arteries [24]. Thesestudies strongly suggest that cytotoxic vascular gene therapycould be effective in preventing restenosis and in the underlyingatherosclerotic disease.

2.2. Cytostatic gene transfer

Cellular proliferation depends upon a plethora of regulatoryelements and is modified by a host of growth peptides,transcription factors, and environmental influences. An exten-sive review of SMC proliferation in the context of vasculardisease was published from our laboratories few years back[25]. Dozens of transgenes that modify SMC proliferation byinhibition of cell cycle have been studied in the setting ofvascular injury. The first such transgene to be studied was amodified retinoblastoma (Rb) gene [26]. In its active form, Rb isnormally bound to DNA elongation factor E2F and inhibitsDNA transcription. Phosphorylated or inactivated Rb releasesE2F, DNA transcription is initiated, and cell cycle progressionoccurs. Adenovirus-mediated transfer of a non-phosphorylableform of Rb or another member of Rb family, pRb/p130, into ratcarotids at the time of injury was associated with a significantdecrease in SMC proliferation and I:M ratio [27,28]. We havealso previously shown that adenovirus-mediated overexpres-sion of E2F1 at sites of balloon injury in mice accelerates

functional endothelial recovery and inhibits neointima forma-tion [29]. A range of other modifiers of cell cycle progression,including cdk inhibitors p21, p27, and p53 [30–34], have alsobeen studied. Additionally, our laboratories have shown thattransfer of the growth arrest homeobox gene gax also inhibitsneointima formation in a rabbit angioplasty model [35,36].Furthermore, a selection of inhibitors of critical intermediatesteps in the induction of proliferation by receptor-mediatedmechanisms has been studied: dominant-negative mutant formsof proto-oncogene H-ras [37] of mitogen-activated proteinkinase (MAPK)-kinase [38], members of MAPK family ERKand JNK kinases [39], truncated fragment of b-adrenergicreceptor kinase (an inhibitor of MAPKs), all reduced neointimaformation after gene transfer into rat carotids.

2.3. Anti-migrational gene transfer

Cell migration is central to the development of neointimalhyperplasia in those animals where no lesion exists prior toinjury and in-stented vessels where cells are to be found afterinjury in sites where there were no cells previously. Migrationinvolves regulated cell attachment and detachment fromextracellular matrix (ECM), contraction of muscle myosin andactin, cytoskeletal plasticity, and requires oxygen and proteinsynthesis. Matrix metalloproteinases (MMPs), a family ofmolecules involved in the degradation and remodeling of ECM,are necessary for SMC migration. Implantation of rat aorticSMC transduced ex vivo with cDNA encoding for tissueinhibitors of metalloproteinases 1 (TIMP1) or the adenovirus-mediated transfer of TIMP1 into injured rat carotid arteriessignificantly reduced the I:M ratio [40,41]. Similar observationswere made with TIMP2 and TIMP3 gene transfer [42,43].Working on the same principle of inhibiting ECM degradation,exposure of atherosclerotic rabbit iliac arteries to the serineelastase inhibitor elafin by liposome-mediated gene transferalso decreased arterial elastase activity and reduced I:M ratio[44]. Other anti-migrational gene transfer approaches haveutilized plasminogen activator inhibitor-1 (PAI-1) and modula-tion of urokinase type plasminogen activator (u-PA) withsignificant success in reducing intimal hyperplasia [45–47].

2.4. Pleiotropic gene transfer strategies

The mechanisms underlying restenosis are clearly complex.His complexity has promoted the suggestion that moleculeswith pleiotropic actions may represent more effective thera-peutic agents than those which simply affect one of the putativemechanisms of lesion formation. The archetypical pleiotropicgene employed in the studies of vascular injury is nitric oxidesynthase (NOS), producing nitric oxide (NO), a vitally im-portant molecule for vascular homeostasis. NO is synthesizedby an endothelial NOS (eNOS) but can be produced in muchlarger quantities by an inducible NO synthase (iNOS). NO inthe vasculature is primarily vasoprotective by inhibiting plateletand leukocyte adhesion, inhibiting SMC proliferation andmigration, and promoting endothelial survival and proliferation[48]. At sites of vascular injury, the endothelium is disrupted

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and NO synthesis is impaired. Hence, augmenting local NOsynthesis through gene therapy may help arrest the proliferativeresponse to vascular injury. In 1995, the first study to delivereNOS to injured rat carotid arteries using HVJ liposomesdemonstrated a 70% reduction in I:M [49]. Chen et al. [50]seeded SMCs engineered to express eNOS using retrovirusesonto injured rat carotid arteries and inhibited neointimaformation by 37%. Others have similarly shown that adenoviraldelivery of eNOS to injured rodent and porcine arteries can limitIH [51,52]. On an equimolar basis, iNOS produces muchgreater levels of NO compared with eNOS. Additionally, incontrast to eNOS, iNOS produces NO in a calcium-independentand sustained manner [53]. This property makes iNOS attractivebecause one of the limiting factors in gene therapy is genetransfer efficiency. In a porcine model of iliac artery injury,iNOS gene transfer reduced IH by 52%, again using much lessvirus (3-to 20-fold less) than other vascular gene deliverystudies [54]. Other pleiotropic gene transfer includes alternateapproaches to modify NO signaling by transferring SGC alpha 1and beta 1 subunits and cyclic GMP-dependent protein kinase[55,56]. Antagonism of another pleiotropic chemokine, MCP1,by transferring dominant-negative cDNA in the femoralmuscles of hypercholestromic rabbits reduced neointimaformation in injured carotid arteries [57]. Similarly genetransfers of other pleiotropic modifiers VEGF [58,59], C-typenatriuretic peptide [60], prostacyclin [61], COX1 [62],uteroglobin [63], dominant-negative Rho kinase [64], andanti-inflammatory cytokine IL-10 [65] have also been shown toinhibit injury-induced neointimal hyperplasia.

3. Pro-reendothelialization gene therapy

Limitations of anti-proliferative strategies that have beenrecognized to many of these approaches discussed above mayrelate in part to belated reendothelialization (ReEndo) becausenone of these strategies includes a specific proactive effect onEC repaving of the mechanically injured arterial segment.Delinquent ReEndo has a permissive, if not facilitatory, impacton smooth muscle cell proliferation. This inverse relation hasbeen attributed to certain functions of the endothelium; thesefunctions include barrier regulation of permeability, thrombo-genicity, and leukocyte adherence, as well as production ofgrowth-inhibitory molecules. Indeed many earlier studiesestablished direct correlation of accelerated endothelial recov-ery to the inhibition of intimal hyperplasia [66]. Furthermore,strategies including endovascular radiation [67] and drug-eluting anti-proliferative stents showing a decrease in neointi-mal growth in animal experiments, however, are accompaniedby delayed healing characterized by persistence of neointimalfibrin (with or without inflammation), a decrease in smoothmuscle cells, and incomplete endothelialization [68]. Thesefindings indicate that the regenerative capacity of the vesselwall endothelial cells seems to be essential for the healingprocess and might be impaired in treatment strategies with anti-proliferative agents.

VEGF is a potent endothelium-specific angiogenic factor.With this property, VEGF may assist in promoting reendothe-

lialization of denuded arterial wall and therefore arrest IHsooner by halting the mitogenic signals that originate at sites ofplatelet and leukocyte attachment. Recombinant VEGF hasbeen administered in animal models of arterial injury. Ourlaboratories were first to report that catheter-mediated, site-specific arterial gene transfer of VEGF165 in balloon-injuredrabbit femoral arteries can accelerate ReEndo at local andremote sites, leading to inhibition of neointimal thickening,reduction in thrombogenicity, and restoration of endothelium-dependent vasomotor reactivity [69]. Further studies from ourand other laboratories using VEGG naked plasmid or VEGFcoated stents have suggested that acceleration of reendothelia-lization can attenuate restenosis and inhibit stent thrombosis[58,70,71]. In reversed autologous vein bypass grafting of thecommon carotid artery in rabbits, Ohno et al. showed that Cnatriuretic peptide (CNP) overexpression in the vein graftresulted in the acceleration of reendothelialization with lessthrombus formation and the suppression of intimal thickening[72]. Similar effects on reendothelialization associated withsuppression of neointimal proliferation have also been demon-strated for estradiol [73], tumor necrosis factor-soluble receptor[74], HMG CoA reductase inhibitors [75], other EC mitogenslike bFGF [76], and PDGF [77] emphasizing the concept thatgene therapy-mediated acceleration of endothelial recovery maybe an alternative strategy for the prevention of neointimalproliferation.

Neointimal thickening has been inferred to result most oftenfrom vascular smoothmuscle cell (VSMC) proliferation [78]. Asa result, intense effort has been applied to discern themechanisms that govern VSMC proliferation after angioplastyand to develop therapies to inhibit VSMC growth. Theenthusiasm for this field is reflected by over 925 manuscriptsthat have been published examining the role of SMCproliferation in restenosis and, most importantly, by the adventof approaches that have demonstrated significant degrees ofclinical success in addressing this aspect of the response to injury[79]. Both brachytherapy and drug-eluting stents targetproliferating VSMCs at the site of injury and have beensuccessful in reducing neointimal lesion formation. However,the fate of the endothelium in humans after radiation or drug-eluting stent is uncertain at present, although evidence exists tosuggest that endothelial recovery may be perturbed. The initialapplications of intravascular brachytherapy were complicated bya significant incidence of stent thrombosis occurring up to9 months after the revascularization procedure [80], despite theusual administration of dual antiplatelet therapy for 1 monthafter stent implantation. Subsequently, the incidence of latethrombosis was reduced by extending the duration of dualantiplatelet therapy for 6 to 12 months [81]. These findingsimply that endothelial recovery was inhibited by the anti-proliferative approach, a hypothesis documented in animalstudies that revealed protracted deendothelialization in irradiat-ed arteries. Moreover, recent data have revealed direct inhibitionof reendothelialization by paclitaxel [82] and inhibition ofendothelial cell (EC) proliferation by rapamycin [83].

Data regarding the relationship between endothelial integrityand neointimal thickening in human arteries, although limited,

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are consistent with the results of animal experiments. Davies etal. [84] harvested coronary arteries from explanted hearts of 6transplant recipients and found that the severity and extent ofEC defects varied directly with the severity and extent of intimaldisease. Schwarcz et al. [85] found foci of, but not complete,reendothelialization in only 6 (38%) of 16 carotid specimens ofpatients with recurrent carotid artery narrowing after an initiallysuccessful endarterectomy. Perhaps most relevant to theproposed investigations, Gravanis and Roubin [86] found noEC regeneration among 11 patients dying within 1 month ofcoronary angioplasty. These studies thus support the notion thatcertain functions of the endothelium are critical to prevention ofluminal narrowing by neointimal thickening. This concept hasstimulated efforts to preserve intact the endothelium of nativeveins used for bypass surgery, accelerate reendothelializationafter balloon-induced arterial injury, and facilitate endothelia-lization of prosthetic conduits or endovascular stents.

Localized delivery of transgenes to the site of arterial injuryis a technically demanding procedure and is further restricted bythe transfection inefficiency. The transfection efficiency to ECcells in the denuded arteries is further compromised by theabrasion of endothelial lining. One emerging strategy may be toutilize endothelial progenitor cells (EPCs) as a means of arterialgene delivery. Our laboratory [87] and others [88] have shownthat ECs adjacent to the site of arterial injury might notconstitute the sole participants in endothelial recovery. Thesestudies have indicated that circulating cells, referred to as EPCs,derived from the bone marrow (BM) and exhibiting certainfeatures consistent with EC identity, are capable of beingrecruited to sites of arterial injury and contributing toreestablishing the neoendothelium. Indeed, several lines ofevidence have demonstrated that BM-derived EPCs, which aremobilized by a variety of factors [72,89,90] as well asexogenously infused cells [91], can significantly contribute toreendothelialization after endothelial denudation followingarterial injury. Ex vivo overexpression of genes in EPCs priorto infusion might be an important therapeutic strategy for theprevention of restenosis.

4. Conclusion—a promise of clinical trial?

Despite promising results from the numerous animal studies,there has been a general failure for such strategies to translateinto clinically relevant treatments and large-scale phase III trialsof human gene therapy in the vasculature remain elusive. Aphase II trial for peripheral vascular disease using VEGF-A viaplasmid/liposome or adenovirus to intrainguinal arteriesreported increased vessel formation at 3 months, but had littleeffect on angiographic restenosis, a secondary endpoint [92].Trials involving the coronary vasculature exist only in thecontext of ex vivo gene transfer (PREVENT I) to coronary veingrafts where moderate success has been reported [93], or as anantisense-based strategy for in-stent restenosis, the ITALICStrial [94], which failed to demonstrate any effect on restenosis asmeasured by IVUS or angiography.

Although more than 10 years have passed since the firststudies on ‘therapeutic’ vascular gene transfer were published,

we are in truth only a little closer now to realizing the clinicalpotential of localized gene transfer for the prevention ofrestenosis. The reasons for this apparent lack of progress aremany: an incomplete understanding of vascular biology, whichin turn leads to a multitude of putative transgenes with fewdemonstrating efficacy beyond pre-clinical studies; safety andtoxicity issues particularly in relation to viral vectors; vectorinefficiencies, particularly non-viral vectors and a necessaryreliance on animal models which cannot provide a complete andaccurate surrogate for human disease. The potential of drug-eluting stents and progenitor/stem cells suggests that local DNAdelivery will hold the key to coronary gene transfer forrestenosis. In pursuing an effective gene therapy clinicallyrelevant delivery techniques must drive design. It also appearsthat most efficacious transgenes will likely be those withpleiotropic effects on the vascular wall—it is doubtful whether asingle hit approach can encompass the complex response toarterial injury. Progress will be required in each of these areas ifthe promise of large-scale clinical trials has to go beyond beingembryonic.

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