Gene Therapy for Restenosis

Getting Nearer the Heart of the Matter

From the Krannert Institute of Cardiology (S.B., K.L.M.), Indiana University School of Medicine, and the Richard L. Roudebush Veterans Administration Medical Center (K.L.M.), Indianapolis, Ind.

Correspondence to Keith L. March, MD, PhD, FACC, Krannert Institute of Cardiology, 1111 West 10th St, Indianapolis, IN 46202. E-mail march{at}kimail.dmed.iupui.edu

	Abstract

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Abstract—Intensive work over the past decade has been directed to the study of vascular gene transfer as an approach to the unresolved problem of restenosis. This effort has resulted in a significant foundation of knowledge relative to the activities of potentially therapeutic gene products as well as the capabilities and limitations of vector systems and mechanical delivery modalities available for effecting the vascular expression of these gene products. In several instances, significant progress has been made by experiments highlighting unexpected difficulties and the need for more comprehensive understanding. It is thus now possible to clearly define and address specific challenges that must be overcome in order to make feasible progress from the preclinical to the clinical arena. The key challenges at present appear to include the evolution of clinically practical delivery methods that meet the kinetic requirements of achieving efficient gene transduction and the availability of vectors that maximize efficiency while minimizing undesirable host responses. Emerging data suggest that approaches to solving each of these issues may have recently been developed. Basic research evaluating these new delivery mechanisms and molecular vectors is essential to establish their true potential for use in the clinical arena.

Key Words: gene therapy • restenosis • local delivery • vectors • transduction efficiency

	Introduction

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He who knows most, knows best how little he knows.

Thomas Jefferson (1790)

Nearly a decade has elapsed since the first descriptions of the site-directed transfer of exogenous genetic material into the vascular system.^{1 2 3} Since these initial experiments, techniques of vascular gene transfer have been successfully used to evaluate hypotheses concerning the function of selected proteins in the vasculature by their targeted overexpression.^{4 5 6} These reports have sparked the anticipation that such approaches would be used for the therapeutic modulation of vascular responses, including stenosis after injury and restenosis. Although there have been many advances in the knowledge of the possibilities and limitations involved in somatic transgenesis of the vascular tree, it still remains to be demonstrated that such an approach is likely to offer practical therapeutic benefits to patient populations at risk for restenosis.

Given the length of time since the initial reports, many question whether gene therapy will ever be useful in the treatment of restenosis. In fact, a significant lag time between the pioneering work in this field and demonstration of clinical utility is not surprising in light of the range of developments that are required to provide a foundation for future clinical trials. This knowledge base is composed of experimental success and of increasingly sharply defined technical limitations, leading in turn to conceptual revision. In several areas, much progress has been made by experiments highlighting unexpected difficulties and resulting in an appreciation for the need for more comprehensive understanding of vascular biology, gene expression, vector design, and catheter-tissue interactions. The eventual demonstration of the efficacy and safety of gene transfer in inhibiting restenosis will clearly depend on the continuation of carefully designed preclinical efforts. It is the purpose of this article, as well as the accompanying article⁷ in this issue of Circulation Research, to provide a balanced overview of progress toward such a therapeutic goal and the possibility of its eventual achievement. This review deliberately takes the position that current data indeed support cautious optimism that genetic approaches will in fact provide for restenosis reduction and emphasizes the experimental success as well as the progress that has been made over the past decade in defining the key problems for solution.

The majority of the present review is devoted to three areas, the ongoing development of which is critical to the desired goal: (1) the selection of potentially therapeutic gene products to express in the vascular wall, (2) the multiple vectors available for introduction of these genes into cells, and (3) the range of catheter-based and other approaches to the mechanical delivery of vectors to the target cells. Any practical gene therapy for restenosis will depend on the appropriate choice of all three of these interdependent elements. The gene product chosen for introduction must affect molecular targets in the vessel wall in a way that produces a desirable response, the vector must be capable of efficient gene transfer with an appropriate safety profile, and the mechanical delivery approach must consistently place the vector in the desired region with relative ease. These requirements correspond precisely with concepts routinely used in the development of classical drug therapies and described using formalisms of pharmacokinetics. According to this terminology, the gene to be delivered is the prodrug, whereas the actual drug(s) is the gene product(s) (whether RNA, a protein, or the product of an enzymatic reaction carried out by the new protein), which arises by a series of biotransformations from the genetic prodrug. The vector itself represents the formulation of the therapeutic agent (as in the case of a coated tablet or capsule), whereas the delivery approach used defines a route of administration. The delivery mechanism actually used to deposit the vector will profoundly affect the distribution and function of the delivered material, just as more classical methods of drug administration (eg, oral, intramuscular, and intravenous) play significant roles in the clinical effects of virtually all medical therapies. Each of these components will now be explored in detail.

	Candidate Gene Products for Local Therapeutic Expression

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Gene products suitable for overexpression to reduce restenosis may be broadly categorized according to the targeted processes as outlined below. Over the past 3 years, encouraging studies have emerged in which the local overexpression of several molecules has been found to reduce neointimal formation after vessel injury in large and small animal models (Table 1).

Cell Proliferation
Most studies targeting restenosis have been based on the paradigm that proliferation plays a major role in its development and thus have focused on strategies to reduce smooth muscle cellular proliferation. These studies have attempted to either inhibit cell cycle entry ("cytostatic" strategy) or to cause the death of cells that have entered the cell cycle ("cytotoxic" strategy). The cytostatic approach is exemplified by the overexpression of a constitutively active mutant form of the retinoblastoma protein RB, which functions to complex with the E2F family of transcription factors and block entry into the cell cycle. This RB mutant has been introduced after balloon injury into the iliofemoral artery of the pig and the carotid artery of the rat by using adenoviral vectors and a "dwell" method, in which all branches are ligated and the vector suspension is placed between proximal and distal balloons or ligatures for 20 minutes (in pig models) to permit adequate transduction.⁸ That study demonstrated significant reductions in the intima-to-media ratio in both rats and pigs. An alternative cytostatic strategy has used the overexpression of the endogenous cyclin kinase inhibitor protein p21 with similar results.⁹ This protein functions to negatively regulate S-phase entry by inhibition of cyclin-dependent kinases.¹⁰ Other genes that have been found to be cytostatic in vivo have included the homeodomain gene gax¹¹ and the dominant-negative mutants of ras.^{12 13}

A cytotoxic approach described by several laboratories involves the intramural expression of enzymes capable of converting nucleoside analogues into toxic metabolites that cause an interruption of DNA replication and the consequent death of transduced cells entering the S phase in the presence of the nucleoside analogue. The best known of these gene/drug combinations is the thymidine kinase isozyme derived from the herpes simplex viral genome, which can phosphorylate the inactive drug ganciclovir to ganciclovir phosphate, which is, in turn, capable of interrupting DNA synthesis. The expression of this enzyme and drug activation have again been tested using adenoviral dwell techniques, and use of this enzyme was found to result in a 35% to 87% reduction of the intima-to-media ratio, depending on the specific model and degree of injury.^{14 15 16} An analogous approach has used the expression of cytosine deaminase and coadministration of 5-fluorocytosine,¹⁷ with a 45% reduction of neointimal proliferation.¹⁸ The recent recognition of substantial frequencies of programmed cell death or apoptosis both in native atherosclerotic plaques and in vascular tissues after balloon injury^{19 20} suggests the induction of apoptosis as a particular cytotoxic approach of potential interest. Whether the expression of genes enhancing the incidence of programmed cell death would be specifically desirable remains to be tested.

Cell Migration
The extent to which postinjury growth in intimal mass is dependent on the centripetal migration of preexisting smooth muscle cells or myofibroblasts proceeding along a differentiation pathway to become smooth muscle–like cells is only recently being investigated.^{21 22 23} Key elements of the migratory process include activation of signaling pathways involving tyrosine kinases, such as the receptors for platelet-derived growth factor^{24 25} and nerve growth factor,²⁶ cytoskeletal reorganization, and the expression and activation of a variety of proteases directed to facilitating migration through the extracellular matrix. These proteases include plasminogen activators^{27 28} as well as the family of matrix metalloproteases.^{29 30} Such observations suggest several potential approaches to the modulation of cell migration, including the local overexpression of protease inhibitors such as the tissue inhibitor of metalloprotease (TIMP) family.^{31 32} Indeed, overexpression of TIMP-1 has reduced intimal formation in injured rat carotid arteries (J. McEwan, unpublished data, 1997). However, such approaches are presumably in opposition to processes involving the migration of endothelial cells required for reendothelialization as well as angiogenesis.^{33 34} As such, genetic (as well as nongenetic) manipulations designed to reduce or enhance migration will require cautious monitoring to ensure that they do not inadvertently promote adverse outcomes.

Antithrombotic Strategies
A number of elegant studies have examined the possibility of seeding stents or grafts with genetically modified prothrombolytic or anticoagulant endothelial cells either before^{3 35 36} or after (S.R. Bailey, unpublished data, 1997) placement. These cells have overexpressed tissue plasminogen activator as well as urokinase plasminogen activator and have been shown to possess enhanced local antithrombotic activity in vivo.³⁵ The local expression of hirudin after adenoviral gene transfer in situ has been found to reduce neointimal formation by 35%.³⁷ Recent experiments have revealed an unexpected adverse effect of the endothelial overexpression of tissue plasminogen activator, in that the transduced cells were demonstrated to show a significantly increased rate of detachment from seeded stent surfaces after their in vivo placement.³⁶ This observation highlights the potential for unexpected and potentially pathological results occurring when the "genetic ecosystem" of the vascular wall is unbalanced by the overexpression of specific genes in the absence of appropriate physiological regulation and feedback controls.³⁸

Another group of genes directed to inhibiting thrombosis as well as associated neointimal proliferation target platelet activation or aggregation rather than coagulant or thrombolytic pathways. These include two enzyme families: the NO synthases (inducible, constitutive, or neuronal isoforms) and the cyclooxygenases (prostaglandin H synthases I and II). The local expression of these enzymes increases the synthesis of small molecules, which are in turn the true active agents, NO and prostacyclin, respectively. The hemagglutinating virus of Japan (HVJ)/liposome–based overexpression of constitutive (endothelial) NO synthase in the setting of balloon injury has reduced neointimal formation after balloon injury.³⁹ The introduction of adenoviral vectors encoding endothelial NO synthase using a catheter capable of direct intramural injection (described below) has been demonstrated to inhibit neointimal accumulation in the porcine coronary balloon overstretch model by up to 50% at 4 weeks after injury.⁴⁰ The latter data are particularly exciting because of the use of a delivery catheter⁴¹ and rapid infusion protocol, which have been successfully implemented for coronary delivery of other substances in patients.⁴² Local transduction of prostaglandin H by adenoviral vectors has resulted in substantial reductions in cyclic flow variations occurring as a consequence of platelet thrombi dynamically forming after arterial injury.⁴³

Therapeutic Reendothelialization
The expression of factors promoting endothelial growth may accelerate the recovery of vascular physiology after mechanical injury by the facilitated restitution of endothelial surface coverage. This, in turn, is potentially associated with reduction in the thrombotic and proliferative environment of the vascular wall. Evidence supporting the utility of this approach has recently been generated by studies in carotid^{44 45} as well as iliofemoral⁴⁶ arteries, in which the local intravascular^{44 46} or extravascular⁴⁵ expression of vascular endothelial cell growth factor (VEGF) was associated with accelerated endothelial coverage and also with significantly diminished medial proliferation. Furthermore, the gene transfer of VEGF has been demonstrated to accelerate stent endothelialization and diminish intimal thickening by 56% in stented rabbit iliac arteries.⁴⁷

Candidate Gene Products: Promises and Problems
The preservation of arterial patency that has been demonstrated in animal injury models after the forced expression of any of several genes as described above provides strong confirmation that the introduction of specific gene products can successfully confer biological effects with the promise of potentially therapeutic activity. It also suggests that the modulation of any one of a range of molecular pathways is sufficient for such effects, at least in these models. As such, even more prominent results might be obtained by combination gene therapy involving either vectors encoding multiple gene products with complementary activities or the coadministration of distinct vectors encoding such products.

Whether promising preclinical results can be extrapolated to human restenosis will depend on the extent to which the current animal models, which involve stenotic responses of normal vessels, mimic molecular pathways that are shared by the human restenotic response of diseased vessels. Concerns about the therapeutic implications of these results are highlighted by recent questions concerning the importance of proliferation in the human restenotic setting.^{48 49} Current evidence from serial ultrasound studies after angioplasty suggests that perhaps only 30% of the luminal loss occurring over 6 months after angioplasty involves growth in the plaque area, whereas the remainder reflects constrictive "remodeling" of all tissues circumscribed by the external elastic lamina.^{50 51} Testing and application of antiproliferative strategies will thus likely occur as an adjunct to stent placement, after which restenosis will predominantly involve proliferation without inward remodeling. As other physiological processes such as remodeling are determined to play important roles in restenosis, additional targets will thus be sought for genetic modulation. At present, particularly favorable consideration may be given to genes encoding products that are secreted by transduced cells, so that the effects of gene transfer would effectively be amplified by a paracrine mechanism.

	Vector Systems for Vascular Gene Transfer

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A multitude of vector systems are currently under evaluation for the introduction of therapeutic gene cassettes into vascular tissues (Table 2). These include (1) plasmid DNA with or without a range of liposome or protein conjugates designed to enhance cell entry and access to the nuclear compartment, (2) integrating viral vectors such as retroviruses and adeno-associated viruses (AAVs), and (3) nonintegrating viral vectors such as adenoviruses. Each of these vector systems has been evaluated in several local delivery settings, and their general properties with respect to vascular gene transfer have been established. A fourth distinct category of therapeutic genetic material is consists of oligonucleotide fragments of coding or regulatory genetic material, which function by altering endogenous gene expression rather than by introducing entire exogenous genes. Such approaches do not constitute actual gene transfer and have been reviewed recently in detail⁵² ; thus, these will not be discussed further in the present article, although they clearly constitute a promising potential genetic approach to restenosis.

Plasmid-Based Gene Transfer
Several early studies of direct in vivo vascular gene delivery used plasmid DNA^{1 53 54 55} in complex with liposome-based carriers. Cationic liposomes interact with negatively charged plasmid DNA to form complexes able to fuse with cell membranes, releasing plasmid DNA into the cytoplasm. Such vectors are relatively inefficient, with a typical maximum of 0.1% to 1% of vascular target cells transduced in vivo by these agents, although recent modifications in lipid composition have achieved up to 4% to 5% transduction efficiency of vascular cells in vivo (R. Grove, unpublished data, 1997). Such low transduction levels would not be expected to be adequate for studies involving molecules that are active only in the cells in which they are overexpressed. However, plasmid-based gene transfer has been accompanied by remarkable biological effects when used to mediate in vivo expression of potent secreted molecules, such as VEGF.^{45 46 47 56} In fact, the initial human experience with vascular-directed local gene transfer has been pioneered in two ongoing trials using plasmid DNA encoding VEGF: one directed at enhancing peripheral angiogenesis⁵⁷ and the other directed at inhibiting peripheral restenosis.⁵⁸

Plasmid DNA transfection may be enhanced by the assembly of nonviral transduction complexes incorporating features of viruses such as ligands, which mediate target cell binding and complex endocytosis, and peptides or viral proteins, which facilitate endosomal lysis and the consequent cytoplasmic access of the plasmid DNA. For example, complexes including adenoviral proteins, transferrin, and polylysine-DNA complexes have demonstrated transferrin receptor–mediated endocytosis and improved transfection efficiency.^{59 60} Furthermore, several reports have described the use of liposomal plasmid vectors incorporating elements of the fusigenic Sendai/HVJ-virus capsid to enhance arterial gene transfer.^{39 61} Such advances in plasmid-based transfer methodology are encouraging, given the less stringent regulatory concerns that surround proposals for nonviral clinical trials.

Retroviral Gene Transfer
Retroviral vectors were also evaluated in the earliest studies of arterial gene transfer.^{1 2} A potentially attractive feature of this vector lies in its ability to integrate into the genome of target cells, thus conferring the long-lasting expression of transgenes. However, use of these vectors for gene transfer has been associated with low efficiencies,⁵³ which are presumably due to both the comparatively low available vector concentrations (10⁶ transducing units/mL) and their requirement for host cell proliferation. Most vascular gene transfer studies have not emphasized retrovirus-based vectors because of these limitations. However, in vivo vascular transduction may be feasible using recently developed pseudotyped retroviral vectors that display the vesicular stomatitis virus G protein on their envelopes^{62 63 64} ; these vectors may be prepared at concentrations of nearly 10¹⁰ transducing units/mL⁶⁴ and, accordingly, are expected to manifest proportionally increased efficiencies.

Adenoviral Gene Transfer
Adenoviral vectors have been described as achieving transgene expression in vivo in >10% to 30% of vascular target cell populations, on the basis of their high concentrations and their ability to transduce nondividing cells; such efficiencies are 100- to 1000-fold higher than reported using retroviral vectors.⁵³ They have therefore been most frequently used for vascular gene transfer and have been instrumental in obtaining the proofs of principle for physiological effects of most of the putative therapeutic genes described above.^{8 10 14 15 37 40 43} However, such successful transduction of vascular tissues has routinely been obtained by the isolation of arterial segments between dual ligatures or catheter balloons in conjunction with blocking all the branch vessels from the selected segments; this has been followed by the introduction of vector suspensions into these target regions and prolonged dwell time periods before the reestablishment of blood flow.^{65 66 67 68} Such techniques have maximized gene transfer as a result of the prolonged time available for binding of vector but, unfortunately, may only be extrapolated to clinical situations in which unbranched target segments may be occluded for prolonged periods. This serious limitation to clinical extension in the coronary arteries will be addressed below with respect to delivery.

Despite the advantages of adenoviral vectors, transduction by the first generation of adenoviral vectors is associated with biological responses that have an impact on their future clinical utility. These vectors retain the majority of the parent virus genome, which in turn is associated with undesired gene expression that results in both immune^{69 70} and vascular inflammatory^{71 72} responses to transduction. Whether purely due to these effects or also related to other phenomena, gene expression from adenoviral vectors in vivo peaks within 1 week and is limited to 2 to 4 weeks in virtually all immunocompetent systems. Such a lack of persistence might be acceptable, given the positive short-term results described with these vectors above, but could limit efficacy against human restenosis, which takes at least 6 months to develop fully.^{50 73} The immune responses to adenoviral vectors involve cellular immunity dependent on CD4+ and CD8+ T cells directed to both vector^{74 75} and transgene-encoded⁷⁶ determinants; they also involve neutralizing antibody to viral capsid proteins in the initial vector inoculum, which can inhibit further gene delivery using the same vector.⁷⁰

Several approaches have been described to reduce or eliminate these immune responses. Three of these approaches involve (1) reduction of the expression of immunogenic viral proteins by introduction of additional functional blocks into the vector genome, such as by insertion of a temperature-sensitive mutation into the E2A region^{77 78 79} or further deletion of the E4 region,^{80 81 82} (2) total elimination of expression of viral proteins, which is also associated with an enlarged capacity for the transport of foreign DNA with sizes nearing that of the viral genome,^{83 84} and (3) transient ablation of T-cell activation to prevent the effector responses of the CD8+ T and B cells. Encouraging results demonstrating enhanced persistence of gene expression and reduced inflammation have been obtained by coadministration of adenoviral vectors with either immunosuppressive agents or one of several targeted immunomodulators, such as CTLA4Ig, and monoclonal antibodies to CD4, CD8, CD11A, and B7.^{85 86 87} A fourth approach is the reduction of input vector, thus reducing the antigenic burden. This may be possible by vector modifications that enhance cell internalization efficiency^{88 89} at lower vector doses or by the use of promoters confirming enhanced expression of the desired therapeutic gene to produce "higher potency" vectors.⁹⁰ Although all of these approaches have promise, it seems likely that the development of vectors with highly ablated viral gene expression holds the greater potential for vascular-targeted genetic therapies. Such "third-generation" adenoviral constructs presently await testing in animal models of arterial gene transfer.

Adeno-Associated Gene Transfer
AAV vectors are quite different from most adenoviral vectors in the absence of expressing viral sequences as well as in their potential for integration into the host genome, although the frequency and consistency of this event is less clear for current vectors than for the parent virus.⁹¹ These features appear to render them capable of producing recombinant in vivo gene expression for prolonged periods of time (at least 6 months) without prominent inflammation in extravascular systems.⁹² Whether these advantages will be borne out in vascular tissues in vivo is not yet clear but is strongly suggested by the consistencies found between vascular and extravascular tissues with respect to effects of other vector systems. Recent developments in the ability to generate highly concentrated AAV vector preparations (10¹⁰ transducing particles/mL) have made possible initial studies showing the feasibility of in vivo vascular delivery.^{93 94} These vectors thus show early, but significant, promise for eventual utility in vascular transduction.

	Mechanical Approaches for Vector Delivery

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The vascular delivery of genetic vectors has used a wide range of devices and methods. Although gene transfer has been demonstrated for each device, most studies of catheter-based gene transfer have revealed low efficiency except in the context of prolonged vessel occlusion with ligated branches, as mentioned above. Accordingly, the development of clinically viable delivery approaches remains a critical challenge to therapeutic extension. The mechanical bases for this hurdle will be considered after summarizing the characteristics of the various approaches, which have been reviewed in detail elsewhere.⁹⁵ The devices may be categorized broadly according to the predominant mode(s) of delivery used and the site of delivery (Table 3). This table lists several approaches and relates these to their expected behavior with regard to immediate localization efficiency, distribution, and local tissue permeation.⁹⁵

Endovascular Delivery Approaches: Convective, Diffusive, and Energy-Assisted
Convective delivery devices are characterized by comparatively high-flow limited-time deliveries, with the predominant delivery mechanism thus expected to be convection rather than diffusion. These include the porous and microporous infusion catheters. The initial porous balloon catheter as described by Wolinsky and Thung⁹⁶ has been used for delivery of a range of vectors, including plasmids, retroviruses, and adenoviruses.^{53 69 97 98 99} Such experiments using porous balloon catheters to rapidly instill vector suspensions onto or into vessel walls from the luminal surface have been associated with transduction efficiencies significantly lower than values easily obtainable in vitro in cell culture experiments.⁵³ This is largely due to the kinetics of vector-cell attachment, which are slow¹⁰⁰ in comparison to the bulk convection of the vector particles on exit from the catheter orifices. These experiments have also demonstrated prominent vectoremia after instillation,¹⁰¹ the direct consequence of inefficient initial vector adsorption at the target site, with "upstream" or "downstream" delivery of the infusate during inflation.¹⁰² Currently under way are experiments investigating approaches to maximize local disposition of vector while minimizing systemic distribution by the use of matrices intended to retain vector particles locally, thus favoring productive vector-cell collisions.¹⁰⁰ Increases in transduction have recently been demonstrated in vivo using hydrogel adjuvants.¹⁰³ Another approach in the restriction of systemic vector effects involves the use of vectors with vascular-specific promoter elements or controllable gene expression; both approaches are currently under active investigation.

A distinct category of delivery devices permits more prolonged endovascular vector contact and thus diffusion. The earliest intravascular delivery approach involved a device with proximal and distal balloons capable of isolation of the intervening arterial segment for subsequent delivery; such a double-balloon catheter has been used to deliver retroviral and adenoviral vectors as well as transfected endothelial cells and smooth muscle cells into nonatherosclerotic porcine femoral arteries.^{1 15 68 98} Recent designs of these devices allow for the maintenance of distal blood flow, thus reducing the potential for ischemic sequelae. One such device, the Dispatch catheter (SciMed/Boston Scientific), has been described as promising for the intramural delivery of adenoviral vectors,^{104 105} but regional as well as systemic distribution may be expected to be substantial in the event of branch vessel delivery.

The hydrogel catheter (SciMed/Boston Scientific) is an angioplasty balloon coated with a hydrophilic polymer that is capable of absorbing fluid containing a range of medications as well as vectors, which may then be delivered as the hydrogel is compressed against the arterial wall. Because the delivery volume is less than that originally contained in the hydrogel coating (0.004 mL), the efficiency of delivery is expected to be quite high, but the absolute amount of material delivered will most likely be quite low. A limitation of this balloon technique is rapid loss of the loaded agent from the hydrogel, which has been documented when the balloon comes in contact with blood within the guiding catheter or the artery before mural apposition.¹⁰⁶ Gene delivery has been achieved by this catheter with minimal or no systemic distribution noted.^{107 108} This catheter is currently undergoing clinical evaluation for the delivery of genes encoding VEGF to patients with severe peripheral vascular disease.^{57 58}

The nipple balloon catheter, or Infiltrator (InterVentional Technologies), was designed to provide direct intramural delivery of agents by mechanical access into the media, which was achieved using sharp-edged injection orifices mounted on the balloon surface.⁴¹ This catheter has been used clinically⁴² and has been demonstrated to yield transduction by adenoviral vectors using reasonable delivery protocols⁴⁴ as described above.

A recent approach to local drug delivery uses energy sources to supplement convective and diffusive forces in achieving agent transfer. One such device (e^-Med Corp) uses iontophoresis to facilitate diffusion of charged ions through a porous membrane covering an infusion balloon into the arterial wall.¹⁰⁹ A potential advantage of this device is enhanced local tissue permeation, as well as delivery efficiency. The utility of this approach in promoting gene transfer is under active investigation.

Perivascular Delivery Approaches: Transvascular and Pericardial
Placement of vector particles within tissues appears to result in enhanced local transduction efficiency compared with that achievable by endoluminal delivery; presumably, this enhanced efficiency is a consequence of the restriction of rapid vector redistribution by connective tissue. Recently, several devices with modified needles capable of direct injection into interstitial tissue of either myocardium or vasculature have been described. Delivery of agents and genes directly into the adventitia, bypassing the delivery barriers represented by the intima and media,¹¹⁰ may provide for relatively rapid and efficient delivery compared with endovascular approaches.¹¹¹ The feasibility of adventitial transduction by direct deposition of adenovirus on the abluminal aspect of the vessel using surgical approaches has been reported,¹¹² and recent experiments have demonstrated adenoviral transduction of the adventitia and perivascular tissue by transvascular needle injection using a delivery protocol requiring only several seconds.¹¹¹

An alternative approach to perivascular delivery involves the instillation of vectors into the pericardial space. The inability of vectors to readily diffuse out of this space results in high frequency transduction of epicardial as well as parietal pericardial mesothelium. We¹¹³ and others¹¹⁴ have found this approach to be efficient in swine and dogs, with expression of both intracellular and secreted proteins localized to the epicardium and pericardium. In addition to serving as a repository for prodrug (vector), we have hypothesized that the pericardial sac may serve as a secretory organ for transduced therapeutic proteins or their metabolites. Agents such as cytokines or growth factors may thus be delivered efficiently as well as locally to the epicardial tissue and vessels.

	Challenges to the Achievement of Optimized Vector Delivery

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For any vector, achievement of an optimized delivery distribution will depend on overcoming key challenges to delivery in several areas, including (1) efficiency of localization, (2) restriction of systemic distribution, and (3) adequacy of permeation into the target tissue. Each of the devices under consideration for gene administration may be assessed according to their ability to provide these delivery characteristics, as well as criteria of clinical feasibility.

Efficiency of Localization
Studies of nearly all delivery devices and agents performed to date have revealed values for delivery efficiency in the range of 0.1% to 1%, when expressed in terms of fractional intramural delivery (FID), defined as the ratio of intramural delivery mass to total delivered mass.^{102 115 116} This indicates that the agent is not primarily distributed to the target vessel but to the region of tissue surrounding the target vessel and/or into the systemic circulation. One key reason for this observation relates to solvent exclusion by the arterial tissue. Because the volume of the vessel may be replaced only fractionally by the delivered liquid, use of total delivery volumes near or in excess of the target vessel volume (0.03 to 0.15 mL) will lead to immediate extratarget distribution (and resultant delivery inefficiency) on a purely volumetric basis. A second basic reason for delivery inefficiency is the complete bypass of the vessel wall by a significant fraction of the infused volume due to siphoning of the fluid into low-resistance pathways, such as branches and dissections. In cases with no branches, and in the absence of transmedial tearing, delivery efficiency is potentially maximized by forcing all delivered volume to pass through the wall, thus rendering agent binding a possibility.

The third phenomenon contributing to low values of FID and rapid systemic distribution after delivery is the finite time period required for agent binding to receptors or sites providing binding affinity in the vessel wall. For the case of adenoviral vector transduction, the rate constants for attachment to vascular smooth muscle cells as well as other cell types have been evaluated and found to be consistent with theoretical predictions based on velocities of particle diffusion within liquid media. These studies demonstrate that when cells are exposed to bulk suspensions (as opposed to very thin layers) of vector particles, a majority of vector will remain unadsorbed at time points less than several hours, regardless of the initial vector concentration.^{100 117} The comparatively rapid linear velocities of flow in fluid exiting delivery catheters (1 to 8 m/s) (C. Enger, unpublished data, 1997) as well as the flow of blood adjacent to the vascular surface accordingly represent a significant competition for diffusion-limited vector absorption and contribute to low transduction efficiency.¹¹⁸ This provides the basis for approaches to restrict vector distribution by infusion in agents of elevated viscosity.

Restriction of Systemic Distribution
Relatively few studies have quantitatively described the fraction of agent infused through a local delivery device that is rapidly distributed systemically, ie, that is localized neither to the target vessel nor to the surrounding region. This fraction represents most of the infused material, depending on the specific mode of delivery. After the infusion of adenoviral vectors into rabbit iliofemoral arteries made focally atherosclerotic by a standard desiccation/lipid feeding protocol,¹¹⁹ evidence for significant systemic vector distribution was indicated by luciferase expression as well as polymerase chain reaction–detected vector DNA in multiple tissues.¹⁰¹ Conversely, after the administration of dried adenovector suspension adsorbed onto a hydrogel catheter, circulation of vector was not detected.¹⁰⁷

Local Permeation Into Normal and Diseased Tissue
Another requirement for a theoretically "ideal delivery" is the achievement of deep and symmetric permeation of arterial tissue by vectors. Although significant depth of penetration has been demonstrated for small molecules, including oligonucleotides, proteins, and plasmid DNA, the density and cohesive nature of the media appear to provide an impediment to effective medial delivery of viral vectors as well as other microparticulates in the absence of tissue damage, including dissections of the internal elastic lamina and separations between cells composing the vascular wall.^{120 121} These observations likely reflect the preferential bulk or convective fluid entry into low-resistance pathways, such as dissections, as well as virtual impenetrability of the media to diffusive entry of these particles, termed solute reflectance. It is also possible that receptor-mediated vector attachment and internalization into vascular wall cells, particularly in the media, are impeded by the components of the basal lamina "shell" surrounding each cell, effectively forming a local barrier for vector access to membrane receptors. A potential approach to overcoming the barriers presented by high medial reflectance as well as the basal laminae might be the adjunctive delivery of enzymes directed to connective tissue components of the vessel wall, such as elastases¹²² or collagenases. The long-term effects of such chemical modification of the integrity of the wall remain to be investigated.

Despite the fact that the vessels targeted for local delivery of genetic material will exhibit pathology, including plaque burden in the intima as well as potential medial thinning and intramural cholesterol and calcium deposits, comparatively little quantitative investigation of delivery into diseased vessels has been performed to date.⁹ It is anticipated that the issues described above will be relevant to diseased vessels, but the results of added anisotropy of physical and biological parameters due to inhomogeneous disease are not yet known. Reports of adenoviral gene transfer to diseased vessels have demonstrated feasibility of plaque and medial transduction¹⁰⁴ as well as therapeutic effect but have indicated substantially lower efficiencies of transduction (0.2% of vessel cells) in diseased vessels in some studies.⁹⁷ Most recently, studies of the endovascular delivery of first-generation adenoviral vectors (10¹⁰ plaque-forming units) into diseased human peripheral arteries before planned amputation have revealed 5% overall transduction efficiency in the diseased vessels, although lower values were observed in the plaques. This was well tolerated without significant adverse sequelae (S. Yla-Herttuala, unpublished data, 1997). Such data are particularly encouraging with respect to the feasibility of strategies involving secreted molecules as mentioned above, although the specific delivery mechanisms that will be found best adapted to address the issues posed by vessel disease remain to be determined. Again, the choice of candidate therapeutic genes that cause the generation of diffusible molecules may be expected to decrease the stringency of the requirements for homogeneous high-efficiency gene transfer.

Several delivery systems now appear to have the potential to address these challenges in ways that are clinically feasible, by facilitating tissue penetration using mechanical, electrical, or other forces or by permitting the introduction of genetic material into the perivascular space. Use of such devices should allow the continuing progression of genetic vascular therapies toward clinical utility.

	Summary

Top Abstract Introduction Candidate Gene Products for... Vector Systems for Vascular... Mechanical Approaches for Vector... Challenges to the Achievement... Summary References

 
Intensive work over the past decade has been directed at the study of vascular gene transfer as an approach to the unresolved problem of restenosis. The substantial time and effort invested in this area to date have not gone without reward. A significant foundation of knowledge has been gained relative to activities of potentially therapeutic gene products, as well as the capabilities and limitations of vector systems and mechanical delivery modalities available for effecting the vascular expression of these gene products. Such results have led to a clear definition of key challenges that must be experimentally addressed in order to make feasible progress from the preclinical to the clinical arena. These challenges include primarily (1) the need for clinically practical methods of delivery capable of achieving consistent therapy of diseased arterial tissues, (2) issues of host immune response to the most efficient adenoviral vectors, (3) comparatively low efficiency typical of less immunogenic vectors, and (4) validation of the biological efficacy of gene products in the context of preexistent arterial disease. Data are beginning to emerge suggesting that these issues may indeed be at least partially addressed by recent specific breakthroughs in delivery methods and vector design.

Clinically reasonable delivery systems with the potential for efficient delivery even in highly branched vascular systems are currently being evaluated. Increases in low-delivery efficiencies are being successfully addressed by attempts to prolong retention of vectors in the locale of target cells in order to permit enhanced attachment. These attempts include incorporation of vectors into a variety of matrices, as well as the use of extraluminal/intramural sites of delivery in an effort to avoid competition of blood flow with vector binding and specific localization. Enhanced homogeneity of distribution of vector (prodrug) or gene product (drug) after the vector delivery may result from the use of new approaches that facilitate vector permeation into tissues. The development of plasmid-based gene transfer systems capable of increasingly efficient in vivo transduction and the advent of efficient viral vectors containing minimal genetic material of viral origin, such as advanced-generation adenoviruses and adeno-associated viruses, should help to overcome the vector-based issues of host immune or inflammatory responses. Finally, several genes have been confirmed as having therapeutic effects in preclinical models of stenosis and are ready for further evaluation in the context of arterial disease using appropriate vectors and delivery approaches. Accordingly, the key hurdles to the advent of genetic medicines for restenosis, although significant, no longer appear insurmountable. Although it is yet too early to identify a protocol that defines a new genetic standard of care for the reduction of the restenotic response, the years of experimentation that have led to these advances have indeed brought the field much nearer the heart of the matter.

	Acknowledgments

 
David Hilton is gratefully acknowledged for his contribution to the title, and the excellent editorial assistance of Cheryl Lockett is much appreciated.

Received August 28, 1997; accepted November 6, 1997.

	References

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