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Cardiovascular Research 1997 35(3):391-404; doi:10.1016/S0008-6363(97)00148-X
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Optimal techniques for arterial gene transfer

Laurent J Feldman* and Gabriel Steg

Cardiology Department and U460 INSERM, Faculté Xavier Bichat, Hôpital Bichat, 46, Rue H. Huchard, 75877 Paris, Cedex, France

* Corresponding author. Tel.: +33 1 40256601; Fax: +33 1 40258865; E-mail: laurent.feldman@bch.ap-hop-paris.fr

Received 11 February 1997; accepted 15 May 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Vectors
 3 Gene delivery techniques
 4 Arterial gene transfer:...
 5 Conclusions and perspectives...
 References
 
Cardiovascular gene therapy is becoming a clinical reality due to improved vectors, delivery systems and careful experimental validation studies. Nearly all cardiovascular diseases are amenable to gene therapy, but the optimal combination of vector, delivery system and therapeutic gene is likely to be unique to each application. Currently, the most efficient vectors available are replication-defective adenoviral vectors, but transgene expression is limited in time due to a strong immune response. Conversely, non-viral vectors or plasmid DNA may be used safely but have very limited efficiency. Percutaneous, catheter-based delivery is feasible for most applications. The ultimate issues that will decide of the future of gene therapy are safety of the transfer and delivery techniques as well as cost/effectiveness comparisons with alternative therapies, including local delivery of drugs, proteins and/or mechanical devices.

KEYWORDS Gene therapy; Atherosclerosis; Adenovirus; Liposomes; Catheter; Restenosis; Angiogenesis; Plaque stabilization


   1 Introduction
Top
 Abstract
 1 Introduction
2 Vectors
 3 Gene delivery techniques
 4 Arterial gene transfer:...
 5 Conclusions and perspectives...
 References
 
Atherosclerosis and its complications represent the first cause of mortality and morbidity in the western world [1]. Major advances have been made in the treatment and prevention of symptomatic atherosclerosis. Improved understanding of the pathophysiology of atherosclerosis and its complications [2], as well as spectacular advances in the molecular biology of the vascular wall [3, 4]may open new perspectives for treatment based upon local delivery of genetic material designed to modify the atherosclerotic plaque at the molecular level [5]. Transfer of a functional gene into arterial wall cells, termed arterial gene therapy, may be used to replace or palliate a defective gene, or to express a protein with a therapeutic effect [6].

Effective arterial gene therapy requires techniques to introduce (transfect) a foreign gene (transgene) into the cells of the arterial wall. These techniques rely on transfer vectors which facilitate cellular penetration and intra-cellular trafficking of the transgene, as well as local delivery systems, either catheter-based or surgical, to deliver the vector to the vicinity of the target cells.


   2 Vectors
Top
 Abstract
 1 Introduction
 2 Vectors
3 Gene delivery techniques
 4 Arterial gene transfer:...
 5 Conclusions and perspectives...
 References
 
Under certain conditions, it is feasible to introduce foreign DNA into the nucleus of eukaryotic cells. This has been used to obtain transient or stable expression of several genes in cell lines. To achieve expression of foreign DNA, however, the transferred gene should enter the cell, escape degradation by lysosomal enzymes, cross the nuclear membrane, escape degradation by endonucleases and, eventually, be expressed. Each of these steps represents a potential limitation to the efficacy of gene transfer, which make spontaneous transfer and expression of foreign DNA into eukaryotic cells a rare phenomenon. Transfer vectors are therefore required to increase the efficiency of the process. These may be viruses, non-viral vectors or mixed systems combining viral and non-viral elements.

2.1 Viral vectors
Viral-based vectors are viral particles which retain their ability to enter target cells and transfer in these cells foreign genes, but have been engineered to incorporate the transgene in their genome and loose their replicative activity. Some of them (retroviruses, lentiviruses and possibly adeno-associated viruses) are the only methods ensuring stable integration of transferred DNA into the chromosomal DNA of the target cell. In order to transform viruses into safe vectors, genomic sequences which are required for viral replication have been deleted.

2.1.1 Retroviral vectors
These are single-stranded RNA viruses that bind to a specific cell surface receptor to get entry into the cell. Their capsid is surrounded by an envelope, the proteins of which mediate adhesion to the cell membrane. Following penetration into the cell, viral RNA is transformed into double-stranded DNA by the reverse transcriptase and the DNA integrates randomly into the host cell genome, creating a ‘provirus’. The viral genome contains 3 structure genes, gag, pol and env, encoding the capsid proteins, the reverse transcriptase and the envelope proteins respectively. The genome also contains assembly sequences, including the {Psi} gene, required for virion encapsidation. The retroviral vectors currently used [7]are derived from the Moloney murine leukemia virus. Schematically, in these vectors, the gag, pol and env genes have been deleted and replaced, by homologous recombination, by the transgene, while the {Psi} sequence is retained. Due to packaging limitations, the size of the transgene in retroviral vectors is limited to 9 kb. In the absence of structure genes, these defective recombinant vectors are unable to replicate. In order to obtain viral stocks, it is therefore required to use ‘packaging’ cell lines, in which the gag, pol and env genes are stably expressed. Transfection of these cells by the defective retroviruses genome leads to transcomplementation of the retroviral genome and to the production of replication-defective retroviral vectors harboring the transgene.

Since the retroviral genome integrates into cell DNA, the transgene is transmitted to daughter cells and its expression remains stable. Therefore, retroviral vectors have been used extensively for gene transfer in general [8], and were the first vectors to be used for arterial gene transfer in particular [9–11]. Transfer is restricted to cells possessing the retroviral specific receptor. The receptors for both amphotropic, i.e., with extended tropism including human cells, and ecotropic, i.e., specific for murine cells, retroviruses have been cloned and are broadly expressed [12]. There are however, several problems associated with the use of retroviral vectors for arterial gene transfer: (1) retroviral vectors can only infect replicating cells [13], which represent only a few percents of vascular cells in normal or atherosclerotic arteries, even following balloon angioplasty [14, 15]; (2) it is difficult to purify and concentrate these vectors in order to achieve the high titer solutions required for efficient gene transfer; (3) retroviral vectors are unstable and, therefore, inappropriate for in vivo gene transfer; (4) finally, integration of the retroviral genome into the host cell DNA carries potential risks of insertional mutagenesis as well as activation of cellular oncogenes following integration, which would argue for the restriction of retroviral vectors to the treatment of life-threatening diseases.

Retroviral vectors remain largely used in current gene therapy protocols, especially when the therapeutic gene is delivered ex vivo (indirect gene transfer) in proliferating cells which are subsequently transplanted back into the body [8]. However, they are poorly suited to in vivo gene transfer (direct gene transfer). Recent developments in the field of retroviral vectors include engineering of lentiviral vectors based on HIV, which have been shown to achieve stable in vivo gene transfer into non-dividing cells [16], as well as high titers pseudotyped vectors [17]. Whether these approaches will make retroviral vectors more effective and safe for clinical application however remains to be demonstrated.

2.1.2 Adenoviral vectors
Adenoviruses (for reviews, see Refs. [18–20]) are non-enveloped viruses carrying a 36 kb double-stranded DNA. There are nearly 50 adenoviral serotypes, but only serotypes 2 and 5 have been used for gene transfer. Adenoviral genome is composed of regions which are expressed ‘early’ (E1–E4) or ‘late’ (L1–L5) relative to viral DNA replication. The expression of adenoviral genes is controlled by cellular transcription factors and by the E1 region which encodes a transactivating factor. Adenoviruses enter the cell through a receptor-mediated endocytosis pathway. Adenoviral particles bind to two receptor-types, including a glycoproteic receptor specific for the adenovirus fiber protein and surface integrins ({alpha}vβ3 and {alpha}vβ5) that serve as receptors for the adenovirus penton protein, and are then internalized by endocytosis. Acidification of the endosomal content results in conformational changes of the viral capsid proteins, which leads to rupture of the endosomal vesicle and liberation of the viral DNA in the cytoplasm, before DNA degradation by lysosomal enzymes. Viral DNA is then transported from the cytoplasm into the transduced cell nucleus, where it remains episomal. Endosomolysis is a key feature of adenoviruses which is largely responsible for the high transfection efficiencies reported with adenoviral vectors in numerous organs [21–26].

First generation recombinant adenoviral vectors currently used (for a review, see [27]) are obtained through homologous recombination between the genome of a serotype 5 or 2 adenovirus, which has been deleted of its leftward part, and a shuttle plasmid in which the transgene has been inserted along with the leftward part of the adenoviral genome, in order to facilitate recombination (Fig. 1). Expression of the transgene is usually driven by a ‘strong’ viral promoter, such as the Rous sarcoma virus long terminal repeat (RSV LTR), the cytomegalovirus (CMV) immediate-early promoter/enhancer, or the major late promoter and tripartite leader of adenovirus 2 or 5. These are constitutive promoters, which therefore lead to synthesis of large amounts of recombinant protein, but lack tissue specificity and allow no regulation once the gene is transferred. The vectors are made replication-defective by deletion of the E1A and E1B sequences from the viral genome. The E3 sequence is also deleted in order to accommodate insertion of long transgenes (up to 7.5 kb). Homologous recombination as well as propagation of recombinant adenoviral vectors are achieved by co-transfection in 293 cells, a complementing cell line which constitutively expresses the E1 gene.


Figure 1
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  		Fig. 1 Construction of recombinant, replication-defective (E1-deleted) adenoviral vectors. The genome of recombinant adenoviral vectors is obtained through homologous recombination between (1) a shuttle plasmid (pAd-RSVβgal) carrying the left end (map units 0 to 17) of the Ad5 genome, except E1 which is replaced by a nuclear localization signal (SV40), the Rous Sarcoma Virus long terminal repeat (LTR) and the β-galactosidase reporter gene. and (2) the rightward part of the Ad5 genome (map units 26 to 100), in which the E3 region has been deleted.

 
Adenoviral vectors allow to circumvent some of the problems encountered with retroviruses: (1) they can infect quiescent as well as replicating cells; (2) high titer stocks can be produced easily, generally ranging from 1011 to 1012 plaque forming units (pfu)/ml; (3) transfection efficiencies of 100%, for endothelial cells [28]and ~5%, for medial smooth muscle cells [29, 30], have been recently reported in animal models of arterial gene transfer; and (4) after infection, the adenovirus genome remains episomal, therefore avoiding the risk of insertional mutagenesis.

Nevertheless, several drawbacks of adenoviral vectors have been identified. First, current adenovectors are associated with only transient, 2- to 4-week, transgene expression [31]. Second, at a high multiplicity of infection (MOI) — i.e., the ratio of infectant viral particles to target cells — replication may be observed in E1-deleted adenoviral particles. This may be related to the presence of proteins with E1A-like activity in host cells [32]. Alternatively, replication competent adenovirus (RCA) may emerge during production of E1-deleted viral stocks through homologous recombination between the transgene and the E1 region of 293 cells. Third, it has been demonstrated that first-generation adenovectors evoke a strong cellular immune response, targeted at viral proteins as well as certain transgene products [33, 34], resulting in destruction of these cells by cytotoxic T lymphocytes [35]. In addition, infection of the target cells by adenoviral vectors leads to an acute inflammatory reaction, characterized by a neutrophil- [36]and macrophage-rich [37]cellular infiltrate. Finally, it has been observed that local delivery of adenoviral vectors in normal arteries upregulates the expression of vascular cell adhesion molecules and may even trigger mild, although significant, neointima formation [38]. The respective roles of the vector, the transgene, RCAs or impurities related to vector processing, in the generation of adverse effects associated with first-generation adenoviral vectors remain unclear.

The main consequences of the immune/inflammatory reaction directed against recombinant adenoviral vectors are rapid extinction of transgene expression (within a few weeks) and the occurrence of inflammatory reactions in the recipient. Repeated injections of adenoviral vectors might not result in prolonged transgene expression [39]due to a humoral immune response targeted at both viral proteins and transgene products [40]. Interestingly, the duration of transgene expression is substantially prolonged when transfer is achieved in newborn animals [41], probably because these are not yet immunocompetent and can therefore tolerate viral proteins, or in animals in which cellular immune response has been pharmacologically depressed [42]. Immunosuppression, however, cannot be used routinely in clinical practice. Thus, new adenoviral vectors, termed second and third-generation vectors, are currently developed [32, 43, 44]. These vectors are modified to prevent in a more efficient fashion residual expression of adenoviral proteins, thereby mitigating adenoviral protein-specific cellular immune response, which in turn leads to less inflammatory reaction as well as protracted transgene expression. It must be borne in mind, however, that transgene expression is transient not only because of the cellular immune response, but also because of the episomal situation of the transgene, ‘extinction’ of promoter sequences and because of transcriptional and post-transcriptional mechanisms which remain largely unknown. In certain indications, however, such as prevention of restenosis, transient gene expression may be sufficient and may even confer a relative safety to adenoviral vector-based strategies for gene therapy, since potential deleterious effects related to transgene expression would be limited to a few weeks.

2.1.3 Other viral vectors
Other viral vectors are being developed for gene transfer but have not been tested in models of arterial transfection. The AAV (adeno-associated viruses) are single-stranded DNA parvoviruses. Wild-type AAV have the ability to integrate stably their genome into chromosome 19q13 of the human cellular genome. Such ‘targeted’ integration could theoretically limit the risk of insertional mutagenesis related to random integration into the host cell genome. However, recombinant AAV [45]do not appear to exhibit the same site specific integration as wild-type virus. Alternatively, herpes viruses would result in protracted transgene expression. They are however, toxic to the transfected cell, even in their replication-defective form [8].

2.2 Non viral vectors
The limitations and risks associated with viral vectors are powerful stimulants for the development of non viral vectors. DNA delivered by nonviral methods is maintained in an extra-chromosomal state and is not integrated into the cellular genome [46].

2.2.1 Naked DNA
It is possible to incubate plasmid DNA with cells and obtain non-specific DNA uptake, which generally does not result in DNA integration into the host cell genome. When integration occurs, a rare phenomenon, it is random and carries a risk of insertional mutagenesis. Although this technique has a low efficiency of transfer, it is simple and safe. It is extremely well suited to ex-vivo transfer when transduced cells can be sorted using a selectable marker gene, but it has also been used clinically [47, 48].

2.2.2 Cationic liposomes
Cationic liposomes are positively charged artificial lipid vesicles, which incorporate negatively-charged plasmid DNA. In contrast with viral vectors, there is no size constraint for the transgene and preparation of the vectors is easy (Fig. 2). DNA-liposomes complexes contain an excess of liposomes and therefore of positive charges, which facilitate fusion between liposomes and the negatively-charged cell membranes [49]. Once in the cytoplasm, most of the DNA-liposome complexes are degraded by lysosomal enzymes, and approximately 1% of the DNA which originally penetrated the cell enter the nucleus where it remains extra-chromosomal. This explains why transgene expression is transient when liposomes are used as vectors. Various cationic liposome preparations are commercially available: DOTMA-DOPE (Lipofectin), DOSPA/DOPE (Lipofectamine), DC-cholesterol, DMRIE/DOPE. The efficiency of cationic liposomes for gene transfer is superior to that of other non-viral methods and varies with the preparation used, the DNA/liposome concentration ratio, the type and the proliferative status of the transfected cells (transfer appears increased in proliferating cells). While in theory, under optimal conditions, in vitro transfection efficiencies can reach up to 90% of target cells, experience with in vivo arterial gene transfer suggests that efficiency remains disturbingly low [50–52]. Compared to viral vectors these vectors are at least 3 logs less efficient, and require extremely high DNA concentrations to achieve successful gene transfer. Finally, cationic liposomes are potentially toxic due to the cellular accumulation of lipids. This untoward effect, however, has not been observed in several studies in which DNA/liposomes complexes were introduced systemically [53].


Figure 2
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  		Fig. 2 Schematic representation of cationic liposomes. Positively charged cationic liposomes are complexed with negatively charged DNA, and penetrate into the cell via membrane fusion, facilitated by the electrostatic interaction between the positive liposome charges and the negative cell membrane charges.

 
2.2.3 Conjugated vectors
Conjugated vectors (for a review, see [54]) represent a heterogeneous class of vectors in which the transgene is conjugated to a polycation (e.g., polylysine) which is chemically bound to a proteic ligand. Polylysine forms a complex with negatively-charged DNA via electrostatic interaction, and condenses the DNA into a macromolecule-like structure. Binding of the ligand to its specific membrane receptor mediates vector internalization into the cell. Several studies have established that selective in vitro gene transfer can be achieved using this method. In these studies, the ligand was either an asialoglycoprotein, which is recognized by hepatocytes, or transferrin, which is recognized by many replicating cell lines, or polylysine-bound osidic residues. The main limit of this method is its poor efficiency, related to transgene degradation by lysosomal enzymes. Several methods have been used to improve somewhat transfection efficiency, such as addition of chloroquine (which prevents acidification of the endosomal content, thereby preventing transgene hydrolysis) or fusion peptides (which disrupt the endosome membrane and release the transgene into the cytoplasm). Such conjugated vectors have been used in vivo to obtain transient and partial correction of the hypercholesterolemia of Watanabe rabbits via transfer into hepatocytes of the gene encoding for the LDL receptor [55].

2.3 Mixed vectors (viral/non viral)
These vectors are designed to combine the benefits of both viral and non viral vectors.

2.3.1 Conjugated vectors associated with defective adenoviruses
The combination of replication-defective adenoviruses and conjugated vectors is designed to increase gene transfer efficiency by using the adenovirus capability for endosomolysis, while retaining the specificity of transfer associated with ligand/membrane receptor interactions. Several methods have been used. The most simple consists in incubating the target cells in the presence of a mixture of conjugated vectors (e.g., DNA-polylysine-transferrin) and defective adenoviruses. This method is 2000-fold more efficient than that using conjugated vectors alone [56]. Efficiency is even greater when defective adenoviruses are coupled to conjugated vectors via a monoclonal antibody targeted at the hexon protein of the adenoviral capsid [57]. In this case, however, transfer specificity is lost due to the interaction between the adenoviral fiber protein and its specific receptor. This problem has been solved by masking the fiber protein, either by a monoclonal antibody, or by oxidation of the fiber protein. These mixed conjugated vectors are very efficient in vitro. In contrast with recombinant adenoviral vectors, the size of the transgene is not a limiting factor, since it is not incorporated into the viral genome. However, in vivo efficiency of these vectors is limited by their instability and the requirement to cross the endothelial barrier in case of systemic injection. For this reason, mixed conjugated vectors are used in models of ex vivo (indirect) gene transfer [58]. Even under these ‘optimal’ conditions, duration of transgene expression is limited to approximately 2 weeks, probably because of non-integration of the transgene into the host cell genome and viral protein-specific immune response.

2.3.2 Conjugated vectors associated with the Hemagglutinating Virus of Japan (HVJ)
These vectors integrate liposomes, DNA and UV-inactivated HVJ particles. This combination has been proved effective in models of liver and kidney transfection, probably due to the ability of HVJ virus particles to penetrate the cell. It is assumed that when one HVJ particle gains access to the cell, via a specific receptor-mediated pathway, one or several liposome/DNA complexes are internalized in the cell and a certain amount of internalized DNA reaches the nucleus where it is expressed. The absence of toxicity of these vectors and their efficiency (~10-fold that of cationic liposomes) for vascular smooth muscle cell transfection, make them an attractive tool for arterial gene transfer [59]. These vectors have been used for a host of cardiovascular applications (for a review, see [60]), including the study of the effect of autocrine-paracrine vasoactive modulators (e.g., the renin-angiotensin system) on vascular smooth muscle cells in vitro [61]as well as the inhibition of intimal hyperplasia using nitric oxide synthase cDNA [62], antisense oligonucleotides directed towards cell-cycle positive regulators [63], or transcription factor ‘decoys’ [64].


   3 Gene delivery techniques
Top
 Abstract
 1 Introduction
 2 Vectors
 3 Gene delivery techniques
4 Arterial gene transfer:...
 5 Conclusions and perspectives...
 References
 
3.1 Percutaneous local delivery systems
Most potential targets of arterial gene therapy would require local expression of the transgene at a specific arterial site. This can be achieved either by delivering the gene vector in the vicinity of the target site, i.e., local delivery, or by incorporating in the vector design a ligand which will drive transgenic expression at that site, even if the vector is injected systemically. Advances in local gene delivery to the arterial wall have largely benefited from progress made in the technology of angioplasty balloon catheters. The simplest method for local arterial gene transfer is the ‘dwell method’, which requires to isolate surgically between two temporary ligatures an arterial segment, withdraw blood, inject into the isolated segment a solution containing the vector, then withdraw this solution after a variable incubation time and reestablish blood flow [28, 65, 66]. Dwelling allows to control several of the parameters which determine gene transfer efficiency: absence of leakage from the transfer compartment, minimization of vector loss in the systemic circulation, shortened transfer handling time, ..., etc. The invasive nature of the method, however, is a major drawback for clinical application. For this reason, various local gene delivery systems have been developed (for reviews, see [67, 68]).

3.1.1 Catheters
An ‘ideal’ local delivery catheter should incorporate the following features to perform optimal arterial gene transfer. First and foremost it should achieve very efficient gene transfer. Second, if it is intended to be used for prevention of restenosis, balloon angioplasty and gene transfer should be performed simultaneously during a one-step straightforward procedure using the same catheter. Third, the local delivery device should be safe, and, in particular, should not induce excessive injury to the arterial wall, in addition to the injury associated with the angioplasty per se. Fourth, gene delivery catheters should incorporate a perfusion design to limit myocardial ischemia during gene incubation. Finally, site-specificity is a key issue for arterial gene therapy. Local delivery catheters should be designed in order to minimize gene leakage in the bloodstream, which may limit local efficacy and result in systemic toxicity, especially when viral vectors are required.

Currently available local delivery catheters are listed in Table 1. It must be stressed, however, that the vast majority of them have only been tested for arterial delivery of pharmacologic agents to normal arteries. Therefore, data obtained from the literature may not be directly applicable to gene delivery, in particular in atherosclerotic arteries. Schematically, local delivery through catheters involves 3 basic, device-related mechanisms: passive diffusion, pressure facilitation, and mechanical facilitation.


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  		Table 1 Currently available local delivery catheters

 
3.1.1.1 Passive diffusion
The double balloon catheter is made of two latex balloons which, when inflated into the target arterial segment, delineate a ‘transfection chamber’ of varying length (usually 15 to 20 mm), into which the transfer vector can be instilled via an infusion port, generally using pressure. A retrieval port is usually available to withdraw the solution at the end of the incubation period. This catheter was the first to be used for catheter-based arterial gene transfer [10]. It has, however, several shortcomings. Passive diffusion of the gene vector requires long incubation times, which may generate tissue ischemia. There is a risk of vector diffusion into the systemic circulation via side branches (such side branches emerge every 2–4 mm in the epicardial coronary arteries), which may result in both decreased transfer efficiency and viral dissemination. Finally, inflation of the two latex balloons is a source of additional arterial trauma both upstream and downstream of the transfected segment.

New catheters represent ‘improved’ designs of the double-balloon catheter. The DispatchTM catheter is a sophisticated catheter allowing simultaneous distal perfusion and isolation of multiple infusion chambers between the catheter and the vessel wall [69]. The main advantage of this device is that even protracted incubations do not induce significant tissue ischemia [70]. This system has been successfully used to achieve substantial gene delivery into the endothelium and superficial medial layers of both normal and atherosclerotic rabbit arteries [71]. However, the infusion chambers do not ensure total isolation from the systemic circulation; therefore the DispatchTM catheter, like most other local delivery catheters, is associated with a substantial risk of vector dissemination, in particular in the liver.

The hydrogel-coated balloon catheter is a conventional angioplasty balloon which is coated with a hydrophilic polymer which swells like a sponge in presence of a solution containing a drug or a gene vector. Upon inflation of the angioplasty balloon in the target vessel, the polymer express the adsorbed materials toward the arterial wall [72]. This balloon was initially designed to cross high-grade complex lesions. It turned out to be a very efficient local delivery system which has been released for clinical use in Europe and United States. This catheter is currently used for intracoronary delivery of urokinase during primary angioplasty for acute myocardial infarction [73], and for local delivery of the VEGF cDNA in the peripheral arteries of patients suffering end-stage arteriopathy [48]. Its main shortcoming is that during exposure of the catheter in the bloodstream, most of the hydrogel content is ‘washed’ off the balloon [69]. However, retention into the polymer may be enhanced by using a protective sheath.

3.1.1.2 Pressure facilitation
The Wolinsky catheter incorporates an angioplasty balloon with 25 µm diameter pores. During balloon angioplasty, inflation pressure drives the solution filling the balloon into the arterial wall [74]. However, at high inflation pressures typically required for optimal angioplasty (5 to 10 atm), porous balloons generate high velocity jets, which result in arterial perforation as well as reactive intimal hyperplasia, and carries a risk of gene dissemination. Therefore, improved porous balloon catheters have been designed to overcome these limitations, principally to dissociate balloon inflation pressure from vector instillation pressure.

The microporous balloon catheter [75]incorporates an internal porous balloon, with 25 µm diameter pores, and a second external membrane with thousands of micropores of less than 1 µm diameter. These micropores tend to limit jetting and wall trauma.

The channeled balloon catheter (Fig. 3) is a conventional angioplasty balloon covered with 24 longitudinal channels, each having a 100 µm diameter pore perfused via a separated lumen, allowing local low pressure instillation during high pressure balloon inflation [76].


Figure 3
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  		Fig. 3 The channelled-balloon catheter. (A) Longitudinal (1) and cross-sectional (2) views of the channelled-balloon. Gene vector solutions are infused through small perforated channels which are located on the surface of the angioplasty balloon. Pressures for balloon inflation and vector infusion can thus be separated. (B) Radiograph showing the channelled-balloon inflated in a rabbit external iliac artery. Balloon-angioplasty and transfection are performed simultaneously. Infusion of contrast medium into the balloon inflation lumen delineates balloon contour.

 
3.1.1.3 Mechanical facilitation
The mechanism used to facilitate local delivery may be either an electrical field, in he case of the iontophoretic catheter, or a physical injury to the vessel wall in the case of the needle catheter or the nipple balloon. The iontophoretic balloon is a porous balloon with an inner electrode which serves as a cathode. An anode is applied on the skin. Electric current drives negatively-charged molecules outside of the balloon into the arterial wall [77]. This system appears particularly well suited to deliver negatively-charged plasmid DNA in the arterial wall.

3.1.2 Intracoronary stents
Clinical use of metallic intracoronary stents has become standard practice during routine PTCA to treat established coronary dissection. Intracoronary stents such as the Palmaz–Schatz stent have led to a reduction in the incidence of restenosis compared to conventional balloon angioplasty, most likely due to a reduction of acute and chronic constrictive remodeling of the vessel wall as well as an optimization of the initial geometric result of PTCA [78, 79].

In addition, intracoronary stents may be used as vehicles for local delivery of drugs or gene vectors into the arterial wall (for reviews see [68, 80]). One of the approaches would be to coat the stent struts with endothelial cells, previously transfected with a therapeutic gene, the product of which may be released locally to exert therapeutic effects. In vitro experiments with metallic stents seeded with normal [81], immortalized [82], or tPA-transfected [83, 84]endothelial cells have established the feasibility of this technique. However, clinical applicability of this technique remains limited due to the requirement of previous isolation and in vitro transfection of endothelial cells — a long and costly procedure — as well as the poor adhesion of these cells to the stent struts following expansion under flow condition. Another strategy is to use polymer-coated stents, which may act as a reservoir for a gene vector [85], or even biodegradable polymeric stents [86].

3.1.3 Polymers
The ‘ideal’ polymer for arterial gene transfer should contain large concentrations of genetic materials to ensure local delivery and protracted residence of the latter into the arterial wall. It should also be biocompatible for the blood/wall interface, and, if possible, biodegradable. Biodegradable polymers could be used for arterial gene transfer in the form of intracoronary stents, micro- or nano-particles injected locally via a catheter [87], gels (‘pavement’) coating the endoluminal aspect of the artery [88], or periadventitial wrapping [89]. The latter method has been used to transfer plasmidic DNA [90]as well as antisense oligonucleotides [91]into the rat carotid artery, in order to prevent intimal thickening following arterial injury. In addition, we recently demonstrated that co-delivery of adenoviral vectors together with the block co-polymer poloxamer 407 facilitates arterial transfection and allows for shorter incubation times [37].

3.2 Myocardial delivery
Pioneered by Wolff et al. [92], intramuscular gene transfer represents an alternative method to target the myocardium. Direct injection of plasmid DNA into the myocardium, although feasible [93–95], has been associated with short-lived (2 to 4 weeks) expression of the transgene in only a small number of cells, as well as with potentially deleterious myocardial fibrosis and inflammation. When intravenous injection of recombinant replication defective adenoviral vectors is used, only minor myocardial uptake is observed [41]. In contrast, direct injection of such vectors in the heart resulted in efficient transfection [26, 96], albeit limited by the previously described shortcomings associated with adenoviral vectors (transient expression of the transgene, immune and inflammatory responses) and by the need for direct access to the myocardium which requires intraoperative, pericardial or perimyocardial injections. Alternatively, at least three studies have reported efficient transfection of the myocardium after intracoronary catheter delivery of adenoviral vectors [97–99]. Pressure conditions during delivery as well as pretreatment of the vessel with various vasoactive agents may impact heavily on the feasibility of myocardial gene transfer via the intracoronary route [99].


   4 Arterial gene transfer: Feasibility and problems
Top
 Abstract
 1 Introduction
 2 Vectors
 3 Gene delivery techniques
 4 Arterial gene transfer:...
5 Conclusions and perspectives...
 References
 
Two strategies can be used for gene transfer: indirect gene transfer involves in vitro transfection of vascular cells which are then implanted back into the vasculature; conversely, direct gene transfer is the direct introduction of a foreign gene into the arterial wall in vivo (Fig. 4).


Figure 4
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  		Fig. 4 Direct vs. indirect gene transfer. (A) Indirect gene transfer: gene transfer is performed in vitro, and the transfected cells are subsequently reimplanted in the target organ. (B) Direct gene transfer: the transgene is directly introduced into the target organ in vivo via systemic or local (surgical or catheter-based) delivery.

 
4.1 Indirect gene transfer
In their seminal experiments, Nabel et al. used recombinant retroviral vectors to transfer the β-galactosidase reporter gene into porcine endothelial cells in vitro [9]. After staining with the specific chromagen X-gal, transfected endothelial cells could be easily recognized and selected for subsequent introduction into the iliac artery in vivo using a double-balloon catheter. Expression of the transgene was found in these arteries several weeks after transfer. A similar strategy was successfully applied to in vitro transfected smooth muscle cells [11]. Other techniques for indirect gene transfer were later suggested such as seeding of genetically modified endothelial cells on the surface of endovascular metallic stents [83, 84], of Dacron arterial prosthetic grafts [100], or of venous grafts [101]. Indirect gene transfer allows for in vitro selection of successfully transduced cells prior to arterial delivery. However, it is only suitable for autologous transfection, i.e., transfected cells can be transplanted only in the same patient from who these cells have been harvested. Moreover, it requires cell isolation and culture prior to transfer, which makes it a cumbersome and costly technique for routine clinical application [5, 8], with the exception of ex vivo transfer into saphenous vein grafts during coronary artery bypass surgery, for which the tissue to be transfected is readily available for ex vivo transfer.

4.2 Direct gene transfer
Direct gene transfer is a one-step method in which the transgene incorporated in a vector is directly delivered into the target arterial site. Again, Nabel et al. were the first to achieve direct arterial gene transfer using replication-defective retroviral vectors and cationic liposomes expressing the LacZ reporter gene, introduced via a double balloon catheter into porcine iliac arteries [10]. β-galactosidase activity was observed in all the transfected animals, up to 21 weeks in those animals transfected using retroviral vectors and 6 weeks when liposomes were used. Other studies using retroviral vectors [102], cationic liposomes [50–52]or plasmid DNA [103, 104], have confirmed these results. However, in all theses studies transfection efficiency was far below 0.1% when nuclear-specific β-galactosidase was used as reporter gene (allowing discrimination from endogenous cytoplasmic β-galactosidase activity).

Low transfection efficiency is a major limitation of these methods, in particular when the transgene encodes a protein which remains intracellular, a frequent feature for many of the candidate genes used in attempts to prevent restenosis [6]. Conversely, when the transgene encodes a secreted protein, such as the growth hormone [105]or a secreted growth factor [106, 107], a substantial biological effect may be observed even when a small number of target cells express the transgene.

Replication-defective adenoviral vectors have been used for gene transfer into the arterial wall, using surgical [28, 36, 37, 65, 66, 108–111]or percutaneous [29, 30, 112–116]transfer techniques. The first generation vectors used in these experiments were replication defective due to deletion of the E1 sequence. When such a vector expressing nuclear-targeted β-galactosidase is delivered in contact with the endothelial layer without previous injury, for example using a surgical ‘dwell’ method or a double balloon catheter, transfer is strictly localized to the endothelium (Fig. 5) [29, 111, 115]. In addition, as previously mentioned, expression of the transgene is confined to the first days or weeks following delivery. Transfection efficiency is related to the concentration of the adenovirus stocks used [111]: efficiency increases with viral concentration when the latter remains ≤5·1010 pfu/ml; it then reaches a plateau, related to direct cytotoxicity of the vector. The magnitude and mechanism of this cytotoxic effect appear to vary with vector design and preparation. When concentration is further increased beyond 1011 pfu/ml, efficiency decreases. Under certain conditions, however, transfection efficiency approximates 100% [28, 29]. In intact vessels, the endothelium is therefore the elective target of replication-defective adenoviral vectors.


Figure 5
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  		Fig. 5 Endothelium-specific arterial gene transfer. An adenoviral vector expressing a nuclear-specific β-galactosidase reporter gene was locally delivered in rat carotid arteries using the dwell technique. Three days following gene transfer, β-galactosidase activity is found exclusively in the endothelial layer (dark nuclei). L, lumen; M, media; A, adventitia. (Feldman et al., unpublished data.)

 
When adenovirus-mediated gene transfer is attempted following abrasion of the endothelial layer, transgene expression is found mostly in smooth muscle cells located in the superficial layers of the media (Fig. 6) [29, 30, 36, 37, 66, 110, 112]. Reported transfection efficiencies, expressed as the percentage of transfected medial cells, range from 2 to 70%. Adenoviral vectors are thus, by far, the most effective vectors for medial transfection. Transfection efficiency in the media depends upon various factors, including virus preparation (concentration of viral stocks, promoter sequences), delivery technique (dwell >catheter-based), catheter-type (hydrogel balloon >double balloon), duration of incubation (long > short), ligation of side-branches, as well as co- or pre-treatment with adjunctive agents. The endothelium and internal elastic lamina are the main barriers to penetration of the adenoviral vectors in the media of non atherosclerotic vessels [29, 115, 117], whereas the neointima is a relatively resistant layer to adenovirus penetration in severely atherosclerotic arteries [30, 118]. Recently, Perlman et al. demonstrated that balloon angioplasty rapidly induces massive apoptosis in the arterial wall, mostly in the superficial layers of the media [119], an anatomic feature which may influence transfection efficiency as well. Low transfection efficiency in atherosclerotic vessels must be considered when potential clinical applications relate to atheromatous arteries. Methods to circumvent low transfer efficiency include the use of therapeutic genes encoding for secreted proteins, which may therefore affect not only transduced but also neighboring untransduced cells [6]. Alternatively, transfection efficiency can be enhanced by using adjunctive agents such as elastase, to permeate the internal elastic lamina [117], or polymers such as poloxamer 407 [37, 120].


Figure 6
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  		Fig. 6 Smooth-muscle cell-specific arterial gene transfer. Percutaneous gene transfer was performed in rabbit iliac arteries. An adenoviral vector expressing the nuclear-specific β-galactosidase reporter gene was locally delivered using a channelled-balloon catheter. In this case, balloon angioplasty and gene transfer are performed simultaneously. Three days following gene transfer, transduced cells (dark nuclei) are located in the superficial as well as deep layers of the media. These cells are easily identified as vascular smooth muscle cells. Note that the endothelial layer has been totally abraded, whereas the internal elastic lamina appears well preserved. L, lumen; M, media; A, adventitia. (Feldman et al., unpublished data.)

 
The delivery device used has also a major impact on the risk of systemic dissemination of viral particles, which is high in the case of amphotropic adenoviral vectors. This may be explored by studying expression of the transgene, or rather its presence (by PCR) in organs remote from the transfer site. Pervious studies have shown that the risk is low when adenoviral vectors are introduced into a surgically isolated arterial segment [28, 37]. When percutaneous delivery is attempted, however, the risk of viral dissemination becomes substantial, most of extra-arterial transfection being located in the liver [29, 37]. The risk is high with the double balloon catheter [29], the porous balloon catheter [121], or the dispatch catheter [71]. It is reduced with the channeled balloon catheter [30], and appears low with hydrogel balloon catheters, at least when a protective sheath is used [29]. The risk is also correlated to the degree of arterial trauma induced by the gene delivery device [114]. Other factors may promote viral spreading such as the use of non-specific viral promoters, high viral titers, as well as the presence of developed vasa vasora in the arterial wall, a typical feature of the atherosclerotic plaque.

Another issue is that in most of currently available transfection systems transgene expression cannot be regulated by physiological or exogeneous signals, which may represent an important limitation for clinical application of gene therapy to some diseases in which precise control over the level of protein production is required to achieve a therapeutic effect and/or prevent toxicity. Several approaches have been tested in transgenic animals and in models of somatic gene transfer to regulate transgene expression [122–124]. The basic element of any system is a pharmacologic agent that modifies the activity of a transcription factor, which is capable of regulating a heterologous promoter that drives transgene expression. For exemple, Bohl et al. have recently designed a system in which two genes packaged into distinct retroviral vectors are transferred in primary myoblasts in vitro: an erythropoietin cDNA driven by a tetO-CMV promoter and a reverse transactivator (rtTA). When doxycycline is added, rtTA binds the tetO-CMV promoter and activates transcription of the erythropoietin cDNA [124]. To date, none of these systems have been tested in models of cardiovascular gene transfer. However, tight regulation of transgene expression may not be as crucial in cardiology as in inheritable metabolic diseases (see below).


   5 Conclusions and perspectives of clinical application
Top
 Abstract
 1 Introduction
 2 Vectors
 3 Gene delivery techniques
 4 Arterial gene transfer:...
 5 Conclusions and perspectives...
References
 
In vivo transfection of foreign genes in the cardiovascular system has broad applications. Retroviral vectors have been used to study cardivoascular development [125]and to learn more on the role of growth factors in the pathophysiology of intimal hyperplasia [106]. Clinical use of transfection techniques to treat cardiovascular disease represents a more challenging issue [5].In vivo application of gene therapy requires a unique combination of appropriate vector, delivery system, target cell and therapeutic gene which is likely to be specifically tailored to each application. Therefore, there is no ‘best’ vector or delivery system. In fact, efforts should be focused on designing the appropriate combination required to approach each clinical situation, which should be tested in the appropriate experimental models [126, 127]. Current indications for arterial gene therapy should fulfill the following requirements: (1) it should be a disease without proven efficient conventional therapy (due to either ineffectiveness or major adverse effects); (2) the arterial lesions to be treated should be amenable to local delivery techniques; (3) pathophysiology should be clear enough for identification of candidate therapeutic genes.

There are currently two major potential indications for arterial gene therapy: prevention of restenosis after angioplasty and therapeutic angiogenesis to treat chronic ischemia, either in the myocardium or in the limbs (for reviews, see [5, 6, 47, 128]). Other potential indications are prevention of degeneration of aortocoronary saphenous vein bypass grafts [101]and atheromatous plaque stabilization [129], but nearly all vascular diseases are theoretically amenable to gene therapy [4, 130], as well as a host of non vascular diseases in which arterial access to the diseased organs is available for local genetic treatment.

Genetic prevention of restenosis best exemplifies the importance of gene delivery techniques in achieving therapeutic success. Indeed, it has long been considered that restenosis — i.e., the recurrence of luminal narrowing in the months following angioplasty — results almost exclusively from intimal hyperplasia, a proliferating process involving medial and intimal layers of vascular smooth muscle cells [131]. Therefore, most of current strategies aimed at preventing restenosis by gene therapy consist in local transluminal delivery of antiproliferative genes in the abluminal arterial smooth muscle cells in order to inhibit intimal hyperplasia [5, 128]. However, recent advances in the understanding of the pathophysiology of restenosis [132]indicate that, both in experimental models [133]and in humans [134], chronic constrictive remodeling (or the absence of compensatory vessel enlargement) plays a major role in late luminal loss and that lumen diameter is strongly correlated to the magnitude of constriction but not to intimal thickness. The mechanism of constrictive remodeling, although still unclear [135], may involve biological changes in the adventitia including inflammation, myocellular proliferation and migration, and fibrosis [136–138]. Should constrictive remodeling become a target for antiproliferative gene therapy, maybe periadventitial gene delivery will be more appropriate than current intraluminal delivery techniques. Adventitial transfection, however, is likely to induce unacceptable arterial trauma which may outweigh the potential benefit of the therapy. Alternatively, endovascular stents are both efficient and safe to prevent arterial constriction. It has been convincingly demonstrated that in-stent restenosis, which occurs in roughly 25% of the patients [78, 79], is almost exclusively composed of intimal hyperplasia [139]. Based on these recent data, the concept of integrated strategies to prevent restenosis arose, in which anti-remodeling stents are combined with antiproliferative genes to achieve optimal prevention of restenosis [140]. If integrated strategies are to be applied to the prevention of restenosis, we should consider developing in-stent gene delivery techniques as a priority. In this regard, preliminary results from a study by Van Belle et al. suggesting that efficient in-stent transfection can be performed percutaneously are encouraging [141].

Time for primary review 31 days.


   References
Top
 Abstract
 1 Introduction
 2 Vectors
 3 Gene delivery techniques
 4 Arterial gene transfer:...
 5 Conclusions and perspectives...
 References
 

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