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Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

Written By

Divya Pankajakshan and Devendra K. Agrawal

Published: February 27th, 2013

DOI: 10.5772/54075

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1. Introduction

Cardiovascular (CV) disease is the most prevalent life-threatening clinical problem and is a major cause of disability and economic burden worldwide [1]. Despite extensive pharmacotherapies, there remain many vascular conditions for which pharmacological interventions are either non-existent or largely ineffective. CV gene therapy offers the benefit of sustained and/or controlled expression of desired proteins in cell types, which makes it more beneficial in providing durable clinical benefits [2]. The therapeutic gene works by either over-expressing therapeutically beneficial proteins, replacing a deficient gene or its expression proteins, or silencing a particular gene whose expression is not beneficial in the clinical scenario [3]. In addition, success of gene therapy also depends on the choice of the vector and the delivery approach. Blood vessels are among the most feasible targets for gene therapy because of ease of access using a catheter or by systemic delivery. The new genetic material should enter the cells in the vasculature overcoming the anatomical, cellular and physiological barriers and induce the expression of the transfected gene in the target tissue. The target cells in the arteries are endothelial cells (EC), smooth muscle cells (SMC) and fibroblasts, which constitute the intimal, medial and adventitial layers, respectively [4]. In the case of atherosclerotic lesions, macrophages also become a target cell. For the treatment of cardiovascular diseases, gene therapy strategies have been designed to enhance re-endothelialization and EC function to reduce thrombosis, inhibit SMC proliferation and migration to prevent neointimal hyperplasia, and to improve therapeutic neo-vascularization to counteract ischemia.

Viral and non-viral vector systems have been evaluated for gene transfer to the vasculature. Lipoplexes, polyplexes and lipopolyplexes as well as naked DNA have been used as non-viral vectors for gene delivery to vascular tissues. Retroviruses, lentiviruses, adenoviruses and adeno-associated viruses have been tested as viral vectors. Both systems have their own advantages and disadvantages that determine its use for a particular subset of CV diseases. Another challenge is the development of delivery approaches that are clinically viable and are capable of achieving consistent therapy for diseased arterial tissues. The efficiency of localization, restriction of systemic distribution and adequacy of permeation into the target tissue are required for the optimal delivery of the vector. It is also dependent on the requirements of a given patho-physiological situation. Systemic, intravascular and perivascular approaches are used for gene delivery to the vasculature.

In this chapter, our goal is to summarize the current understanding of gene therapy strategies used to treat CV diseases, specifically the therapies targeting thrombosis, atherogenesis, SMC proliferation and migration, modification of extracellular matrix (ECM) and regeneration of the endothelial cell layer. We will discuss various vectors and delivery approaches used in the CV gene therapy and describe, in detail, the challenges associated with each approach.

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2. Vectors in vascular gene therapy

The ideal vector for clinical application would target the specific cell, offer the capacity to transfer large DNA sequences, result in therapeutic levels of transgene expression that are not attenuated by the host immune response, express transgene for a duration required to alleviate the clinical problem, pose no risk of toxicity either acutely (as a result of immunogenicity or unregulated transgene expression) or in the long-term (such as oncogenesis), and be cost-effective and easy to produce in therapeutically applicable quantity [5]. Currently, no available vector fulfils all these criteria; therefore, a perfect vector for vascular gene therapy does not exist. Nonetheless, viral and non-viral vector systems have been evaluated for gene transfer to the vasculature.

2.1. Viral vectors

Retroviruses, adenoviruses (Ad) and adeno-associated viruses (AAV) are used as viral vectors in vascular gene transfer. Recombinant retroviruses are RNA viruses that are capable of integrating transgene into the target genome. Disadvantages of this vector include instability, the requirement of cell division for gene transfer and the inability to attain high titers. Since the majority of vascular cells are not undergoing mitosis at the time of exposure to the viral vector, the efficiency of gene delivery to vascular cells by such vectors may be as low as 1% to 2% [6]. Attempts have been made to increase the transduction efficiency in endothelial cell using multiple viral exposures [7] or increasing viral titers by ultracentrifugation [8]. Murine leukemia retroviral vectors (MuLV) pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) have the capacity to transfect human ECs and SMCs in vitrowith significant improvement in stability and transduction efficiency [9]. Unlike other retroviruses, lentiviruses are able to transduce non-dividing cells, which is an attractive characteristic for CV gene therapy. These vectors demonstrate significantly broadened tropism and high stability and have been used to demonstrate efficient transgene delivery in vitrointo SMCs and ECs from human saphenous vein [10], human coronary artery SMCs and ECs [11], and cardiomyocytes [12].

Ad vectors are the most commonly used viral vectors in the CV system. They transfect non-dividing cells efficiently [Figure 1], but sustained gene expression is limited to approximately 2 weeks because the gene is kept episomal [2]. The administration of the Ad vectors is almost invariably associated with the development of systemic neutralizing antibodies directed against the vector [13]. Therefore, lowering the immunogenicity of the Ad virus is desirable and can be achieved by deleting genes that encode viral proteins [14]. Another method of reducing the inflammatory reaction to gene transfer by Ad vectors is to preserve the E3 region, which is supposed to modulate the host immune response in vivo[15]. When systemically administered, Ad5 poorly transduced ECs but could effectively transduce medial SMCs during endothelial denudation [5]. Efficient myocardial transduction was observed following local delivery of Ad5 vectors in porcine heart, where almost 80% of cardiomyocytes were transduced [16].

AAV vectors have emerged as versatile vehicles for gene delivery due to their efficient infection of dividing and non-dividing cells in the presence of helper virus, sustained maintenance of viral genome leading to long-term expression of the transgene, and a strong clinical safety profile [17]. AAV is non-pathogenic since it cannot replicate without the assistance of a helper virus. Recombinant AAV (rAAV) vectors have almost the entire viral genome removed, thereby yielding a delivery vehicle with enhanced safety and reduced immunogenicity [18]. The AAV Repand Capgenes, which are required for viral replication and packaging, are supplied by a helper plasmid during the production process. Wild type AAV preferentially integrates to a specific locus of human chromosome 19. The rAAV has mechanisms for sustained episomal maintenance or semi-randomly integrates at a low rate [19]. Problems with AAV vectors include limited tissue tropism for serotypes that bind heparan sulphate, challenges with preexisting immunity due to prior exposure, and also substantially delayed onset of transgene expression compared to other vectors.

Figure 1.

Transduction using adenoviral vectors. Recombinant adenovirus enters cells via CAR-mediated binding allowing internalization via receptor-mediated endocytosis through clathrin-coated vesicles. Inside the cytoplasm, the endocytosed adenoviral vector escapes from the endosomes, disassembles the capsid and the viral DNA enter into the nucleus through the nuclear envelope pore complex. The viral DNA is not incorporated into the host cell genome, but rather assumes an epichromosomal location, where it can still use the transcriptional and translational machinery of the host cell to synthesize recombinant protein. [CAR; Coxsackievirus and adenovirus receptor]

2.2. Non-viral vectors

Even though the transfection efficiency of non-viral vectors are lower than that of their viral counterparts, they are associated with many advantages such as low immunogenic response, the capacity to carry large inserts of DNA (52Kb), the possibility of selective modification using ligand and large scale manufacture [20]. Ideal non-viral vectors should be degradable into low molecular weight components in response to biological stimuli for lower toxicity and effective systemic clearance. They should also be efficient in overcoming extracellular and intracellular barriers and tissue/cell-targeted for specific accumulations [21]. In this group of vectors, naked DNA, cationic liposomes and cationic polymers have been used for vascular gene transfer.

Gene transfer with naked DNA is attractive because of its simplicity and lack of toxicity [22]. However, the efficiency of gene transfer with naked DNA is low due to its negative charge conferred by the phosphate groups, making cellular uptake difficult by the negatively charged cell surface, rapid degradation by nucleases in the serum and clearance by the mononuclear phagocyte system in the systemic circulation. However, site-specific arterial gene transfer of vascular endothelial growth factor (VEGF)-165 could yield efficient gene transfection resulting in accelerated re-endothelialization, inhibition of neointimal thickening, reduced thrombogenicity, and restoration of endothelium-dependent vasomotor reactivity after injury due to balloon angioplasty in a rabbit model [23]. Physical approaches have been explored for plasmid gene transfer into vascular cells in vitroand in vivo. Ultrasound exposure can induce transient pore formation in the cell membrane, thereby increasing the plasmid DNA uptake. Indeed, microbubble-enhanced ultrasound can achieve transgene expression levels in vitroat approximately 300-fold than that of naked plasmid DNA alone in porcine VSMCs [24]. The non-invasive nature of this technique makes it more feasible for clinical use. Local administration of plasmid DNA, coupled with application of brief electric pulses to cells or tissues to increase cellular permeability-- also called electroporation--yields high levels of transgene expression in the arteries [25]. However this technique is limited by its invasive nature and tissue damage associated with high voltages applied [26].

To increase the efficiency of gene transfer by naked DNA, they are complexed with cationic lipids (liposomes or lipoplexes) or polymers (polyplexes). The resulting net positive charge of the cationic lipid/polymer DNA complexes facilitates fusion with the negatively charged cell membrane and also reduces susceptibility to circulating nucleases. Transfection efficiency of cationic lipoplexes varies dramatically depending on the structure of the cationic lipids (the overall geometric shape, the number of charged groups per molecules, the nature of lipid anchors, and linker bonds), the charge ratio used to form DNA–lipid complexes, and the properties of the co-lipid [22]. Although transfection efficiencies of liposomes are generally seen lower in vascular cells [22], the LID vector system, consisting of a liposome (L), an integrin targeting peptide (I), and plasmid DNA (D), transfects primary porcine vascular SMCs and porcine aortic ECs with efficiency levels of 40% and 35%, respectively, under in vitroconditions [27]. Some of the cationic lipids have been found to negatively affect cell function. Cationic lipid-mediated transfection of bovine aortic ECs inhibits their attachment [28].

The DNA packaging efficiency and in vivostability are higher for cationic polymers compared to cationic lipids. Furthermore, these complexes can be surface-modified with antibodies or other targeting ligands to deliver nucleic acids to specific cells [29]. Several cationic polymers have been evaluated for their ability to form complexes with DNA, the most significant being poly-lysine (PLL) and polyethylene-imine (PEI) [30]. PEI affects EC function [31]; however, when conjugated with fractured polyamidoamine (PAMAM) dendrimers, less toxic effects were observed on vascular cells in addition to the enhanced transfection efficiencies [32]. Brito et al. [33] developed lipo-polyplex nanovector systems that can transfect EC and SMCs with reasonably high efficiency. They used a combination of a cationic biodegradable polymer, poly(beta-amino ester) (PBAE), and a cationic phospholipid, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and obtained 20% and 33% transfection efficiencies in vitroin SMC and ECs, respectively. Molecular tuning of non-viral vectors via stimuli responsive degradation is another novel approach that can be adopted in vascular gene transfer [21]. Schematic representation of non-viral gene delivery is given in Figure 2.

Figure 2.

Non-viral gene delivery using lipoplexes:DNA is complexed with cationic liposomes and is internalized through receptor mediated endocytosis. After their internalization large amounts of complexes are degraded in the endolysosomal compartments. Only a small fraction enters into the nucleus and elicits desired gene expression.

2.3. Stem cells

One of the recent approaches is to use stem cells as gene delivery vehicles. Stem cell-based gene therapy approaches are currently being employed in recent studies as an alternative strategy to promote myocardial angiogenesis and regeneration. Indeed, the injection of genetically modified bone marrow-derived mesenchymal stem cells to express angiopoietin-1 improved arteriogenesis and increased collateral blood flow in porcine model of chronic myocardial ischemia [34]. Nanofiber-expanded hematopoietic stem cells over-expressing VEGF and platelet-derived growth factor (PDGF) had a favorable impact on the improvement of rat myocardial function accompanied by upregulation of tissue connexin 43 and pro-angiogenic molecules after infarction [35].

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3. Major targets in vascular gene therapy

3.1. Promotion of re-endothelialization

EC loss because of vascular injury is a major contributing factor to the local activation of patho-physiological events leading to the development of neo-intimal hyperplasia [36]. Previous reports have shown that transplantation of autologous endothelial progenitor cells (EPCs) onto balloon-injured carotid artery leads to rapid re-endothelialization of the denuded vessels [37]. EPCs can be genetically manipulated ex vivo, expanded, and reintroduced in vivo, where at least a proportion will contribute to a long-lasting pool that can provide therapeutically relevant levels of transgene expression. Chemokine receptor, CXCR4, is a key molecule in regulating EPC homing [38]. Chen et al. [38] reported that CXCR4 gene transfer to EPCs contributes to their enhanced in vivore-endothelialization capacity. In another study, Ohno and colleagues over-expressed C-type natriuretic peptide by gene transfer in rabbit jugular vein grafts and observed accelerated re-endothelialization [39]. EPCs over-expressing endothelial nitric oxide synthase (eNOS) further enhance the vasculo-protective properties of these cells [40]. Local intravascular and extra-vascular expression of VEGF, using plasmid DNA, accelerated re-endothelialization and decreased intimal thickening after arterial injury in rabbit models [23, 41].

3.2. Promotion of endothelial cell function

Antithrombotic and anticoagulation therapy generally involves the systemic administration of agents that target a small region of the vasculature. Localized and controlled delivery of specific genes could allow sustained antithrombotic or anticoagulant treatment when prolonged systemic administration is undesirable. Antithrombotic gene therapy strategies could include inhibition of coagulation factors, over-expression of anticoagulant factors, or modulation of EC biology to make thrombus formation or propagation unfavorable [42]. Ad gene transfer of thrombomodulin decreased arterial thrombosis to 28% compared to 86% in control rabbit model [43]. Hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer of tissue factor pathway inhibitor (TFPI), a primary inhibitor of TF-induced coagulation, significantly reduced/inhibited thrombosis after angioplasty in atherosclerotic arteries without any significant adverse effects [44]. Ad gene transfer of many mediators, including hirudin to inhibit thrombin [45], tissue plasminogen activator (tPA) to enhance fibrinolysis [43], cyclo-oxygenase to augment prostacyclin synthesis [46], prevents arterial thrombosis and promotes local thromboresistance. Vascular gene delivery of anticoagulants by local infusion of retrovirally-transduced EPCs with tPA and hirudin genes has also been attempted [37].

3.3. Inhibition of atherogenesis

The extensive cross-talk between the immune system and vasculature leading to the infiltration of immune cells into the vascular wall is a major step in atherogenesis. In this process, reactive oxygen species play a crucial role, by inducing the oxidation of low-density lipoprotein (LDL) and the formation of foam cells, and by activating a number of redox-sensitive transcriptional factors, such as nuclear factor kappa B (NFκB), Nuclear factor E2-related factor-2 (Nrf2) [47], or activating protein 1 (AP1) that regulate the expression of multiple pro-and anti-inflammatory genes involved in atherogenesis [48]. Delivery of genes encoding antioxidant defense enzymes, like extracellular superoxide dismutase [49, 50], catalase [51], glutathione peroxidase [51] or heme oxygenase-1 [52], suppresses atherogenesis in animal models.

Apolipoprotein E (ApoE), a blood circulating protein with pleiotropic atheroprotective properties, has emerged as a strong candidate for treating hypercholesterolemia and CV disease. The gene transfer of ApoE Ad vectors produced substantial amounts of plasma ApoE following intravenous injection into ApoE-/- mice, which lowered plasma cholesterol, and after 1 month, slowed aortic atherogenesis [53]. Hepatic expression of human ApoE3 using a second-generation recombinant Ad vector directly induced regression of pre-existing atherosclerotic lesions without reducing plasma cholesterol or altering lipoprotein distribution [54]. High concentrations of atherogenic apolipoprotein (apo) B100 could also be lowered by hepatic gene transfer with the catalytic subunit of apoB mRNA editing enzyme [55].

3.4. Inhibition of SMC proliferation and migration

SMC migration and proliferation as well as deposition and turnover of ECM proteins contribute to the process of Intimal hyperplasia. Several different approaches were introduced to inhibit SMC proliferation during restenosis. Most of the approaches targeted inhibition of cell cycle, where cell cycle inhibitor genes are over-expressed. Non-phosphorylated retinoblastoma gene (Rb) [56]; p21 [57, 58]; p27-p16 fusion gene [59, 60] ; cyclin-dependent kinase inhibitor p57Kip2 [61]; and the growth-arrest homeobox gene gax [62] are few of the genes over-expressed to inhibit cell proliferation and neo-intimal formation. Genes that have a beneficial influence on various aspects of vessel wall physiology also inhibit SMC proliferation. Nitric oxide generation by endothelial nitric oxide synthase inhibits SMC proliferation in vitroand modulates vascular tone locally in vivo[63].

Another approach was to inhibit growth factor signaling by the introduction of nucleic acid constructs that interfere with mRNA stability, such as antisense oligonucleotides, hammer head ribozymes and siRNA [64]. Gene transfer of a truncated form of fibroblast growth factor (FGF) receptor using Ad vector suppressed SMC proliferation in vitro[65]. Hammerhead ribozymes directed against PDGF-A chain [66] and transforming growth factor-β [67] inhibited SMC proliferation and neointima formation in rat carotid artery after balloon injury.

The regulation of a target gene can influence the level of transcription, either by decoy oligonucleotides, which are either short double-stranded oligonucleotides or dumb-bell shaped circular oligonucleotides that represent transcription factor binding sites, and thus compete for binding of a specific transcription factor that is relevant for the respective gene [64]. Administration of AP-1 decoy ODNs in vivousing HVJ-liposome method virtually abolished neointimal formation after balloon injury to the rat carotid artery [68]. Transfection of vein grafts with a decoy antisense oligonucleotide to block transcription factor E2F imparted long-term resistance to neointimal hyperplasia and atherosclerosis in rabbits on a cholesterol diet [69]. Another approach was to drive SMC into apoptosis during the process of proliferation and migration. Transduction of rabbit iliac arteries with recombinant Ad vectors for Fas ligand (L) reduced neointima formation, which occurred through the killing of Fas expressing neighboring SMC by FasL-transduced cells [70].

The regulation of SMC migration is mediated partly through the action of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs) [71]. AAV-mediated TIMP1 transduction in SMCs of injured rat carotid arteries significantly reduced the ratio of intima to media (52.4%) after two months of treatment [72]. Overexpression of TIMP-2 [73], TIMP-3 [74] and TIMP-4 [75] has also been demonstrated to inhibit SMC migration and neo-intimal proliferation in human vein grafts and porcine vascular injury models. Gurjar et al.[76] demonstrated that eNOS gene transfer inhibits SMC migration and MMP-2 and MMP-9 activities in SMCs in vitro. A combination approach of TIMP-1 and plasminogen activator system inhibited vein graft thickening in hypercholesterolemic mice, when plasmids encoding TIMP-1-ATF (amino terminal fragment of urokinase) were incorporated to the vein graft by intravascular electroporation [77].

3.5. Enhancement of therapeutic angiogenesis

Ischemic diseases, including acute myocardial infarction and chronic cardiac ischemia, are characterized by an impaired supply of blood resulting from narrowed or blocked arteries that starve tissues of needed nutrients and oxygen [78]. Delivery of genes encoding angiogenic factors or the whole protein has been shown to induce angiogenesis in numerous animal models with the expression of a functioning product [79]. The successful application of recombinant protein and gene transfer for the treatment of myocardial ischemia was reported by Losordo and colleagues [80] by direct intra-myocardial gene transfer of naked plasmid DNA encoding VEGF-165 in porcine model. These results were confirmed in phase 1 assessment of direct intra-myocardial administration of Ad vector expressing VEGF-121 cDNA in patients with severe coronary artery disease [81]. Ad-mediated FGF-4 gene transfer improved cardiac contractile function and regional blood flow in the ischemic region during stress in pig model [82]. Placebo-controlled trials in humans with chronic stable angina indicate that Ad5FGF-4 increased treadmill exercise duration and improved stress-related ischemia [82]. In another study, following coronary artery occlusion, rabbits treated with Ad vector containing acidic FGF showed a 50% reduction in the risk region for myocardial infarction [83].

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4. Challenges in gene therapy

4.1. Cellular and extracellular barriers in gene delivery

Viruses have highly evolved mechanisms for obtaining optimized receptor-mediated internalization, efficient cytosolic release, directed and fast intracellular transport towards compartments and readily disassemble. In contrast, non-viral vectors must overcome multiple extracellular and intracellular barriers [21]. These barriers include binding to the cell surface, traversing the plasma membrane, escaping lysosomal degradation, and overcoming the nuclear envelope. To overcome the delivery barriers in non-viral gene transfer, various strategies have been employed to enhance the circulation time, improve intracellular delivery, and enhance endosomal escape and nuclear import. Lipoplexes have shown rapid hepatic clearance during systemic administration. Modification of lipoplexes with hydrophilic molecules like polyethylene glycol (PEG) and polyethyleneimine (PEI) causes steric hinderance between opsonins and the delivery vectors, increasing their circulation time in the blood. PEGylation of PLL decreases interparticle aggregation, resulting in high transfection efficiency in the presence of serum [29]. One study has demonstrated that when artery wall binding peptide (AWBP), a core peptide of apo B100 -- a major protein component of LDL -- was conjugated to PLL with PEG as the linker, the PLL-PEG-AWBP protected the plasmid DNA from nucleases for more than 120 min in circulation and also showed 100 times higher transfection efficiency when compared to PLL and PLL-g-PEG in bovine aortic ECs and SMCs [84]. In an innovative approach, micellar nanovectors made of PEG-block-polycation, carrying ethylenediamine units in the side chain [PEG-PAsp(DET)], complexed with plasmid DNA to form polyplex micelle effectively transfected vascular smooth muscle cells in vascular lesions without any vessel occlusion by thrombus [85] in rabbit carotid arteries. However, PEI-mediated gene delivery can affect EC function and viability [31].

The size and charge of the lipoplex/polyplex play an important role in their intracellular delivery. Lipoplexes and polyplexes are generally formulated into particles with net positive charges to trigger endocytosis by non-specific electrostatic interaction between the positively charged complexes and negatively charged cell surface [29]. Since drug carriers with a smaller particle size have resulted in higher arterial uptake compared to carriers with larger size, the size of the complexes was expected to be a dominating factor in the arterial wall lesions because of the rapid blood flow which could wash out most of the drugs or therapeutic chemical agents from the arterial wall lesions within 20–30 min. Song et al. [86] reported a potentially useful particle size of 70∼160 nm for local intraluminal therapy of restenosis.

By taking advantage of high expression levels of receptors or antigens in diseased conditions, gene complexes can be targeted using specific ligands, such as antibodies, peptides and proteins. Cyclic RGD (cRGD) peptide recognizes α(v)β(3) and α(v)β(5) integrins, which are abundantly expressed in vascular lesions. When cRGD was conjugated to PEG-PAsp(DET) to form polyplex micelles through complexing with plasmid DNA, the micelles achieved significantly more efficient gene expression and cellular uptake as compared to PEG-PAsp(DET) micelles in ECs and SMCs [87]. PAMAM dendrimers with E/P-selectin antibody was used for gene targeting to activated vascular ECs [88]. The lectin-like oxidized LDL receptor (LOX-1) is expressed selectively at low levels on ECs but is strongly upregulated in dysfunctional ECs associated with hypertension and atherogenesis. White and colleagues [89] confirmed the selectivity to LOX-1 for peptides LSIPPKA, FQTPPQL, and LTPATAI, which could be potential targets to dysfunctional ECs expressing LOX-1 receptor. Another approach to increase intracellular delivery is to use cell penetrating peptides (CPPs). CPPs consist of short peptide sequences that are able to translocate large molecules into the cells and increase the transfection efficiency [90].

Following internalization of lipoplexes and polyplexes via endocytosis, endosomal entrapment and subsequent lysosomal degradation are the major hurdles that limit transfection efficiency [29]. Lipoplexes are modified with dioleoylphosphatidylethanolamine (DOPE) or other helper lipids due to its fusogenic functionality and its ability to destabilize endosomal membranes. Small PLLs with cationic lipid DOCSPER [1,3-dioleoyloxy-2-(N(5)-carbamoyl-spermine)-propane] enhanced gene transfer in primary porcine SMCs in vitroand in vivoin porcine femoral arteries [91]. Polyplexes, PEI and PAMAM are cationic polymers of high efficiency partly because of their ability to burst the endosomal membrane due to ‘proton sponge effect’.

A promising new delivery strategy is to use synthetic peptide carriers containing a nuclear localization signal to facilitate nuclear uptake of plasmid DNA. Nuclear import of plasmid DNA is more challenging for transfecting non-dividing cells. Strategies to increase the nuclear import of genes involve tagging the nuclear localization sequence (NLS) with DNA vectors. NLS is a major player that shuttles protein-plasmid complexes through the nuclear pore by interaction with importins and transportin [92, 93]. Incorporation of DNA nuclear targeting sequence SV40 into expression plasmids results in 10-40 fold increases in vascular gene expression in rat mesenteric arteries [94], confirming the function of DNA nuclear targeting sequences in vivo.

4.2. Challenges associated with the vectors

4.2.1. Insertional mutagenesis

Insertional mutagenesis is a major concern in gene therapy involving viral vectors. These vectors integrate randomly or quasi-randomly into the host cell’s genome, to stably transfect the target cell. The variable site and frequency of integration of the transgene can induce mutagenesis in the host genome, resulting in devastating consequences for the cell and for the organism. [95, 96]. Another disadvantage of the random integration of a transgene is the unpredictability of its stability and its expression. The genomic locus in which the vector integrates can have profound effects on the level of transgene expression, as it can completely silence the transgene, or it can increase or decrease its expression. These effects could not be avoided by sophisticated vector design or inclusion of the gene’s own promoter and/or enhancer region in the transgenic vector construct, as the surrounding chromatin can override the activity of the original regulatory regions. Gene targeting by homologous recombination, however, lacks many of these shortcomings [96]. In this process, the transgene recombines with its natural locus in the host genome, thereby ensuring correct transcription. Also, after homologous recombination, the targeted modification of the chromosomal locus is stable, whereas randomly integrated sequences might be lost over time. In their seminal paper, Russel and Hirata [97] reported that DNA vectors based on the AAV could target homologous chromosomal DNA sequences and allow high-fidelity, non-mutagenic gene repair in a host cell. Although the laborious vector design and low transfection efficiencies of AAV vectors compared to the other viral vectors still remains a concern, statistical information neatly outlines the advantage of rAAV gene replacement system over standard viral vectors, which induce strong immune response.

4.2.2. Tissue-specific targeting

The promiscuous tropism of vectors resulting in high-level transgene expression in multiple tissues is another major challenge in vascular gene therapy. After systemic application, most viral vectors are trapped by the liver, hampering delivery to target CV tissues. Approaches to restrict gene delivery to desired cell types in vivorelied mostly on cell surface targeting or cell-specific promoters.

The cis-acting regulatory elements of the SM (smooth muscle)22α [98-100], telokin [101], smooth muscle myosin heavy chain [102], smooth muscle α- [100] and γ-actin [103], and desmin [104] genes have been shown to direct reporter gene expression to smooth muscle tissues in transgenic mice. In our studies, specific gene transfer to the SMC layer was achieved in swine coronary and peripheral arteries using SM22α promoter in AAV [17]. Although the efficiency of transduction was low when compared to a similar study using AAV vectors with cytomegalovirus (CMV) promoter [105], the use of SM22α promoter caused specific transduction of SMCs in vivo. An interesting approach to enhance the transduction efficiency of SM22α -containing plasmid was to incorporate chimeric transcriptional cassettes containing a SM-myosin heavy chain enhancer element combined with the SM22α promoter [106]. The transfection levels obtained using these chimeric constructs in Ad vector were similar to that with CMV promoter when tested in rat carotid arteries. Certain DNA nuclear targeting sequences can be used to restrict DNA nuclear import to specific cell types. Young et al.[107] improved the efficiency of transduction in SMCs of rat vasculature using a SMC-specific DNA nuclear targeting sequence.

EC specific gene expression was obtained when promoters of fms-like tyrosine kinase-1 (FLT-1) [108], intercellular adhesion molecule (ICAM) -2 [109], angiopoietin-2 [110], eNOS [111], vascular cell adhesion molecule-1 (VCAM-1) [112], von Willebrand factor [113], tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Tie) [114], kinase-like domain receptor [115] were used in transgenic mouse models. Other EC-specific promoters include the oxidized LDL receptor LOX-1 [116] and ICAM-1 [117], which exhibit upregulation upon cytokine stimulation, a possible advantage depending on the application in inflammatory conditions [118]. With the possible exception of the mouse Tie-2 and human ICAM-2 genes, most of EC–specific promoters tested to-date have been shown to direct expression in distinct and restricted sites of the vascular tree [119]. A combination approach of the Tie2 promoter and enhancer (Tshort) by Minami and collegues [119] directed widespread EC expression in vivo.

Another challenge was in generating an EC-specific promoter with comparable efficiency as the CMV promoter. White et al.[120] examined several novel Ad expression cassettes for EC-specific gene transfer with CMV, Tshort, ICAM-2, ICAM-1, FLT-1 promoters, respectively and found that LOX-1 promoter elements significantly increased reporter gene expression in carotid arteries compared to other promoters. The efficacy of these novel expression cassettes in large animal models have yet to be established.

An increasingly important area to in-tissue specific targeting is to engineer viral vectors Ads and AAVs with altered cell tropisms to narrow or broaden its efficiency in tissues refractory to infection [19, 121]. Non-genetic approaches typically utilize bispecific antibodies that both neutralize wild-type virus tropism and provide a new cell binding capacity [122]. For genetic targeting strategies, the virus capsid are engineered to express foreign ligands that target selected receptors in the absence or presence of additional modification to ablate the natural tropism of the virus [122, 123]. Ad homing to target endothelial cells at specific sites of the body can be achieved by deleting the ability of the virus to interact with its natural receptor, Coxsackievirus and adenovirus receptor (CAR), and a simultaneous addition of a ligand that directs the virus to the angiotensin converting enzyme on the ECs. Retargeting of AAV-2 with novel peptides could increase both transduction efficiency and selectivity [124] in vascular ECs [125] and SMCs [126] in vitro.

4.3. Challenges associated with the mode and route of gene delivery

4.3.1. Systemic gene delivery

The vascular system represents an ideal route of substance transport for reaching a specific site for therapeutic intervention. However, in the case of non-viral vectors, which are cationic polymers in most cases, it has been found that electrostatic interactions between the sulphated glycosaminoglycans in the serum as well as those expressed on the cell surface cause premature release of plasmid DNA leading to its inactivation and extracellular degradation by serum DNAses [21]. Also, after systemic vascular application, non-specific distribution of plasmid DNA throughout the vasculature would result in undesired side effects because of accumulation at non-specific sites. Intravenous administration of cationic polymers resulted in their localization to liver, lung, kidney, and spleen in pigs and rabbits [127-129]. Other barriers to systemic delivery include rapid clearance of the lipoplexes by the reticulo-endothelial system and target specificity.

Most Ad vectors are trapped by the liver, hampering delivery to target CV tissues after systemic application. Systemic tail vein injection of Ad vector in mice resulted in virus DNA deposition liver, lung, kidney and testis [130]. Furthermore, the use of a heterologous viral promoter CMV in the majority of vascular gene transfers causes systemic organ toxicity resulting from unrestricted transgene expression [131]. Retargeting of vectors and use of tissue specific promoters offers an enhanced safety profile by reducing ectopic expression in vital organs including the liver and lungs.

4.3.2. Endovascular gene delivery

Endovascular catheter-based gene delivery allows localization of vectors to the vessel wall and has the advantage that smaller quantities of viral vectors can be used when compared to those used in systemic delivery. The localized delivery minimizes widespread bio-distribution of vectors and simultaneously increases the local vector concentration. Several catheters are used for vascular gene delivery [132], and the efficiency of gene transfer depends on multiple physical parameters during the delivery process, including balloon pressure, vessel wall exposure time, concentration, and injection force [133]. Diffusive balloon catheters that include double balloon, channel, microporous and hydrogel balloons, facilitate passive diffusion of the vector to reach only the innermost layers of the artery (intima and inner media) [134]. Although this system has the advantage of causing relatively minor damage to the vessel media and intima, the major drawbacks include tissue ischemia caused due to blood flow blockage following balloon inflation and relatively low gene transfection rates owing to the short exposure time to the vessel wall. The pressure-driven balloon catheters [135], like the circumferential needle injection balloon catheter and the porous balloon catheter, are thought to efficiently delivery vectors to the deeper medial and adventitial layers of the artery compared to passive diffusion catheters, but they increase the risk of vascular injury. Damage to the endothelial lining promotes SMC proliferation and may lead to restenosis. The localized vascular injury can also cause increased inflammatory response. Iontophoretic catheters, a mechanically assisted injection catheter, enhance the vector penetration across the EC lining by generating an electrical current gradient to drive charged or hydrophilic molecules as deep as the adventitial layer of the artery wall, but depends on the charge, size, and concentration of the delivered compound [136]. Despite the theoretical aspects, in most cases of catheter-based gene transfer the vector is not distributed to the target vessels but to the region of tissue surrounding the target vessel or into the systemic circulation.

Gene eluting stents are attractive alternatives for localized gene delivery as they provide a platform for prolonged gene elution and efficient transduction of opposed arterial walls, especially in the treatment of in stent restenosis [132]. Local delivery of naked plasmid DNA encoding for human VEGF-2 via gene-eluting stent could decrease neointima formation while accelerating re-endothelialization in rabbit model [137]. Stents coated with lipoplexes containing eNOS plasmid accelerated re-endothelialization in hypercholesterolemic rabbits [138]. The same research group also demonstrated successful Ad and AAV delivery to the vessel wall by gene eluting stents with no systemic dissemination of the viral vectors [139]. Stents are often coated with synthetic or naturally occurring biopolymers for prolonged release of the gene to the vessel wall [140]. Recently, fully biodegradable stents have shown great promise in the treatment of peripheral arterial disease [141]. A combination approach of therapeutic gene delivery and fully biodegradable stents would be a novel approach to gene therapy.

4.3.3. Perivascular gene delivery

In endovascular approach, most catheters require prolonged total vascular occlusion for efficient gene delivery to the vasculature increasing the risk of ischemia. Delivery of genes directly into the adventitia bypassing intima and media may facilitate relatively rapid and efficient delivery compared to endovascular approaches [132]. The advantages of perivascular gene transfer are that the blood flow and endothelium are not disrupted and the placement of vector particles within tissues will result in enhanced local transduction efficiency compared to that achievable by endoluminal delivery [142]. Moreover, the local gene delivery through this ‘outside in’ approach has received increased attention due to important findings on the capacity of adventitia to influence neointima formation and vascular remodeling [143]. Localized adventitial delivery of a replication-deficient Ad construct containing a fibroblast-active promoter with the gp19ds portion of NADPH inhibitor was effective in reducing overall vascular superoxide anion O2- and neointima formation after angioplasty in rat common carotid artery [144]. Shneider et al.[145] showed that the infusion of Ad vectors into the carotid artery adventitia achieved recombinant gene expression at a level equivalent to that achieved by means of intraluminal vector infusion. Further, perivascular approach has been reported to minimize the pro-inflammatory effects of Ad vectors [145]. Adventitial gene delivery are also reported to be performed with silastic or biodegradable collars [146] which act as reservoirs of the vector.

The endovascular access is comparatively difficult in the case of coronary arteries, and the numerous side branches will also permit the run-off of the infused volume. An alternative delivery approach for coronary arteries is the expression of diffusible gene products into the pericardial space surrounding the heart and coronary arteries [147]. Transvascular needle injections of Ad vectors to the adventitia and perivascular tissue of coronary arteries have also been reported [148].

4.4. Immunological barriers to gene transfer

The immune system has evolved to eliminate foreign material and therefore, constrains the successful use of gene-replacement therapy based on viral vectors. There are several reports that suggest innate and adaptive immune responses to gene transfer [149, 150]. The vector dose, the route of administration, the nature of the transgene, and host-related factors responsible for inter-individual variability influence the immune response [151]. The early responses involve mechanisms that include the detection of pathogen-associated molecular patterns (PAMPs) present on the viral structural proteins containing the transgene by pattern recognition receptors (PRRs) on cells of the innate immune system (i.e., macrophages and dendritic cells) and the subsequent elaboration of pro-inflammatory cytokines that can up-regulate later adaptive immune responses [152]. The most studied family of PRRs are the toll-like receptors (TLRs), of which TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 have been implicated in initiating inflammatory responses to viruses [153]. The adaptive responses can include: the generation of antibodies to the transgene delivery vehicle compromising vector administration, or the generation of antibodies to the transgene product which nullifies transgene expression, or cytotoxicity to vector and/or transgene product which leads to the loss of transduced cells. It also results in a CD8+ memory T cell response that thwarts further efforts to use the same vector or transgene.

Ad vector particles can elicit strong innate and adaptive immune responses. The interplay of both systems activates CD4+ and CD8+ T cells and B cells as well as facilitates the induction of transgene-specific immune responses. The innate immune responses after systemic administration of Ad vectors are due to several processes: complement system activation, anaphylotoxin release, macrophage activation, release of cytokines and chemokines, including Interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, macrophage inhibitory protein-2, and RANTES (regulated and normal T cell expressed and secreted); EC activation, generalized transcriptome dysregulation in multiple tissues, activation of macrophages and dendritic cells, mobilization of granulocyte and mast cells, and thrombocytopenia [154]. These responses are due to activation of multiple PRRs including RIG-I-like receptors and Toll-like receptors: TLR-2, TLR-4 and TLR-9 [155]. In vivoadministration of higher doses of Ad vectors can result in one or all of these innate responses or may even lead to mortality in small animal models [156]. Ad infection of ECs is followed by expression of adhesion molecules such as ICAM-1 and VCAM-1 leading to increased leukocyte infiltration within transduced tissues [157]. Kupffer cells, the resident macrophages of the liver, rapidly scavenge and eliminate Ad5-based vectors from the circulation in mice [158], and this interaction contributes to the induction of pro-inflammatory cytokines and chemokines [159]. It has been reported that increasing the dose of Ad vector would probably fail to increase transgene expression, as the CAR adenoviral receptors would become saturated; in addition, the higher dose would induce a stronger inflammatory response responsible for increased elimination of the infected cells expressing the transgene [151].

Ad-based gene transfers can be hindered due to adaptive immune responses to the virus or the transgene it encodes. Ad viruses can induce a cytotoxic T-cell response as well as infiltration by CD4+ and CD8+ T cells. The mechanism involves internalization and priming by dendritic cells of capsid antigens associated with Class II Major histocompatibility complex (MHC) antigens, presentation of these antigens to CD4+ T cells, which become activated, and in turn CD8+ T cell activation by these CD4+ T cells [151]. These adaptive immune responses can limit the duration of transgene expression, and/or limit the ability to re-administer the vector.

Development of new large capacity or gutless (devoid of all viral genes) vectors [160] or modification of capsid sequences [161] are a few of the various strategies devised to reduce the immunogenicity of the Ad viral vectors. Adaptive immunity against these vectors has been substantially reduced through the development of helper-dependent Ad vectors that contain no Ad genes. However, these gutless Ad vectors can efficiently transduce antigen presenting cells (APCs) [162], which readily triggered innate immune responses and further augmented the induction of adaptive immune responses to the transgene product. This problem led to the introduction of tissue-specific promoters in gutless Ad vectors to restrict transgene expression in target cells but not in APCs [162]. Genome modification, capsid modification by Ad capsid-display of immuno-evasive proteins, chimeric Ad vectors and Ad vectors derived from alternative Ad serotypes are few techniques adopted for eluding Ad vector immunity [161]. The tropism modification strategies for targeted gene delivery using Ad vectors have been extensively reviewed [163]. Another method to decrease the immune response is to modify the route of delivery of the vector. In the adventitial delivery of Ad vectors to rabbit carotid arteries, recombinant gene expression was achieved at a level equivalent to that achieved by intraluminal vector infusion. Despite the generation of a systemic immune response, adventitial infusion had no detectable pathologic effects on the vascular intima or media [145]

Pre-existing immunity due to neutralizing antibodies against endemic Ad serotypes in human populations can contribute to pre-existing Ad specific adaptive immune responses [154]. These cellular responses may be more challenging than humoral immune responses, as these cellular adaptive immune responses to Ads have been shown to recognize multiple diverse, cross-clade Ad serotypes subsequent to exposure to only a single Ad serotype [154]. Arterial gene transfer with type 5 Ad vectors did not cause significant levels of gene expression in the majority of humans. Both immune-suppression and further engineering of the vector genome to decrease expression of viral genes show promise in circumventing barriers to Ad-mediated arterial gene transfer [164].

The innate immune response to the AAV capsid has received limited attention due to the minimal responses that AAV2 elicits [162]. According to recent reports by Herzog and others [165], innate immune system also plays important roles in activation of immunity by AAV mediated gene transfer, both in inducing the initial response to the vector and in promoting a deleterious adaptive immune responses. The initial innate immune responses were mediated by the TLR9-MyD88 pathway via a traditional NF-κB pathway to induce type 1 interferon production. Subsequently, alternative NF-κB pathway is triggered, prompting adaptive immune responses [166]. In vivo, intravenous injection of AAV-lacZ rapidly induces the expression of messenger RNAs (mRNAs) for the cytokines TNF-α, RANTES, interferon-γ-induced protein 10, macrophage inflammatory protein(MIP)-1β, monocyte chemotactic protein-1, and MIP-2. However, this effect lasts only 6 h, compared to more than 24 h with Ad infection [151]. The adaptive cell-mediated response is far weaker with AAV vectors than with adenoviral vectors probably due to the inability of AAVs to efficiently infect APC, including dendritic cells and macrophages. AAV vectors may be capable of infecting immature dendritic cells, but only when large doses of vector are used. In addition, even though a modest amount of dendritic cells are present at sites of AAV infection in vivo,they usually fail to induce a T-cell response of sufficient magnitude to eliminate the infected cells and, therefore, to decrease the duration of transgene expression [151].

Cytotoxic T-cell responses to AAV capsid antigen especially in patients with pre-existing neutralizing antibodies against AAV remain a major road block to achieve persistent therapeutic correction for clinical application. Natural, asymptomatic AAV infection in humans is common, and it estimates that up to 80% of humans possess neutralizing antibodies to some AAV serotypes, especially AAV-2 [167]. Recently, multiple serotypes of AAV in addition to AAV2 have been developed; these serotypes carry different capsid proteins and exhibit different tropism towards different organs [18]. However, changing serotypes may only lead to partial success due to the strong conservation of immune-dominant capsid epitopes in AAVs. In patients with high titers of neutralizing antibodies to gene therapy vectors such as AAV and Ad vectors, IgGs can be removed from blood by plasmapheresis, double filtration plasmapheresis and immune-absorbant plasmapheresis before gene transfer procedure to increase transduction rates of target tissues [168].

Plasmids alone or in combination with naked bacterial DNA can stimulate innate immune responses [152]. Plasmids, composed chiefly of bacterial DNA, contain far greater amounts of unmethylated CpG motifs than do the DNA in eukaryotic cells. DNA devoid of CpG motifs does not induce proinflammatory cytokine synthesis by macrophages in vitro. TLR 9 recognizes the unmethylated CpG motifs in immunostimulatory sequences of bacterial DNA which activate the cells responsible for innate immune responses (for example macrophages) after penetration of bacteria into the body [169]. Indeed, elimination or methylation of these sequences could be a method for suppressing the inflammatory response induced by unmethylated CpG sequences in plasmids [168].

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5. Conclusion

An enormous amount of research has been done in the past few decades on the choice of the therapeutic gene, vectors and delivery approaches for effective vascular gene transfer. The low efficiency of gene transfer to vascular tissues still remains a major drawback.. Of the several approaches used so far, Ad-mediated gene transfer has been found to be the most efficient when compared to other methods. However, gene transfer using viral vectors has often caused ectopic expression and also an increased immunological response. The use of tropism modified vectors and plasmids with cell specific promoters are solutions for reducing the ectopic expression. Using “gutless” viral vectors devoid of the immunogenic regions of viral plasmid is an attractive option to reduce the immunologic response, but we have to wait for more in vivodata using these third-generation vectors to reach a conclusive result [160]. Non-viral methods have more barriers to overcome to successfully transfect the cell; however, with the advent of innovative technologies like nanobots [170], stimuli responsive polymers [171], novel erythrocyte based carriers [172], magnetically targeted delivery [173] and focused in vivoplasmid DNA delivery to the vascular wall via intravascular ultrasound destruction of microbubbles [174]; we expect enhanced transgene expression in vascular cells in future studies. This will also be a possible solution to tackle with the immune response associated with the viral vectors. Site specific biodegradable stent based gene delivery approach [175] and modified percutaneous gene delivery systems offer new opportunities for enhanced gene delivery to vascular cells.

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Acknowledgments

This work was supported by research grants from the National Institute of Health, R01 HL104516, R01 HL112597 and R01 HL116042 to DKA. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung, and Blood Institute or the National Institutes of Health. Both authors declare no conflict of any sort with the content in this paper.

References

  1. 1. Roger VL, Go AS, Lloyd-Jones DM, Benjamin EJ, Berry JD, Borden WB, Bravata DM, Dai S, Ford ES, Fox CS, Fullerton HJ, Gillespie C, Hailpern SM, Heit JA, Howard VJ, Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Makuc DM, Marcus GM, Marelli A, Matchar DB, Moy CS, Mozaffarian D, Mussolino ME, Nichol G, Paynter NP, Soliman EZ, Sorlie PD, Sotoodehnia N, Turan TN, Virani SS, Wong ND, Woo D, Turner MB. Heart disease and stroke statistics--2012 update: a report from the American Heart Association. Circulation. 2012;125(1) e2-e220.
  2. 2. Brewster LP, Brey EM, Greisler HP. Cardiovascular gene delivery: The good road is awaiting. Adv Drug Deliv Rev. 2006;58(4) 604-629.
  3. 3. Yla-Herttuala S, Martin JF. Cardiovascular gene therapy. Lancet. 2000;355(9199) 213-222.
  4. 4. Pankajakshan D, Agrawal DK. Scaffolds in tissue engineering of blood vessels. Can J Physiol Pharmacol. 2010;88(9) 855-873.
  5. 5. Williams PD, Ranjzad P, Kakar SJ, Kingston PA. Development of viral vectors for use in cardiovascular gene therapy. Viruses. 2010;2(2) 334-371.
  6. 6. Kahn ML, Lee SW, Dichek DA. Optimization of retroviral vector-mediated gene transfer into endothelial cells in vitro. Circ Res. 1992;71(6) 1508-1517.
  7. 7. Inaba M, Toninelli E, Vanmeter G, Bender JR, Conte MS. Retroviral gene transfer: effects on endothelial cell phenotype. J Surg Res. 1998;78(1) 31-36.
  8. 8. Zelenock JA, Welling TH, Sarkar R, Gordon DG, Messina LM. Improved retroviral transduction efficiency of vascular cells in vitro and in vivo during clinically relevant incubation periods using centrifugation to increase viral titers. J Vasc Surg. 1997;26(1) 119-127.
  9. 9. Yu H, Eton D, Wang Y, Kumar SR, Tang L, Terramani TT, Benedict C, Hung G, Anderson WF. High efficiency in vitro gene transfer into vascular tissues using a pseudotyped retroviral vector without pseudotransduction. Gene Ther. 1999;6(11) 1876-1883.
  10. 10. Dishart KL, Denby L, George SJ, Nicklin SA, Yendluri S, Tuerk MJ, Kelley MP, Donahue BA, Newby AC, Harding T, Baker AH. Third-generation lentivirus vectors efficiently transduce and phenotypically modify vascular cells: implications for gene therapy. J Mol Cell Cardiol. 2003;35(7) 739-748.
  11. 11. Cefai D, Simeoni E, Ludunge KM, Driscoll R, von Segesser LK, Kappenberger L, Vassalli G. Multiply attenuated, self-inactivating lentiviral vectors efficiently transduce human coronary artery cells in vitro and rat arteries in vivo. J Mol Cell Cardiol. 2005;38(2) 333-344.
  12. 12. Bonci D, Cittadini A, Latronico MV, Borello U, Aycock JK, Drusco A, Innocenzi A, Follenzi A, Lavitrano M, Monti MG, Ross J, Jr., Naldini L, Peschle C, Cossu G, Condorelli G. 'Advanced' generation lentiviruses as efficient vectors for cardiomyocyte gene transduction in vitro and in vivo. Gene Ther. 2003;10(8) 630-636.
  13. 13. Harvey BG, Hackett NR, El-Sawy T, Rosengart TK, Hirschowitz EA, Lieberman MD, Lesser ML, Crystal RG. Variability of human systemic humoral immune responses to adenovirus gene transfer vectors administered to different organs. J Virol. 1999;73(8) 6729-6742.
  14. 14. Mastrangeli A, Harvey BG, Yao J, Wolff G, Kovesdi I, Crystal RG, Falck-Pedersen E. "Sero-switch" adenovirus-mediated in vivo gene transfer: circumvention of anti-adenovirus humoral immune defenses against repeat adenovirus vector administration by changing the adenovirus serotype. Hum Gene Ther. 1996;7(1) 79-87.
  15. 15. Wen S, Driscoll RM, Schneider DB, Dichek DA. Inclusion of the E3 region in an adenoviral vector decreases inflammation and neointima formation after arterial gene transfer. Arterioscler Thromb Vasc Biol. 2001;21(11) 1777-1782.
  16. 16. Sasano T, Kikuchi K, McDonald AD, Lai S, Donahue JK. Targeted high-efficiency, homogeneous myocardial gene transfer. J Mol Cell Cardiol. 2007;42(5) 954-961.
  17. 17. Pankajakshan D, Makinde TO, Gaurav R, Del Core M, Hatzoudis G, Pipinos I, Agrawal DK. Successful transfection of genes using AAV-2/9 vector in swine coronary and peripheral arteries. J Surg Res. 2012;175(1) 169-175.
  18. 18. Choi VW, McCarty DM, Samulski RJ. AAV hybrid serotypes: improved vectors for gene delivery. Curr Gene Ther. 2005;5(3) 299-310.
  19. 19. Kwon I, Schaffer DV. Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer. Pharm Res. 2008;25(3) 489-499.
  20. 20. Morris VB, Sharma CP. Folate mediated in vitro targeting of depolymerised trimethylated chitosan having arginine functionality. J Colloid Interface Sci. 2010;348(2) 360-368.
  21. 21. Shim MS, Kwon YJ. Stimuli-responsive polymers and nanomaterials for gene delivery and imaging applications. Adv Drug Deliv Rev. 2012;64(11) 1046-1059.
  22. 22. Al-Dosari MS, Gao X. Nonviral gene delivery: principle, limitations, and recent progress. Aaps J. 2009;11(4) 671-681.
  23. 23. Asahara T, Chen D, Tsurumi Y, Kearney M, Rossow S, Passeri J, Symes JF, Isner JM. Accelerated restitution of endothelial integrity and endothelium-dependent function after phVEGF165 gene transfer. Circulation. 1996;94(12) 3291-3302.
  24. 24. Lawrie A, Brisken AF, Francis SE, Cumberland DC, Crossman DC, Newman CM. Microbubble-enhanced ultrasound for vascular gene delivery. Gene Ther. 2000;7(23) 2023-2027.
  25. 25. Nishi T, Yoshizato K, Yamashiro S, Takeshima H, Sato K, Hamada K, Kitamura I, Yoshimura T, Saya H, Kuratsu J, Ushio Y. High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation. Cancer Res. 1996;56(5) 1050-1055.
  26. 26. Williams PD, Kingston PA. Plasmid-mediated gene therapy for cardiovascular disease. Cardiovasc Res. 2011;91(4) 565-576.
  27. 27. Parkes R, Meng QH, Siapati KE, McEwan JR, Hart SL. High efficiency transfection of porcine vascular cells in vitro with a synthetic vector system. J Gene Med. 2002;4(3) 292-299.
  28. 28. Kader KN, Sweany JM, Bellamkonda RV. Cationic lipid-mediated transfection of bovine aortic endothelial cells inhibits their attachment. J Biomed Mater Res. 2002;60(3) 405-410.
  29. 29. Wang T, Upponi JR, Torchilin VP. Design of multifunctional non-viral gene vectors to overcome physiological barriers: dilemmas and strategies. Int J Pharm. 2012;427(1) 3-20.
  30. 30. Zaric V, Weltin D, Erbacher P, Remy JS, Behr JP, Stephan D. Effective polyethylenimine-mediated gene transfer into human endothelial cells. J Gene Med. 2004;6(2) 176-184.
  31. 31. Godbey WT, Wu KK, Mikos AG. Poly(ethylenimine)-mediated gene delivery affects endothelial cell function and viability. Biomaterials. 2001;22(5) 471-480.
  32. 32. Turunen MP, Hiltunen MO, Ruponen M, Virkamaki L, Szoka FC, Jr., Urtti A, Yla-Herttuala S. Efficient adventitial gene delivery to rabbit carotid artery with cationic polymer-plasmid complexes. Gene Ther. 1999;6(1) 6-11.
  33. 33. Brito L, Little S, Langer R, Amiji M. Poly(beta-amino ester) and cationic phospholipid-based lipopolyplexes for gene delivery and transfection in human aortic endothelial and smooth muscle cells. Biomacromolecules. 2008;9(4) 1179-1187.
  34. 34. Chen SL, Zhu CC, Liu YQ, Tang LJ, Yi L, Yu BJ, Wang DJ. Mesenchymal stem cells genetically modified with the angiopoietin-1 gene enhanced arteriogenesis in a porcine model of chronic myocardial ischaemia. J Int Med Res. 2009;37(1) 68-78.
  35. 35. Das H, George JC, Joseph M, Das M, Abdulhameed N, Blitz A, Khan M, Sakthivel R, Mao HQ, Hoit BD, Kuppusamy P, Pompili VJ. Stem cell therapy with overexpressed VEGF and PDGF genes improves cardiac function in a rat infarct model. PLoS One. 2009;4(10) e7325.
  36. 36. Behrendt D, Ganz P. Endothelial function. From vascular biology to clinical applications. Am J Cardiol. 2002;90(10C) 40L-48L.
  37. 37. Griese DP, Achatz S, Batzlsperger CA, Strauch UG, Grumbeck B, Weil J, Riegger GA. Vascular gene delivery of anticoagulants by transplantation of retrovirally-transduced endothelial progenitor cells. Cardiovasc Res. 2003;58(2) 469-477.
  38. 38. Chen L, Wu F, Xia WH, Zhang YY, Xu SY, Cheng F, Liu X, Zhang XY, Wang SM, Tao J. CXCR4 gene transfer contributes to in vivo reendothelialization capacity of endothelial progenitor cells. Cardiovasc Res. 2010;88(3) 462-470.
  39. 39. Ohno N, Itoh H, Ikeda T, Ueyama K, Yamahara K, Doi K, Yamashita J, Inoue M, Masatsugu K, Sawada N, Fukunaga Y, Sakaguchi S, Sone M, Yurugi T, Kook H, Komeda M, Nakao K. Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide. Circulation. 2002;105(14) 1623-1626.
  40. 40. Kong D, Melo LG, Mangi AA, Zhang L, Lopez-Ilasaca M, Perrella MA, Liew CC, Pratt RE, Dzau VJ. Enhanced inhibition of neointimal hyperplasia by genetically engineered endothelial progenitor cells. Circulation. 2004;109(14) 1769-1775.
  41. 41. Laitinen M, Hartikainen J, Hiltunen MO, Eranen J, Kiviniemi M, Narvanen O, Makinen K, Manninen H, Syvanne M, Martin JF, Laakso M, Yla-Herttuala S. Catheter-mediated vascular endothelial growth factor gene transfer to human coronary arteries after angioplasty. Hum Gene Ther. 2000;11(2) 263-270.
  42. 42. Channon KM, Annex BH. Antithrombotic strategies in gene therapy. Curr Cardiol Rep. 2000;2(1) 34-38.
  43. 43. Waugh JM, Yuksel E, Li J, Kuo MD, Kattash M, Saxena R, Geske R, Thung SN, Shenaq SM, Woo SL. Local overexpression of thrombomodulin for in vivo prevention of arterial thrombosis in a rabbit model. Circ Res. 1999;84(1) 84-92.
  44. 44. Yin X, Yutani C, Ikeda Y, Enjyoji K, Ishibashi-Ueda H, Yasuda S, Tsukamoto Y, Nonogi H, Kaneda Y, Kato H. Tissue factor pathway inhibitor gene delivery using HVJ-AVE liposomes markedly reduces restenosis in atherosclerotic arteries. Cardiovasc Res. 2002;56(3) 454-463.
  45. 45. Rade JJ, Schulick AH, Virmani R, Dichek DA. Local adenoviral-mediated expression of recombinant hirudin reduces neointima formation after arterial injury. Nat Med. 1996;2(3) 293-298.
  46. 46. Zoldhelyi P, McNatt J, Xu XM, Loose-Mitchell D, Meidell RS, Clubb FJ, Jr., Buja LM, Willerson JT, Wu KK. Prevention of arterial thrombosis by adenovirus-mediated transfer of cyclooxygenase gene. Circulation. 1996;93(1) 10-17.
  47. 47. Levonen AL, Inkala M, Heikura T, Jauhiainen S, Jyrkkanen HK, Kansanen E, Maatta K, Romppanen E, Turunen P, Rutanen J, Yla-Herttuala S. Nrf2 gene transfer induces antioxidant enzymes and suppresses smooth muscle cell growth in vitro and reduces oxidative stress in rabbit aorta in vivo. Arterioscler Thromb Vasc Biol. 2007;27(4) 741-747.
  48. 48. Levonen AL, Vahakangas E, Koponen JK, Yla-Herttuala S. Antioxidant gene therapy for cardiovascular disease: current status and future perspectives. Circulation. 2008;117(16) 2142-2150.
  49. 49. Li Q, Bolli R, Qiu Y, Tang XL, Guo Y, French BA. Gene therapy with extracellular superoxide dismutase protects conscious rabbits against myocardial infarction. Circulation. 2001;103(14) 1893-1898.
  50. 50. Laukkanen MO, Kivela A, Rissanen T, Rutanen J, Karkkainen MK, Leppanen O, Brasen JH, Yla-Herttuala S. Adenovirus-mediated extracellular superoxide dismutase gene therapy reduces neointima formation in balloon-denuded rabbit aorta. Circulation. 2002;106(15) 1999-2003.
  51. 51. Woo YJ, Zhang JC, Vijayasarathy C, Zwacka RM, Englehardt JF, Gardner TJ, Sweeney HL. Recombinant adenovirus-mediated cardiac gene transfer of superoxide dismutase and catalase attenuates postischemic contractile dysfunction. Circulation. 1998;98(19 Suppl) II255-260; discussion II260-251.
  52. 52. Duckers HJ, Boehm M, True AL, Yet SF, San H, Park JL, Clinton Webb R, Lee ME, Nabel GJ, Nabel EG. Heme oxygenase-1 protects against vascular constriction and proliferation. Nat Med. 2001;7(6) 693-698.
  53. 53. Kashyap VS, Santamarina-Fojo S, Brown DR, Parrott CL, Applebaum-Bowden D, Meyn S, Talley G, Paigen B, Maeda N, Brewer HB, Jr. Apolipoprotein E deficiency in mice: gene replacement and prevention of atherosclerosis using adenovirus vectors. J Clin Invest. 1995;96(3) 1612-1620.
  54. 54. Tsukamoto K, Tangirala R, Chun SH, Pure E, Rader DJ. Rapid regression of atherosclerosis induced by liver-directed gene transfer of ApoE in ApoE-deficient mice. Arterioscler Thromb Vasc Biol. 1999;19(9) 2162-2170.
  55. 55. Greeve J, Jona VK, Chowdhury NR, Horwitz MS, Chowdhury JR. Hepatic gene transfer of the catalytic subunit of the apolipoprotein B mRNA editing enzyme results in a reduction of plasma LDL levels in normal and watanabe heritable hyperlipidemic rabbits. J Lipid Res. 1996;37(9) 2001-2017.
  56. 56. Khurana R, Martin JF, Zachary I. Gene therapy for cardiovascular disease: a case for cautious optimism. Hypertension. 2001;38(5) 1210-1216.
  57. 57. Granada JF, Ensenat D, Keswani AN, Kaluza GL, Raizner AE, Liu XM, Peyton KJ, Azam MA, Wang H, Durante W. Single perivascular delivery of mitomycin C stimulates p21 expression and inhibits neointima formation in rat arteries. Arterioscler Thromb Vasc Biol. 2005;25(11) 2343-2348.
  58. 58. Chang MW, Barr E, Lu MM, Barton K, Leiden JM. Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest. 1995;96(5) 2260-2268.
  59. 59. McArthur JG, Qian H, Citron D, Banik GG, Lamphere L, Gyuris J, Tsui L, George SE. p27-p16 Chimera: a superior antiproliferative for the prevention of neointimal hyperplasia. Mol Ther. 2001;3(1) 8-13.
  60. 60. Tsui LV, Camrud A, Mondesire J, Carlson P, Zayek N, Camrud L, Donahue B, Bauer S, Lin A, Frey D, Rivkin M, Subramanian A, Falotico R, Gyuris J, Schwartz R, McArthur JG. p27-p16 fusion gene inhibits angioplasty-induced neointimal hyperplasia and coronary artery occlusion. Circ Res. 2001;89(4) 323-328.
  61. 61. Takagi Y. Adenovirus-mediated overexpression of a cyclin-dependent kinase inhibitor, p57Kip2, suppressed vascular smooth muscle cell proliferation. Hokkaido Igaku Zasshi. 2002;77(3) 221-230.
  62. 62. Maillard L, Van Belle E, Smith RC, Le Roux A, Denefle P, Steg G, Barry JJ, Branellec D, Isner JM, Walsh K. Percutaneous delivery of the gax gene inhibits vessel stenosis in a rabbit model of balloon angioplasty. Cardiovasc Res. 1997;35(3) 536-546.
  63. 63. Chen L, Daum G, Forough R, Clowes M, Walter U, Clowes AW. Overexpression of human endothelial nitric oxide synthase in rat vascular smooth muscle cells and in balloon-injured carotid artery. Circ Res. 1998;82(8) 862-870.
  64. 64. Kopp CW, de Martin R. Gene therapy approaches for the prevention of restenosis. Curr Vasc Pharmacol. 2004;2(2) 183-189.
  65. 65. Yukawa H, Miyatake SI, Saiki M, Takahashi JC, Mima T, Ueno H, Nagata I, Kikuchi H, Hashimoto N. In vitro growth suppression of vascular smooth muscle cells using adenovirus-mediated gene transfer of a truncated form of fibroblast growth factor receptor. Atherosclerosis. 1998;141(1) 125-132.
  66. 66. Kotani M, Fukuda N, Ando H, Hu WY, Kunimoto S, Saito S, Kanmatsuse K. Chimeric DNA-RNA hammerhead ribozyme targeting PDGF A-chain mRNA specifically inhibits neointima formation in rat carotid artery after balloon injury. Cardiovasc Res. 2003;57(1) 265-276.
  67. 67. Ando H, Fukuda N, Kotani M, Yokoyama S, Kunimoto S, Matsumoto K, Saito S, Kanmatsuse K, Mugishima H. Chimeric DNA-RNA hammerhead ribozyme targeting transforming growth factor-beta 1 mRNA inhibits neointima formation in rat carotid artery after balloon injury. Eur J Pharmacol. 2004;483(2-3) 207-214.
  68. 68. Ahn JD, Morishita R, Kaneda Y, Lee SJ, Kwon KY, Choi SY, Lee KU, Park JY, Moon IJ, Park JG, Yoshizumi M, Ouchi Y, Lee IK. Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo. Circ Res. 2002;90(12) 1325-1332.
  69. 69. Ehsan A, Mann MJ, Dell'Acqua G, Dzau VJ. Long-term stabilization of vein graft wall architecture and prolonged resistance to experimental atherosclerosis after E2F decoy oligonucleotide gene therapy. J Thorac Cardiovasc Surg. 2001;121(4) 714-722.
  70. 70. Luo Z, Garron T, Palasis M, Lu H, Belanger AJ, Scaria A, Vincent KA, Date T, Akita GY, Cheng SH, Barry J, Gregory RJ, Jiang C. Enhancement of Fas ligand-induced inhibition of neointimal formation in rabbit femoral and iliac arteries by coexpression of p35. Hum Gene Ther. 2001;12(18) 2191-2202.
  71. 71. Newby AC. Matrix metalloproteinases regulate migration, proliferation, and death of vascular smooth muscle cells by degrading matrix and non-matrix substrates. Cardiovasc Res. 2006;69(3) 614-624.
  72. 72. Ramirez Correa GA, Zacchigna S, Arsic N, Zentilin L, Salvi A, Sinagra G, Giacca M. Potent inhibition of arterial intimal hyperplasia by TIMP1 gene transfer using AAV vectors. Mol Ther. 2004;9(6) 876-884.
  73. 73. George SJ, Baker AH, Angelini GD, Newby AC. Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins. Gene Ther. 1998;5(11) 1552-1560.
  74. 74. Johnson TW, Wu YX, Herdeg C, Baumbach A, Newby AC, Karsch KR, Oberhoff M. Stent-based delivery of tissue inhibitor of metalloproteinase-3 adenovirus inhibits neointimal formation in porcine coronary arteries. Arterioscler Thromb Vasc Biol. 2005;25(4) 754-759.
  75. 75. Guo YH, Gao W, Li Q, Li PF, Yao PY, Chen K. Tissue inhibitor of metalloproteinases-4 suppresses vascular smooth muscle cell migration and induces cell apoptosis. Life Sci. 2004;75(20) 2483-2493.
  76. 76. Gurjar MV, Sharma RV, Bhalla RC. eNOS gene transfer inhibits smooth muscle cell migration and MMP-2 and MMP-9 activity. Arterioscler Thromb Vasc Biol. 1999;19(12) 2871-2877.
  77. 77. Eefting D, de Vries MR, Grimbergen JM, Karper JC, van Bockel JH, Quax PH. In vivo suppression of vein graft disease by nonviral, electroporation-mediated, gene transfer of tissue inhibitor of metalloproteinase-1 linked to the amino terminal fragment of urokinase (TIMP-1.ATF), a cell-surface directed matrix metalloproteinase inhibitor. J Vasc Surg. 2010;51(2) 429-437.
  78. 78. Emanueli C, Madeddu P. Angiogenesis gene therapy to rescue ischaemic tissues: achievements and future directions. Br J Pharmacol. 2001;133(7) 951-958.
  79. 79. Syed IS, Sanborn TA, Rosengart TK. Therapeutic angiogenesis: a biologic bypass. Cardiology. 2004;101(1-3) 131-143.
  80. 80. Losordo DW, Vale PR, Isner JM. Gene therapy for myocardial angiogenesis. Am Heart J. 1999;138(2 Pt 2) S132-141.
  81. 81. Rosengart TK, Lee LY, Patel SR, Kligfield PD, Okin PM, Hackett NR, Isom OW, Crystal RG. Six-month assessment of a phase I trial of angiogenic gene therapy for the treatment of coronary artery disease using direct intramyocardial administration of an adenovirus vector expressing the VEGF121 cDNA. Ann Surg. 1999;230(4) 466-470; discussion 470-462.
  82. 82. Grines C, Rubanyi GM, Kleiman NS, Marrott P, Watkins MW. Angiogenic gene therapy with adenovirus 5 fibroblast growth factor-4 (Ad5FGF-4): a new option for the treatment of coronary artery disease. Am J Cardiol. 2003;92(9B) 24N-31N.
  83. 83. Safi J, Jr., DiPaula AF, Jr., Riccioni T, Kajstura J, Ambrosio G, Becker LC, Anversa P, Capogrossi MC. Adenovirus-mediated acidic fibroblast growth factor gene transfer induces angiogenesis in the nonischemic rabbit heart. Microvasc Res. 1999;58(3) 238-249.
  84. 84. Nah JW, Yu L, Han SO, Ahn CH, Kim SW. Artery wall binding peptide-poly(ethylene glycol)-grafted-poly(L-lysine)-based gene delivery to artery wall cells. J Control Release. 2002;78(1-3) 273-284.
  85. 85. Akagi D, Oba M, Koyama H, Nishiyama N, Fukushima S, Miyata T, Nagawa H, Kataoka K. Biocompatible micellar nanovectors achieve efficient gene transfer to vascular lesions without cytotoxicity and thrombus formation. Gene Ther. 2007;14(13) 1029-1038.
  86. 86. Song C, Labhasetwar V, Cui X, Underwood T, Levy RJ. Arterial uptake of biodegradable nanoparticles for intravascular local drug delivery: results with an acute dog model. J Control Release. 1998;54(2) 201-211.
  87. 87. Kagaya H, Oba M, Miura Y, Koyama H, Ishii T, Shimada T, Takato T, Kataoka K, Miyata T. Impact of polyplex micelles installed with cyclic RGD peptide as ligand on gene delivery to vascular lesions. Gene Ther. 2012;19(1) 61-69.
  88. 88. Theoharis S, Krueger U, Tan PH, Haskard DO, Weber M, George AJ. Targeting gene delivery to activated vascular endothelium using anti E/P-Selectin antibody linked to PAMAM dendrimers. J Immunol Methods. 2009;343(2) 79-90.
  89. 89. White SJ, Nicklin SA, Sawamura T, Baker AH. Identification of peptides that target the endothelial cell-specific LOX-1 receptor. Hypertension. 2001;37(2 Part 2) 449-455.
  90. 90. Jarver P, Langel K, El-Andaloussi S, Langel U. Applications of cell-penetrating peptides in regulation of gene expression. Biochem Soc Trans. 2007;35(Pt 4) 770-774.
  91. 91. Golda A, Pelisek J, Klocke R, Engelmann MG, Rolland PH, Mekkaoui C, Nikol S. Small poly-L-lysines improve cationic lipid-mediated gene transfer in vascular cells in vitro and in vivo. J Vasc Res. 2007;44(4) 273-282.
  92. 92. Cartier R, Reszka R. Utilization of synthetic peptides containing nuclear localization signals for nonviral gene transfer systems. Gene Ther. 2002;9(3) 157-167.
  93. 93. Hebert E. Improvement of exogenous DNA nuclear importation by nuclear localization signal-bearing vectors: a promising way for non-viral gene therapy? Biol Cell. 2003;95(2) 59-68.
  94. 94. Young JL, Benoit JN, Dean DA. Effect of a DNA nuclear targeting sequence on gene transfer and expression of plasmids in the intact vasculature. Gene Ther. 2003;10(17) 1465-1470.
  95. 95. Vasileva A, Jessberger R. Precise hit: adeno-associated virus in gene targeting. Nat Rev Microbiol. 2005;3(11) 837-847.
  96. 96. Vasileva A, Linden RM, Jessberger R. Homologous recombination is required for AAV-mediated gene targeting. Nucleic Acids Res. 2006;34(11) 3345-3360.
  97. 97. Russell DW, Hirata RK. Human gene targeting by viral vectors. Nat Genet. 1998;18(4) 325-330.
  98. 98. Li L, Miano JM, Mercer B, Olson EN. Expression of the SM22alpha promoter in transgenic mice provides evidence for distinct transcriptional regulatory programs in vascular and visceral smooth muscle cells. J Cell Biol. 1996;132(5) 849-859.
  99. 99. Mack CP, Owens GK. Regulation of smooth muscle alpha-actin expression in vivo is dependent on CArG elements within the 5' and first intron promoter regions. Circ Res. 1999;84(7) 852-861.
  100. 100. Mack CP, Thompson MM, Lawrenz-Smith S, Owens GK. Smooth muscle alpha-actin CArG elements coordinate formation of a smooth muscle cell-selective, serum response factor-containing activation complex. Circ Res. 2000;86(2) 221-232.
  101. 101. Hoggatt AM, Simon GM, Herring BP. Cell-specific regulatory modules control expression of genes in vascular and visceral smooth muscle tissues. Circ Res. 2002;91(12) 1151-1159.
  102. 102. Madsen CS, Regan CP, Hungerford JE, White SL, Manabe I, Owens GK. Smooth muscle-specific expression of the smooth muscle myosin heavy chain gene in transgenic mice requires 5'-flanking and first intronic DNA sequence. Circ Res. 1998;82(8) 908-917.
  103. 103. Qian J, Kumar A, Szucsik JC, Lessard JL. Tissue and developmental specific expression of murine smooth muscle gamma-actin fusion genes in transgenic mice. Dev Dyn. 1996;207(2) 135-144.
  104. 104. Mericskay M, Parlakian A, Porteu A, Dandre F, Bonnet J, Paulin D, Li Z. An overlapping CArG/octamer element is required for regulation of desmin gene transcription in arterial smooth muscle cells. Dev Biol. 2000;226(2) 192-208.
  105. 105. Su H, Yeghiazarians Y, Lee A, Huang Y, Arakawa-Hoyt J, Ye J, Orcino G, Grossman W, Kan YW. AAV serotype 1 mediates more efficient gene transfer to pig myocardium than AAV serotype 2 and plasmid. J Gene Med. 2008;10(1) 33-41.
  106. 106. Ribault S, Neuville P, Mechine-Neuville A, Auge F, Parlakian A, Gabbiani G, Paulin D, Calenda V. Chimeric smooth muscle-specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy. Circ Res. 2001;88(5) 468-475.
  107. 107. Young JL, Zimmer WE, Dean DA. Smooth muscle-specific gene delivery in the vasculature based on restriction of DNA nuclear import. Exp Biol Med (Maywood). 2008;233(7) 840-848.
  108. 108. Morishita K, Johnson DE, Williams LT. A novel promoter for vascular endothelial growth factor receptor (flt-1) that confers endothelial-specific gene expression. J Biol Chem. 1995;270(46) 27948-27953.
  109. 109. Cowan PJ, Shinkel TA, Witort EJ, Barlow H, Pearse MJ, d'Apice AJ. Targeting gene expression to endothelial cells in transgenic mice using the human intercellular adhesion molecule 2 promoter. Transplantation. 1996;62(2) 155-160.
  110. 110. Hegen A, Koidl S, Weindel K, Marme D, Augustin HG, Fiedler U. Expression of angiopoietin-2 in endothelial cells is controlled by positive and negative regulatory promoter elements. Arterioscler Thromb Vasc Biol. 2004;24(10) 1803-1809.
  111. 111. Karantzoulis-Fegaras F, Antoniou H, Lai SL, Kulkarni G, D'Abreo C, Wong GK, Miller TL, Chan Y, Atkins J, Wang Y, Marsden PA. Characterization of the human endothelial nitric-oxide synthase promoter. J Biol Chem. 1999;274(5) 3076-3093.
  112. 112. Neish AS, Williams AJ, Palmer HJ, Whitley MZ, Collins T. Functional analysis of the human vascular cell adhesion molecule 1 promoter. J Exp Med. 1992;176(6) 1583-1593.
  113. 113. Jahroudi N, Lynch DC. Endothelial-cell-specific regulation of von Willebrand factor gene expression. Mol Cell Biol. 1994;14(2) 999-1008.
  114. 114. Korhonen J, Lahtinen I, Halmekyto M, Alhonen L, Janne J, Dumont D, Alitalo K. Endothelial-specific gene expression directed by the tie gene promoter in vivo. Blood. 1995;86(5) 1828-1835.
  115. 115. Patterson C, Perrella MA, Hsieh CM, Yoshizumi M, Lee ME, Haber E. Cloning and functional analysis of the promoter for KDR/flk-1, a receptor for vascular endothelial growth factor. J Biol Chem. 1995;270(39) 23111-23118.
  116. 116. Aoyama T, Sawamura T, Furutani Y, Matsuoka R, Yoshida MC, Fujiwara H, Masaki T. Structure and chromosomal assignment of the human lectin-like oxidized low-density-lipoprotein receptor-1 (LOX-1) gene. Biochem J. 1999;339 ( Pt 1) 177-184.
  117. 117. Hou J, Baichwal V, Cao Z. Regulatory elements and transcription factors controlling basal and cytokine-induced expression of the gene encoding intercellular adhesion molecule 1. Proc Natl Acad Sci U S A. 1994;91(24) 11641-11645.
  118. 118. Tessitore A, Pastore L, Rispoli A, Cilenti L, Toniato E, Flati V, Farina AR, Frati L, Gulino A, Martinotti S. Two gamma-interferon-activation sites (GAS) on the promoter of the human intercellular adhesion molecule (ICAM-1) gene are required for induction of transcription by IFN-gamma. Eur J Biochem. 1998;258(3) 968-975.
  119. 119. Minami T, Kuivenhoven JA, Evans V, Kodama T, Rosenberg RD, Aird WC. Ets motifs are necessary for endothelial cell-specific expression of a 723-bp Tie-2 promoter/enhancer in Hprt targeted transgenic mice. Arterioscler Thromb Vasc Biol. 2003;23(11) 2041-2047.
  120. 120. White SJ, Papadakis ED, Rogers CA, Johnson JL, Biessen EA, Newby AC. In vitro and in vivo analysis of expression cassettes designed for vascular gene transfer. Gene Ther. 2008;15(5) 340-346.
  121. 121. Gigout L, Rebollo P, Clement N, Warrington KH, Jr., Muzyczka N, Linden RM, Weber T. Altering AAV tropism with mosaic viral capsids. Mol Ther. 2005;11(6) 856-865.
  122. 122. Nicklin SA, Baker AH. Tropism-modified adenoviral and adeno-associated viral vectors for gene therapy. Curr Gene Ther. 2002;2(3) 273-293.
  123. 123. Baker AH. Designing gene delivery vectors for cardiovascular gene therapy. Prog Biophys Mol Biol. 2004;84(2-3) 279-299.
  124. 124. White SJ, Nicklin SA, Buning H, Brosnan MJ, Leike K, Papadakis ED, Hallek M, Baker AH. Targeted gene delivery to vascular tissue in vivo by tropism-modified adeno-associated virus vectors. Circulation. 2004;109(4) 513-519.
  125. 125. Nicklin SA, Buening H, Dishart KL, de Alwis M, Girod A, Hacker U, Thrasher AJ, Ali RR, Hallek M, Baker AH. Efficient and selective AAV2-mediated gene transfer directed to human vascular endothelial cells. Mol Ther. 2001;4(3) 174-181.
  126. 126. Work LM, Nicklin SA, Brain NJ, Dishart KL, Von Seggern DJ, Hallek M, Buning H, Baker AH. Development of efficient viral vectors selective for vascular smooth muscle cells. Mol Ther. 2004;9(2) 198-208.
  127. 127. Takakura Y, Nishikawa M, Yamashita F, Hashida M. Influence of physicochemical properties on pharmacokinetics of non-viral vectors for gene delivery. J Drug Target. 2002;10(2) 99-104.
  128. 128. Mahato RI, Kawabata K, Takakura Y, Hashida M. In vivo disposition characteristics of plasmid DNA complexed with cationic liposomes. J Drug Target. 1995;3(2) 149-157.
  129. 129. Gonin P, Gaillard C. Gene transfer vector biodistribution: pivotal safety studies in clinical gene therapy development. Gene Ther. 2004;11 Suppl 1 S98-S108.
  130. 130. Ye X, Gao GP, Pabin C, Raper SE, Wilson JM. Evaluating the potential of germ line transmission after intravenous administration of recombinant adenovirus in the C3H mouse. Hum Gene Ther. 1998;9(14) 2135-2142.
  131. 131. Beck C, Uramoto H, Boren J, Akyurek LM. Tissue-specific targeting for cardiovascular gene transfer. Potential vectors and future challenges. Curr Gene Ther. 2004;4(4) 457-467.
  132. 132. Sharif F, Daly K, Crowley J, O'Brien T. Current status of catheter- and stent-based gene therapy. Cardiovasc Res. 2004;64(2) 208-216.
  133. 133. Fram DB, Aretz T, Azrin MA, Mitchel JF, Samady H, Gillam LD, Sahatjian R, Waters D, McKay RG. Localized intramural drug delivery during balloon angioplasty using hydrogel-coated balloons and pressure-augmented diffusion. J Am Coll Cardiol. 1994;23(7) 1570-1577.
  134. 134. Opie SR, Dib N. Local endovascular delivery, gene therapy, and cell transplantation for peripheral arterial disease. J Endovasc Ther. 2004;11 Suppl 2 II151-162.
  135. 135. Barath P, Popov A, Dillehay GL, Matos G, McKiernan T. Infiltrator Angioplasty Balloon Catheter: a device for combined angioplasty and intramural site-specific treatment. Cathet Cardiovasc Diagn. 1997;41(3) 333-341.
  136. 136. Fernandez-Ortiz A, Meyer BJ, Mailhac A, Falk E, Badimon L, Fallon JT, Fuster V, Chesebro JH, Badimon JJ. A new approach for local intravascular drug delivery. Iontophoretic balloon. Circulation. 1994;89(4) 1518-1522.
  137. 137. Walter DH, Cejna M, Diaz-Sandoval L, Willis S, Kirkwood L, Stratford PW, Tietz AB, Kirchmair R, Silver M, Curry C, Wecker A, Yoon YS, Heidenreich R, Hanley A, Kearney M, Tio FO, Kuenzler P, Isner JM, Losordo DW. Local gene transfer of phVEGF-2 plasmid by gene-eluting stents: an alternative strategy for inhibition of restenosis. Circulation. 2004;110(1) 36-45.
  138. 138. Sharif F, Hynes SO, McCullagh KJ, Ganley S, Greiser U, McHugh P, Crowley J, Barry F, O'Brien T. Gene-eluting stents: non-viral, liposome-based gene delivery of eNOS to the blood vessel wall in vivo results in enhanced endothelialization but does not reduce restenosis in a hypercholesterolemic model. Gene Ther. 2012;19(3) 321-328.
  139. 139. Sharif F, Hynes SO, McMahon J, Cooney R, Conroy S, Dockery P, Duffy G, Daly K, Crowley J, Bartlett JS, O'Brien T. Gene-eluting stents: comparison of adenoviral and adeno- associated viral gene delivery to the blood vessel wall in vivo. Hum Gene Ther. 2006;17(7) 741-750.
  140. 140. Klugherz BD, Song C, DeFelice S, Cui X, Lu Z, Connolly J, Hinson JT, Wilensky RL, Levy RJ. Gene delivery to pig coronary arteries from stents carrying antibody-tethered adenovirus. Hum Gene Ther. 2002;13(3) 443-454.
  141. 141. Nishio S, Kosuga K, Igaki K, Okada M, Kyo E, Tsuji T, Takeuchi E, Inuzuka Y, Takeda S, Hata T, Takeuchi Y, Kawada Y, Harita T, Seki J, Akamatsu S, Hasegawa S, Bruining N, Brugaletta S, de Winter S, Muramatsu T, Onuma Y, Serruys PW, Ikeguchi S. Long-Term (>10 Years) clinical outcomes of first-in-human biodegradable poly-l-lactic acid coronary stents: Igaki-Tamai stents. Circulation. 2012;125(19) 2343-2353.
  142. 142. George SJ, Baker AH. Gene transfer to the vasculature: historical perspective and implication for future research objectives. Mol Biotechnol. 2002;22(2) 153-164.
  143. 143. Siow RC, Churchman AT. Adventitial growth factor signalling and vascular remodelling: potential of perivascular gene transfer from the outside-in. Cardiovasc Res. 2007;75(4) 659-668.
  144. 144. Dourron HM, Jacobson GM, Park JL, Liu J, Reddy DJ, Scheel ML, Pagano PJ. Perivascular gene transfer of NADPH oxidase inhibitor suppresses angioplasty-induced neointimal proliferation of rat carotid artery. Am J Physiol Heart Circ Physiol. 2005;288(2) H946-953.
  145. 145. Schneider DB, Sassani AB, Vassalli G, Driscoll RM, Dichek DA. Adventitial delivery minimizes the proinflammatory effects of adenoviral vectors. J Vasc Surg. 1999;29(3) 543-550.
  146. 146. Laitinen M, Pakkanen T, Donetti E, Baetta R, Luoma J, Lehtolainen P, Viita H, Agrawal R, Miyanohara A, Friedmann T, Risau W, Martin JF, Soma M, Yla-Herttuala S. Gene transfer into the carotid artery using an adventitial collar: comparison of the effectiveness of the plasmid-liposome complexes, retroviruses, pseudotyped retroviruses, and adenoviruses. Hum Gene Ther. 1997;8(14) 1645-1650.
  147. 147. March KL, Woody M, Mehdi K, Zipes DP, Brantly M, Trapnell BC. Efficient in vivo catheter-based pericardial gene transfer mediated by adenoviral vectors. Clin Cardiol. 1999;22(1 Suppl 1) I23-29.
  148. 148. Baek S, March KL. Gene therapy for restenosis: getting nearer the heart of the matter. Circ Res. 1998;82(3) 295-305.
  149. 149. Marshall E. Gene therapy death prompts review of adenovirus vector. Science. 1999;286(5448) 2244-2245.
  150. 150. Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ, Ozelo MC, Hoots K, Blatt P, Konkle B, Dake M, Kaye R, Razavi M, Zajko A, Zehnder J, Rustagi PK, Nakai H, Chew A, Leonard D, Wright JF, Lessard RR, Sommer JM, Tigges M, Sabatino D, Luk A, Jiang H, Mingozzi F, Couto L, Ertl HC, High KA, Kay MA. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med. 2006;12(3) 342-347.
  151. 151. Bessis N, GarciaCozar FJ, Boissier MC. Immune responses to gene therapy vectors: influence on vector function and effector mechanisms. Gene Ther. 2004;11 Suppl 1 S10-17.
  152. 152. Waters B, Lillicrap D. The molecular mechanisms of immunomodulation and tolerance induction to factor VIII. J Thromb Haemost. 2009;7(9) 1446-1456.
  153. 153. Hartman ZC, Appledorn DM, Amalfitano A. Adenovirus vector induced innate immune responses: impact upon efficacy and toxicity in gene therapy and vaccine applications. Virus Res. 2008;132(1-2) 1-14.
  154. 154. Seregin SS, Amalfitano A. Improving adenovirus based gene transfer: strategies to accomplish immune evasion. Viruses. 2010;2(9) 2013-2036.
  155. 155. Kawai T, Akira S. Toll-like receptor and RIG-I-like receptor signaling. Ann N Y Acad Sci. 2008;1143 1-20.
  156. 156. Varnavski AN, Calcedo R, Bove M, Gao G, Wilson JM. Evaluation of toxicity from high-dose systemic administration of recombinant adenovirus vector in vector-naive and pre-immunized mice. Gene Ther. 2005;12(5) 427-436.
  157. 157. Rafii S, Dias S, Meeus S, Hattori K, Ramachandran R, Feuerback F, Worgall S, Hackett NR, Crystal RG. Infection of endothelium with E1(-)E4(+), but not E1(-)E4(-), adenovirus gene transfer vectors enhances leukocyte adhesion and migration by modulation of ICAM-1, VCAM-1, CD34, and chemokine expression. Circ Res. 2001;88(9) 903-910.
  158. 158. Lieber A, He CY, Meuse L, Schowalter D, Kirillova I, Winther B, Kay MA. The role of Kupffer cell activation and viral gene expression in early liver toxicity after infusion of recombinant adenovirus vectors. J Virol. 1997;71(11) 8798-8807.
  159. 159. Shayakhmetov DM, Gaggar A, Ni S, Li ZY, Lieber A. Adenovirus binding to blood factors results in liver cell infection and hepatotoxicity. J Virol. 2005;79(12) 7478-7491.
  160. 160. Alba R, Bosch A, Chillon M. Gutless adenovirus: last-generation adenovirus for gene therapy. Gene Ther. 2005;12 Suppl 1 S18-27.
  161. 161. Bangari DS, Mittal SK. Current strategies and future directions for eluding adenoviral vector immunity. Curr Gene Ther. 2006;6(2) 215-226.
  162. 162. Miao CH. Advances in Overcoming Immune Responses following Hemophilia Gene Therapy. J Genet Syndr Gene Ther. 2011;S1.
  163. 163. Coughlan L, Alba R, Parker AL, Bradshaw AC, McNeish IA, Nicklin SA, Baker AH. Tropism-modification strategies for targeted gene delivery using adenoviral vectors. Viruses. 2010;2(10) 2290-2355.
  164. 164. Schulick AH, Vassalli G, Dunn PF, Dong G, Rade JJ, Zamarron C, Dichek DA. Established immunity precludes adenovirus-mediated gene transfer in rat carotid arteries. Potential for immunosuppression and vector engineering to overcome barriers of immunity. J Clin Invest. 1997;99(2) 209-219.
  165. 165. Herzog RW, Dobrzynski E. Immune implications of gene therapy for hemophilia. Semin Thromb Hemost. 2004;30(2) 215-226.
  166. 166. Jayandharan GR, Aslanidi G, Martino AT, Jahn SC, Perrin GQ, Herzog RW, Srivastava A. Activation of the NF-kappaB pathway by adeno-associated virus (AAV) vectors and its implications in immune response and gene therapy. Proc Natl Acad Sci U S A. 2011;108(9) 3743-3748.
  167. 167. Moskalenko M, Chen L, van Roey M, Donahue BA, Snyder RO, McArthur JG, Patel SD. Epitope mapping of human anti-adeno-associated virus type 2 neutralizing antibodies: implications for gene therapy and virus structure. J Virol. 2000;74(4) 1761-1766.
  168. 168. Wu TL, Ertl HC. Immune barriers to successful gene therapy. Trends Mol Med. 2009;15(1) 32-39.
  169. 169. Verthelyi D. Adjuvant properties of CpG oligonucleotides in primates. Methods Mol Med. 2006;127 139-158.
  170. 170. Jacob T, Hemavathy K, Jacob J, Hingorani A, Marks N, Ascher E. A nanotechnology-based delivery system: Nanobots. Novel vehicles for molecular medicine. J Cardiovasc Surg (Torino). 2011;52(2) 159-167.
  171. 171. Piskin E. Stimuli-responsive polymers in gene delivery. Expert Rev Med Devices. 2005;2(4) 501-509.
  172. 172. Lande C, Cecchettini A, Tedeschi L, Taranta M, Naldi I, Citti L, Trivella MG, Grimaldi S, Cinti C. Innovative Erythrocyte-based Carriers for Gene Delivery in Porcine Vascular Smooth Muscle Cells: Basis for Local Therapy to Prevent Restenosis. Cardiovasc Hematol Disord Drug Targets. 2012;12(1) 68-75.
  173. 173. Chorny M, Fishbein I, Adamo RF, Forbes SP, Folchman-Wagner Z, Alferiev IS. Magnetically targeted delivery of therapeutic agents to injured blood vessels for prevention of in-stent stenosis. Methodist Debakey Cardiovasc J. 2012;8(1) 23-27.
  174. 174. Phillips LC, Klibanov AL, Bowles DK, Ragosta M, Hossack JA, Wamhoff BR. Focused in vivo delivery of plasmid DNA to the porcine vascular wall via intravascular ultrasound destruction of microbubbles. J Vasc Res. 2010;47(3) 270-274.
  175. 175. Fishbein I, Chorny M, Levy RJ. Site-specific gene therapy for cardiovascular disease. Curr Opin Drug Discov Devel. 2010;13(2) 203-213.

Written By

Divya Pankajakshan and Devendra K. Agrawal

Published: February 27th, 2013