Current Status of Cardiovascular Gene Therapy

review

© The American Society of Gene Therapy

Current Status of Cardiovascular Gene Therapy Tuomas T Rissanen1 and Seppo Ylä-Herttuala1,2,3 Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute, Kuopio University, Kuopio, Finland; 2Department of Medicine, Kuopio University, Kuopio, Finland; 3Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland 1

Gene transfer for the therapeutic modulation of cardiovascular diseases is an expanding area of gene therapy. During the last decade several approaches have been designed for the treatment of hyperlipidemias, postangioplasty restenosis, hypertension, and heart failure, and for protection of vascular by-pass grafts and promotion of therapeutic angiogenesis. Adenoviruses (Ads) and adeno-associated viruses (AAVs) are currently the most efficient vectors for delivering therapeutic genes into the cardiovascular system. Gene transfer using local gene delivery techniques have been shown to be superior to less-targeted intra-arterial or intra-venous applications. To date, no gene therapy drugs have been approved for clinical use in cardiovascular applications. In preclinical studies of therapeutic angiogenesis, various growth factors such as vascular endothelial growth factors (VEGFs) and fibroblast growth factors (FGFs), have shown positive results. Gene therapy also appears to have potential clinical applications in improving the patency of vascular grafts and in treating heart failure. Post-angioplasty restenosis, hypertension, and hyperlipidemias (excluding homozygotic familial hypercholesterolemia) can usually be managed satisfactorily by conventional approaches, and are therefore less favored areas for gene therapy. The development of technologies that can ensure long-term, targeted, and regulated gene transfer, and a careful selection of target patient populations, will be very important for the progress of cardiovascular gene therapy in clinical applications. Received 4 January 2007; accepted 11 March 2007; published online 8 May 2007. doi:10.1038/sj.mt.6300175

Introduction Cardiovascular diseases are still the leading cause of death in the Western world in spite of significantly improved management of risk factors, the availability of conventional medical therapies including invasive treatments for coronary artery disease (CAD) and peripheral arterial disease, and also improved prognosis of heart failure. This is why cardiovascular gene therapy has been under intensive research in recent years.1 The history of therapeutic angiogenesis reflects the slow but progressive developments in this field. Thirty years ago it was proposed that an angiogenic factor from tumors could be used for inducing therapeutic vascular growth in the ischemic myocardium.2 However, it took 15 years before sufficient amounts of growth factors could to be tested in preclinical experiments.3 In spite of the promising results in animal experiments, randomized placebo-controlled double-blind clinical trials of recombinant vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in CAD and peripheral arterial disease yielded disappointing results.4–6 Because growth factors have a short half-life7 it became obvious that the gene transfer approach could be useful for sustaining the effects of angiogenic and other therapeutic factors. Currently, gene therapy for therapeutic angiogenesis is the most advanced application in clinical testing,8 whereas the other areas of cardiovascular gene therapy, such as the prevention of atherogenesis through the management of severe hyperlipidemias, and treatment of heart failure, hypertension,

post-­angioplasty restenosis, and occlusion of vascular by-pass grafts have progressed more slowly.1 In this review we summarize the current status of cardiovascular gene therapy including the most promising treatment genes and gene transfer vectors as well as recent advances in preclinical and clinical trials.

Gene transfer vectors in the cardiovascular system Early experimental and clinical studies of cardiovascular gene therapy predominantly utilized plasmid DNA because of its easy production and safety. However, though the initial results of using plasmid DNA encoding VEGF and other angiogenic factors in animal models and in small open-label uncontrolled clinical trials were promising,9–13 larger randomized controlled trials have not supported the use of plasmid DNA, because of the low gene transfer efficacy.14–18 Liposome complexes and cationic polymers do not significantly improve the gene transfer efficacy of plasmid DNA in vivo.19,20 Quantitative methods, such as enzyme-linked immunosorbent assay, show that plasmid DNA-mediated gene transfer does not easily generate measurable quantities of protein in target tissues of large mammals.21 In the rabbit heart, naked plasmid DNA-mediated gene transfer resulted in transfection of only a few cells.20 Furthermore, in contrast to the results obtained with adenovirus (Ad), there is often a lack of a dose response effect with plasmid DNA.21,22 Physical methods such as electroporation or ultrasound may improve the efficacy of plasmid DNA

Correspondence: Seppo Ylä-Herttuala, Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. E-mail: [email protected] Molecular Therapy vol. 15 no. 7, 1233–1247 july 2007

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Table 1  Vectors used for cardiovascular gene transfer Vector

Advantages

Disadvantages

Naked plasmid DNA

Easy to produce safe

Very low transduction efficiency Transient expression

Adenovirus

High transduction efficiency Relatively high transgene capacity Easy to produce in high titers Transduces quiescent cells Tropism for multiple cells

Inflammation with high doses Transient expression

Adeno-associated virus (AAV-1, -2, 5, 6, 8, 9)

Long-term gene expression Moderate immune response Transduces quiescent cells High tropism for skeletal muscle (AAV-1, -6) and myocardium (AAV-8 and -9) Wild type does not cause disease in humans

Limited transgene capacity Difficult to produce in large quantities

Lentivirus

Long-term gene expression Transduces quiescent cells Relatively high transgene capacity Low immune response

Non-specific integration Low transduction efficiency Limited tropism Difficult to produce in large quantities

Retrovirus

Long-term gene expression Relatively easy to produce Low immune response

Non-specific integration Transduces only dividing cells Low transfection efficiency Limited tropism Difficult to produce in large quantities

Herpes simplex-virus (HSV-1)

High transduction efficiency High transgene capacity Tropism for neuronal cells

Transduces only dividing cells Cytotoxicity Limited tropism

Epstein-Barr-virus

High transduction efficiency High transgene capacity Extrachromosomal replication

Transduces only dividing cells Difficult to produce in large quantities

Baculovirus

High transgene capacity Easy to produce in high titers Rapid construction of recombinant baculoviruses Wild type does not cause disease in humans

Transient expression Inflammation with high doses Limited tropism

Antisense oligonucleotides

Easy to produce

Limited efficacy High gene transfer efficiency required

siRNA

More potent than antisense approach

High gene transfer efficiency required

Abbreviation: siRNA, small interfering RNA.

by helping it to penetrate cell membranes,23,24 but it is uncertain whether clinically relevant gene transfer efficacy can be achieved with these methods. Although considered safe, plasmid DNA can cause transient fever, inflammation and even infarction in skeletal muscle and myocardium.15,25,26 It therefore appears that viral gene transfer vectors will be preferred for efficient cardiovascular gene therapy (Table 1). Ad and adeno-associated virus (AAV) vectors have shown high transduction efficacy in blood vessel wall, skeletal muscle, heart and liver, which are frequent targets of cardiovascular gene therapy. In the myocardium, they are several orders of magnitude more efficient than naked plasmid DNA.20 Retroviruses were among the first vectors to be used for in vivo cardiovascular gene transfer19 but safety problems have reduced the interest in these vectors. Lentiviruses have been recently used in cardiovascular gene therapy e.g., for the treatment of familial hypercholesterolemia.27 Lentiviruses have been shown to transduce vascular smooth muscle cells (SMCs) also28–30 but further vector engineering is needed to improve their efficacy. Sendaiviruses31 and ­herpesviruses32 have also been utilized in some ­ cardiovascular 1234

gene therapy applications. Baculoviruses transduce blood vessels transiently with a moderate efficiency.33 Currently, more potent baculovirus vectors are being developed that would yield higher and more long-term gene expression with less inflammation. Antisense oligonucleotides came into the picture more than 10 years ago but their efficacy appears to be insufficient for efficient clinical use.34,35 In contrast, small interfering RNAs (­siRNAs) have shown more potential, and are replacing antisense oligonucleotides in inhibition studies.36 However, siRNAs too are fundamentally limited by the intracellular mechanism of action which requires very high transfection efficiency. Therefore, gene transfer of secreted decoy receptors, antibodies or aptamers may be more feasible for blocking the functions of harmful factors in the cardiovascular system. First generation Ads produce a very high initial level of gene expression with biological effects peaking a few days after the gene transfer but, unfortunately, diminishing thereafter in 2 weeks.21,37,38 Thus, adenoviral gene transfer offers a gene expression boost which may be enough in certain situations such as therapeutic angiogenesis in skin wounds. Unfortunately, repeated administration of Ads www.moleculartherapy.org vol. 15 no. 7 july 2007

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of the same serotype is not effective in large mammals, because of the generation of neutralizing antibodies.39 Although considered quite immunogenic, optimal low doses of Ads produced according to good manufacturing practice protocols induce very little inflammation in skeletal muscle and myocardium.21,38,40 Second and, especially, third generation Ads may be even less immunogenic and promote longer-lasting gene expression, but their potential in gene therapy in humans remains unknown.41 Ads have better natural tropism towards myocardium and regenerating skeletal myocytes than towards mature quiescent skeletal muscle.42 In skeletal muscle, other cells, e.g., interstitial fibroblasts, are more effectively transduced.43 Vascular SMCs and endothelial cells display high adenoviral transduction in vivo.19,44 There seems to be a significant interspecies variation in the efficacy of adenoviral vector. Mouse, rat and pig tissues are much less prone to adenoviral gene transfer than human and rabbit tissues.21,45–47 Even though theoretically having very promising potential,48 targeted adenoviral vectors have so far been used only rarely in cardiovascular applications in vivo, probably because of a lower efficiency when compared with the wild-type vectors. The overall safety profile of Ads has been very good in clinical trials, transient fever being the most important side-effect.14,15 The death caused by adenoviral gene transfer some years ago was due to a vast over-dosing in a patient with a defective immune defense.49 AAV has many characteristics that are potentially useful for gene therapy. It has a natural tropism towards vascular SMCs, cardiac myocytes and skeletal muscle, it transduces quiescent cells, and drives a long-term gene expression lasting several months. It is also thought to generate only a restricted inflammatory reaction, and the wild-type virus is not known to cause any diseases in humans. The long-lasting gene expression pattern of AAV with lower maximal gene expression levels than those induced by Ads, is very attractive.50 Importantly, the peak expression of AAVs does not necessarily take weeks but can be achieved in just a few days after the transduction.51 The wild-type AAV incorporates in the human genome, but recombinant AAV vectors do not seem to integrate, and this is important for safety.52 The new AAV serotypes 1, 6, and 8, 9 have been reported to be even more effective in skeletal muscle and myocardium, respectively, than the most commonly used serotype 2.53–55 Targeting of AAVs may be important in order to avoid ectopic gene transfer.56 This is because gene expression lasting at least 3.7 years has been reported to occur with AAV-2 in human skeletal muscle.57 AAVs can also be transcriptionally targeted to the myocardium through regulatory sequences of genes encoding e.g., α-myosin heavy chain, myosin light chain-2v, or β-actin promoters. However, this is at the cost of gene transfer efficiency when compared with the cytomegalovirus promoter.54,58,59

needle catheters have been developed, to improve the penetration of vectors through the intimal layer.45,60 Despite these improvements in catheter technology, an advanced lesion with a thick neointima, calcification, cholesterol deposits, and infiltrates of inflammatory cells is a very challenging target for effective gene transfer. A recently introduced method of intravascular gene transfer is a gene-eluting stent,61,62 which is a very timely approach considering the extensive use of drug-eluting stents. Gene transfer in vascular grafts can be done either by incubating gene transfer solution inside the graft in situ or by immersion (ex vivo) gene transfer.50,63 Both these methods lead to high gene transfer efficiency because viruses are able to transduce the target segment in the absence of blood flow, components of the innate immune system, and neutralizing antibodies. Administration of viral vectors through intramuscular (IM) or intramyocardial injections is currently by far the most efficient way to produce efficient gene expression in the target tissue. Many experimental studies have shown that the intraarterial route of Ad administration is ineffective unless the permeability properties of the endothelium are modulated, or a high pressure gradient is utilized.46,64,65 Furthermore, more extensive biodistribution and ectopic gene expression in distant tissues can be expected after administration of Ads into circulation than with direct injections into tissues. Pericardial delivery does not result in effective transmural transduction of the myocardium without concomitant treatment with proteases.7 An intravenously injected targeted vector is an intriguing idea but the amounts of viruses needed in large mammals for this approach would be probably too large, considering the safety aspects and limitations in vector production. In the skeletal muscles of the lower limbs, IM injections are theoretically easy to perform. However, as most of the effects of angiogenic growth factors are local,38,46 injections must be carefully directed inside the target muscles, and this might be best accomplished e.g., with ultrasound guidance. A misdirected injection between muscles will cause angiogenesis mostly in muscle fascias and not in the muscle itself. Intramyocardial injections are much more challenging to carry out than injections into skeletal muscles. Thoracotomy performed only for intramyocardial gene transfer might be too risky, unless it is needed for other therapeutic purposes such as bypass surgery. Percutaneous catheter-mediated intramyocardial injections guided with a 3D mapping system have recently proven safe and feasible.12,16,21 IM injection of cells transduced ex vivo to constitutively express therapeutic factors has also been used but this approach appears to be hampered by the suboptimal spreading of cells inside the target tissue, which may lead to severe side-effects.66,67 In the liver, the intravascular gene transfer route appears to be effective and yields high hepatic gene transfer efficiency.27,68

Gene transfer routes in the cardiovascular system

Applications of cardiovascular gene transfer Therapeutic vascular growth

The main target organs for cardiovascular gene therapy, in addition to arteries and veins, are the myocardium, skeletal muscles of the lower limbs, and liver. Cardiovascular gene transfer can be ­performed at least by intravascular gene transfer, ex vivo gene transfer, and direct intraorgan injection. For intravascular gene transfer many different catheter systems such as infusion-­perfusion and Molecular Therapy vol. 15 no. 7 july 2007

Currently, gene transfer for therapeutic blood vessel growth i.e., therapeutic angiogenesis is the most explored area of cardiovascular gene therapy, with several clinical trials having been completed.8 Several laboratories have shown that therapeutic vascular growth in vivo can be achieved e.g., by gene transfer of the ­members of the 1235

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Table 2  Factors with therapeutic potential in cardiovascular gene therapy Therapeutic [target] application

Objective

Overexpression

Inhibition of function

Promotion of growth of blood vessels and lymphatic vessels

Stimulation of capillary growth (angiogenesis), collateral artery growth (arteriogenesis) and lymphatic vessel growth (lymphangiogenesis)

VEGF (VEGF-A), PlGF, VEGF-B, -C, -D and –E, EG-VEGF, FGF-1, -2, -4 and -5, Ang-1 and -2, HGF, PDGF-A, -B, C- and –D, IGF-1 and -2, HIF-1α, MCP-1, GM-CSF, eNOS, kallikrein, EGR-1, Ets-1, Del-1, PR39, Id1, stromal cell-derived factor-1α, platelet-derived endothelial cell growth factor/thymidine phosphorylase, adrenomedullin, sonic hedgehog, secretoneurin, thrombopoietin, netrin-1 and -4

Ezrin

Vascular protection, and prevention of restenosis, in-stent restenosis, and graft failure

Enhanced endothelial function (vasodilatation and anti-thrombosis), endothelial regrowth/repair, reduced SMC proliferation and migration, anti-inflammation, inhibition of excess matrix production, induction of apoptosis

VEGFs, eNOS, iNOS, prostacyclin, ecSOD, hemeoxygenase-1, catalase, TIMPs, HGF, p53, p21, p27-p16 Chimera, RB2/p130, Ras, Fas ligand, thymidine kinase, β-interferon, lipoprotein-associated phospholipase A2, C-type natriuretic peptide, PPARγ, Forkhead, β-adrenergic receptor kinase, CGRP, RAD50, TGF-β3, soluble TGF-β type II receptor, kallikrein, homeobox gene Gax

PDGF-B, PDGFR-β, FGF-2, E2F, COX, ICAM, VCAM, midkine, activator protein-1, PAI-1, Rho kinase, Gβγ, CDC2 kinase, cyclin B1, cyclin G1, MCP-1, TNFα

Management of vulnerable plaques and aneurysms

Inhibition of cholesterol accumulation in lesions, inhibition of inflammation and matrix degradation, increased fibrosis within lesions

LDL receptor in liver, TGF-β, Ets-1, TIMPs and soluble scavenger-receptor decoy, PDGFs

COX, MCP-1, TNFα and other chemokines

Control of hyperlipidemias (homozygous familial hyperlipidemia)

Inhibition of cholesterol accumulation in lesions

LDL or VLDL receptor, apoE, apoA-1, soluble scavenger-receptor decoy

apoB-100

Inhibition of blood clotting

Inhibition of platelet aggregation and formation of fibrin, and inhibition of synthesis of blood coagulation factors

tPA, COX, thrombomodulin, C-type natriuretic peptide, ectonucleoside triphosphate diphosphohydrolase

Tissue factor

Management of hypertension

Inhibition of renin–angiotensin system and β-adrenergic receptor, promotion of vasodilatation

eNOS, kallikrein, adrenomedullin, atrial natriuretic peptide

Angiotensinogen, ACE, ATII R1, β-adrenergic receptor

Management of heart failure

Improvement of cardiomyocyte contraction, inhibition of hypertrophy and fibrosis

VEGFs, p38 kinase, p35, β2-adrenergic receptor, cyclin A2, SERCA1, SERCA2a, S100A1, Ang-1, HGF, ecSOD, adenylyl cyclase VI, sorcin, kallikrein, inhibitor-2, hemeoxygenase-1, Bcl-2, norepinephrine transporter uptake-1, phosphoinositide kinase domain, leukemia inhibitory factor, TIMP-1, parvalbumin, V(2) vasopressin receptors

PKCα, phospholamban, β-adrenergic receptor kinase, SEK-1, TGF-β, MCP-1

Biological pacemaker

Promotion of spontaneous rhythmic electrical activity in certain cardiomyocytes of the heart

β2-adrenergic receptor, hyperpolarization-activated cyclic nucleotide-gated (HCN) genes 1-4, hyperpolarization-activated nonselective channel

Kir2.1

Abbreviations: ACE, angiotensin-converting enzyme; Ang, angiopoietin; COX, cyclo-oxygenase; ecSOD, extracellular superoxide dismutase; EGR-1, early growth response factor-1; EG-VEGF, endocrine gland-derived VEGF; eNOS, endothelial nitric oxide synthase; FGF, fibroblast growth factor; GM-CSF, granulocyte macrophage colonystimulating factor; HGF, hepatocyte growth factor; ICAM, intercellular adhesion molecule; IGF, insulin-like growth factor; LDL, low density lipoprotein; MCP-1, monocyte chemoattractant protein 1; PAI-1, plasminogen activator inhibitor-1; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PKC, protein kinase C; PlGF, placenta growth factor; PPARγ, peroxisome proliferator-activated receptor; SEK-1, stress-signaling kinase; SERCA2a, sarcoplasmic reticulum Ca2+ ATPase; SMC, smooth muscle cell; TGF, transforming growth factor; TIMP-1, tissue inhibitor of metalloproteinase 1; TNFα, tumor necrosis factor; tpa, tissue plasminogen activator; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor; VLDL, very-low-density-lipoprotein.

VEGF family, FGF family, hepatocyte growth factor (HGF), and hypoxia-inducible factor-1α (HIF-1α). There are also several other angiogenic molecules currently under investigation (Table 2). There is accumulating evidence that VEGF (VEGF-A) is the master switch of physiological and pathological angiogenesis, and therefore also a prototype angiogenic growth factor in gene therapy.69 The absolute requirement for the presence of both VEGF alleles for embryonic vasculogenesis, hypoxia-inducible expression, efficient induction of angiogenesis and vascular permeability make VEGF an extraordinary growth factor. Of the four main VEGF isoforms, 121, 165, 189, and 206, VEGF165 is the most promising for therapeutic promotion of vascular growth, because it is approximately 1236

100-fold more potent biologically than VEGF121, and is able to create growth factor gradients in the tissues that are necessary for proper patterning of new vessels.69 In addition to myocardial and peripheral ischemia, new areas of application of VEGF gene transfer could be strokes, non-healing diabetic ulcers, osteoporotic and avascular bone fractures, and skin-flaps.70–72 Overexpression of VEGF via viral vectors has been shown to stimulate ­excellent angiogenesis, and supraphysiological perfusion increases both in ischemic and healthy skeletal muscle and myocardium because of sprouting angiogenesis and capillary enlargement i.e., arterialization.21,40,73–78 This kind of histological data showing active proliferation of both endothelial cells and SMCs www.moleculartherapy.org vol. 15 no. 7 july 2007

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and remodeling of the capillary network have not been documented in the case of naked plasmid VEGF gene transfer.9,22 With adenoviral gene transfer, most of these changes are transient, with the exception of enlarged collaterals (arteriogenesis), which may persist.40,43,46,79 VEGF-induced arteriogenesis resembles that promoted by a model of a surgical arteriovenous fistula, wherein very high rates of blood flow via collaterals cause extraordinarily powerful arteriogenesis.80 Endothelial progenitor cells do not appear to contribute significantly to the therapeutic vascular growth after VEGF gene transfer.81 Recently, constructs expressing genomic VEGF with an emphasis on the long isoforms or VEGF-inducing transcription factors have been reported to be effective in hindlimb ischemia models.82–84 The most important side effect of adenoviral gene transfers of VEGFs and FGF-4 is transient edema and accumulation of free fluid between skeletal muscles and in the pericardial sac.21,38,46,75 The edema is dose-dependent, and coincides with the peak perfusion 5–6 days after the adenoviral gene transfer.69 In transgenic animals, a chimeric VEGF-E/PlGF construct has been reported to cause less edema than VEGF overexpression does.85 Strong corticosteroids such as dexamethasone can be used for counteracting increased vascular permeability, but unfortunately they also inhibit therapeutic vascular growth (P. Korpisalo, unpublished results). It is also possible that the stimulation of lymphatic vessel growth by simultaneous gene transfer of VEGF receptor 3 (VEGFR-3) ligands may prevent the formation of excess edema. However, the strongest body of evidence suggests that angiogenesis-related edema can be best prevented by using only moderate but long-lasting expression of VEGFs, resulting in the stabilization of growing vessels.86 However, even in this situation it appears that it will be difficult to completely avoid tissue edema, and it can be considered as a surrogate marker of successful therapeutic vascular growth. Other members of the VEGF family also have interesting potential as vascular therapeutic agents.69 Gene transfer of the long forms of VEGF-C and VEGF-D binding to VEGFR-3 could be used for stimulating lymphangiogenesis and thus alleviate congenital or acquired tissue edema.87,88 A mutant form of VEGF-C, VEGF-C156S, binds only to VEGFR-3 and exclusively promotes lymphangiogenesis.38 In contrast, proteolytically processed forms of VEGF-C and -D∆N∆C activate both VEGFR-2 and VEGFR3, and this makes them growth factors that are both angiogenic and lymphangiogenic.21,38 When compared with VEGF165, VEGFD∆N∆C is not sequestered by extracellular matrix, and therefore it induces more diffuse angiogenesis in target tissues and leaks into the circulation.21,38 The viral VEGFs, collectively called VEGF-E, bind only to VEGFR-2, and thus have clear angiogenic potential.89 Recently, VEGF members binding to VEGFR-1, VEGF-B and placenta growth factor have quite unexpectedly also turned out to be angiogenic in non-ischemic tissues, likely via indirect signals.90–92 Our recent observations also suggest tissue-specific patterns of VEGF-B-induced angiogenesis (J. Markkanen, ­ unpublished results). The FGF-family of growth factors consists of 23 members which bind to four tyrosine kinase receptors. The theoretical ability of FGFs to directly stimulate mesenchymal cells in addition to endothelial cells could be useful. However, the biology of FGFs is Molecular Therapy vol. 15 no. 7 july 2007

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not very well understood and the benefits of these properties for therapeutic angiogenesis are still largely unknown. FGF-1 (aFGF) and FGF-2 (bFGF) are the prototypic members of the family and, although not crucial for the formation of blood vasculature in the embryo,93 they were the first that were shown to induce blood vessel growth by viral gene transfer.94,95 FGF-4 and FGF-5 are efficiently secreted, which may offer an advantage to them over intracellular FGFs (FGF-1 and FGF-2) in therapeutic angiogenesis.46,96 Adenoviral IM FGF-4 gene transfer induces angiogenesis with marked increases in perfusion, vascular permeability and collateral growth in a rabbit hindlimb ischemia model, at least partially via endogenous VEGF upregulation.46 The role of platelet-derived growth factors (PDGFs), especially PDGF-B, in the recruitment of pericytes to growing vessels97 makes PDGFs attractive candidates for combined use with VEGFs. Substantiating this hypothesis, recombinant PDGF-B protein given in combination with VEGF has been reported to result in more mature and stable blood vessels than monotherapy with either of the factors alone.98 Furthermore, the administration of PDGF-B protein together with FGF-2 has been shown to produce vessel net­works that remain stable in rat cornea for more than a year after the withdrawal of the growth factors.99 Recombinant PDGFC protein was reported to mobilize endothelial progenitor cells in ischemic conditions.100 PDGF-C also induced differentiation of bone marrow cells into endothelial cells and SMCs, and enhanced revascularization of ischemic heart and limbs in mice.100 However, these results have to be confirmed in other models because there are known limitations in recombinant protein therapy, and because PDGF-C and -D overexpression was shown to lead to cardiac fibrosis and heart failure.101 Another growth factor with potential pleiotropic and angiogenic properties is HGF which is a ligand for a receptor encoded by the c-met protooncogene.102 There is growing evidence that many growth factors such as HGF, insulin-like growth factor 1, PDGF-B, placenta growth factor and FGFs upregulate VEGF, which may at least partially explain their angiogenic actions and provide further evidence that VEGF is the master regulator of angiogenesis.69 Angiopoietin-1 and -2 (Ang-1 and Ang-2) form ­an ­agonist– antagonist pair of molecules that modulate the maturation of blood vessels via binding to Tie-2 receptor. Ang-1 has been shown to be anti-inflammatory, to stabilize vessels, and counteract VEGF-induced vascular permeability.77,103 However, the antipermeability effect of Ang-1 is currently poorly understood and the results seem to be model-dependent. Theoretically, it would not be enough for Ang-1 merely to normalize the permeability properties of the endothelium; it would also need to make the endothelium hyperresistant to plasma protein leakage to counterbalance biophysical forces such as elevated capillary pressure, that drive plasma proteins into the extravascular space. COMP-Ang is a soluble and stable tetrameric variant of Ang-1, and is suggested to be more potent than monomeric Ang-1.104 Ang-2 behaves as a natural antagonist of Ang-1 by binding to but not activating the Tie-2 receptor, thereby resulting in the disruption of angiogenesis in the absence of VEGF.105 The gene transfer of transcription factors, such as the constitutively active form of HIF-1α or zinc finger transcription factors, could induce simultaneous expression of many growth factors 1237

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and cytokines, leading to a more natural angiogenic response.83,106 However, the angiogenic potency of e.g., adenoviral HIF-1α appears to be at least an order of magnitude lower than that of adenoviral VEGF (H. Karvinen, unpublished results). Gene transfer of endothelial nitric oxide synthase (endothelial NOS) has been reported to promote angiogenesis in a rat hindlimb ischemia model,107 whereas angiogenesis has not been observed in hindlimbs of rabbits after gene transfer of adenoviral endothelial NOS, thereby indicating that NO is necessary but not sufficient for angiogenesis (T.T. Rissanen, unpublished results). Cytokines, such as monocyte chemoattractant protein-1, granulocyte macrophage colony-stimulating factor and granulocyte colony-stimulating factor, are indirect vascular growth factors thought to act via circulating monocytes and endothelial progenitor cells.108 Other factors reported to be angiogenic in ischemic tissues of experimental animals include early growth response-1, Ets-1, Del-1, PR39, insulinlike growth factors-1 and -2, endocrine-gland-derived-VEGF, Id1, stromal cell-derived factor-1α, adrenomedullin and Sonic hedgehog109–114 (Table 2). However, these findings are mostly based on experiments in single laboratories and further studies are needed to confirm their potency in therapeutic angiogenesis in large animal models and in humans.

Current status of clinical trials of gene therapy for therapeutic angiogenesis Among cardiovascular gene therapy trials, the vast majority have been designed to study therapeutic blood vessel growth (Tables 3 and 4). Recombinant protein (VEGF, FGF-2 and granulocyte macrophage colony-stimulating factor) therapies have been investigated in randomized controlled studies in peripheral and myocardial ischemia, but there were no convincing improvements in clinical outcomes.4–6,115 Proangiogenic VEGF gene therapy was initially tested with naked plasmid DNA delivery and was reported to result in the resolution of rest pain, increased collateral vasculature, and an increase in ankle brachial pressure index.10,11,116 Naked VEGF165 plasmid DNA as intramyocardial injections via throracotomy in small cohorts of “no-option” patients was reported to significantly improve the clinical status, myocardial perfusion, collateral growth and myocardial contractile function.117–119 However, naked plasmid VEGF165 administered together with granulocyte macrophage colony-stimulating factor did not lead to improved myocardial performance.120 In other trials naked VEGF-C (also called VEGF-2) plasmid DNA injected percutaneously into ischemic myocardium with the NOGA catheter or via thoracotomy alleviated angina and reduced the area of ischemic myocardium.12,121 In phase I trials, intra-arterial and IM delivery of AdVEGF165 and AdVEGF121 to human peripheral arteries and skeletal muscles, respectively, was safe and well tolerated.45,122 In a phase I trial of 34 no-option patients with critical limb ischemia AdHIF-1α treatment was shown to be safe and well tolerated.123 Only a few randomized, placebo-controlled angiogenic gene therapy trials have been reported on patients with intermittent claudication or critical limb ischemia (Table 3). In a randomized controlled phase II trial (n = 54) of catheter-mediated VEGF165 gene delivery using plasmid-liposomes or Ads, VEGF treatment improved the vascularity on digital ­subtraction 1238

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angiography at 3 months without differences in clinical outcomes.14 In the RAVE trial using 2 doses (4 × 109 particle units or 4 × 1010 particle units) of AdVEGF121 no significant differences were found between the groups in the change in peak walking time at 3 months or in ankle brachial pressure index and quality of life measures.124 No severe side effects were noted, although transient edema was associated with IM AdVEGF121 gene transfer. In a ­double-blind, placebo-controlled study of naked plasmid VEGF in 54 adult diabetic patients with critical limb ischemia, there was no difference in the primary endpoint, namely, the amputation rate at 100 days but significant improvements were achieved in secondary endpoints (ankle brachial index and clinical condition).17 There are a few published phase II/III trials using adenoviral gene therapy in patients with CAD (Table 4). The study of Laitinen et al. demonstrated the safety and feasibility of VEGF165 gene transfer in patients with stable CAD undergoing percutaneous coronary intervention (n = 15).125 This study was followed by the phase II Kuopio Angiogenesis Trial (n = 103) aimed at investigating the effect of VEGF165 gene therapy on restenosis rate and on myocardial perfusion given during the angioplasty and stenting procedure with an infusion-perfusion catheter.15 There was no difference in the restenosis rate (6%) between the study groups (plasmid/liposome VEGF165, AdVEGF165 or placebo) but myocardial perfusion showed a significant improvement in the AdVEGF165 group at 6 months. Although intracoronary AdFGF4 tended to improve exercise time at 4 weeks in 79 stable angina pectoris patients (Canadian Cardiovascular Society (CCS) class II/III) in the phase I/II AGENT-1 trial and resulted in a significant reduction of ischemic defect size in the phase II AGENT-2 trial,126,127 a larger multinational, multicenter double blind phase III AGENT-4 trial conducted in Europe documented no significant effect on exercise tolerance at 12 weeks in 116 patients.128 Also, the recruitment of patients was prematurely terminated in the AGENT-3 trial conducted in the United States, because of the failure to achieve the primary endpoint.129 In a subsequent posthoc analysis, however, significant efficacy was noted in patients over 55 years of age who had more severe angina symptoms.129 An ongoing phase III AWARE trial (n = 300) is testing the efficacy of intracoronary AdFGF-4 in women patients.130 Percutaneous intramyocardial NOGA-delivery of VEGF-C naked plasmid in no-option patients (n = 19) was reported to result in a significant reduction in CCS angina class after a 12-week follow-up.131 In the Euroinject One study a total of 80 no-option CCS angina class III–IV patients were assigned to intramyocardial injections of naked plasmid VEGF165 or placebo with the NOGA catheter.16 After a 3-month follow-up no significant differences were found between the groups in the CCS class, or in the size of perfusion defect at rest or during exercise. However, local wall motion as assessed by ventriculography or local linear shortening mapping with NOGA were better in the plasmid VEGF165 treated group than in the placebo group. In a phase II trial, no-option CAD patients (n = 67) with CCS class II–IV angina were randomized to either continue optimal medical therapy or to receive AdVEGF121 (4 × 1010 particle units) into the myocardium via thoracotomy.132 Time to 1 mm ST-depression during exercise test was significantly improved at 26 weeks, though not at 12 weeks, in patients who received AdVEGF121, as compared to www.moleculartherapy.org vol. 15 no. 7 july 2007

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Table 3 Clinical phase II/III randomized controlled gene therapy trials in PAD

Trial

Therapeutic [target] application

VEGF peripheral vascular disease trial

Control treatment

Primary endpoint

a

Reference

Increased vascularity in angiography at 3 months

Positive

14

105

PWT at 12 weeks

Negative

124

Vehicle

300

PWT at 6 months

Ongoing

133 (Unpublished)

Intramuscular injections

Vehicle

157

PWT at 3 months

Negative

135 (Unpublished)

Naked VEGF165 Plasmid

Intramuscular injections

Saline

54

Decrease in amputation rate

Negative 17 (secondary endpoints positive)

Therapeutic angiogenesis in PAD (CLI)

Naked HGF plasmid

Intramuscular injections

Saline

48 (planned)

Wound healing, amputation rate, rest pain, ABI

Ongoing

TALISMAN 201

Therapeutic angiogenesis in PAD (CLI)

Naked FGF-1 plasmid

Intramuscular injections

Vehicle

107

Ulcer healing at 6 months

Negative 136 (secondary (Unpublished) endpoints positive)

PM 202

Therapeutic angiogenesis in PAD (CLI)

Naked FGF-1 plasmid

Intramuscular injections

Vehicle

71

Change in transcutaneous pO2

Negative

Prevent III

Vein graft failure in PAD (CLI)

Edifoligide (an E2F transcription factor decoy)

Ex vivo Buffered pressure-mediated saline delivery

1,404

Time to graft reintervention or major amputation due to graft failure

Negative 35 (secondary endpoint positive)

Therapeutic agent

Administration

n

Therapeutic angiogenesis in PAD (claudication)

AdVEGF165 or Plasmid/liposome VEGF165

Intraarterial injection at the angioplasty site

Ringer’s lactate

54

RAVE trial

Therapeutic angiogenesis in PAD (claudication)

AdVEGF121

Intramuscular injections

Vehicle (no virus)

WALK

Therapeutic angiogenesis in PAD (claudication)

AdHIF-1α/VP16

Intramuscular injections

DELTA-1

Therapeutic angiogenesis in PAD (claudication)

Plasmid-expressing Del-1 formulated with poloxamer 188

Groningen trial

Therapeutic angiogenesis in PAD (CLI)

HGF-STAT

Results

134 (Unpublished)

137 (Unpublished)

Abbreviations: ABI, ankle brachial index; Ad, adenovirus; CLI, critical limb ischemic; FGF, fibroblast growth factor; HIF-1α, hypoxia inducible factor-1α; HGF, hepatocyte growth factor; PAD, peripheral arterial disease; PWT, peak walking time; vascular endothelial growth factor. a A measure of the efficacy in relation to the study protocol-defined primary or secondary endpoint.

controls. ­Secondary endpoints, CCS class, and total exercise tolerance were improved at both time points. However, the significant ­contribution that thoracotomy could have made to the placebo effect should not be excluded from consideration. In addition to AGENT-3 and AGENT-4 trials, there are some other yet unpublished trials of gene transfer for ­ therapeutic angiogenesis (Tables 3 and 4). Currently, a phase II randomized, double-blind, placebo-controlled WALK trial is under way to assess the positive impact of AdHIF-1α on peak walking time at 6 months in approximately 300 peripheral arterial disease patients.133 An ongoing HGF-STAT trial seeks to assess the efficacy of naked plasmid-mediated HGF gene transfer on tissue perfusion in patients with unreconstructable critical ischemia.134 The phase II study addressing the effect of IM administered Del-1 plasmid formulated with poloxamer 188 on the change in the peak walking time at 3 months in 100 patients with claudication showed no efficacy.135 The phase II trials using IM FGF-1 naked plasmid gene transfer in critical limb ischemic patients failed to achieve the primary endpoints, namely, ulcer healing and increased transcutaneous pO2, but the TALISMAN trial resulted in a significant reduction in amputation rate.136,137 The phase Molecular Therapy vol. 15 no. 7 july 2007

II GENASIS trial, originally designed to enroll 404 no-option CAD patients with CCS III–IV angina to study the efficacy of per­cutaneous, intramyocardial Stiletto catheter-mediated naked VEGF-C (VEGF-2) plasmid gene transfer on exercise tolerance time at 3 months, was recently prematurely stopped after only 295 patients, because of complications related to the catheter and a high likehood of the lack of positive impact on the primary endpoint.18 The ongoing phase II/III NORTHERN trial (planned n = 120) is testing the efficacy of intramyocardial NOGAmediated delivery of AdVEGF121 in no-option CAD patients.138 The NOVA trial with a very similar design was recently stopped.139

Vascular protection and prevention of restenosis and graft failures Vascular gene therapy to alleviate post-angioplasty restenosis has experienced a significant change in the past few years. From being one of the most popular, it has now become a less attractive area of gene therapy because sirolimus and paclitaxel-eluting stents have offered a significant relief to this clinical problem. T­herefore, future studies should aim at investigating how gene therapy can be combined with drug-eluting stents to further improve the 1239

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© The American Society of Gene Therapy

Table 4 Clinical phase II/III randomized controlled gene therapy trials in CAD Therapeutic [target] application

Therapeutic agent

KAT

Therapeutic angiogenesis in CAD (CCS class II–III)

AdVEGF165 or plasmid/ liposome VEGF165

Intracoronary injection at the angioplasty site

REVASC trial

Therapeutic angiogenesis in CAD (CCS II–IV)

AdVEGF121

Intramyocardial Best medical 67 injection via treatment mini-thoracotomy (no placebo treatment)

Time to 1 mm Positive ST-segment depression on ETT at 26 weeks

132

Euroinject one trial

Therapeutic angiogenesis in CAD (CCS III–IV)

Naked VEGF165 Plasmid

Percutaneous Intramyocardial injections

Placebo plasmid

74

Improved myocardial perfusion at 3 months

Negative

16

Genasis

Therapeutic angiogenesis in CAD (CCS III–IV)

Naked VEGF-2 (VEGF-C) plasmid

Percutaneous Intramyocardial injections

Vehicle

295 (404 planned)

ETT at 3 months

Negative at interim analysis, stopped

18 (Unpublished)

Northern

Therapeutic angiogenesis in CAD (CCS III–IV)

AdVEGF121

Percutaneous Intramyocardial injections

Vehicle

120 Change in (planned) myocardial perfusion in stress/rest at 12 weeks

Ongoing

138 (Unpublished)

NOVA

Therapeutic angiogenesis in CAD (CCS II–IV)

AdVEGF121

Percutaneous Intramyocardial injections

Vehicle

129 ETT at (planned) 26 weeks

Stopped

139 (Unpublished)

AGENT-2

Therapeutic angiogenesis in CAD (CCS II–IV)

AdFGF-4

Intracoronary injection

Vehicle

52

SPECT at 8 weeks

Positive

127

AGENT-3

Therapeutic angiogenesis in CAD (CCS II–IV)

AdFGF-4

Intracoronary injection

Vehicle

416

ETT at 12 weeks

Negative (subgroup of >55 yr with CCS III-IV positive)

129 (Unpublished)

AGENT-4

Therapeutic angiogenesis in CAD (CCS II–IV)

AdFGF-4

Intracoronary injection

Vehicle

116

ETT at 12 weeks

Negative

128 (Unpublished)

AWARE

Therapeutic angiogenesis in CAD (CCS III–IV)

AdFGF-4

Intracoronary injection

Vehicle

300 (women)

ETT at 6 months

Ongoing

130 (Unpublished)

Italics

In-stent restenosis in CAD

Anti-sense Local delivery oligonucleotide after stent against c-myc implantation

Saline

85

% neointimal volume obstruction at 6 months

Negative

147

Prevent IV

Vein graft failure in CAD

edifoligide (an E2F transcription factor decoy)

Ex vivo Buffered pressure-mediated saline delivery

2,400

Death or vein graft stenosis of >75% at 12–18 months

Negative

34

ISIS

Familial hypercholesterolemia

ISIS 301012 (antisense oligonucleotide inhibitor of apoB)

Intravenous followed by weekly subcutaneous injections for 4 weeks

36

Percent reduction in LDL-cholesterol from baseline to 12 weeks

Positive (35% 170 reduction of LDL-cholesterol at 39 days)

Trial

Administration

Control treatment

Ringer’s lactate

Unknown

n

103

Primary endpoint

Improved myocardial perfusion at 6 months

a

Reference

Positive (adenovirus group only)

15

Results

Abbreviations: Ad, adenovirus; apoB, apolipoprotein B; CAD, coronary artery disease; CCS, Canadian cardiovascular society; ETT, exercise tolerance testing; FGF, fibroblast growth factor; HIF-1α, hypoxia inducible factor-1α; LDL, low density lipoprotein; SPECT, single-photon emission computed tomography; VEGF, vascular endothelial growth factor. a A measure of the efficacy in relation to the study protocol-defined primary or secondary endpoint α.

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© The American Society of Gene Therapy

results. One target for gene therapy could be the acceleration of re-endotheliazation impaired by these anti-proliferation drugs, although the animal data on re-endotheliazation by overexpression of VEGFs has been conflicting.44,140–143 Other therapeutic genes that could possibly further improve the effects of drugeluting stents include anti-oxidative genes such as extracellular superoxide dismutase, and vasoprotective and anti-thrombotic factors such as like NOS and prostacyclin synthase, that have been effective in animal models of restenosis.144–146 While there is no drug-gene combination stent yet, gene-eluting stents have already been introduced.61,62 A vast number of additional factors have been tested in preclinical models of balloon-angioplasty restenosis since the beginning of the 1990s (Table 2) but none has so far proven effective in clinical studies.15,147 Gene therapy may be more applicable in the treatment of vein graft stenosis than of post-angioplasty restenosis because there is currently no established pharmacological treatment aimed at inhibiting vein graft occlusions. Ex vivo gene transfer offers a feasible method for the transduction of the whole vein graft prior to implantation. This approach is eligible both for coronary artery and peripheral bypass vein grafts.63,148 Also, the failure of arteriovenous grafts in hemodialysis patients is a significant clinical problem and these grafts could be genetically modified.149,150 Gene transfer of NOS, or of other factors causing increased NO production within the grafts could be effective in achieving vasodilatation, anti-thrombotic effects, and diminished SMC proliferation.151,152 Normal physiological and moderately elevated levels of VEGF are likely beneficial for blood vascular homeostasis i.e., are vasculoprotective.153 This hypothesis is supported by the findings from clinical studies that when VEGF function is blocked by antibody treatment, cardiovascular complications occur.154 On the other hand, while many animal models of intimal thickening and arteriosclerosis have indicated that too much of VEGF may enhance atherogenesis,142,143,155 clinical studies using intra-arterial adenoviral VEGF gene transfer have not shown such effects so far.14,15 VEGF or NOS gene transfer could also improve erectile function caused by diabetes or hypercholesterolemia.156 Angs could also function as vasculoprotective factors e.g., in cardiac allograft arteriosclerosis157 but the current data on their therapeutic potential is controversial and even their basic biology is not yet very well understood.158 Other strategies for the prevention of intimal thickening include factors that inhibit SMC proliferation, oxidative stress, inflammation or the degradation of extracellular matrix.152,159–161 For example, Ad-mediated ex vivo gene transfer of extracellular superoxide dismutase, alone or in combination with tissue inhibitor of metalloproteinase-1 or vaccinia virus antiinflammatory protein 35K, prevented vein graft stenosis in a jugular vein graft model in normocholesterolemic rabbits.63 Although Ads are very effective during the first 2 weeks after gene transfer, the treatment for vein graft stenosis may require long-term gene expression, which is currently best achieved with AAVs.162 Despite early positive reports, an elongation factor 2 transcription factor decoy was ineffective in preventing vein graft failure in patients undergoing coronary artery bypass graft or peripheral bypass in the lower extremities, in randomized controlled trials Molecular Therapy vol. 15 no. 7 july 2007

Cardiovascular Gene Therapy

involving 3,804 patients.34,35,163 These results may indicate that the antisense oligonucleotide strategy may not be efficient in human gene therapy. siRNA technology may be the next step to block expression of harmful factors in vein grafts, but currently there are very few published reports of experiments. In one study, siRNA targeting a heparin-binding growth factor midkine was reported to attenuate intimal hyperplasia up to 90% in jugular vein-tocarotid artery interposition vein grafts in rabbits.164 The experience in ­ siRNA technology is limited as yet, and it remains to be seen whether this approach is any more effective than the antisense strategy. This is because siRNAs too must be delivered to a significant number of cells in the graft in order to be clinically effective.

Hyperlipidemias, hypertension, atherogenesis and anti-thrombosis Over the past decade, conventional pharmacological treatment of the major risk factors of atherosclerosis has improved significantly. For example, practically all cases of Western-typediet-related hypercholesterolemia and heterozygous familial hypercholesterolemia can be managed with lipid-lowering drugs, especially by using the most potent statins combined with ezetimibe when necessary. There is also a diversity of treatment options available for patients with essential hypertension. Thus, considering the risk-to-benefit ratio of current gene therapy vectors and approaches, it appears likely that only a quite low number of patients with a very difficult lipid disorders, such as homozygotic familial hypercholesterolemia or drug-resistant hypertension, would be potential candidates for gene therapy. Current anti-thrombotic drug treatments are also very effective but may have significant shortcomings, such as those of warfarin. Only vectors capable of long-term gene expression can be considered for the treatment of cardio­vascular risk factors. It would be useful if such a vector could have regulatory elements that detect changes in cholesterol or in blood pressure levels, and adjust gene expression to an appropriate level accordingly. Homozygotic familial hypercholesterolemia, with a prevalence of 1:1,000,000 persons, is a potential target for gene therapy because: (i) the current treatment options (low density lipoprotein (LDL) apheresis or liver transplantation) are very laborious, (ii) the disease leads to premature cardiovascular death in young adulthood, and (iii) functional LDL receptor (LDLR) expression in the liver can be reconstituted by gene transfer. The initial approach was an ex vivo strategy wherein autologous hepatocytes were retrovirally transduced in vitro and injected back via the portal vein. However, pilot clinical experiments showed that, although it is safe and feasible, the therapy resulted only in a modest LDL lowering effect because of the limited number of transduced hepatocytes.165 The first significant long-term effect on plasma cholesterol levels was achieved by an in vivo gene therapy approach using Moloney murine leukemia virus-derived retroviruses carrying LDLR into the livers of spontaneously hypercholesterolemic LDLR-deficient watanabe heriditary hyperlipidemic rabbits. The plasma total cholesterol in the animals was reduced by 35% 2–3 months after the gene transfer.68 However, the requirement for cell proliferation, and therefore for partial liver resection and/or treatment with a cytotoxic agent as well as insertional mutagenesis with retroviruses, likely preclude the clinical use of this approach. Recent 1241

Cardiovascular Gene Therapy

experiments have utilized lentiviruses and AAV. Gene transfer of human LDLR via AAV serotypes 7 and 8 injected into the portal veins of LDLR-deficient mice on a high-fat diet resulted in nearly complete normalization of serum lipids lasting at least 21 weeks, and also significantly alleviated atherosclerosis.166 Long-term liverspecific gene transfer using an intraportally injected third-generation lentiviral vector encoding LDLR in watanabe heriditary hyperlipidemic rabbits has been shown to decrease serum total cholesterol levels by 14% at 1 month, 44% at 1 year and 34% at 2 years as compared to the controls, with no major safety issues during the follow up.27 Gene transfer of a soluble macrophage scavenger receptor or apolipoprotein E has been shown to lower cholesterol levels and lesion development in mouse models.167,168 However, excessive adenoviral overexpression of LDLR may induce an imbalance between the speed of LDL uptake and metabolism, leading to pathological accumulation of lipids and cholesterol in hepatocytes,169 thereby suggesting that regulated gene expression might be useful for the treatment of hypercholesterolemia. No clinical trials have yet been performed using the in vivo gene transfer strategy for homozygotic familial hypercholesterolemia. In a small phase II double-blind, randomized, placebocontrolled, dose-escalation clinical trial 36 volunteers with mild dyslipidemia were treated with a 4-week multiple-dosing regimen of antisense oligonucleotide against apolipoprotein B administered intravenously followed by weekly subcutaneous injections.170 The treatment was found to be safe, though erythema was commonly observed at the injection site. The therapy resulted in up to 50% reduction in apolipoprotein B and a maximum 35% reduction in LDL cholesterol, both of which remained significantly below baseline up to 3 months after the last dose. However, there were only four patients in the placebo group and these results need to be confirmed in larger trials. Aspirin, clopidogrel, glycoprotein IIb/IIIa receptor inhibitors, and warfarin are effective anti-thrombotic drugs, but the use of warfarin, especially, has led to problems. Experimentally tested anti-thrombotic gene therapy approaches include the use of tissue plasminogen activator, thrombomodulin, cyclo-oxygenase-1, tissue factor pathway inhibitor, C-type natriuretic peptide and ectonucleoside triphosphate diphosphohydrolase.171–176 Also, increased production of NO by the use of endothelial NOS could be an option as a potent anti-thrombotic agent. There is yet no clinical experience in the area of anti-thrombotic gene transfer approaches. A potential therapeutic strategy for control of hypertension is the inhibition of genes that have been implicated in the elevation of blood pressure, particularly members of the renin–­angiotensin system, such as angiotensinogen, angiotensin-converting enzyme, angiotensin II type-1 receptor and β-adrenergic receptor.177 Another way of decreasing blood pressure is to overexpress genes that induce vasodilation e.g., kallikrein, adrenomedullin, atrial natriuretic ­peptide and endothelial NOS.178 Because repeated administration of antisense oligonucleotides hardly offers any significant benefit over conventional drug therapy, gene expression should be longterm and optimally regulated by the blood pressure level. By the use of vectors capable of long-term gene expression, both of the above mentioned strategies have been shown to be ­ effective in rodent models of hypertension.179–181 In addition to these approaches, one interesting possibility could be a permanent gene transfer of soluble 1242

© The American Society of Gene Therapy

angiotensin II type-1 receptor or an antibody against angiotensin converting enzyme, which would circumvent problems related to the requirement of an intracellular mechanism of action of the antisense approach. Gene therapy of pulmonary hypertension has also been investigated, because there is a lack of an optimal conventional treatment and also a poor prognosis for the disease. ­Factors that inhibit SMC proliferation and induce SMC apoptosis as well as overexpression of vasodilatators like prostacyclin synthase and NOS have been utilized in experimental studies.182,183 Clinical experience is currently lacking in the use of gene therapy in both systemic and pulmonary ­hypertension.

Heart failure and modulation of the electrical activity in the myocardium Heart failure remains one of the most important causes of morbi­ dity and mortality in the Western world. Though there have been significant advances in treatment, and greater knowledge of the molecular pathophysiology of the failing myocardium is now ­available, there is a clear need to find ways to modify, and possibly even reverse, the underlying pathophysiology. The main focus areas of gene therapy for heart failure are: improvement of the contractile function of cardiomyocytes by improving perfusion, enhancing Ca2+ transients, inhibiting cardiac hypertrophy, remodeling and fibrosis, and prevention of arrhythmias (Table 2). Ischemic cardiovascular disease is the most important cause of congestive heart failure and therefore angiogenesis using gene therapy could be a novel treatment option in patients who are not suitable candidates for conventional revascularization. VEGF gene transfer has improved systolic function of the left ventricle in animal models of ischemic and pacing-induced heart failure.73,184 Perhaps one of the most promising approaches for improving the contractility of cardiac myocytes is to amend Ca2+ metabolism. This could be done by gene transfer of sarcoplasmic reticulum Ca2+ ATPase, calcium-binding proteins parvalbumin or S100A1, or inhibition of phospholamban, a regulator of cardiac ­sarcoplasmic reticulum Ca2+ cycling.185–188 Manipulation of the myo­cardial β-adrenergic receptor system, which is impaired in heart failure, is also an interesting target of myocardial gene therapy.189 Gene therapy studies that take advantage of the modification of the renin–angiotensin system are still to be explored in the treatment of heart failure. In addition to all these, which are also important areas of conventional drug treatment for heart failure, there are also some completely new potential areas of focus for gene therapy, such as many protein kinases. For example, the inhibition of protein kinase C-α by a dominant negative protein kinase C-α, or overexpression of p38 kinase, have both been shown to enhance myocardial contractility in rat models of postinfarction cardiomyopathy.190,191 Other ways to promote cardiac myocyte survival and mitosis in the failing heart may be overexpression of Bcl-2 and cyclin A2 or the inhibition of caspases.192–194 Intracoronary gene transfer of Ad encoding adenyl cyclase VI also improved left ventricular function in a pig model of congestive heart failure.195 AAV-mediated gene transfer of hemeoxygenase-1 into the rat myocardium conferred cardioprotective effects against myocardial ischemia.196 To date, there have been no published clinical studies specifically designed for treating heart failure using gene therapy. www.moleculartherapy.org vol. 15 no. 7 july 2007

© The American Society of Gene Therapy

An exciting focus area for gene therapy in cardiology is the modulation of the electrical activity of the heart. This could pot­en­ tially be an alternative to implantable electronic pacemaker ­devices. Approaches that have been experimentally tested include β2adrenergic receptor overexpression, downregulation of the inward rectifier current, overexpression of the pacemaker current, and engineered stem cells.197 The most recent advances include overexpression of modulated ion channels, such as a bioengineered hyper­polarization-activated nonselective channel, or a hyper­ polarization-activated, cyclic nucleotide-gated construct with the capability of tuning the frequency of oscillation.198,199 The identification of gene defects that cause congenital long QT syndrome and Brugada syndrome might open the doors for a cure using long-term gene transfer of the functional ion channel in the ­myocardium.

Insights into cardiovascular gene therapy in the future In clinical cardiovascular gene therapy, the emphasis so far has been on developing proangiogenic therapy for patients who are not suitable candidates for conventional revascularization. Because of this, other areas of application are still lagging behind. A major characteristic of the field has been the disappointing fact that, whereas preclinical trials as well as all published proangiogenic gene and protein therapy trials without controls have yielded positive results, none of the randomized controlled trials have shown convincing and clinically relevant results. Firstly, this indicates that only randomized, placebo-controlled trials can properly judge the efficacy of angiogenic therapies.153 Secondly, there are a few key issues that need to be addressed in order to improve the design and outcome of future studies. The large number of publications and experiments done in laboratories around the world highlight the biological potency of overexpression of VEGFs and some other angiogenic growth factors such as FGFs. Thus, it is likely that technical and pharmacological shortcomings in the current treatment approaches have caused the failures.153 One obvious confounding factor is a strong placebo effect, and further complications are introduced by the use of quite subjective endpoints such as treadmill exercise tolerance test. More objective endpoints that address perfusion, collateral flow and, ultimately, survival should be used. Animal experiments have now shown that one likely explanation for the lack of efficacy and biological effects in clinical proangiogenic trials is that growth factor concentration in target tissues has not reached sufficient levels and/or has not persisted long enough to trigger relevant vascular growth. The short half-life of recombinant growth factors, low gene transfer with naked plasmid, low dose of Ad, and compromised delivery routes such as an intra-arterial injection, together with suboptimal time points in relation to the duration of gene expression, have likely contributed to the disappointing outcomes. For example, although both gene expression and perfusion peak at around 1 week and diminishes by 2 weeks after adenoviral gene transfer, the clinical studies have omitted this important time point and only focused on long-term time points of 2–6 months, which is likely post festum. Although gene expression kinetics of Ad and naked plasmid are not very well understood in humans, the preclinical data on gene ­expression kinetics should be taken into consideration when planning future clinical trials. Molecular Therapy vol. 15 no. 7 july 2007

Cardiovascular Gene Therapy

The optimal cardiovascular gene therapy application would fulfil most of the following requirements: the therapeutic protein is biologically very powerful, the product is secreted, gene transfer can be physically (i.e., by surgery or via a catheter) and possibly also genetically targeted, gene transfer vector is highly efficient, and gene expression time is long enough and can be regulated. There are several different factors reported to have therapeutic potential but often the findings have not been confirmed by observations in independent laboratories. It appears that in many cases the approaches that are being developed are too complicated. For instance, it is uncertain whether hypoxia-specific promoters have any benefit over unspecific promoters in therapeutic angiogenesis, because angiogenesis and especially arteriogenesis should also be induced in normoxic tissues. Targeting of vectors, which usually leads to diminished gene expression efficiency, may be unnecessary unless untargeted vectors cause adverse effects. Clinical trials with transcription factors and antisense oligonucleotides have so far been disappointing.34,35,147 Almost 100% gene transfer efficiency will be required in the absence of a bystander effect to make these approaches effective. Thus, despite the enthusiasm they have aroused, it is somewhat uncertain whether siRNA approaches will yield any better clinical results. The intra-arterial route is much less effective than IM injections, and the areas with a perfusion deficit, and therefore in the most urgent need for neovascularization, may not be effectively reached by the intra-arterial route. The efficacy of a gene transfer vector and the method of its administration are of crucial importance. Overall, the rules of conventional pharmaceutical development should also be applied for gene therapy trials.153 In experimental gene therapy studies this means that the levels of the therapeutic protein in the target tissue (not only in plasma) should be shown quantitatively. Moreover, the kinetics of the therapeutic protein expression and downstream biological effects should be demonstrated. Excluding some vaccination applications, naked plasmid DNA has failed in almost all randomized, controlled phase II/III clinical trials and the most likely reason isthat gene transfer efficacy is too low.21 Although it is very potent, Ad may not be an optimal vector for most clinical applications of cardiovascular gene therapy. Instead, it may serve as a tool to study the biology of therapeutic proteins while other vectors could be used for clinical gene therapy. However, e.g., an angiogenic boost with adenoviral VEGF gene transfer could still be useful as an adjuvant therapy in combination with bypass surgery or angioplasty rather than as a sole therapy.69 Similar boosts of therapeutic vascular growth could be beneficial in orthopedics and trauma surgery as well as in plastic surgery. For example, bone formation may be promoted by adenoviral VEGF gene transfer, and this suggests a novel treatment method for avascular and osteoporotic fractures.70 Furthermore, the surgical repair of soft tissue traumas, such as rupture of the degenerated rotator cuff or the Achilles tendon, might benefit from increased perfusion to the injured area. Diabetic wounds and surgical skin flaps are other possible areas of application of angiogenic gene transfer.71,88 It is likely that overinterpretations have been made in many preclinical studies, and this has prompted clinical testing of factors and approaches with minor biological relevance and effects. For example, a persistent belief that angiogenesis can be induced only in ischemic tissues has led to the routine use of small animal 1243

Cardiovascular Gene Therapy

models of severe ischemia, causing difficulties in the separation of endogenous and exogenous components of angiogenesis. According to the current knowledge it appears that potent angiogenic factors induce blood vessel growth both in non-ischemic and ischemic tissues.21,38,40,46,74,75 Thus, it is proposed that non-ischemic normal tissues should be preferred in the first proof-of-principle experiments in order to avoid misinterpretation caused by endogenous angiogenesis, necrosis, and inflammation in ischemic tissues. Only therafter should the potency of a given factor be evaluated in ischemia models. Large animal models should be used instead of mice and rats where e.g., the reliable measurement of systolic heart function using echocardiography is usually not technically feasible. The large differences in the transduction efficacy of Ads between species and tissues should be kept in mind. It should not be acceptable to just show “more black dots” in between myocytes in angiogenesis studies. Mechanistic data on how the number of capillaries was actually increased should also be provided. In conclusion, there are several viable indications for cardiovascular gene therapy, factors with biological potential have been identified, and efficient gene transfer vectors have been constructed. However, when compared against the achievements in other areas of gene therapy,200 there is no clear demonstration of a clinical benefit in the area of cardiovascular gene therapy. The first possible clinical applications are expected to be therapeutic vascular growth, prevention of vein graft stenosis and treatment of homozygous familial hypercholesterolemia. Acknowledgments

© The American Society of Gene Therapy

13.

14.

15.

16.

17.

18. 19.

20. 21.

22. 23. 24.

This study was supported by grants from the Academy of Finland, ­Finnish Foundation for Cardiovascular Research, Emil Aaltonen ­ Foundation, Maud Kuistila Foundation, Sigrid Juselius Foundation, EU Vascular ­Genomics Network, EVGN (LSHM-CT-2003-503254) and EU CliniGene network (LSH-CT-2004-018933).

26.

References

27.

1. Yla-Herttuala, S and Martin, JF (2000). Cardiovascular gene therapy. Lancet 355: 213–222. 2. Svet-Moldavsky, GJ and Chimishkyan, KL (1977). Tumour angiogenesis factor for revascularisation in ischaemia and myocardial infarction. Lancet 1: 913. 3. Yanagisawa-Miwa, A, Uchida, Y, Nakamura, F, Tomaru, T, Kido, H, Kamijo, T et al. (1992). Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor. Science 257: 1401–1403. 4. Henry, TD, Annex, BH, McKendall, GR, Azrin, MA, Lopez, JJ, Giordano, FJ et al. (2003). The VIVA trial: vascular endothelial growth factor in Ischemia for Vascular Angiogenesis. Circulation 107: 1359–1365. 5. Lederman, RJ, Mendelsohn, FO, Anderson, RD, Saucedo, JF, Tenaglia, AN, Hermiller, JB et al. (2002). Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (the TRAFFIC study): a randomised trial. Lancet 359: 2053–2058. 6. Simons, M, Annex, BH, Laham, RJ, Kleiman, N, Henry, T, Dauerman, H et al. (2002). Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double–blind, randomized, controlled clinical trial. Circulation 105: 788–793. 7. Lazarous, DF, Shou, M, Stiber, JA, Hodge, E, Thirumurti, V, Goncalves, L et al. (1999). Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis. Cardiovasc Res 44: 294–302. 8. Yla-Herttuala, S and Alitalo, K (2003). Gene transfer as a tool to induce therapeutic vascular growth. Nat Med 9: 694–701. 9. Tsurumi, Y, Takeshita, S, Chen, D, Kearney, M, Rossow, ST, Passeri, J et al. (1996). Direct intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor augments collateral development and tissue perfusion. Circulation 94: 3281–3290. 10. Isner, JM, Baumgartner, I, Rauh, G, Schainfeld, R, Blair, R, Manor, O et al. (1998). Treatment of thromboangitis obliterans (Buerger’s disease) by intramuscular gene transfer of vascular endothelial growth factor: preliminary clinical results. J Vasc Surg 28: 964–973. 11. Baumgartner, I, Pieczek, A, Manor, O, Blair, R, Kearney, M, Walsh, K et al. (1998). Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation 97: 1114–1123. 12. Vale, PR, Losordo, DW, Milliken, CE, McDonald, MC, Gravelin, LM, Curry, CM et al. (2001). Randomized, single-blind, placebo-controlled pilot study of

1244

25.

28. 29.

30. 31. 32. 33. 34.

35. 36. 37.

38.

catheter-based myocardial gene transfer for therapeutic angiogenesis using left ventricular electromechanical mapping in patients with chronic myocardial ischemia. Circulation 103: 2138–2143. Fortuin, FD, Vale, P, Losordo, DW, Symes, J, DeLaria, GA, Tyner, JJ et al. (2003). One-year follow-up of direct myocardial gene transfer of vascular endothelial growth factor-2 using naked plasmid deoxyribonucleic acid by way of thoracotomy in no-option patients. Am J Cardiol 92: 436–439. Makinen, K, Manninen, H, Hedman, M, Matsi, P, Mussalo, H, Alhava, E et al. (2002). Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study. Mol Ther 6: 127–133. Hedman, M, Hartikainen, J, Syvanne, M, Stjernvall, J, Hedman, A, Kivela, A et al. (2003). Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT). Circulation 107: 2677–2683. Kastrup, J, Jorgensen, E, Ruck, A, Tagil, K, Glogar, D, Ruzyllo, W et al. (2005). Direct intramyocardial plasmid vascular endothelial growth factor-A165 gene therapy in patients with stable severe angina pectoris A randomized double-blind placebo-controlled study: the Euroinject One trial. J Am Coll Cardiol 45: 982–988. Kusumanto, YH, Weel, VV, Mulder, NH, Smit, AJ, Dungen, JJ, Hooymans, JM et al. (2006). Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: a double-blind randomized trial. Hum Gene Ther 17: 683–691. Corautus Genetics (2006). Corautus announces termination of patient enrollment in GENASIS severe angina clinical trial. Corautus Genetics. . Laitinen, M, Pakkanen, T, Donetti, E, Baetta, R, Luoma, J, Lehtolainen, P et al. (1997). 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 8: 1645–1650. Wright, MJ, Wightman, LM, Lilley, C, de Alwis, M, Hart, SL, Miller, A et al. (2001). In vivo myocardial gene transfer: optimization, evaluation and direct comparison of gene transfer vectors. Basic Res Cardiol 96: 227–236. Rutanen, J, Rissanen, TT, Markkanen, JE, Gruchala, M, Silvennoinen, P, Kivela, A et al. (2004). Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart. Circulation 109: 1029–1035. Hao, X, Mansson-Broberg, A, Grinnemo, KH, Siddiqui, AJ, Dellgren, G, Brodin, LA et al. (2007). Myocardial angiogenesis after plasmid or adenoviral VEGF-A(165) gene transfer in rat myocardial infarction model. Cardiovasc Res 73: 481–487. Mathiesen, I (1999). Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Ther 6: 508–514. Yamashita, Y, Shimada, M, Tachibana, K, Harimoto, N, Tsujita, E, Shirabe, K et al. (2002). In vivo gene transfer into muscle via electro-sonoporation. Hum Gene Ther 13: 2079–2084. Wright, MJ, Rosenthal, E, Stewart, L, Wightman, LM, Miller, AD, Latchman, DS et al. (1998). β-Galactosidase staining following intracoronary infusion of cationic liposomes in the in vivo rabbit heart is produced by microinfarction rather than effective gene transfer: a cautionary tale. Gene Ther 5: 301–308. McMahon, JM, Wells, KE, Bamfo, JE, Cartwright, MA and Wells, DJ (1998). Inflammatory responses following direct injection of plasmid DNA into skeletal muscle. Gene Ther 5: 1283–1290. Kankkonen, HM, Vahakangas, E, Marr, RA, Pakkanen, T, Laurema, A, Leppanen, P et al. (2004). Long-term lowering of plasma cholesterol levels in LDL-receptor-deficient WHHL rabbits by gene therapy. Mol Ther 9: 548–556. Dishart, KL, Denby, L, George, SJ, Nicklin, SA, Yendluri, S, Tuerk, MJ et al. (2003). Third-generation lentivirus vectors efficiently transduce and phenotypically modify vascular cells: implications for gene therapy. J Mol Cell Cardiol 35: 739–748. Kankkonen, HM, Turunen, MP, Hiltunen, MO, Lehtolainen, P, Koponen, J, Leppanen, P et al. (2004). Feline immunodeficiency virus and retrovirus-mediated adventitial ex vivo gene transfer to rabbit carotid artery using autologous vascular smooth muscle cells. J Mol Cell Cardiol 36: 333–341. Cefai, D, Simeoni, E, Ludunge, KM, Driscoll, R, von Segesser, LK, Kappenberger, L et al. (2005). Multiply attenuated, self-inactivating lentiviral vectors efficiently transduce human coronary artery cells in vitro and rat arteries in vivo. J Mol Cell Cardiol 38: 333–344. Masaki, I, Yonemitsu, Y, Komori, K, Ueno, H, Nakashima, Y, Nakagawa, K et al. (2001). Recombinant Sendai virus-mediated gene transfer to vasculature: a new class of efficient gene transfer vector to the vascular system. FASEB J 15: 1294–1296. Coffin, RS, Howard, MK, Cumming, DV, Dollery, CM, McEwan, J, Yellon, DM et al. (1996). Gene delivery to the heart in vivo and to cardiac myocytes and vascular smooth muscle cells in vitro using herpes virus vectors. Gene Ther 3: 560–566. Airenne, KJ, Hiltunen, MO, Turunen, MP, Turunen, AM, Laitinen, OH, Kulomaa, MS et al. (2000). Baculovirus-mediated periadventitial gene transfer to rabbit carotid artery. Gene Ther 7: 1499–1504. Alexander, JH, Hafley, G, Harrington, RA, Peterson, ED, Ferguson, TB Jr., Lorenz, TJ et al. (2005). Efficacy and safety of edifoligide, an E2F transcription factor decoy, for prevention of vein graft failure following coronary artery bypass graft surgery: PREVENT IV: a randomized controlled trial. JAMA 294: 2446–2454. Conte, MS, Bandyk, DF, Clowes, AW, Moneta, GL, Seely, L, Lorenz, TJ et al. (2006). Results of PREVENT III: a multicenter, randomized trial of edifoligide for the prevention of vein graft failure in lower extremity bypass surgery. J Vasc Surg 43: 742–751. Fichou, Y and Ferec, C (2006). The potential of oligonucleotides for therapeutic applications. Trends Biotechnol 24: 563–570. Poliakova, L, Kovesdi, I, Wang, X, Capogrossi, MC and Talan, M (1999). Vascular permeability effect of adenovirus-mediated vascular endothelial growth factor gene transfer to the rabbit and rat skeletal muscle. J Thorac Cardiovasc Surg 118: 339–347. Rissanen, TT, Markkanen, JE, Gruchala, M, Heikura, T, Puranen, A, Kettunen, MI et al. (2003). VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses. Circ Res 92: 1098–1106.

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39. Zoltick, PW, Chirmule, N, Schnell, MA, Gao, GP, Hughes, JV and Wilson, JM (2001). Biology of E1-deleted adenovirus vectors in nonhuman primate muscle. J Virol 75: 5222–5229. 40. Rissanen, TT, Korpisalo, P, Markkanen, JE, Liimatainen, T, Orden, MR, Kholova, I et al. (2005). Blood flow remodels growing vasculature during vascular endothelial growth factor gene therapy and determines between capillary arterialization and sprouting angiogenesis. Circulation 112: 3937–3946. 41. Wen, S, Graf, S, Massey, PG and Dichek, DA (2004). Improved vascular gene transfer with a helper-dependent adenoviral vector. Circulation 110: 1484–1491. 42. Nalbantoglu, J, Pari, G, Karpati, G and Holland, PC (1999). Expression of the primary coxsackie and adenovirus receptor is downregulated during skeletal muscle maturation and limits the efficacy of adenovirus-mediated gene delivery to muscle cells. Hum Gene Ther 10: 1009–1019. 43. Vajanto, I, Rissanen, TT, Rutanen, J, Hiltunen, MO, Tuomisto, TT, Arve, K et al. (2002). Evaluation of angiogenesis and side effects in ischemic rabbit hindlimbs after intramuscular injection of adenoviral vectors encoding VEGF and LacZ. J Gene Med 4: 371–380. 44. Hiltunen, MO, Laitinen, M, Turunen, MP, Jeltsch, M, Hartikainen, J, Rissanen, TT et al. (2000). Intravascular adenovirus-mediated VEGF-C gene transfer reduces neointima formation in balloon-denuded rabbit aorta. Circulation 102: 2262–2268. 45. Laitinen, M, Mäkinen, K, Manninen, H, Matsi, P, Kossila, M, Agrawal, RS et al. (1998). Adenovirus-mediated gene transfer to lower limb artery of patients with chronic critical leg ischemia. Hum Gene Ther 9: 1481–1486. 46. Rissanen, TT, Markkanen, JE, Arve, K, Rutanen, J, Kettunen, MI, Vajanto, I et al. (2003). Fibroblast growth factor 4 induces vascular permeability, angiogenesis and arteriogenesis in a rabbit hindlimb ischemia model. FASEB J 17: 100–102. 47. Thirion, C, Lochmuller, H, Ruzsics, Z, Boelhauve, M, Konig, C, Thedieck, C et al. (2006). Adenovirus vectors based on human adenovirus type 19a have high potential for human muscle-directed gene therapy. Hum Gene Ther 17: 193–205. 48. Nicklin, SA, White, SJ, Nicol, CG, Von Seggern, DJ and Baker, AH (2004). In vitro and in vivo characterisation of endothelial cell selective adenoviral vectors. J Gene Med 6: 300–308. 49. Raper, SE, Chirmule, N, Lee, FS, Wivel, NA, Bagg, A, Gao, GP et al. (2003). Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab 80: 148–158. 50. Gruchala, M, Bhardwaj, S, Pajusola, K, Roy, H, Rissanen, TT, Kokina, I et al. (2004). Gene transfer into rabbit arteries with adeno-associated virus and adenovirus vectors. J Gene Med 6: 545–554. 51. Su, H, Huang, Y, Takagawa, J, Barcena, A, Arakawa-Hoyt, J, Ye, J et al. (2006). AAV serotype-1 mediates early onset of gene expression in mouse hearts and results in better therapeutic effect. Gene Ther 13: 1495–1502. 52. Schnepp, BC, Clark, KR, Klemanski, DL, Pacak, CA and Johnson, PR (2003). Genetic fate of recombinant adeno-associated virus vector genomes in muscle. J Virol 77: 3495–3504. 53. Wang, Z, Zhu, T, Qiao, C, Zhou, L, Wang, B, Zhang, J et al. (2005). Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart. Nat Biotechnol 23: 321–328. 54. Kawamoto, S, Shi, Q, Nitta, Y, Miyazaki, J and Allen, MD (2005). Widespread and early myocardial gene expression by adeno-associated virus vector type 6 with a beta-actin hybrid promoter. Mol Ther 11: 980–985. 55. Pacak, CA, Mah, CS, Thattaliyath, BD, Conlon, TJ, Lewis, MA, Cloutier, DE et al. (2006). Recombinant adeno-associated virus serotype 9 leads to preferential cardiac transduction in vivo. Circ Res 99: e3–e9. 56. Work, LM, Buning, H, Hunt, E, Nicklin, SA, Denby, L, Britton, N et al. (2006). Vascular bed-targeted in vivo gene delivery using tropism-modified adeno-associated viruses. Mol Ther 13: 683–693. 57. Jiang, H, Pierce, GF, Ozelo, MC, de Paula, EV, Vargas, JA, Smith, P et al. (2006). Evidence of multiyear factor IX expression by AAV-mediated gene transfer to skeletal muscle in an individual with severe hemophilia B. Mol Ther 14: 452–455. 58. Aikawa, R, Huggins, GS and Snyder, RO (2002). Cardiomyocyte-specific gene expression following recombinant adeno-associated viral vector transduction. J Biol Chem 277: 18979–18985. 59. Su, H, Joho, S, Huang, Y, Barcena, A, Arakawa-Hoyt, J, Grossman, W et al. (2004). Adeno-associated viral vector delivers cardiac-specific and hypoxia-inducible VEGF expression in ischemic mouse hearts. Proc Natl Acad Sci USA 101: 16280–16285. 60. Varenne, O, Pislaru, S, Gillijns, H, Van Pelt, N, Gerard, RD, Zoldhelyi, P et al. (1998). Local adenovirus-mediated transfer of human endothelial nitric oxide synthase reduces luminal narrowing after coronary angioplasty in pigs. Circulation 98: 919–926. 61. Walter, DH, Cejna, M, Diaz-Sandoval, L, Willis, S, Kirkwood, L, Stratford, PW et al. (2004). Local gene transfer of phVEGF-2 plasmid by gene-eluting stents: an alternative strategy for inhibition of restenosis. Circulation 110: 36–45. 62. Sharif, F, Hynes, SO, McMahon, J, Cooney, R, Conroy, S, Dockery, P et al. (2006). Gene-eluting stents: comparison of adenoviral and adeno- associated viral gene delivery to the blood vessel wall in vivo. Hum Gene Ther 17: 741–750. 63. Turunen, P, Puhakka, HL, Heikura, T, Romppanen, E, Inkala, M, Leppanen, O et al. (2006). Extracellular superoxide dismutase with vaccinia virus anti-inflammatory protein 35K or tissue inhibitor of metalloproteinase-1: combination gene therapy in the treatment of vein graft stenosis in rabbits. Hum Gene Ther 17: 405–414. 64. Wright, MJ, Wightman, LM, Latchman, DS and Marber, MS (2001). In vivo myocardial gene transfer: optimization and evaluation of intracoronary gene delivery in vivo. Gene Ther 8: 1833–1839. 65. Lee, LY, Patel, SR, Hackett, NR, Mack, CA, Polce, DR, El-Sawy, T et al. (2000). Focal angiogen therapy using intramyocardial delivery of an adenovirus vector coding for vascular endothelial growth factor 121. Ann Thorac Surg 69: 14–23. 66. Springer, ML, Chen, AS, Kraft, PE, Bednarski, M and Blau, HM (1998). VEGF gene delivery to muscle: potential role for vasculogenesis in adults. Mol Cell 2: 549–558. 67. Lee, RJ, Springer, ML, Blanco-Bose, WE, Shaw, R, Ursell, PC and Blau, HM (2000). VEGF gene delivery to myocardium: deleterious effects of unregulated expression. Circulation 102: 898–901.

Molecular Therapy vol. 15 no. 7 july 2007

Cardiovascular Gene Therapy

68. Pakkanen, TM, Laitinen, M, Hippelainen, M, Kallionpaa, H, Lehtolainen, P, Leppanen, P et al. (1999). Enhanced plasma cholesterol lowering effect of retrovirus-mediated LDL receptor gene transfer to WHHL rabbit liver after improved surgical technique and stimulation of hepatocyte proliferation by combined partial liver resection and thymidine kinase ganciclovir treatment. Gene Ther 6: 34–41. 69. Yla-Herttuala, S, Rissanen, TT, Vajanto, I and Hartikainen, J (2006). Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine. J Am Coll Cardiol 49: 1015–1026. 70. Hiltunen, MO, Ruuskanen, M, Huuskonen, J, Mahonen, AJ, Ahonen, M, Rutanen, J et al. (2003). Adenovirus-mediated VEGF-A gene transfer induces bone formation in vivo. FASEB J 17: 1147–1149. 71. Zacchigna, S, Papa, G, Antonini, A, Novati, F, Moimas, S, Carrer, A et al. (2005). Improved survival of ischemic cutaneous and musculocutaneous flaps after vascular endothelial growth factor gene transfer using adeno-associated virus vectors. Am J Pathol 167: 981–991. 72. Shen, F, Su, H, Fan, Y, Chen, Y, Zhu, Y, Liu, W et al. (2006). Adeno-associated viral-vector-mediated hypoxia-inducible vascular endothelial growth factor gene expression attenuates ischemic brain injury after focal cerebral ischemia in mice. Stroke 37: 2601–2606. 73. Mack, CA, Patel, SR, Schwarz, EA, Zanzonico, P, Hahn, RT, Ilercil, A et al. (1998). Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J Thorac Cardiovasc Surg 115: 168–176. 74. Gowdak, LH, Poliakova, L, Wang, X, Kovesdi, I, Fishbein, KW, Zacheo, A et al. (2000). Adenovirus-mediated VEGF(121) gene transfer stimulates angiogenesis in normoperfused skeletal muscle and preserves tissue perfusion after induction of ischemia. Circulation 102: 565–571. 75. Pettersson, A, Nagy, JA, Brown, LF, Sundberg, C, Morgan, E, Jungles, S et al. (2000). Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor. Lab Invest 80: 99–115. 76. Springer, ML, Ozawa, CR, Banfi, A, Kraft, PE, Ip TK, Brazelton, TR et al. (2003). Localized arteriole formation directly adjacent to the site of VEGF-induced angiogenesis in muscle. Mol Ther 7: 441–449. 77. Arsic, N, Zentilin, L, Zacchigna, S, Santoro, D, Stanta, G, Salvi, A et al. (2003). Induction of functional neovascularization by combined VEGF and angiopoietin-1 gene transfer using AAV vectors. Mol Ther 7: 450–459. 78. Parsons-Wingerter, P, Chandrasekharan, UM, McKay, TL, Radhakrishnan, K, DiCorleto, PE, Albarran, B et al. (2006). A VEGF165-induced phenotypic switch from increased vessel density to increased vessel diameter and increased endothelial NOS activity. Microvasc Res 72: 91–100. 79. Greve, JM, Chico, TJ, Goldman, H, Bunting, S, Peale, FV Jr., Daugherty, A et al. (2006). Magnetic resonance angiography reveals therapeutic enlargement of collateral vessels induced by VEGF in a murine model of peripheral arterial disease. J Magn Reson Imaging 24: 1124–1132. 80. Eitenmuller, I, Volger, O, Kluge, A, Troidl, K, Barancik, M, Cai, WJ et al. (2006). The range of adaptation by collateral vessels after femoral artery occlusion. Circ Res 99: 656–662. 81. Zentilin, L, Tafuro, S, Zacchigna, S, Arsic, N, Pattarini, L, Sinigaglia, M et al. (2006). Bone marrow mononuclear cells are recruited to the sites of VEGF-induced neovascularization but are not incorporated into the newly formed vessels. Blood 107: 3546–3554. 82. Vincent, KA, Shyu, KG, Luo, Y, Magner, M, Tio, RA, Jiang, C et al. (2000). Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1α/VP16 hybrid transcription factor. Circulation 102: 2255–2261. 83. Dai, Q, Huang, J, Klitzman, B, Dong, C, Goldschmidt-Clermont, PJ, March, KL et al. (2004). Engineered zinc finger-activating vascular endothelial growth factor transcription factor plasmd DiNA induces therapeutic angiogenesis in rabbits with hindlimb ischemia. Circulation 110: 2467–2475. 84. Whitlock, PR, Hackett, NR, Leopold, PL, Rosengart, TK and Crystal, RG (2004). Adenovirus-mediated transfer of a minigene expressing multiple isoforms of VEGF is more effective at inducing angiogenesis than comparable vectors expressing individual VEGF cDNAs. Mol Ther 9: 67–75. 85. Zheng, Y, Murakami, M, Takahashi, H, Yamauchi, M, Kiba, A, Yamaguchi, S et al. (2006). Chimeric VEGF-E(NZ7)/PlGF promotes angiogenesis via VEGFR-2 without significant enhancement of vascular permeability and inflammation. Arterioscler Thromb Vasc Biol 26: 2019–2026. 86. Dor, Y, Djonov, V, Abramovitch, R, Itin, A, Fishman, GI, Carmeliet, P et al. (2002). Conditional switching of VEGF provides new insights into adult neovascularization and pro-angiogenic therapy. EMBO J 21: 1939–1947. 87. Joukov, V, Pajusola, K, Kaipainen, A, Chilov, D, Lahtinen, I, Kukk, E et al. (1996). A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J 15: 290–298. 88. Saaristo, A, Tammela, T, Timonen, J, Yla-Herttuala, S, Tukiainen, E, Asko-Seljavaara, S et al. (2004). Vascular endothelial growth factor-C gene therapy restores lymphatic flow across incision wounds. FASEB J 18: 1707–1709. 89. Ogawa, S, Oku, A, Sawano, A, Yamaguchi, S, Yazaki, Y and Shibuya, M (1998). A novel type of vascular endothelial growth factor, VEGF-E (NZ-7 VEGF), preferentially utilizes KDR/Flk-1 receptor and carries a potent mitotic activity without heparin-binding domain. J Biol Chem 273: 31273–31282. 90. Odorisio, T, Schietroma, C, Zaccaria, ML, Cianfarani, F, Tiveron, C, Tatangelo, L et al. (2002). Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability. J Cell Sci 115: 2559–2567. 91. Silvestre, JS, Tamarat, R, Ebrahimian, TG, Le-Roux, A, Clergue, M, Emmanuel, F et al. (2003). Vascular endothelial growth factor-B promotes in vivo angiogenesis. Circ Res 93: 114–123. 92. Roy, H, Bhardwaj, S, Babu, M, Jauhiainen, S, Herzig, KH, Bellu, AR et al. (2005). Adenovirus-mediated gene transfer of placental growth factor to perivascular tissue induces angiogenesis via upregulation of the expression of endogenous vascular endothelial growth factor-A. Hum Gene Ther 16: 1422–1428.

1245

Cardiovascular Gene Therapy

93. Miller, DL, Ortega, S, Bashayan, O, Basch, R and Basilico, C (2000). Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice. Mol Cell Biol 20: 2260–2268. 94. Muhlhauser, J, Pili, R, Merrill, MJ, Maeda, H, Passaniti, A, Crystal, RG et al. (1995). In vivo angiogenesis induced by recombinant adenovirus vectors coding either for secreted or nonsecreted forms of acidic fibroblast growth factor. Hum Gene Ther 6: 1457–1465. 95. Ueno, H, Li, JJ, Masuda, S, Qi, Z, Yamamoto, H and Takeshita, A (1997). Adenovirus-mediated expression of the secreted form of basic fibroblast growth factor (FGF-2) induces cellular proliferation and angiogenesis in vivo. Arterioscler Thromb Vasc Biol 17: 2453–2460. 96. Giordano, FJ, Ping, P, McKirnan, MD, Nozaki, S, DeMaria, AN, Dillmann, WH et al. (1996). Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. Nat Med 2: 534–539. 97. Lindahl, P, Johansson, BR, Leveen, P and Betsholtz, C (1997). Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 277: 242–245. 98. Richardson, TP, Peters, MC, Ennett, AB and Mooney, DJ (2001). Polymeric system for dual growth factor delivery. Nat Biotechnol 19: 1029–1034. 99. Cao, R, Brakenhielm, E, Pawliuk, R, Wariaro, D, Post, MJ, Wahlberg, E et al. (2003). Angiogenic synergism, vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2. Nat Med 9: 604–613. 100. Li, X, Tjwa, M, Moons, L, Fons, P, Noel, A, Ny, A et al. (2005). Revascularization of ischemic tissues by PDGF-CC via effects on endothelial cells and their progenitors. J Clin Invest 115: 118–127. 101. Ponten, A, Li, X, Thoren, P, Aase, K, Sjoblom, T, Ostman, A et al. (2003). Transgenic overexpression of platelet-derived growth factor-C in the mouse heart induces cardiac fibrosis, hypertrophy, and dilated cardiomyopathy. Am J Pathol 163: 673–682. 102. Aoki, M, Morishita, R, Taniyama, Y, Kida, I, Moriguchi, A, Matsumoto, K et al. (2000). Angiogenesis induced by hepatocyte growth factor in non-infarcted myocardium and infarcted myocardium: up-regulation of essential transcription factor for angiogenesis, ets. Gene Ther 7: 417–427. 103. Thurston, G, Rudge, JS, Ioffe, E, Zhou, H, Ross, L, Croll, SD et al. (2000). Angiopoietin1 protects the adult vasculature against plasma leakage. Nat Med 6: 460–463. 104. Cho, CH, Kim, KE, Byun, J, Jang, HS, Kim, DK, Baluk, P et al. (2005). Long-term and sustained COMP-Ang1 induces long-lasting vascular enlargement and enhanced blood flow. Circ Res 97: 86–94. 105. Lobov, IB, Brooks, PC and Lang, RA (2002). Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo. Proc Natl Acad Sci USA 99: 11205–11210. 106. Patel, TH, Kimura, H, Weiss, CR, Semenza, GL and Hofmann, LV (2005). Constitutively active HIF-1alpha improves perfusion and arterial remodeling in an endovascular model of limb ischemia. Cardiovasc Res 68: 144–154. 107. Smith, RS Jr., Lin, KF, Agata, J, Chao, L and Chao, J (2002). Human endothelial nitric oxide synthase gene delivery promotes angiogenesis in a rat model of hindlimb ischemia. Arterioscler Thromb Vasc Biol 22: 1279–1285. 108. Ito, WD, Arras, M, Winkler, B, Scholz, D, Schaper, J and Schaper, W (1997). Monocyte chemotactic protein-1 increases collateral and peripheral conductance after femoral artery occlusion. Circ Res 80: 829–837. 109. Zhong, J, Eliceiri, B, Stupack, D, Penta, K, Sakamoto, G, Quertermous, T et al. (2003). Neovascularization of ischemic tissues by gene delivery of the extracellular matrix protein Del-1. J Clin Invest 112: 30–41. 110. Hiasa, K, Ishibashi, M, Ohtani, K, Inoue, S, Zhao, Q, Kitamoto, S et al. (2004). Gene transfer of stromal cell-derived factor-1α enhances ischemic vasculogenesis and angiogenesis via vascular endothelial growth factor/endothelial nitric oxide synthase-related pathway: next-generation chemokine therapy for therapeutic neovascularization. Circulation 109: 2454–2461. 111. Tokunaga, N, Nagaya, N, Shirai, M, Tanaka, E, Ishibashi-Ueda, H, Harada-Shiba, M et al. (2004). Adrenomedullin gene transfer induces therapeutic angiogenesis in a rabbit model of chronic hind limb ischemia: benefits of a novel nonviral vector, gelatin. Circulation 109: 526–531. 112. Nishiyama, K, Takaji, K, Kataoka, K, Kurihara, Y, Yoshimura, M, Kato, A et al. (2005). Id1 gene transfer confers angiogenic property on fully differentiated endothelial cells and contributes to therapeutic angiogenesis. Circulation 112: 2840–2850. 113. Kusano, KF, Pola, R, Murayama, T, Curry, C, Kawamoto, A, Iwakura, A et al. (2005). Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic signaling. Nat Med 11: 1197–1204. 114. Wilson, BD, Ii, M, Park, KW, Suli, A, Sorensen, LK, Larrieu-Lahargue, F et al. (2006). Netrins promote developmental and therapeutic angiogenesis. Science 313: 640–644. 115. van Royen, N, Schirmer, SH, Atasever, B, Behrens, CY, Ubbink, D, Buschmann, EE et al. (2005). START Trial: a pilot study on STimulation of ARTeriogenesis using subcutaneous application of granulocyte-macrophage colony-stimulating factor as a new treatment for peripheral vascular disease. Circulation 112: 1040–1046. 116. Shyu, KG, Chang, H, Wang, BW and Kuan, P (2003). Intramuscular vascular endothelial growth factor gene therapy in patients with chronic critical leg ischemia. Am J Med 114: 85–92. 117. Losordo, DW, Vale, PR, Symes, JF, Dunnington, CH, Esakof, DD, Maysky, M et al. (1998). Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF(165) as sole therapy for myocardial ischemia. Circulation 98: 2800– 2804. 118. Vale, PR, Losordo, DW, Milliken, CE, Maysky, M, Esakof, DD, Symes, JF et al. (2000). Left ventricular electromechanical mapping to assess efficacy of phVEGF(165) gene transfer for therapeutic angiogenesis in chronic myocardial ischemia. Circulation 102: 965–974. 119. Sylven, C, Sarkar, N, Ruck, A, Drvota, V, Hassan, SY, Lind, B et al. (2001). Myocardial Doppler tissue velocity improves following myocardial gene therapy with VEGF-A165 plasmid in patients with inoperable angina pectoris. Coron Artery Dis 12: 239–243. 120. Ripa, RS, Wang, Y, Jorgensen, E, Johnsen, HE, Hesse, B and Kastrup, J (2006). Intramyocardial injection of vascular endothelial growth factor-A165 plasmid followed by granulocyte-colony stimulating factor to induce angiogenesis in patients with severe chronic ischaemic heart disease. Eur Heart J 27: 1785–1792.

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121. Reilly, JP, Grise, MA, Fortuin, FD, Vale, PR, Schaer, GL, Lopez, J et al. (2005). Long-term (2-year) clinical events following transthoracic intramyocardial gene transfer of VEGF-2 in no-option patients. J Interv Cardiol 18: 27–31. 122. Rajagopalan, S, Shah, M, Luciano, A, Crystal, R and Nabel, EG (2001). Adenovirusmediated gene transfer of VEGF(121) improves lower-extremity endothelial function and flow reserve. Circulation 104: 753–755. 123. Rajagopalan, S, Olin, J, Deitcher, S, Pieczek, A, Laird, J, Grossman, PM et al. (2007). Use of a constitutively active hypoxia-inducible factor-1{α} transgene as a therapeutic strategy in no-option critical limb ischemia patients. Phase I dose-escalation experience. Circulation 115: 1234–1243. 124. Rajagopalan, S, Mohler, ER 3rd, Lederman, RJ, Mendelsohn, FO, Saucedo, JF, Goldman, CK et al. (2003). Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: a phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication. Circulation 108: 1933–1938. 125. Laitinen, M, Hartikainen, J, Hiltunen, MO, Eränen, J, Kiviniemi, M, Närvänen, O et al. (2000). Catheter-mediated vascular endothelial growth factor gene transfer to human coronary arteries after angioplasty. Hum Gene Ther 11: 263–270. 126. Grines, CL, Watkins, MW, Helmer, G, Penny, W, Brinker, J, Marmur, JD et al. (2002). Angiogenic Gene Therapy (AGENT) trial in patients with stable angina pectoris. Circulation 105: 1291–1297. 127. Grines, CL, Watkins, MW, Mahmarian, JJ, Iskandrian, AE, Rade, JJ, Marrott, P et al. (2003). A randomized, double-blind, placebo-controlled trial of Ad5FGF-4 gene therapy and its effect on myocardial perfusion in patients with stable angina. J Am Coll Cardiol 42: 1339–1347. 128. Barbeau, G, Beatt, K, Betriu, A, Janssens, S, Martinez-Rios, M, Mautner, B et al. (2006). Adenoviral fibroblast growth factor-4 gene therapy in patients with stable angina. 12-month results of a double blind randomized multicenter trial. J Am Coll Cardiol 47 (Suppl 1): 305A. 129. Cardium Therapeutics (2006). Summary of generx clinical development. Cardium Therapeutics. . 130. ClinicalTrials.gov (2007). Angiogenesis in women with angina pectoris who are not candidates for revascularization (AWARE). ClinicalTrials.gov. . 131. Losordo, DW, Vale, PR, Hendel, RC, Milliken, CE, Fortuin, FD, Cummings, N et al. (2002). Phase 1/2 placebo-controlled, double-blind, dose-escalating trial of myocardial vascular endothelial growth factor 2 gene transfer by catheter delivery in patients with chronic myocardial ischemia. Circulation 105: 2012–2018. 132. Stewart, DJ, Hilton, JD, Arnold, JM, Gregoire, J, Rivard, A, Archer, SL et al. (2006). Angiogenic gene therapy in patients with nonrevascularizable ischemic heart disease: a phase 2 randomized, controlled trial of AdVEGF(121) (AdVEGF121) versus maximum medical treatment. Gene Ther 13: 1503–1511. 133. ClinicalTrials.gov (2006). Safety and efficacy study of Ad2/hypoxia inducible factor (HIF)-1a/VP16 gene transfer in patients with intermittent claudication. ClinicalTrials.gov. . 134. Powell, RJ, Dormandy, J, Simons, M, Morishita, R and Annex, BH (2004). Therapeutic angiogenesis for critical limb ischemia: design of the hepatocyte growth factor therapeutic angiogenesis clinical trial. Vasc Med 9: 193–198. 135. Valentis (2006). Valentis announces results of VLTS 934 phase IIb trial. Valentis. . 136. Nikol, S, Baumgartner, I, Van Belle, E, Diehm, C, Visona, A, Capogrossi, MC et al. (2006). Therapeutic angiogenesis with intramuscular NV1FGF improves amputation-free survival in patients with critical limb ischemia. Late breaking clinical trial. American College of Cardiology 55th Annual Scientific Session. 137. Henry, TD, Mendelsohn, F, Comerota, A, Pham, E, Grek, V, Coleman, M et al. (2006). Dose and regimen effects of intramuscular NV1FGF in patients with critical limb ischemia: a randomized, double-blind, placebo controlled study. Eur Heart J 27 (Suppl 1): 235. 138. ClinicalTrials.gov (2006). NOGA angiogenesis revascularization therapy: evaluation by radionuclide imaging—the northern trial. ClinicalTrials.gov. . 139. ClinicalTrials.gov (2006). A study to treat patients whose chronic angina symptoms are not relieved by medication and have an area of the heart that cannot be treated by standard therapies. ClinicalTrials.gov. . 140. Leppanen, O, Rutanen, J, Hiltunen, MO, Rissanen, TT, Turunen, MP, Sjoblom, T et al. (2004). Oral imatinib mesylate (STI571/gleevec) improves the efficacy of local intravascular vascular endothelial growth factor-C gene transfer in reducing neointimal growth in hypercholesterolemic rabbits. Circulation 109: 1140–1146. 141. Rutanen, J, Turunen, AM, Teittinen, M, Rissanen, TT, Heikura, T, Koponen, JK et al. (2005). Gene transfer using the mature form of VEGF-D reduces neointimal thickening through nitric oxide-dependent mechanism. Gene Ther 12: 980–987. 142. Khurana, R, Zhuang, Z, Bhardwaj, S, Murakami, M, De Muinck, E, Yla-Herttuala, S et al. (2004). Angiogenesis-dependent and independent phases of intimal hyperplasia. Circulation 110: 2436–2443. 143. Bhardwaj, S, Roy, H, Heikura, T and Yla-Herttuala, S (2005). VEGF-A, VEGF-D and VEGF-D(DeltaNDeltaC) induced intimal hyperplasia in carotid arteries. Eur J Clin Invest 35: 669–676. 144. Janssens, S, Flaherty, D, Nong, Z, Varenne, O, Van Pelt, N, Haustermans,C et al. (1998). Human endothelial nitric oxide synthase gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation after balloon injury in rats. Circulation 97: 1274–1281. 145. Numaguchi, Y, Naruse, K, Harada, M, Osanai, H, Mokuno, S, Murase, K et al. (1999). Prostacyclin synthase gene transfer accelerates reendothelialization and inhibits neointimal formation in rat carotid arteries after balloon injury. Arterioscler Thromb Vasc Biol 19: 727–733. 146. Laukkanen, MO, Kivela, A, Rissanen, T, Rutanen, J, Karkkainen, MK, Leppanen, O et al. (2002). Adenovirus-mediated extracellular superoxide dismutase gene therapy reduces neointima formation in balloon-denuded rabbit aorta. Circulation 106: 1999–2003. 147. Kutryk, MJ, Foley, DP, van den Brand, M, Hamburger, JN, van der Giessen, WJ, deFeyter, PJ et al. (2002). Local intracoronary administration of antisense

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oligonucleotide against c-myc for the prevention of in-stent restenosis: results of the randomized investigation by the thoraxcenter of antisense DNA using local delivery and IVUS after coronary stenting (ITALICS) trial. J Am Coll Cardiol 39: 281–287. 148. Chiu-Pinheiro, CK, O’Brien, T, Katusic, ZS, Bonilla, LF, Hamner, CE and Schaff, HV (2002). Gene transfer to coronary artery bypass conduits. Ann Thorac Surg 74: 1161–1166. 149. Fuster, V, Charlton, P and Boyd, A (2001). Clinical protocol. A phase IIb, randomized, multicenter, double-blind study of the efficacy and safety of Trinam (EG004) in stenosis prevention at the graft-vein anastomosis site in dialysis patients. Hum Gene Ther 12: 2025–2027. 150. Luo, Z, Akita, GY, Date, T, Treleaven, C, Vincent, KA, Woodcock, D et al. (2005). Adenovirus-mediated expression of beta-adrenergic receptor kinase C-terminus reduces intimal hyperplasia and luminal stenosis of arteriovenous polytetrafluoroethylene grafts in pigs. Circulation 111: 1679–1684. 151. Kibbe, MR, Tzeng, E, Gleixner, SL, Watkins, SC, Kovesdi, I, Lizonova, A et al. (2001). Adenovirus-mediated gene transfer of human inducible nitric oxide synthase in porcine vein grafts inhibits intimal hyperplasia. J Vasc Surg 34: 156–165. 152. Puhakka, HL, Turunen, P, Rutanen, J, Hiltunen, MO, Turunen, MP and Yla-Herttuala, S (2005). Tissue inhibitor of metalloproteinase 1 adenoviral gene therapy alone is equally effective in reducing restenosis as combination gene therapy in a rabbit restenosis model. J Vasc Res 42: 361–367. 153. Yla-Herttuala, S, Markkanen, JE and Rissanen, TT (2004). Gene therapy for ischemic cardiovascular diseases: some lessons learned from the first clinical trials. Trends Cardiovasc Med 14: 295–300. 154. Ratner, M (2004). Genentech discloses safety concerns over Avastin. Nat Biotechnol 22: 1198. 155. Lemstrom, KB, Krebs, R, Nykanen, AI, Tikkanen, JM, Sihvola, RK, Aaltola, EM et al. (2002). Vascular endothelial growth factor enhances cardiac allograft arteriosclerosis. Circulation 105: 2524–2530. 156. Ryu, JK, Cho, CH, Shin, HY, Song, SU, Oh, SM, Lee, M et al. (2006). Combined angiopoietin-1 and vascular endothelial growth factor gene transfer restores cavernous angiogenesis and erectile function in a rat model of hypercholesterolemia. Mol Ther 13: 705–715. 157. Nykanen, AI, Krebs, R, Saaristo, A, Turunen, P, Alitalo, K, Yla-Herttuala, S et al. (2003). Angiopoietin-1 protects against the development of cardiac allograft arteriosclerosis. Circulation 107: 1308–1314. 158. Nykanen, AI, Pajusola, K, Krebs, R, Keranen, MA, Raisky, O, Koskinen, PK et al. (2006). Common protective and diverse smooth muscle cell effects of AAV-mediated angiopoietin-1 and -2 expression in rat cardiac allograft vasculopathy. Circ Res 98: 1373–1380. 159. Chen, SJ, Wilson, JM and Muller, DW (1994). Adenovirus-mediated gene transfer of soluble vascular cell adhesion molecule to porcine interposition vein grafts. Circulation 89: 1922–1928. 160. Ali, ZA, Bursill, CA, Hu, Y, Choudhury, RP, Xu, Q, Greaves, DR et al. (2005). Gene transfer of a broad spectrum CC-chemokine inhibitor reduces vein graft atherosclerosis in apolipoprotein E-knockout mice. Circulation 112 (Suppl 9): I235–I241. 161. Schepers, A, Eefting, D, Bonta, PI, Grimbergen, JM, de Vries, MR, van Weel, V et al. (2006). Anti-MCP-1 gene therapy inhibits vascular smooth muscle cells proliferation and attenuates vein graft thickening both in vitro and in vivo. Arterioscler Thromb Vasc Biol 26: 2063–2069. 162. Eslami, MH, Gangadharan, SP, Sui, X, Rhynhart, KK, Snyder, RO and Conte, MS (2000). Gene delivery to in situ veins: differential effects of adenovirus and adeno-associated viral vectors. J Vasc Surg 31: 1149–1159. 163. Mann, MJ, Whittemore, AD, Donaldson, MC, Belkin, M, Conte, MS, Polak, JF et al. (1999). Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomised, controlled trial. Lancet 354: 1493–1498. 164. Banno, H, Takei, Y, Muramatsu, T, Komori, K and Kadomatsu, K (2006). Controlled release of small interfering RNA targeting midkine attenuates intimal hyperplasia in vein grafts. J Vasc Surg 44: 633–641. 165. Grossman, M, Rader, DJ, Muller, DW, Kolansky, DM, Kozarsky, K, Clark, BJ 3rd et al. (1995). A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolaemia. Nat Med 1: 1148–1154. 166. Lebherz, C, Gao, G, Louboutin, JP, Millar, J, Rader, D and Wilson, JM (2004). Gene therapy with novel adeno-associated virus vectors substantially diminishes atherosclerosis in a murine model of familial hypercholesterolemia. J Gene Med 6: 663–672. 167. Jalkanen, J, Leppanen, P, Pajusola, K, Narvanen, O, Mahonen, A, Vahakangas, E et al. (2003). Adeno-associated virus-mediated gene transfer of a secreted decoy human macrophage scavenger receptor reduces atherosclerotic lesion formation in LDL receptor knockout mice. Mol Ther 8: 903–910. 168. Kitajima, K, Marchadier, DH, Miller, GC, Gao, GP, Wilson, JM and Rader, DJ (2006). Complete prevention of atherosclerosis in apoE-deficient mice by hepatic human apoE gene transfer with adeno-associated virus serotypes 7 and 8. Arterioscler Thromb Vasc Biol 26: 1852–1857. 169. Cichon, G, Willnow, T, Herwig, S, Uckert, W, Loser, P, Schmidt, HH et al. (2004). Non-physiological overexpression of the low density lipoprotein receptor (LDLr) gene in the liver induces pathological intracellular lipid and cholesterol storage. J Gene Med 6: 166–175. 170. Kastelein, JJ, Wedel, MK, Baker, BF, Su, J, Bradley, JD, Yu, RZ et al. (2006). Potent reduction of apolipoprotein B and low-density lipoprotein cholesterol by short-term administration of an antisense inhibitor of apolipoprotein B. Circulation 114: 1729–1735. 171. Zoldhelyi, P, McNatt, J, Xu, XM, Loose-Mitchell, D, Meidell, RS, Clubb, FJ Jr. et al. (1996). Prevention of arterial thrombosis by adenovirus-mediated transfer of cyclooxygenase gene. Circulation 93: 10–17. 172. Waugh, JM, Kattash, M, Li, J, Yuksel, E, Kuo, MD, Lussier, M et al. (1999). Gene therapy to promote thromboresistance: local overexpression of tissue plasminogen activator to prevent arterial thrombosis in an in vivo rabbit model. Proc Natl Acad Sci USA 96: 1065–1070.

Molecular Therapy vol. 15 no. 7 july 2007

Cardiovascular Gene Therapy

173. Waugh, JM, Yuksel, E, Li, J, Kuo, MD, Kattash, M, Saxena, R et al. (1999). Local overexpression of thrombomodulin for in vivo prevention of arterial thrombosis in a rabbit model. Circ Res 84: 84–92. 174. Zoldhelyi, P, McNatt, J, Shelat, HS, Yamamoto, Y, Chen, ZQ and Willerson, JT (2000). Thromboresistance of balloon-injured porcine carotid arteries after local gene transfer of human tissue factor pathway inhibitor. Circulation 101: 289–295. 175. Ohno, N, Itoh, H, Ikeda, T, Ueyama, K, Yamahara, K, Doi, K et al. (2002). 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 105: 1623–1626. 176. Furukoji, E, Matsumoto, M, Yamashita, A, Yagi, H, Sakurai, Y, Marutsuka, K et al. (2005). Adenovirus-mediated transfer of human placental ectonucleoside triphosphate diphosphohydrolase to vascular smooth muscle cells suppresses platelet aggregation in vitro and arterial thrombus formation in vivo. Circulation 111: 808–815. 177. Raizada, MK and Der Sarkissian, S (2006). Potential of gene therapy strategy for the treatment of hypertension. Hypertension 47: 6–9. 178. Miller, WH, Brosnan, MJ, Graham, D, Nicol, CG, Morecroft, I, Channon, KM et al. (2005). Targeting endothelial cells with adenovirus expressing nitric oxide synthase prevents elevation of blood pressure in stroke-prone spontaneously hypertensive rats. Mol Ther 12: 321–327. 179. Wang, HW, Katovich, MJ, Gelband, CH, Reaves, PY, Phillips, MI and Raizada, MK (1999). Sustained inhibition of angiotensin I-converting enzyme (ACE) expression and long-term antihypertensive action by virally mediated delivery of ACE antisense cDNA. Circ Res 85: 614–622. 180. Wang, T, Li, H, Zhao, C, Chen, C, Li, J, Chao, J et al. (2004). Recombinant adeno-associated virus-mediated kallikrein gene therapy reduces hypertension and attenuates its cardiovascular injuries. Gene Ther 11: 1342–1350. 181. Schillinger, KJ, Tsai, SY, Taffet, GE, Reddy, AK, Marian, AJ, Entman, ML et al. (2005). Regulatable atrial natriuretic peptide gene therapy for hypertension. Proc Natl Acad Sci USA 102: 13789–13794. 182. Nagaya, N, Yokoyama, C, Kyotani, S, Shimonishi, M, Morishita, R, Uematsu, M et al. (2000). Gene transfer of human prostacyclin synthase ameliorates monocrotaline-induced pulmonary hypertension in rats. Circulation 102: 2005–2010. 183. McMurtry, MS, Archer, SL, Altieri, DC, Bonnet, S, Haromy, A, Harry, G et al. (2005). Gene therapy targeting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary arterial hypertension. J Clin Invest 115: 1479–1491. 184. Leotta, E, Patejunas, G, Murphy, G, Szokol, J, McGregor, L, Carbray, J et al. (2002). Gene therapy with adenovirus-mediated myocardial transfer of vascular endothelial growth factor 121 improves cardiac performance in a pacing model of congestive heart failure. J Thorac Cardiovasc Surg 123: 1101–1113. 185. Miyamoto, MI, del Monte, F, Schmidt, U, DiSalvo, TS, Kang, ZB, Matsui, T et al. (2000). Adenoviral gene transfer of SERCA2a improves left-ventricular function in aortic-banded rats in transition to heart failure. Proc Natl Acad Sci USA 97: 793–798. 186. Szatkowski, ML, Westfall, MV, Gomez, CA, Wahr, PA, Michele, DE, DelloRusso, C et al. (2001). In vivo acceleration of heart relaxation performance by parvalbumin gene delivery. J Clin Invest 107: 191–198. 187. Most, P, Pleger, ST, Volkers, M, Heidt, B, Boerries, M, Weichenhan, D et al. (2004). Cardiac adenoviral S100A1 gene delivery rescues failing myocardium. J Clin Invest 114: 1550–1563. 188. Hoshijima, M, Ikeda, Y, Iwanaga, Y, Minamisawa, S, Date, MO, Gu, Y et al. (2002). Chronic suppression of heart-failure progression by a pseudophosphorylated mutant of phospholamban via in vivo cardiac rAAV gene delivery. Nat Med 8: 864–871. 189. Maurice, JP, Hata, JA, Shah, AS, White, DC, McDonald, PH, Dolber, PC et al. (1999). Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary beta2-adrenergic receptor gene delivery. J Clin Invest 104: 21–29. 190. Hambleton, M, Hahn, H, Pleger, ST, Kuhn, MC, Klevitsky, R, Carr, AN et al. (2006). Pharmacological- and gene therapy-based inhibition of protein kinase Calpha/beta enhances cardiac contractility and attenuates heart failure. Circulation 114: 574–582. 191. Tenhunen, O, Soini, Y, Ilves, M, Rysa, J, Tuukkanen, J, Serpi, R et al. (2006). p38 Kinase rescues failing myocardium after myocardial infarction: evidence for angiogenic and anti-apoptotic mechanisms. FASEB J 20: 1907–1909. 192. Chatterjee, S, Stewart, AS, Bish, LT, Jayasankar, V, Kim, EM, Pirolli, T et al. (2002). Viral gene transfer of the antiapoptotic factor Bcl-2 protects against chronic postischemic heart failure. Circulation 106 (Suppl 1): I212–I217. 193. Woo, YJ, Panlilio, CM, Cheng, RK, Liao, GP, Atluri, P, Hsu, VM et al. (2006). Therapeutic delivery of cyclin A2 induces myocardial regeneration and enhances cardiac function in ischemic heart failure. Circulation 114 (Suppl 1): I206–I213. 194. Laugwitz, KL, Moretti, A, Weig, HJ, Gillitzer, A, Pinkernell, K, Ott, T et al. (2001). Blocking caspase-activated apoptosis improves contractility in failing myocardium. Hum Gene Ther 12: 2051–2063. 195. Lai, NC, Roth, DM, Gao, MH, Tang, T, Dalton, N, Lai, YY et al. (2004). Intracoronary adenovirus encoding adenylyl cyclase VI increases left ventricular function in heart failure. Circulation 110: 330–336. 196. Melo, LG, Agrawal, R, Zhang, L, Rezvani, M, Mangi, AA, Ehsan, A et al. (2002). Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene. Circulation 105: 602–607. 197. Rosen, MR, Brink, PR, Cohen, IS and Robinson, RB (2004). Genes, stem cells and biological pacemakers. Cardiovasc Res 64: 12–23. 198. Kashiwakura, Y, Cho, HC, Barth, AS, Azene, E and Marban, E (2006). Gene transfer of a synthetic pacemaker channel into the heart: a novel strategy for biological pacing. Circulation 114: 1682–1686. 199. Tse, HF, Xue, T, Lau, CP, Siu, CW, Wang, K, Zhang, QY et al. (2006). Bioartificial sinus node constructed via in vivo gene transfer of an engineered pacemaker HCN Channel reduces the dependence on electronic pacemaker in a sick-sinus syndrome model. Circulation 114: 1000–1011. 200. Immonen, A, Vapalahti, M, Tyynela, K, Hurskainen, H, Sandmair, A, Vanninen, R et al. (2004). AdvHSV-tk gene therapy with intravenous ganciclovir improves survival in human malignant glioma: a randomised, controlled study. Mol Ther 10: 967–972.

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