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Nature Medicine  9, 694 - 701 (2003)
doi:10.1038/nm0603-694

Gene transfer as a tool to induce therapeutic vascular growth

Seppo Ylä-Herttuala1 & Kari Alitalo2

1 A.I. Virtanen Institute and Department of Medicine, University of Kuopio and Gene Therapy Unit, Kuopio University Hospital, P.O. Box 1627, FIN-70211 Kuopio, Finland. Seppo.Ylaherttuala@uku.fi

2 Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, The Haartman Institute and Helsinki University Central Hospital, Biomedicum Helsinki, P.O. Box 63, FIN-00014 Helsinki, Finland. Kari.Alitalo@Helsinki.fi

Therapeutic induction of vascular growth may provide a treatment option for those patients with myocardial or peripheral ischemia who are not suited to conventional revascularization therapies. Some lymphatic vascular disorders may also be amenable to this therapy. However, clear evidence of efficacy must be obtained from phase 2 and 3 clinical trials before these new treatments can be entered into clinical practice. Apart from the clinical applications, gene transfer aimed at stimulating or blocking vascular growth with various growth factors, cytokines, transcription factors and receptors or their antagonists is useful for analyzing the effects of those molecules on the vasculature, especially when gene targeting results in lethality or when large animal models are required.
Gene therapy can be defined as the transfer of nucleic acids to somatic cells of an individual with a resulting therapeutic effect1. One of the most promising areas of gene therapy is the stimulation of vascular growth for treatment of myocardial ischemia and peripheral vascular disease1. In these applications, current gene delivery technologies seem to result in therapeutic benefits at relatively low doses of the gene transfer vectors. Advantages of gene therapy over existing therapeutic modalities include (i) selective local treatment of affected tissues (ii) the possibility of using endogenous proteins locally in cases where systemic delivery would lead to severe side effects and (iii) the possibility of long-term therapeutic effects after a single application. Treatment of genetic diseases has been very difficult, and although gene therapy could lead to correction of the genetic cause of a disease, formidable problems still exist when considering the development of safe long-term treatments for monogenic disorders2.

Gene transfer vectors
Only a small amount of naked plasmid DNA will be taken into a cell, leading to a very low gene transfer efficiency3. An exception may be muscle tissue, where naked plasmids have been reported to lead to relatively high gene expression4. Carrier molecules have therefore been used to increase plasmid-based gene transfer efficiency. Liposomes or polymer complexes improve plasmid delivery to the cytoplasm, although only a small fraction enters the nucleus, where the plasmids remain extrachromosomal1. Plasmids are expressed transiently in the target cells, usually for 1−2 weeks. Antisense and decoy oligonucleotides and inhibitory RNA (RNAi) can also be used for gene transfer. An advantage of plasmids, oligonucleotides and carrier molecules is that they are easier to manufacture in large quantities than viral vectors. The use of plasmid vectors for clinical trials has been approved in several countries.

Because gene transfer efficiency with plasmid-based systems is usually relatively low, viral vectors have been used to increase the efficiency. The viral life cycle involves specific mechanisms that deliver viral genes into host cells1, 2. For therapeutic angiogenesis, the most commonly used viral vector is adenovirus, which can transduce both dividing and non-dividing cells. Adenoviruses enter cells through the Coxsackie-adenovirus receptor (CAR)5 and through alphavbeta3 and alphavbeta5 integrins6. The adenoviruses can escape from endosomes, which helps to release the transgenes into the cytoplasm where they are transported into the nucleus. In target cells, adenoviruses remain extrachromosomal and provide transient gene expression for 1−2 weeks in immunocompetent hosts7.

In human trials, adenoviral vectors have caused inflammatory reactions, formation of antibodies to adenovirus, transient fever and increases in liver transaminases, but they have not been linked to any human malignancies. However, the first gene therapy−associated death occurred when a high dose of adenovirus vector was given intraportally into a patient who suffered from a genetic defect causing ornithine transcarbamylase deficiency. In retrospect, the death seems to have been a result of toxicity caused by the adenoviral vector in conjunction with the underlying disease8. In spite of those adverse effects, the safety profile of adenoviruses is already well established and their use in clinical trials has been approved by US Food and Drug Administration and other regulatory authorities.

Other vectors used for angiogenic gene transfer include retroviruses, lentiviruses and adeno-associated viruses (AAVs), which all integrate into the host genome and cause long-lasting transgene expression1, 2. In general, retroviral gene transfer efficiency in vivo is very low3 and it has mostly been used in applications where cells are transfected ex vivo and then returned to the host. Unlike the retroviruses, lentiviruses and AAVs can also transduce non-dividing cells. Lentiviruses have shown relatively high transduction efficiencies in the central nervous system and liver9, 10 whereas AAVs can also efficiently transduce skeletal muscle, myocardium and blood vessels11, 12. Other vectors used for gene transfer include herpes simplex virus13, Sendai virus14 and baculovirus15. The usefulness of integrating vectors for therapeutic vascular growth remains unclear, as long-lasting expression of potent growth factors may cause deleterious side effects such as hemangiomas16. It is evident that regulated vector systems are needed if integrating vectors are to be used to induce therapeutic vascular growth17, 18, 19. Potential applications for integrating vectors include the use of small interfering RNA technology for targeted inactivation of cellular functions20.

Severe safety problems have occurred in one retroviral gene therapy trial for severe combined immunodeficiency syndrome21. In this trial, CD34+ bone marrow cells from young children were transduced ex vivo with a retrovirus encoding the common gammac chain of the interleukin receptor. The clinical condition of the patients improved substantially, but two children developed leukemia, apparently as a result of retroviral integration into chromosome 11 in a region encoding the LMO2 gene, which is known to be associated with T-cell leukemias21. These findings underline the safety issues in the use of integrating vectors. In this regard, it is noteworthy that therapeutic vascular growth does not require stable transduction; instead, transient gene expression by extrachromosomal adenoviral or plasmid vectors should be sufficient. Also, angiogenic gene therapy usually uses secreted growth factors, which can be effective even if the transfection efficiency of the target tissue is low. This situation is clearly different from gene therapy applications used for the treatment of genetic diseases such as muscular dystrophy or cystic fibrosis, where long-term gene expression is required.

Gene delivery for vascular disease
Therapeutic potency is affected by the efficiency of gene delivery into the target tissue, the entry of the new genetic material into cells and nuclei, and expression of the transgene in the target cells. When specific physical or biological targeting methods are available, they usually improve transgene expression (Fig. 1). An example of physical targeting is catheter-mediated gene transfer to the blood vessels of the heart or peripheral circulation, where effective gene delivery has been documented in humans22. Intramuscular injection can lead to sustained transgene expression and seems to provide a simple and efficient way to induce therapeutic vascular growth1. Another approach for local delivery to small arterioles and capillaries is injection of biodegradable microspheres or nanoparticles coated with recombinant growth factors or expression vectors23. Ultrasonography techniques may further improve the efficiency of gene transfer24. Growth factors or expression vectors can also be impregnated into vascular grafts or other surgical materials. Several studies suggest that the endothelium in various organs expresses specific molecular markers that could be used for targeting of vectors to certain vascular beds25.

Figure 1. Clinically feasible gene delivery routes to induce therapeutic vascular growth.
Figure 1 thumbnail

(a) Gene delivery through major arteries. (b) Myocardium can be approached from ventricles, through coronary arteries, through epicardial surface or during bypass surgery. (c) Peripheral vascular disease and some lymphatic disorders can be treated through intravascular or intramuscular routes or during surgery.



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Therapeutic vascular growth
Therapeutic vascular growth includes stimulation of angiogenesis, arteriogenesis and lymphangiogenesis1, 26. Angiogenesis is the sprouting of new blood vessels from pre-existing ones, whereas arteriogenesis is the in situ enlargement of muscular collateral vessels from pre-existing arteriolar anastomoses. Lymphangiogenesis is the generation of new lymphatic vessels from pre-existing ones27, 28, 29. Various strategies have also been presented for the use of vasculogenesis, that is, the de novo differentiation and formation of vascular structures from endothelial progenitor cells (EPCs) or stem cells.

Factors used to stimulate therapeutic vascular growth
Vascular endothelial growth factors (VEGF). VEGF30, 31 has been widely used for vascular therapeutic purposes (Table 1). It has several splice variants, of which two (VEGF165 and VEGF121) have been consistently angiogenic in several animal models. VEGF121 does not bind to heparan sulphate and is readily diffusible, whereas VEGF165 binds to matrix components and remains confined to the pericellular matrix after its secretion from the cells that produce it. Both VEGF165 and VEGF121 have been used in clinical trials1. VEGF-B32, VEGF-C33, VEGF-D34, VEGF-E35 and placental growth factor (PLGF)36 have also shown angiogenic activity in preclinical animal models. PLGF binds to VEGFR-1 (ref. 37) and has been implicated in angiogenesis under pathological conditions38, 39. The binding profile of VEGF-B is similar to that of PLGF37, and its therapeutic potential is currently under intensive study. The VEGF-E produced by the Orf virus binds to VEGFR-2 (ref. 35), but interest in its clinical use has been attenuated by its viral origin.

Table 1. Factors with potential for therapeutic vascular growth
Table 1 thumbnail

Full TableFull Table
An important property of VEGF, VEGF-B and PLGF is their chemotactic effect on circulating monocyte-macrophages through activation of VEGFR-1 expressed on these cells. Macrophages, in turn, are a rich source of growth factors, cytokines and proteases that can induce vascular growth40. VEGFR-1-positive monocytes migrating into tissues are an integral part of many types of pathological angiogenesis that involve an inflammatory component. PLGF also promotes angiogenesis and arteriogenesis and mobilizes hematopoietic stem cells and EPCs from the bone marrow38, 39. The clinical significance of VEGF-B, PLGF and EPCs in the treatment of ischemia remains unknown.

In general, VEGF-induced capillaries tend to regress soon after the cessation of transgene expression41, 42. It is not yet known what factors are needed to stabilize the newly formed vessels, but clinical experience regarding collateral growth indicates that blood flow promotes vessel persistence. It has become increasingly clear that arterialization of the new conduits and recruitment of pericytes are important for the generation of functional, persistent blood vessels41, 42, 43. Optimization of the dose and duration of the expression of VEGF and other growth factors is of key importance for clinically successful vascular growth factor therapy.

VEGF-C and VEGF-D bind to VEGFR-2 and VEGFR-3 and show both lymphangiogenic and angiogenic activity33, 34. These factors are synthesized as long precursor molecules that bind preferentially to VEGFR-3. Their mature processed forms, generated by proteolytic cleavage, can also activate VEGFR-2, which is probably responsible for their angiogenic activity33, 34. VEGF-C gene delivery induces significant lymphangiogenesis27, 44, 45 and has also been used in clinical trials for angiogenesis46. The VEGF-D long form is mostly lymphangiogenic in skeletal muscle, whereas its proteolytically activated short form also causes significant angiogenesis41. VEGF-D has been recently approved for clinical trials (S.Y.-H. et al., unpublished data).

Angiopoietins. Angiopoietin-1 (Ang-1)47 and angiopoietin-2 (Ang-2)48 modify VEGF responses by affecting vessel maturation and stability. Ang-1 binds to the Tie-2 receptor tyrosine kinase on endothelial cells, whereas Ang-2 acts in general as an antagonist of Ang-1. Ang-1, but not Ang-2, has been reported to augment angiogenesis in vivo49. A combination of ANG-1 and VEGF gene transfer has been reported to result in larger vessels, although Ang-1 does not stimulate endothelial proliferation50, 51. At the moment, the value of Ang-1 and Ang-2 in vascular gene therapy remains unclear.

Fibroblast growth factor (FGF). The FGF family has 23 known members, which share 30−70 % identical primary sequences. Of these, FGF-1, FGF-2, FGF-4 and FGF-5 have been used for angiogenesis studies52. FGFs are multifunctional proteins that bind to various alternatively spliced isoforms of the receptors FGFR-1, FGFR-2 and FGFR-3; only one isoform is known for FGFR-4 (ref. 52). The FGFs stimulate the proliferation of cells of mesodermal and neuroectodermal origin, including endothelial cells, smooth muscle cells and myoblasts53. In vitro, FGF-1, FGF-2, FGF-4 and FGF-9 have the highest mitogenic activity52. FGF-1 binds to all FGFRs, whereas FGF-2, FGF-4 and FGF-9 show preferential activation of only one receptor isoform. Although both FGF-1 and FGF-2 are highly angiogenic in vivo, mice lacking these factors have no vascular phenotype54. In contrast, targeted disruption of several other FGFs or their receptors (FGF-3, FGF-4, FGF-8, FGFR-1 and FGFR-2) results in embryonic lethality55.

FGF-1 and FGF-2 lack a signal peptide but they are released by an alternative secretion pathway52. They bind to the basement membrane and extracellular matrix until they are liberated by tissue injury. Blood vessels produced by FGF-1, FGF-2 and FGF-4 seem to be less enlarged than those induced by VEGFs56 (Fig. 2). FGFs and VEGFs have synergistic effects; for example, angiogenesis induced by FGF-4 may be at least partially mediated by VEGF56. FGF-2 has also been reported to induce lymphangiogenesis by stimulating VEGF-C expression57. FGF-5 has shown promising angiogenic effects in preclinical studies58. FGF-1, FGF-2 and FGF-4 are produced in adult human tissues54 and have been used in clinical trials. One of the side effects that has emerged from the clinical trials using FGF-2 recombinant proteins is proteinuria59. However, FGFs still constitute a promising group of growth factors for the induction of therapeutic vascular growth.

Figure 2. Therapeutic vascular growth achieved by gene transfer in vivo.
Figure 2 thumbnail

(a−f), Examples of increased vascularity after adenovirus-Vegf (AdVegf; b,e) and adenovirus-Fgf4 (AdFgf4; c,f) gene delivery in rabbit skeletal muscle. Control muscle treated with LacZ adenovirus (AdLacZ; a,d) shows normal capillaries. Muscle treated with AdVegf (b) and AdFgf4 (c) shows increased capillary staining. Insets in a−c show endothelial cell proliferation with bromodeoxyuridine (brown) and CD31 (blue) staining. d−f, Magnetic resonance imaging using T2* sequences and gadolinium contrast agent in the thigh region of the muscles shown in a−c. Increased permeability correlates with angiogenesis and is shown as white color (arrowheads). Control muscle treated with lacZ adenovirus shows no increase in permeability (d). Permeability increases after AdVegf (e) and AdFgf4 (f) treatment. Scale bars = 25 mum in a−c (50 mum in insets). a−f from ref. 56, by permission. (g−i), Vegfc gene therapy for the treatment of lymphedema. Chy mice have a missense point mutation in the Vegfr3 gene, and they serve as a genetic model for congenital human lymphedema27. g, The cutaneous lymphatic vessels in the Chy mice are hypoplastic and do not function normally, as analyzed by fluorescent microlymphography. h, AAV-Vegfc gene therapy in the skin of the Chy mice induces growth of new functional lymphatic vessels (arrows). i, Lymphatic vascular function in a wild-type (WT) littermate is shown for comparison. Magnification, times200 in g−i (modified from ref. 29, with permission).



Full FigureFull Figure and legend (80K)
Other factors involved in vascular growth.
Hepatocyte growth factor (HGF) induces angiogenesis in various animal models60. An HGF plasmid is currently in phase 1 clinical trials for peripheral vascular disease. As a multifunctional protein, HGF can stimulate several cell types other than endothelial cells. In addition, monocyte chemotactic protein-1 (MCP-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) can stimulate angiogenesis and arteriogenesis61, 62, probably resulting from an enhancement of the influx of monocyte-macrophages and other bone marrow−derived cells into the tissue. Recombinant MCP-1 protein is currently in phase 1 clinical trials for peripheral vascular disease. Platelet-derived growth factors (PDGFs), especially PDGF-BB, may be useful when stimulating microvascular proliferation, as they appear to recruit pericytes to growing blood vessels63. Insulin-like growth factor (IGF)-1 and IGF-2 are upregulated in chronically ischemic human skeletal muscles64. They may be useful for the treatment of muscle ischemia, as IGF-1 decreases age-related loss of skeletal muscle function by enhancing muscle regeneration through the activation of satellite cells65. Other secreted factors with angiogenic and therapeutic potential66, 67, 68, 69, 70 are listed in Table 1. The gene encoding Del-1 is currently in phase 1 clinical trial.

Another strategy for the induction of therapeutic vascular growth is to take advantage of cellular genes that induce angiogenic factors. This strategy could employ various signal transduction proteins or transcription factors. Nitric oxide produced by endothelial nitric oxide synthetase (eNOS) and inducible NOS (iNOS) is an important mediator of angiogenesis and arteriogenesis (but not lymphangiogenesis41) induced by several growth factors, including the VEGFs. Although nitric oxide has several potentially useful effects in ischemic tissues71, the usefulness of local transduction with eNOS or iNOS genes has remained unclear. Hypoxia-inducible transcription factor (HIF)-1alpha can activate several genes involved in angiogenic processes, such as VEGF, VEGFR-2, IGF-2 and erythropoietin72. At least short-term activation of angiogenesis has been reported in tissues transduced with vectors expressing various stabilized forms of HIF-1alpha, and adenovirus-mediated HIF-1alpha gene therapy is currently in clinical testing for myocardial ischemia. Among other therapeutic candidate transcription factors, the early growth response factor-1 (EGR-1) stimulates angiogenesis and tissue repair through transcriptional activation of several growth factors73, and the Prox-1 homeobox transcription factor induces lymphatic endothelial reprogramming of vascular endothelial cells74. A synthetic leucine zipper transcription factor that can activate VEGF expression has also been described75.

Experience from preclinical models
The concept of therapeutic vascular growth has been tested in several preclinical models, including myocardial and skeletal muscle ischemia, lymphedema and wound healing, as well as ex vivo transduction of stem cells or EPCs. Improvement of the healing of skin wounds has been reported after VEGF gene transfer76, 77. The efficacy of adenovirus-mediated VEGF and FGF delivery and resulting vascular growth has been demonstrated in the myocardium and skeletal muscle (Fig. 2). Remarkable enlargement and proliferation of capillaries has been obtained, although their significance for muscle remains to be elucidated. In a transgenic system, conditionally switching on VEGF expression in the short term (<2 weeks) resulted in a massive and highly disruptive edema and formation of irregularly shaped sac-like vessels42. Cessation of the VEGF stimulus led to regression of most of these vessels. Upon longer expression (>4 weeks) a critical transition point was evident, after which remodelling occurred and the new vessels persisted for months after withdrawing VEGF, conferring a long-term improvement of organ perfusion42. These findings thus highlight a possible solution for one of the main challenges of local angiogenic therapies, implying that prolonged transgene expression (>4 weeks) might be needed to achieve a persistent therapeutic effect. In most models, hemodynamic factors seem to be particularly important for stabilization of the newly formed vessels.

Lymphedema, a disease characterized by insufficient lymphatic drainage, is an attractive target for therapeutic lymphangiogenesis78. Two to five liters of lymph are formed in humans each day, and if the lymphatic drainage is obstructed by infection, surgery, trauma or a genetic defect, the fluid that accumulates in the tissues manifests itself in gross, disfiguring swelling of the affected limbs. Inherited lymphedema is relatively rare, whereas the noninherited forms of the condition are relatively common; it has been estimated that there are 3 to 5 million lymphedema patients in the USA. Tyrosine kinase−inactivating VEGFR3 missense mutations have been implicated in congenital lymphedema, suggesting an important role of VEGFR-3 signaling in the pathogenesis of this disease79. In preclinical models of lymphedema and lymphatic vessel hypoplasia, the lymphatic vessels could be regenerated by using adenovirus- or AAV-mediated transduction of VEGF-C or its VEGFR-3-specific point mutant form (VEGF-C156S; Fig. 2)27, 44. The newly generated lymphatic vessels were stable and functional. Improvement of lymphedema and restoration of normal tissue architecture was also obtained with recombinant VEGF-C in a rabbit model of postsurgical secondary lymphedema28, and consistent results were more recently published from studies using plasmid transfer45.

When considering the predictive value of preclinical models, it is important to realize that gene transfer efficiency is usually inversely related to host size (Fig. 3). Several applications and vector systems work in mice, but it is much more difficult to obtain equal treatment efficacy in larger animals such as pigs. This is because of limited tissue diffusion of the gene transfer vectors and larger volumes of the transfected tissues, such as skeletal and myocardial muscles. Also, tissue damage caused by local manipulations of small animals, especially in the myocardium and skeletal muscle, can significantly increase transduction efficiency when compared to intact tissues80, 81. An additional concern is that preclinical work has been done in healthy young animals that are able to mount an effective therapeutic response, whereas such a capacity may not be present in elderly patients with atherosclerotic blood vessels, diabetes or other chronic disease processes26. Therapeutic vascular growth is poor in diabetic and elderly animals82. Accordingly, it is anticipated that many of the beneficial biological effects reported in rodents might not be achievable in humans, at least when employing the current vector and gene delivery technology.

Figure 3. Low gene transfer efficiency is a major problem in human gene therapy.
Figure 3 thumbnail

Figure shows inverse transfection efficiency relative to body weight, as determined by adenovirus-mediated LacZ transfection in vivo with intravascular and periadventitial gene transfer methods (data collected from refs. 3,22,108,109, 101 and unpublished observations (S.Y.-H. et al.)).



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Towards improved clinical trial protocols
Optimally, stimulation of both angiogenesis and arteriogenesis is required for an improvement of muscle perfusion1, 43. Thus far, gene therapy has not delivered the expected results, and convincing clinical efficacy has been difficult to demonstrate. Although, great difficulties have been encountered in classical gene therapy for the treatment of monogenic disorders2, significant progress has still been made in proangiogenic gene therapy. This is because long-term gene expression is not required for therapeutic vascular growth, and the transient effects obtained with secreted growth factors using current vectors seem to induce at least some physiological improvement.

Three different 'phases' of clinical angiogenesis studies can be recognized. The first set of clinical trials involved pioneering attempts to test gene therapy with naked plasmids83, 84, 85, 86 and adenoviruses22, 87. The second phase involved several, usually small, uncontrolled trials with frequent positive results88, 89, 90, 91, 92, 93, 94, 95, 96. These trials revealed no major safety problems in patients suffering from severe vascular diseases. During these studies, it became evident that for the documentation of potential clinical benefits, one may need to treat patients who have not progressed to an end-state disease, and that it is essential to use predefined and clinically meaningful endpoints. It is also increasingly evident that placebo effects are significant in the treated patients, probably for a variety of reasons such as early mobilization, altered hemodynamics and better overall care59. Only recently, the third set of clinical trials have begun to test the real potential of gene therapy in a manner similar to the protocols used in pharmaceutical drug development. These randomized, controlled and blinded trials have involved larger numbers of patients and predefined primary and secondary endpoints in reasonable clinical settings97, 98, 99, 100, 101, 102, 103, 104, 105, 106 (Table 2).

Table 2. Phase 2 and 3 angiogenesis trials
Table 2 thumbnail

Full TableFull Table
Because safety issues have emerged in viral gene therapy trials8, 21, it is interesting to compare results obtained when the same growth factors have been given as recombinant proteins. VEGF recombinant protein delivery for the treatment of coronary heart disease did not meet its primary endpoint based on the exercise tolerance test (ETT) 60 days after VEGF administration (the VIVA (vascular endothelial growth factor in ischemia for vascular angiogenesis) trial)97. Recombinant FGF-2 protein delivery was ineffective at improving ETT in myocardial ischaemia patients 90 days after FGF delivery (the FIRST (FGF Initiating revascularization) trial)98, whereas the same growth factor showed efficacy in peripheral vascular disease in a secondary intention-to-treat analysis (the TRAFFIC (therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermitent claudication) trial)99. Recombinant GM-CSF protein improved myocardial perfusion as measured by invasive collateral flow index at a 2-week time point100. Adenovirus-mediated FGF-4 gene delivery into coronary arteries produced an improvement in ETT at 4 weeks in one dose group (the AGENT (angiogenic gene therapy) trial)101. Adenovirus- and plasmid-mediated VEGF gene delivery into ischemic legs during angioplasty operations improved the vascularity of the treated limbs 3 months after VEGF gene therapy (Fig. 4)102, and improved myocardial perfusion was documented in VEGF-adenovirus-treated coronary heart disease patients 6 months after the therapy (the KAT (Kuopip angiogenesis) trial)103. As a secondary endpoint, adenoviral delivery of VEGF121 into the myocardium during thoracotomy improved time to 1 mm ST segment depression during ETT at the 26-week time point104, whereas the same VEGF121 adenovirus was inefficient in the treatment of peripheral vascular disease105. NOGA-catheter-mediated intramyocardial delivery of VEGF165 plasmid (the Euroinject One Trial) was inefficient in improving myocardial perfusion at the 3-month time point, but positive results were reported in a subsequent subgroup analysis106.

Figure 4. Increased vascularity after adenovirus-mediated VEGF gene therapy in human limb.
Figure 4 thumbnail

Adenovirus was given by intra-arterial catheter delivery after angioplasty (arrows indicate site of gene transfer). Digital subtraction angiography was used to analyze the vasculature 3 months after therapy. (a) Angiogram before the therapy. (b) Angiogram immediately after the therapy. (c) Angiogram at 3-month time point showing increased vasculature in the distal limb (from ref. 102, by permission).



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Although several trials in Table 2 have been judged positive according to the primary or secondary endpoints defined in the study protocols, one must realize that the positive results may not always be directly transferable into a clear-cut clinical benefit. For example, it is not known why the same adenoviral VEGF121 product gave positive results in the myocardium104 trial but failed in the peripheral vascular disease trial. Reasons for both failures and positive results should be thoroughly analyzed, as this may reveal key strengths and weaknesses of the current trial design, vectors and gene delivery methods. Pharmacokinetics and pharmacodynamics of various types of gene transfer vectors and treatment gene products are also largely unknown107. At the moment, we still do not know the optimal dose or the best gene delivery route for angiogenic gene therapeutics in each application. Furthermore, many trials have not been adequately powered to test clinical efficacy. Surrogate endpoints, while useful in suggesting clinical efficacy, cannot substitute for hard clinical endpoints such as mortality, myocardial infarction, need for revascularization or amputation. Data from long-term follow-up is also lacking, partly because of the very sick, no-option patients treated in several trials.

Safety records from the above-mentioned phase 2 gene therapy trials, however, indicate no major problems. Many of the potential side effects apparent from experiments using transgenic and knockout animals, such as worsening of atherosclerosis or retinopathy, have not been detected in clinical trials90, 101, 102, 103. The incidence of cancer in patients undergoing angiogenic gene therapy has been the same as or lower than that in the general population of the same age101, 102, 103. It is obvious, however, that more patients and longer follow-up times will be required before the safety and efficacy of gene-based treatments can be fully evaluated. Therapeutic lymphangiogenesis is an area where no clinical data is yet available, although it may be a potential treatment for some severely affected individuals and there are plans to proceed to the clinical phase.

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