Therapeutic angiogenesis is a rapidly developing procedure with high promise for the treatment of peripheral limb ischemia and myocardial ischemia, diseases that may affect more than 50 percent of patients with coexisting diabetes. However many of these patients have been excluded from clinical trails of therapeutic angiogenesis because of safety issues resulting from the delivery and expression of unregulated angiogenic growth factors that may result in spillover with adverse effects on distal tissues including for example the retina, causing exacerbated retinopathy. In this application we propose to test a novel method of delivering angiogenic growth factors that will contain the factors physically and temporarily within the target ischemic tissue, and provide a safer treatment that can be used on diabetic as well as cancer patients with complicating ischemic disorders. Stimulation of collateral vessel production in ischemic limbs and the myocardium by treatments with VEGF and FGF proteins and genes has been demonstrated to occur in animal models and patients. Intramuscular injections or intracoronary infusions of naked and adenoviral DNA, have been shown to induce angiogenesis, stimulate collateral circulation, and improve tissue function. The therapeutic potential of this approach for treating ischemic disorders looks encouraging. In all of the studies so far, pro-angiogenic genes have been delivered under the direction of strong viral promoters. These promoters are used because they are powerful and produce high level expression. The principal disadvantages are: (1) There is no regulation, and production of the factors is determined by the injection dose, half life of the protein, and rate of clearance; this precludes a more permanent delivery vehicle such as the potentially more useful adeno associated virus (AAV). (2) Large amounts of plasmid DNA or virus are administered and there is a strong risk of spill-over to other tissues where pro-angiogenic factors may be harmful. Spill-over has been demonstrated for proteins as well as both plasmid and viral vectors. To minimize these side effects we have developed tissue-specific promoters that are strongly regulated by hypoxia and silencer elements. We propose here to test the hypothesis that constitutive promoters can be replaced by these highly regulated promoters in AAV vectors with improved therapeutic benefit and minimized risk of side effects.
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