Abstract 5116: Stable Arteriogenesis of Ischemic Hind Limbs by a Conditionally Silenced AAV9 Gene Therapy Vector Expressing Human VEGF
INTRODUCTION: Therapeutic angiogenesis to treat coronary and peripheral artery disease with genes or cells has not been successful.
RATIONALE: Previous protocols focused on angiogenesis with two major flaws
Inadequate delivery vehicles
Use of unregulated genes with no directional cues for new vessel growth.
HYPOTHESIS: VEGF gene delivery in a semipermanent muscle-tropic AAV9 vector with tight hypoxia-regulation supports directional angiogenesis, arteriogenesis and stable reperfusion.
METHODS: The human (h)VEGF gene driven by a PGK promoter containing tandem arrays of HIF-1α and NSF (silencer) binding sites was cloned into AAV9. Gene expression and regulation were quantified in myocytes and ischemic hind limbs of normal and atherosclerosis-prone mice. Arteriogenesis was evaluated in ischemic hind limbs with intramuscular delivery.
RESULTS: Systemic delivery of AAV9-HR-VEGF to mice showed high tropism for muscle with a 3-fold greater preference for cardiac over skeletal muscle. VEGF expression from AAV9-HR-hVEGF was negligible in aerobic myocytes or normally perfused hind limb and displayed 50 –100-fold induction by hypoxia. Concurrent atherosclerosis muted transgene expression. Delivery of AAV9-HR-hVEGF to ischemic hind limbs supported a progressive increase of limb perfusion that reached 87% of the contra lateral limb after 12 weeks and was sustained at 40 weeks. Limps treated with PBS or adenoviral-hVEGF auto-amputated at 12 weeks. Immunohistology of hind limbs at 2, 4, 12, 16 and 30 weeks revealed significantly larger vessels containing smooth muscle actin in limbs treated with AAV9-HR-VEGF compared with an unregulated vector. DiI imaging combined with scanning confocal microscopy revealed a progressive longitudinal (hip to foot) growth of vessels with the appearance of arteries at 16-weeks. HVEGF expression peaked at 12 weeks and declined thereafter, confirming regulation by ischemia.
CONCLUSION: Sustained, ischemia-regulated VEGF expression supports early angiogenesis that evolves into arteriogenesis with the generation of stable vessels, muscle perfusion and limb salvage. Down-regulation of the transgene coincident with therapy provides a safety switch that will allow the clinical translation of this strategy.