Vascular Regeneration in Ischemic Hindlimb by Adeno‐Associated Virus Expressing Conditionally Silenced Vascular Endothelial Growth Factor

Abstract

BackgroundCritical limb ischemia (CLI) is the extreme manifestation of peripheral artery disease, a major unmet clinical need for which lower limb amputation is the only option for many patients. After 2 decades in development, therapeutic angiogenesis has been tested clinically via intramuscular delivery of proangiogenic proteins, genes, and stem cells. Efficacy has been modest to absent, and the largest phase 3 trial of gene therapy for CLI reported a worsening trend of plasmid fibroblast growth factor. In all clinical trials to date, gene therapy has used unregulated vectors with limited duration of expression. Only unregulated extended expression vectors such as adeno‐associated virus (AAV) and lentivirus have been tested in preclinical models. Methods and ResultsWe present preclinical results of ischemia (hypoxia)‐regulated conditionally silenced (CS) AAV–human vascular endothelial growth factor (hVEGF) gene delivery that shows efficacy and safety in a setting where other strategies fail. In a BALB/c mouse model of CLI, we show that gene therapy with AAV‐CS‐hVEGF, but not unregulated AAV or plasmid, vectors conferred limb salvage, protection from necrosis, and vascular regeneration when delivered via intramuscular or intra‐arterial routes. All vector treatments conferred increased capillary density, but organized longitudinal arteries were selectively generated by AAV‐CS‐hVEGF. AAV‐CS‐hVEGF therapy reversibly activated angiogenic and vasculogenic genes, including Notch, SDF1, Angiopoietin, and Ephrin‐B2. Reoxygenation extinguished VEGF expression and inactivated the program with no apparent adverse side effects. ConclusionsRestriction of angiogenic growth factor expression to regions of ischemia supports the safe and stable reperfusion of hindlimbs in a clinically relevant murine model of CLI.

Keywords

• gene therapy
• hypoxia
• ischemia
• peripheral vascular disease
• revascularization