Abstract 14001: Adeno-Associated Virus Serotype 9 Efficiently Targets Ischemic Skeletal Muscle Following Systemic Delivery in a Mouse Model of Peripheral Arterial Disease
Introduction: Adeno-associated viral (AAV) vectors can be targeted to skeletal muscle following intravenous (IV) delivery. While this approach has potential for treating peripheral arterial disease (PAD), the reduction in blood flow to the ischemic limb may limit transduction.
Methods: Hindlimb ischemia (HLI) was induced by ligation/excision of the femoral artery. AAV vectors expressing luciferase or eGFP from the muscle-specific CK6 promoter were cross-packaged into AAV9 capsids and injected IV (4.15x10e11 vp/mouse) into 15-20 wk-old C57Bl/6 mice 7-8 days after HLI. On day 10 post-AAV, luciferase expression was assessed by bioluminescence imaging prior to euthanasia for tissue harvest (n=4). The magnitude and specificity of transgene expression was then quantitated by in vitro luciferase assay. On day 14 post-AAV, eGFP expression in representative ischemic muscles was assessed by fluorescence microscopy (>600 myofibers counted/section). To assess vascular permeability, Evans blue dye was injected IP into mice (n=6) 7 days after HLI. Tissues were harvested 24 h later for quantitative comparison.
Results: Bioluminescence signals were stronger in ischemic vs. non-ischemic limbs on post-AAV day 10 (Panel A). CK6 promoter-driven luciferase activity in ischemic gastrocnemius (GA) muscle was 34±2-, 28±1-, and 150±3-fold higher than in contralateral GA, heart, and liver, respectively, 10 days after IV delivery (p<0.05, Panel B). Using eGFP, a transduction rate of 54% was found in ischemic tibialis anterior (TA) muscles (Panel C). Ischemic TA had significantly higher Evans blue dye content (12.7 ± 0.6 % injected / g tissue) than non-ischemic TA (2.6 ± 0.3, p<0.05, Panel D).
Conclusions: Transgene expression is targeted to ischemic muscle following IV delivery of AAV9 in a mouse model of HLI. The specificity of ischemic skeletal muscle transduction can be further improved with the CK6 promoter and increased vascular permeability may be a mechanism for this effect.
- © 2011 by American Heart Association, Inc.