Abstract 14698: Self-Complementary AAV9 Provides No Advantage Over Single-Stranded AAV9 in shRNA Knockdown of Ubiquitously Expressed GFP in Heart or Liver after Systemic Delivery

Abstract

Introduction: AAV9 is a potent gene delivery system that has been shown to preferentially transduce the heart over other tissues. We previously used AAV9 to achieve highly-efficient knockdown of GFP in heart but not liver of transgenic mice that express GFP from the human ubiquitin C promoter, and hypothesized that lack of liver knockdown was due to inefficient conversion of the single-stranded genomes into double-stranded DNA after AAV9-mediated delivery. Methods: An expression cassette containing a mouse U6 promoter driving shRNA against GFP was constructed using wild-type AAV2 ITRs (ssAAV) or a mutated 5’ ITR for packaging of self-complementary (scAAV) genomes into AAV9 capsids. ssAAV9 or scAAV9 vectors were administered to 10-day old mice via intraventricular chamber injection at a dose of 8х10^10 vg/g. Uninjected mice were used as controls. Cardiac and liver tissues were collected 7 weeks after injection for protein, RNA and genomic DNA. GFP expression was analyzed by qRT-PCR and western blotting. Results: Compared to controls (n=5) in cardiac tissue, the ssAAV group (n=8) demonstrated an 82% decrease in GAPDH-normalized GFP mRNA (p<0.0001) while the scAAV group (n=7) provided a 79% knockdown (p<0.0001) (Panel A). In liver, the ssAAV group showed a 35% reduction in GFP mRNA versus controls (p<0.01) and the scAAV group showed a 27% knockdown (p<0.05). Western blot analysis showed that GAPDH-normalized GFP expression was reduced by 51% in the ssAAV group (p<0.0001) and by 45% in the scAAV group (p<0.001) compared to controls in heart, but there was no reduction in GFP in the liver (Panel B, n=4 for all groups). Genomic DNA analysis demonstrated a 6-fold and 1.9-fold increase in viral genomes in liver vs. heart for the ssAAV group (p<0.0001) and scAAV group (p<0.05), respectively. Conclusions: AAV9 vectors expressing shRNA provide similar knockdown in cardiac and liver tissues regardless of whether they are packaged as single-stranded or self-complementary genomes.