Systemic Delivery of shRNA by AAV9 Provides Highly Efficient Knockdown of Ubiquitously Expressed GFP in Mouse Heart, but Not Liver

Abstract

AAV9 is a powerful gene delivery vehicle capable of providing long-term gene expression in a variety of cell types, particularly cardiomyocytes. The use of AAV-delivery for RNA interference is an intense area of research, but a comprehensive analysis of knockdown in cardiac and liver tissues after systemic delivery of AAV9 has yet to be reported. We sought to address this question by using AAV9 to deliver a short-hairpin RNA targeting the enhanced green fluorescent protein (GFP) in transgenic mice that constitutively overexpress GFP in all tissues. The expression cassette was initially tested in vitro and we demonstrated a 61% reduction in mRNA and a 90% reduction in GFP protein in dual-transfected 293 cells. Next, the expression cassette was packaged as single-stranded genomes in AAV9 capsids to test cardiac GFP knockdown with several doses ranging from 1.8×1010 to 1.8×1011 viral genomes per mouse and a dose-dependent response was obtained. We then analyzed GFP expression in both heart and liver after delivery of 4.4×1011 viral genomes per mouse. We found that while cardiac knockdown was highly efficient, with a 77% reduction in GFP mRNA and a 71% reduction in protein versus control-treated mice, there was no change in liver expression. This was despite a 4.5-fold greater number of viral genomes in the liver than in the heart. This study demonstrates that single-stranded AAV9 vectors expressing shRNA can be used to achieve highly efficient cardiac-selective knockdown of GFP expression that is sustained for at least 7 weeks after the systemic injection of 8 day old mice, with no change in liver expression and no evidence of liver damage despite high viral genome presence in the liver.

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

“…However, the knockdown efficiency (36% reduction of mRNA level) in the liver is much lower than that in the heart (68% reduction of mRNA level). This is consistent with the previous study showing that, despite higher viral genome presence in the liver, systemic delivery of shRNA by rAAV9 provides more efficient gene knockdown in the heart 35 . It is possible that hepatocytes are more proliferative than cardiomyocytes and are more actively undergoing cell division after rAAV9 administration at the neonatal ages, which results in substantial vector genome dilution in the liver tissue.…”

“…Although the U6 promoter is ubiquitously active in mouse tissues, a most striking inhibition of target mRNA (Trbp) by rAAV9.U6::shRNA was observed in the heart, but not in other organs (Figure 5). The liver is the most common organ transduced by different serotypes of rAAV 35,36 . However, the knockdown efficiency (36% reduction of mRNA level) in the liver is much lower than that in the heart (68% reduction of mRNA level).…”

Cardiac Stim1 Silencing Impairs Adaptive Hypertrophy and Promotes Heart Failure Through Inactivation of mTORC2/Akt Signaling

“…We used AAV9 because it shows strong tropism for the heart, especially for cardiomyocytes, after a single tail vein injection 4, 24 . We found that cardiac STIM1 expression is reduced by 75% three weeks after administration of AAV9.sh Stim1 as compared to controls (Supplemental figure 1B-C).…”

Chemical Endoplasmic Reticulum Chaperone Alleviates Doxorubicin-Induced Cardiac Dysfunction

“…13 We replaced the luciferase gene with the mouse GRP78 gene. 13 AAV9 vectors were purified, and their titers were determined as previously reported. 13 …”

“…13 All bioluminescence imaging was performed with the IVIS 100 system (Caliper Life Sciences, Hopkinton, MA).…”