Key result
Systemic delivery of AAV9-shGFP in neonatal mice resulted in a 77% reduction in cardiac GFP mRNA and a 71% reduction in cardiac GFP protein, with no significant knockdown in the liver.
Effect estimate: 77% reduction
p-value: p=<0.0001
Systemic delivery of AAV9-shRNA achieves highly efficient and sustained cardiac-selective knockdown without affecting liver expression or causing liver damage in a mouse model.
Supports cardiac-selective AAV9-shRNA knockdown in mice; leaves open translation to human cardiovascular gene therapy.
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 1.8×10(10) to 1.8×10(11) viral genomes per mouse and a dose-dependent response was obtained. We then analyzed GFP expression in both heart and liver after delivery of 4.4×10(11) 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.
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Piras et al. (2013) studied Ubiquitous GFP expression in transgenic mice. AAV9-shGFP vs. AAV9-shControl was evaluated on Cardiac GFP mRNA expression (77% reduction, p=<0.0001). Systemic delivery of AAV9-shGFP in neonatal mice resulted in a 77% reduction in cardiac GFP mRNA and a 71% reduction in cardiac GFP protein, with no significant knockdown in the liver.
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