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.
Highlights
A wide variety of adeno-associated viral (AAV) serotypes have been isolated from multiple species [1]
RNA interference technology is an intense area of research for the development of new therapies, and a number of studies have previously demonstrated the utility of AAV for delivering short hairpin RNA (shRNA) in vivo [6,7,8,9]
A short hairpin RNA containing a target sequence for green fluorescent protein (GFP) described by Tiscornia et al [11] was synthesized and inserted downstream of the U6 promoter. This plasmid, “pAUSiG,” was later modified by inserting a reporter cassette containing firefly luciferase driven by the cardiac troponin T promoter downstream of the U6-shGFP cassette to generate the plasmid “pAUSiGTL.” A control plasmid was made by replacing the shRNA targeting GFP with an shRNA against an off-target gene
Summary
A wide variety of adeno-associated viral (AAV) serotypes have been isolated from multiple species [1]. AAV2 is the most widely studied serotype for direct gene transfer, but it has a low transduction rate and a long lag phase (6 weeks in the heart) prior to maximal gene expression compared to more recently discovered serotypes [2,3]. As a result, these newer serotypes are being examined for their ability to more efficiently transduce tissues and quickly reach steady-state expression levels. AAV delivery of RNAi provides temporal control over gene knockdown and is less subject to compensatory mechanisms that may develop over generations of selection in knockout mice. RNA interference technology is an intense area of research for the development of new therapies, and a number of studies have previously demonstrated the utility of AAV for delivering shRNA in vivo [6,7,8,9]
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