Abstract 19697: Highly-Efficient in vivo Knock-Down of Ubiquitously Expressed eGFP in the Murine Heart with siRNA Delivered by a Pseudotyped AAV9 Vector
Introduction: AAV9 is a potent gene delivery system that has been shown to preferentially transduce the heart over other tissues. We hypothesized that systemic administration of an AAV9 vector expressing siRNA against eGFP would inhibit gene expression in transgenic mice that express eGFP from the human ubiquitin C promoter (ubc-GFP).
Methods: An AAV genome expressing siRNA against eGFP from the mouse U6 promoter was constructed and tested in vitro by cotransfecting it into 293 cells with a plasmid expressing eGFP. Microscopy, fluorimetric analysis using an Optima plate reader, real-time RT-PCR, and Western blotting were used to assess eGFP expression in vitro. The same AAV genome was cross-packaged into AAV9 capsids and injected intracardiac into 8-day old ubc-GFP mice in doses ranging from 1010 to 1011 vp/mouse (2-3 mice/group in 3 groups). Control ubc-GFP mice were injected with saline (n=2). Hearts were collected 4 wks after injection, frozen, sectioned and examined by microscopy. To compare eGFP expression, mean fluorescence intensity was measured with ImageJ in 16 distinct regions of interest per mouse heart (>1000 myocytes assessed per heart).
Results: In vitro results (Panels A-B) indicated a 55% knock-down as assessed by real-time PCR (p<0.01), but a 90% knock-down as determined by semi-quantitative blot analysis (p<0.01) and an 82% decrease in fluorescence as determined by fluorimetry (p<0.01). In contrast, controls cotransfected with the eGFP plasmid and a plasmid expressing siRNA against iNOS showed abundant eGFP expression. In vivo results (Panels C-D) indicated a dose-dependent knock-down that was highly significant at every dose evaluated (p < 0.01 vs. control) and reached 75% efficiency at the highest dose tested (Panel E).
Conclusion: AAV9 vectors expressing siRNA can be used to achieve cardiac-selective and highly-efficient knock-down of cardiac gene expression in vivo.
- © 2010 by American Heart Association, Inc.