Abstract 19465: Cardiac Gene Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound

Background RNA interference (RNAi) is a cellular process that regulates gene expression via sequence-specific mRNA degradation and translational blockage. The potent gene knockdown effects of RNAi could have broad therapeutic applications in cardiovascular disease, but is currently limited by inefficient delivery strategies, off-target effects, and nuclease activity which degrades RNAi molecules such as small inhibitory RNA (siRNA). We hypothesized that cardiac ultrasound (US)-targeted destruction of microbubbles (MB) bearing siRNA would selectively knock down target cardiac genes. Methods A transgenic mouse model of cardiac restricted luciferase expression was used. RNA binding-MBs were prepared by amalgamation of cationic and neutral lipids in the presence of siRNA specific to luciferase or negative control siRNA. RNA binding was confirmed by serial washing of loaded MBs followed by electrophoresis. RNAse resistance was determined by exposure of siRNA-loaded MBs to RNAseA, followed by electrophoresis. siRNA-loaded MBs (5 µg per 7x10 8 MBs) were infused through the internal jugular of transgenic mice over 15 min during concurrent MB-destructive transthoracic US (1.3 MHz, MI 1.6). The US pulse sequence was designed to allow complete myocardial MB replenishment between each destruction sequence, as confirmed by contrast-specific US imaging. Luciferase expression was determined 3 days later by luciferase assay and RT PCR. Results MBs bound ∼7 µg siRNA per 10 9 MBs and protected siRNA from RNAseA digestion at concentrations sufficient for digestion of siRNA in the absence of MBs. US-targeted cardiac delivery of MBs bearing luciferase siRNA resulted in significant knockdown of luciferase expression in transgenic mice 3 days after treatment compared to controls (53% knockdown vs. no treatment, p=0.03; 65% knockdown vs. control siRNA+MBs+US, p=0.02) and by RT PCR (68% knockdown vs. no treatment, p=0.05). Conclusion siRNA delivery using custom MBs and US silences cardiac reporter gene expression. These data establish general principles that can be extended to US-MB theranostic platforms for image-guided targeted gene knockdown in cardiovascular diseases for which specific gene silencing represents a therapeutic approach.