535 Development of a Novel microRNA Delivery Platform Using Targeted Ultrasound and Carrier Microbubbles

BACKGROUND: Efficient and targeted delivery strategies for microRNA (miR) are currently under investigation. Ultrasoundmediated gene delivery (UMGD) is a method for delivery of plasmid DNA and siRNA in numerous pre-clinical models; however, UMGD has not been used with microRNA (miR). We aimed to develop a method of conjugating microRNA to cationic microbubbles, and to test it for the purpose of ultrasound-mediated microRNA delivery in vivo. METHODS: Cationic microbubbles were incubated with increasing concentrations (0, 1, 2.5, 5 ig/ml) of miR-126. After incubation, conjugated microbubbles were assessed for clumping, size and amount of bound microRNA. Time course of circulating miR-126 concentration was then assessed using qRTPCR, without and with microbubble conjugation at various time points after intravenous injection in rats. Ultrasound-mediated gene delivery (UMGD) of miR-126 (1x109 cationic microbubbles 2.5 ig of miR-126) (n 6) or scrambled miR (miR-SCR) (n 4) was performed in a subset of animals. Control animals (n 4) receiving no treatment. RESULTS: Binding of miR-126 to microbubbles (1x109) saturated at approximately 120,000 miR-126 molecules per microbubble with the addition of 2.5ig miR (n 5). Upon visual inspection and size analysis, there was no microbubble clumping before or after miR-126 conjugation. Upon intravenous administration, greater concentrations of miR-126 were found in the plasma after injection of miR-126 conjugated microbubbles as compared to miR-126 alone, microbubbles alone or sham. PCR results for miR-126 levels after UMGD into the distal hindlimb showed an approximate 15-fold increase over control at 3h, with persistent expression up to day3 and normalizing by day7 (n 4 per time point). MiR-126 targets (SPRED1, PIK3R2) were knocked down at 3h (SPRED1; 72 10% and PIK3R2 73 13%, p 0.05 v. Control), day1 (SPRED1; 48 5% and PIK3R2 44 11%, p 0.01 v. Control) and day3 (SPRED1; 69 13% and PIK3R2 80 20%, p 0.05 v. Control) with normalization by day 7. MiR-SCR delivery had no effect on SPRED1 or PIK3R2 levels as compared to controls and there was no detectable transfection seen in remote organs (liver, lung, kidney and spleen). CONCLUSION: MiR-126 had avid binding to cationic microbubbles, and miR-conjugated microbubbles led to prolonged circulation times of miR-126. In vivo transfection data shows robust transfection of miR-126 in hindlimb skeletal muscle after UMGD, with significant downregulation of targets, SPRED1 and PIK3R2. UMGD of miR results in targeted transfection and is a promising in vivo miR delivery technique. Canadian Cardiovascular Society (CCS) CCS246 Oral HERITABLE ARRHYTHMIAS AND SUDDEN DEATH Monday, October 29, 2012