Abstract 15404: Efficient and Persistent Transgene Expression by Minicircle Plasmid versus Standard Plasmid Using Ultrasound Targeted Microbubble Destruction

Background: Ultrasound-targeted microbubble destruction (UTMD) is a non-invasive gene transfection technique using carrier microbubbles and targeted high power ultrasound, primarily used to deliver plasmid DNA (pDNA). Minicircle-DNA (mcDNA) is a novel gene vector, which has recently been shown to have an improved and persistent transfection, compared to conventional pDNA. Hypothesis: We hypothesized that UTMD of mcDNA in a hind-limb model would exhibit a more potent and prolonged gene expression compared to conventional pDNA. Methods: In vitro , HUVECs, fibroblasts (3T3) and neonatal cardiomyocytes were transfected with molar equivalents of GFP-minicircle and GFP-plasmid. GFP expression was measured by RT-PCR and fluorescent microscopy for 28 days. We then performed a comparison of bubble binding capacities of both vectors to cationic lipid microbubbles. In vivo , for UTMD to the left proximal hind-limb adductor muscle, 500μg and 214μg of GFP-plasmid and GFP-minicircle respectively were charge-coupled with 1x10 9 cationic microbubbles and delivered via UMGD into Sprague-Dawley rats (n=30). The animals were followed for 28 days with GFP measured by RT-PCR and immunohistochemistry. Results: In vitro results showed greater GFP expression by mcDNA across all cell lines, with 7-10 fold transfection efficacy compared to pDNA. mcDNA demonstrated greater binding capacity to cationic microbubbles compared to pDNA. For plasmid and minicircle DNA, binding saturations were reached at ~6000 copies per microbubble, and ~20000 copies per microbubble respectively, suggesting a higher minicircle bubble binding efficiency. In vivo results showed higher GFP levels as early as 6 hours post minicircle UTMD, demonstrating minicircle to be a faster acting therapeutic agent over conventional plasmid. A significantly greater (p<0.01) expression of GFP was also evident at day 28 in minicircle UTMD-treated group, proving mcDNA to be a better choice for longer-term gene expression. Conclusions: In summary, UTMD using mcDNA results in more rapid and sustained transfection compared to conventional pDNA, and may be a more effective vector for translational studies of UTMD.