266. Development of rAAV-Based Skeletal and Cardiac Muscle Regulatory Cassettes for Gene Therapy of Duchenne Muscular Dystrophy

Abstract

Top of pageAbstract A promising gene therapy approach for treatment of DMD involves systemic delivery of rAAV vectors encoding microdystrophin. This method was shown to mediate efficient transduction of all striated muscles and high-level transgene expression driven by the ubiquitous CMV promoter. However, the use of viral promoters increases the risk of an immune response, largely due to transgene expression in antigen-presenting cells, and may thus be inadequate for long-term therapy. An ideal regulatory cassette for gene therapy of DMD thus needs to direct high-level, tissue-specific expression in skeletal and cardiac muscle and be short enough to package into the rAAV microdystrophin construct. Our lab has developed a series of muscle-specific regulatory cassettes based on the murine muscle creatine kinase (MCK) gene enhancer and promoter regions. The previous |[ldquo]|best|[rdquo]| small cassette, CK6 (580 bp) drives microdystrophin expression in skeletal muscle following systemic rAAV delivery, at 10% of the level of the CMV promoter. However, expression in heart and diaphragm is very low. To improve expression, we combined the wild type MCK enhancer with a 190 bp enhancer region from the murine alpha myosin heavy chain (MyHC) gene, which is expressed at high levels in cardiac muscle. Addition of the MyHC enhancer increased activity in MM14 skeletal myocytes by 5-fold as assessed by alkaline phosphatase reporter gene activity. Furthermore, high-level expression was detected in skeletal muscle, as well as heart and diaphragm after systemic delivery in mice of rAAV6 vectors encoding these constructs. The activity of the hybrid cassette was 7-fold higher in heart and 3-fold higher in diaphragm and soleus (predominantly slow-twitch muscle fibers) when compared to the wild type MCK cassette, while the activity in tibialis anterioris (predominantly fast-twitch muscle fibers) was unchanged. Expression was restricted to muscle tissue, as evidenced by nearly undetectable levels in the liver, spleen, lung, and aorta. Additional cassettes comprising the MyHC enhancer and various modifications of the minimal MCK cassette have also been tested in MM14 skeletal myocytes. The strongest cassettes were shown to possess 12-fold higher activity than the wild type MCK cassette, and were about 55% as active as the CMV promoter. These contained a 63 bp deletion within the MCK enhancer, addition of a 50 bp region downstream of the transcription initiation site, and a mutation that creates a consensus initiator-binding site from the terminal transferase promoter. We are currently testing activity of these constructs in mouse muscle and non-muscle tissues following systemic delivery of rAAV6 vectors. The strongest tissue-specific cassette will be evaluated for long-term expression of therapeutic levels of microdystrophin in the mdx mouse model for DMD.