561. Novel Tissue-Specific Regulatory Cassettes Direct High-Level Transgene Expression in Skeletal and Cardiac Muscle
Authors: Maja Z. Salva, Charis L. Himeda, Phillip Tai, Eiko Nishiuchi, Eric E. Finn, James M. Allen, Paul Gregorevic, Michael J. Blankinship, Leonard A. Meuse, Jeffrey S. Chamberlain, Stephen D. Hauschka
Journal: Molecular Therapy (2006)

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Systemic delivery of rAAV6 vectors can achieve efficient transduction of the entire striated musculature, making this an attractive strategy for gene therapy of Duchenne Muscular Dystrophy. However, this delivery method also transduces cells in many non-muscle tissues, which may cause therapeutic problems including toxicity and a transgene-directed immune response. These problems could be avoided by limiting transgene expression through the use of muscle-specific promoters. Unfortunately, the expression levels of such promoters following systemic delivery of rAAV vectors are lower than viral promoters in most skeletal muscles and negligible in cardiac muscle. The goal of this project is to design regulatory cassettes that drive high levels of skeletal and cardiac muscle-specific transgene expression while also being sufficiently short (<800bp) to fit into rAAV vectors containing microdystrophin cDNA (3.8-kb). Our lab has developed a series of muscle-specific regulatory cassettes based on the enhancer and promoter of the murine muscle creatine kinase (MCK) gene. The CK6 cassette, previously thought to be the strongest modification, has higher activity than the wild type cassette in skeletal muscle, but very low activity in cardiac and diaphragm muscle. We have designed new cassettes to increase expression, and their activities have been evaluated in various muscles and non-muscle tissues following systemic delivery of rAAV6 vectors expressing a human placental alkaline phosphatase reporter gene. First, we designed a 570-bp MCK-based cassette, CK7, which exhibited activity levels similar to CK6 in quadriceps, a predominantly fast-twitch muscle, but had significantly higher activity in cardiac muscle, diaphragm, and soleus, a predominantly slow-twitch muscle. To further improve expression, especially in cardiac muscle, we added a 190-bp enhancer from the murine alpha-myosin heavy chain gene (|[alpha]|MHC) to the CK7 cassette to produce MHCK7, a 770-bp cassette that has the highest overall activity. When compared to CK6, the activity of MHCK7 was about 400-fold higher in cardiac muscle and 10-fold higher in soleus, which was equivalent to the activity of the CMV promoter. Staining for AP activity in cross-sections of both muscles revealed strong expression in virtually all fibers. The activity in diaphragm was about 40-fold higher, but was still considerably lower than the CMV promoter, with only about 20% of the fibers staining positive. Finally, the activity in quadriceps muscle was equivalent to CK6. The activity of MHCK7 in kidney, aorta, brain, testes, and intestine was at background levels, while a very slight increase in activity, about 50|[ndash]|200-fold lower than in muscle, was detected in liver, spleen, and lung. Based on these results, the MHCK7 cassette is currently being tested with respect to its capacity for driving microdystrophin expression in mdx mice following intramuscular and systemic injections of rAAV6 vectors.