Optimizing and validation of gene therapy vectors to treat limb girdle muscular dystophy

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PROJECT SUMMARY We aim to develop a gene therapy for limb girdle muscular dystrophy type 2A (LGMD2A), an autosomal recessive (AR) muscle wasting disorder due to mutations in CAPN3. LGMD2A is considered to be the most prevalent of the AR LGMDs and yet there is currently no treatment for patients, who are usually wheelchair dependent a decade after diagnosis. We and others have shown that overexpression of CAPN3 can be accomplished in skeletal muscle without toxicity; a finding which makes the feasibility of gene therapy for LGMD2A a realistic goal; however, LGMD2A is unique from most other LGMDs and this fact warrants careful development of gene therapy vectors. One consideration is that CAPN3 cannot be expressed in the heart, due to cardiac toxicity, which is not the case with Duchenne muscular dystrophy and other LGMDs. Although a few pre-clinical, proof of concept studies have successfully accomplished AAV-Capn3 overexpression in mice, there has not been a systematic optimization of any gene therapy construct for humans with LGMD2A, especially one that considers the relative skeletal muscle vs cardiac gene expression issues. Furthermore, because LGMD2A preferentially impacts slow fibers, it is critical that the therapeutic construct that is ultimately carried into clinical trials is optimized for slow fiber expression. Because each patient can only be dosed one time, it is imperative that the construct used for gene delivery is ideal. In this application, we will use an iterative and systematic approach to optimize these vectors. We have assembled an expert, collaborative team with deep experience in the area of regulatory cassettes, AAV vector development and LGMD2A pathogenesis and calpainopathy animal models. The team will work together to generate and test the safety and efficacy of a series of AAV vectors to develop a treatment for LGMD2A. These vectors will be optimized for slow fiber expression while avoiding cardiac toxicity. One team member, Dr. Hauschka, is largely credited with creating the vast majority of regulatory cassettes being used in the current AAV-gene therapy trials for Duchenne muscular dystrophy. Dr. Chamberlain designed and optimized the first micro and mini dystrophins, which formed the basis for all constructs currently in gene therapy clinical trials for DMD. Drs. Spencer and Kramerova generated numerous mouse models that have led to the identification of outcome measures useful for testing therapeutic interventions for LGMD2A. Dr. Cannon, is an internationally known muscle physiologist who will carry out physiological assessments of contractile function. The team will apply their extensive and cumulative knowledge of LGMD2A, AAV vectors, regulatory cassettes and mouse muscle testing to create this gene therapy for LGMD2A.