Trans-splicing as a Therapeutic Approach for Dysferlin-deficient Muscular Dystrophies (P3.154)

OBJECTIVE: We aimed to establish a feasible pre-mRNA trans-splicing strategy as an RNA-based therapy for muscular dystrophies caused by mutations in the dysferlin gene (DYSF). BACKGROUND: Mutations in DYSF predominantly cause Limb girdle muscular dystrophy type 2B (LGMD2B) and the allelic disease Miyoshi Myopathy. No therapy exists for dysferlinopathies and gene replacement therapy is not suitable for these diseases as DYSF overexpression has been shown to induce a progressive myopathy in the murine animal model. We attempted therapeutic mRNA repair by developing spliceosome-mediated precursor-mRNA (pre-mRNA) trans-splicing (SmaRT) of the DYSF 3’ pre-mRNA terminus. DESIGN/METHODS: We have targeted four different DYSF pre-mRNA target introns by pre-mRNA trans-splicing molecules (PTMs) expressed from recombinant adeno-associated viral vectors in order to manipulate target intron cis-splicing and provoke trans-splicing to replace the dysferlin 3’ pre-mRNA moiety. Read-out on mRNA and protein level was performed in vitro in immortalized LGMD2B patient myoblasts as well as in vivo in the Dysferlin-deficient B6.129Dysftm1cKam/J mouse model. RESULTS: PTMs targeting dysferlin introns that encoded weak 3’ splice sites led to efficient dysferlin pre-mRNA correction in vitro and in vivo as well as to Dysferlin protein rescue in injected murine muscles in vivo. CONCLUSIONS: Although the level of Dysferlin protein recovery obtained by SmaRT was moderate, weak 3’ splice sites were identified as the crucial qualities of successfully targeted dysferlin introns as well as one of the concordant qualities among successfully targeted introns in trans-splicing strategies for other diseases. Study Supported by: The German Research Foundation (DFG) and The French Muscular Dystrophy Association (AFM). Disclosure: Dr. Philippi has nothing to disclose. Dr. Lorain has nothing to disclose. Dr. Precigout has nothing to disclose. Dr. Spuler has nothing to disclose. Dr. Garcia has nothing to disclose.