In vivo investigation of the mechanisms regulating truncated dystrophin protein turnover in intact cardiac and skeletal muscle



Poster Number: T335


Addeli Angulski Spies, PhD, University of Minnesota, Nora Hosny, PhD, University of Minnesota, John Bauer, University of Minnesota, Joseph M. Metzger, PhD, University of Minnesota

This study addresses the opportunities and challenges of advancing efficient gene-based therapies to treat Duchenne muscular dystrophy (DMD) patients. We hypothesize that the recent failures to reach functional milestones in micro-dystrophin clinical trials is due, at least in part, to unstable truncated dystrophins that limit the amount of therapeutic dystrophin content. We implemented here a gene regulated system for accessing clinically relevant human micro-dystrophin half-life in heart and skeletal muscle in vivo, including determining the influence of dystrophin domains on protein stability. We investigated complementary strategies to improve the in vivo biological half-life of micro-dystrophins as a way to advance functional outcomes. Our data showed (WB and immunostaining), in both heart and skeletal muscles a marked decay of therapeutic micro-dystrophin content (Sarepta’s clinical trial construct), with very fast turnover rates in vivo (5-8 days). We have evidence of the rapid emergence of myocardial fibrosis as micro-dystrophin content decays from 100% to 50% over 10 days. Interestingly, E3 ligases Murf1 and Atrogin 1 were upregulated at the message and protein levels in micro-dystrophin-treated skeletal muscle tissues compared to full length dystrophin. Moreover, micro-dystrophin protein content increased in skeletal muscle tissue in vivo after treatment with the UPS inhibitor bortezomib, evidencing the involvement of UPS pathway in micro-dystrophin degradation in vivo. In addition, we investigated cell extrinsic mechanisms to stabilize and improve the functional efficacy of therapeutic micro-dystrophin constructs in vivo. To this end we tested the first-in-class membrane interfacing synthetic copolymer Poloxamer 188 (P188). Preliminary evidence shows that at 10 days after gene excision, the amount of micro-dystrophin is higher in the quadriceps of P188 treated animals compared to saline controls. Aiming to understand structural elements of dystrophin that are key for governing half-life in vivo we engineered a Tg mouse model that parallels the internal deletion found in some BMD patients (Δ45-55) and a third mouse model containing a non-therapeutic truncated dystrophin (NTermdys). Collectively, these results will have significant potential impact on the field by deciphering key dystrophin structure-function elements required for long-term effective micro-dystrophin therapies.