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.