Investigations of the in vivo stability and function of full length and truncated dystrophins in dystrophin deficient skeletal muscles and heart.



Poster Number: 107


Addeli Bez Batti Angulski Spies PhD, John Bauer , Jeffrey S. Chamberlain PhD, Joseph M. Metzger PhD


1. University of Minnesota, 2. University of Minnesota, 3. University of Washington, 4. University of Minnesota

The biological half-life of shortened therapeutic dystrophin proteins impacts significantly on the clinical efficacy of gene therapy and gene-editing-based treatment strategies targeting gene replacement in Duchene Muscular Dystrophy (DMD) patients. Significant unknowns are the physiologically relevant half-life of full-length and internally deleted dystrophin molecules in striated muscles in vivo. This is important, as we recently showed evidence that some truncated dystrophins can be highly unstable in muscle in vivo, with unexpectedly very fast turnover rates. Therefore, insight into the turnover and stability of dystrophin under physiological in vivo conditions is critical to ultimately defining key clinical parameters of delivery frequency and dosing regimens in DMD patients. The hypothesis of this study is that clinically guided gene edited/exon skipped truncated dystrophin molecules will have significantly shorter half-lives than the intact full-length dystrophin in vivo. To begin to address that, we implemented a validated methodology for precise temporal-spatial control of shortened and full-length dystrophin excision in vivo. We used a floxed allele approach together with a cardiac directed (αMHC Mer-Cre-Mer) or skeletal muscle directed (HSA Mer-Cre-Mer) inducible Cre for precise control of full-length dystrophin or therapeutic shortened gene excision. Using mdx engineered with floxed full-length dystrophin cDNA, we examined the time course of full-length dystrophin mRNA as a biologically relevant surrogate for intact dystrophin gene excision efficiency. In contrast to the poor efficiency we reported for intact dystrophin locus gene excision efficiency, preliminary data shows evidence of significant intact full-length dystrophin cDNA gene excision, ranging from ~ 80% in heart muscle to ~ 90% in skeletal muscles in vivo. We can attribute this marked improvement in gene deletion efficiency via dramatically shortening the intragenic sequence to be excised from over 2 Mb to ~ 12kb in the case of the full-length cDNA for human dystrophin. Studies of in vivo full-length dystrophin protein stability, post floxed gene excision, are ongoing. In addition, complementary approaches using therapeutic and non-therapeutic truncated and internally deleted dystrophins in vivo are ongoing. Collectively, these studies should provide key information for future long-term successes of ongoing DMD treatments featuring gene therapies with internally deleted dystrophins