direct-reprogramming-human-fibroblasts-into-myoblasts-to-investigate

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

The Research Institute at Nationwide Children’s Hospital

The Ohio State University

Duchenne muscular dystrophy (DMD) is associated with the loss of dystrophin, which plays an important role in myofiber integrity via interactions with β-dystroglycan and other members of the transmembrane dystrophin-associated protein complex. The ZZ domain, a cysteine-rich zinc-finger domain near the dystrophin C-terminus, is implicated in forming a stable interaction between dystrophin and β-dystroglycan, but the mechanism of pathogenesis of ZZ missense mutations has remained unclear because not all such mutations have been shown to alter β-dystroglycan binding in previous experimental systems. We engineered three ZZ mutations (p.Cys3313Phe, p.Asp3335His, and p.Cys3340Tyr) into a short construct similar to the Dp71 dystrophin isoform for in vitro and in vivo studies and delineated their effect on protein expression, folding properties, and binding partners. Our results demonstrate two distinct pathogenic mechanisms for ZZ missense mutations. The cysteine mutations result in diminished or absent subsarcolemmal expression because of protein instability, likely due to misfolding. In contrast, the aspartic acid mutation disrupts binding with β-dystroglycan despite an almost normal expression at the membrane, confirming a role for the ZZ domain in β-dystroglycan binding but surprisingly demonstrating that such binding is not required for subsarcolemmal localization of dystrophin, even in the absence of actin binding domains. 

The ZZ domain of dystrophin in DMD: making sense of missense mutations.

Duchenne muscular dystrophy (DMD) is a devastating X-linked recessive genetic myopathy. DMD physiopathology is still not fully understood and a prenatal onset is suspected but difficult to address.

Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells.

Dystrophinopathies are diseases caused by mutations in the Duchenne Muscular Dystrophy gene (DMD) encoding the dystrophin protein. Depending on the type of mutation, patients develop either the severe DMD or the milder Becker Muscular Dystrophy. Although substantial effort was made, the pathophysiology and variation in disease severity are still poorly understood. During the last two decades, relentless efforts were made to develop therapeutic strategies. Among these, gene therapy and cell replacement therapy appear very promising. These approaches are based on the replacement and/or repair of the mutated DMD gene or transcript at the molecular level, or at the cellular level via replacement of the damaged muscle cells. While highly successful in animal models, these therapies showed only modest efficacy in human clinical trials. More importantly, variable effects were observed in patients carrying the same mutation, suggesting that several factors (e.g., genetic modifiers, environmental factors) can affect treatment outcomes. In this review, we will describe recent advancements and new approaches of gene and cell therapies for dystrophinopathies that pave the way for a medicine "à la carte".

Personalized gene and cell therapy for Duchenne Muscular Dystrophy.

Therapeutic exon skipping as a treatment for Duchenne muscular dystrophy (DMD) has largely concentrated on delivery of antisense oligomers to treat out-of-frame exon deletions. Here we report on the preclinical development of an AAV-encapsidated viral vector containing four copies of the non-coding U7 small nuclear RNA (U7snRNA), each targeted to either the splice donor or the splice acceptor sites of DMD exon 2. We have previously shown that delivery of this vector (rAAV9.U7.ACCA) to the Dup2 mouse model results in expression of full-length dystrophin from wild-type DMD mRNA, as well as an IRES-driven isoform translated only in the absence of exon 2 (Del2 mRNA). Here we present the data from a rigorous dose-escalation toxicity study in nonhuman primates, encompassing two doses (3 x 1013 vg/kg and 8 x 1013 vg/kg) and two time points (3- and 6- months post-injection). No evidence for significant toxicity was seen by biochemical, histopathologic, or clinical measures, providing evidence for safety that led to initiation of a first-in-human clinical trial.

Nicolas Wein

Center for Gene Therapy

The Research Institute at Nationwide Children’s Hospital

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders