Mohammadsharif Tabebordbar

In vivo gene editing in dystrophic mouse muscle and muscle stem cells.

Editing can help build stronger muscles Much of the controversy surrounding the gene-editing technology called CRISPR/Cas9 centers on the ethics of germline editing of human embryos to correct disease-causing mutations. For certain disorders such as muscular dystrophy, it may be possible to achieve therapeutic benefit by editing the faulty gene in somatic cells. In proof-of-concept studies, Long et al., Nelson et al., and Tabebordbar et al. used adeno-associated virus-9 to deliver the CRISPR/Cas9 gene-editing system to young mice with a mutation in the gene coding for dystrophin, a muscle protein deficient in patients with Duchenne muscular dystrophy. Gene editing partially restored dystrophin protein expression in skeletal and cardiac muscle and improved skeletal muscle function. Science, this issue p. 400, p. 403, p. 407 Gene editing via CRISPR-Cas9 restores dystrophin protein and improves muscle function in mouse models of muscular dystrophy. Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction

In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) results in the expansion and directed differentiation of endogenous heart progenitors in a mouse myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors and temporal control with VEGF inhibitors revealed the greatly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long-term organ repair.

A multifunctional AAV-CRISPR-Cas9 and its host response

CRISPR–Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV–split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV–CRISPR–Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV–CRISPR–Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce extensive cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics.

A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species.

Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3β inhibitors, two calpain inhibitors, and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin, and the GSK3β inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle.

Skeletal Muscle Degenerative Diseases and Strategies for Therapeutic Muscle Repair

Skeletal muscle is a highly specialized, postmitotic tissue that must withstand chronic mechanical and physiological stress throughout life to maintain proper contractile function. Muscle damage or disease leads to progressive weakness and disability, and manifests in more than 100 different human disorders. Current therapies to treat muscle degenerative diseases are limited mostly to the amelioration of symptoms, although promising new therapeutic directions are emerging. In this review, we discuss the pathological basis for the most common muscle degenerative diseases and highlight new and encouraging experimental and clinical opportunities to prevent or reverse these afflictions.

Therapeutic Gene Editing in Muscles and Muscle Stem Cells

Duchenne muscular dystrophy (DMD) is a devastating, degenerative muscle disease that affects ~1 in every 3500 male births. DMD arises from mutations in the DMD gene that prevent expression of its encoded protein, Dystrophin (Burghes et al. Nature 328:434–437, 1987). Interestingly, patients with Dmd mutations that delete certain segments of the Dystrophin coding region, but maintain protein reading frame, have a much milder form of the disease, known as Becker Muscular Dystrophy (BMD). This observation has spurred interest in developing “exon skipping” strategies in which certain mutation-containing or mutation-adjacent Dmd exons are intentionally removed in order to restore protein reading frame, and thereby Dystrophin expression, in DMD patients (Beroud et al. Hum Mutat 28:196–202, 2007; Yokota et al. Expert Opin Biol Ther 7:831–842, 2007).

483. In Vivo DMD Gene Editing in Muscles and Muscle Stem Cells of Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a X-linked genetic disorder that arises from frame-disrupting mutations in the DMD gene, encoding DYSTROPHIN. Lack of DYSTROPHIN expression destabilizes muscle fiber membranes, increases susceptibility to contraction-induced injury and drives muscle degeneration. Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated but still functional protein. In this study, we develop and test a direct gene editing strategy to recover DYSTROPHIN expression in the mdx mouse model of DMD. Coupling clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases delivered via adeno-associated virus (AAV) with paired guide RNAs flanking the mutated Dmd exon 23, we demonstrate precise excision of intervening DNA and restoration of Dystrophin reading frame and protein expression in vivo in both skeletal and cardiac muscles following local or systemic delivery. DYSTROPHIN expression in AAV Dmd-CRISPR treated mdx mice was sufficient to partially recover functional deficiencies of dystrophic muscle. Finally, we demonstrate in vivo targeting of the mdx mutation in endogenous muscle stem cells, suggesting that AAV-CRISPR may provide a means to support ongoing repair of dystrophic fibers with corrected muscle precursors. This study provides proof-of-concept evidence supporting the feasibility and efficacy of in vivo genome editing to correct frame-disrupting mutations in DMD.View Large Image | Download PowerPoint Slide

570. Efficient In Vitro Expansion and Gene Correction of Dystrophic Mouse Muscle Stem Cells

Duchenne Muscular Dystrophy (DMD) is a progressive muscle wasting disease caused by mutations in the DYSTROPHIN gene. Gene correction and autologous transplantation of muscle stem cells (i.e., satellite cells) presents a promising therapeutic approach for restoring DYSTROPHIN expression to muscle fibers in these patients; however the low frequency of satellite cells in adult muscle is an obstacle for isolating sufficient numbers of cells for engraftment. To address this issue, we have sought culture conditions that would support the ex-vivo expansion of satellite cells and potentially provide more cells for transplantation. We found that forskolin, an adenylyl cyclase activator, dramatically expanded mouse satellite cells in culture. Forskolin-treated cultured cells retained the immunophenotypic characteristics of engraftable satellite cells, and transplantation of compound-treated cells into dystrophic muscle yielded a significantly higher level of engraftment compared to control cells. Forskolin also dramatically expands satellite cells from mdx mouse model of DMD in culture. As a proof of concept for a combined gene and cell therapy approach for treating DMD, we targeted the mutated Dystrophin locus in ex-vivo expanded forskolin treated dystrophic satellite cells using CRISPR/Cas9 gene editing technology and enriched for the targeted cells using an endogenous fluorescent reporter system. Expansion and gene correction of muscle satellite cells in culture as described here provides the possibility of improving combined gene and cell-based therapies for neuromuscular disorders.