482. Local and Systemic Gene Editing in a Mouse Model of Duchenne Muscular Dystrophy
Authors: Christopher Nelson, Matthew Gemberling, Chady H. Hakim, David G. Ousterout, Pratiksha I. Thakore, Ruth M. Castellanos, Sarina Madhavan, Xiufang Pan, F. Ann Ran, Winston Yan, Aravind Asokan, Feng Zhang, Dongsheng Duan, Charles A. Gersbach
Journal: Molecular Therapy (2016)

Duchenne muscular dystrophy (DMD) is a highly prevalent genetic disorder leading to muscle wasting, loss of ambulation, and premature death by the third decade of life. DMD is caused by gene deletions, duplications, or nonsense mutations leading to the loss of dystrophin, an essential musculoskeletal protein. Gene therapy has held tremendous promise for the treatment of monogenic disorders, yet an effective gene replacement therapy has been elusive. Genome editing has been established as a potential approach to correct the dystrophin gene in cultured human cells by excising non-essential exons from the dystrophin gene producing a shortened yet in-frame dystrophin protein (1). In contrast to gene replacement therapy, genome editing repairs the causative mutation in the native genomic context with the potential for permanent gene repair. Recently, we and others have demonstrated that CRISPR/Cas9 genome editing in neonatal and adult mouse models of DMD restores dystrophin expression, improves muscle biochemistry, and strengthens muscle force generation (2-4). However, further optimization of the approach for systemic gene correction is still needed. To target the dystrophin gene in the mdx mouse, the 3.2kb S. aureus Cas9 and two guide RNAs (gRNA) targeting intronic regions surrounding exon 23 were packaged into an adeno-associated virus (AAV). Double stranded breaks created by Cas9 were repaired with the relatively efficient non-homologous end joining pathway leading to excision of the nonsense mutation in exon 23. AAV vectors were injected intramuscularly into the tibialis anterior muscle in adult mice and intravenously into neonatal mice and adult mice and characterized for gene deletions, dystrophin restoration, and improvements in muscle physiology. Local correction restored overall dystrophin levels to 8% by western blot with 67% of muscle fibers positive for dystrophin by immunofluorescence. Repeated cycles of eccentric contraction showed 60% resistance to damage compared to sham-treated mice. Systemic correction was achieved through IP injection into P2 neonates with dystrophin restoration primarily in the cardiac muscle and skeletal muscle surrounding the peritoneal cavity. IV administration in adult mice restored dystrophin expression in the cardiac muscle. Improved systemic distribution and correction was achieved with intravenous administration into P2 neonates with AAV8 or AAV9 (Fig. 1). This study establishes CRISPR/Cas9-based gene editing as a promising approach for the treatment of DMD. Ongoing work to improve the efficiency and safety of in vivo gene editing includes the incorporation of muscle specific promoters, minimization of vector packaging, and optimization of AAV serotype. 1. Ousterout et al. Nat Comm 2015. | 2. Nelson et al. Science 2015 | 3. Tabebordbar et al. Science 2015 | 4. Long et al. Science 2015