Toward the correction of muscular dystrophy by gene editing

Abstract

Recent advances in gene editing technologies are enabling the potential correction of devastating monogenic disorders through elimination of underlying genetic mutations. Duchenne muscular dystrophy (DMD) is an especially severe genetic disorder caused by mutations in the gene encoding dystrophin, a membrane-associated protein required for maintenance of muscle structure and function. Patients with DMD succumb to loss of mobility early in life, culminating in premature death from cardiac and respiratory failure. The disease has thus far defied all curative strategies. CRISPR gene editing has provided new opportunities to ameliorate the disease by eliminating DMD mutations and thereby restore dystrophin expression throughout skeletal and cardiac muscle. Proof-of-concept studies in rodents, large mammals, and human cells have validated the potential of this approach, but numerous challenges remain to be addressed, including optimization of gene editing, delivery of gene editing components throughout the musculature, and mitigation of possible immune responses. This paper provides an overview of recent work from our laboratory and others toward the genetic correction of DMD and considers the opportunities and challenges in the path to clinical translation. Lessons learned from these studies will undoubtedly enable further applications of gene editing to numerous other diseases of muscle and other tissues.

Keywords: CRISPR; gene editing; myoediting.

Fig. 1.

Dystrophin gene and protein. (A) Schematic of dystrophin protein showing functional domains. Hinge (H), WW, cysteine-rich (CR), and carboxyl-terminal (CT) domains are shown. Myoediting of a DMD gene lacking exon 50 restores the ORF beginning at rod domain 20. Rod domains are numbered 2 through 24. (B) Splicing of 79 exons of the human dystrophin gene. Colors correspond to functional domains of the protein in A. Shapes of exons indicate whether splicing between adjacent exons maintains the contiguous ORF of the protein when the shapes fit together like pieces of a puzzle. Reproduced with permission from ref. . The Annual Review of Medicine © 2019 by Annual Reviews.

Fig. 2.

Single-cut gene editing of an exon 50 DMD deletion. Normal DMD gene and mRNA from exon 49 to 53 is shown (Left). An exon 50 DMD mutation is shown (Middle). Splicing of exon 49 to exon 51 places the dystrophin protein out of frame due to a stop codon. Single-cut gene editing (Right) with an sgRNA directed against a sequence within exon 51 can restore dystrophin expression by exon skipping or reframing, depending on the type of INDEL introduced. Dystrophin protein is schematized as a shock absorber.

Fig. 3.

Dystrophin immunostaining in muscle tissues of a canine model of DMD. (Upper) Dystrophin in muscle tissues from wild type. (Middle) Absence of dystrophin in ΔEx50 dog. (Lower) Restoration of dystrophin expression in ΔEx50 dog 8 wk after AAV9 delivery of gene editing components. Adapted from ref. , with permission from AAAS.