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.

Therapeutic Application of Extracellular Vesicles-Capsulated Adeno-Associated Virus Vector via nSMase2/Smpd3, Satellite, and Immune Cells in Duchenne Muscular Dystrophy

“…(dystriphin-targeted therapies), such as adeno-associated virus (AAV)-mediated micro/minidystrophin gene delivery, synthetic antisense oligonucleotides for exon skipping, nonsense readthrough, CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9)-mediated genome editing, protein replacement therapies, and primarily utropin; (2) the therapy of downstream pathological changes, such as transplantation of muscular stem cells, corticosteroids (prednisolone or deflazacort) with highly effective in therapy of concomitant destructive processes (namely inflammation) and an improvement in calcium homeostasis, leading to a decrease in oxidative stress in muscle tissue, and in mitochondrial function and biogenesis [45][46][47][48][49][50][51][52][53][54][55] (Figure 1). Currently, gene editing is attracting attention as a therapeutic strategy for DMD because of the restoration of the dystrophin reading frame in more than 40% of all patients with DMD [56][57][58][59][60][61][62][63]. However, it has been reported that clinical trials with this strategy have to be discontinued because of off-target effects that cause chromosomal abnormalities.…”

CRISPR Therapeutics for Duchenne Muscular Dystrophy

“…The N and C termini are essential to maintain proper dystrophin function and membrane integrity. However, the central rod domain contains redundant repeats and thus can be shortened while still maintaining functionality [6]. Such internally truncated forms of dystrophin may be expressed in BMD patients, thereby providing evidence that skipping the associated exons is a promising treatment strategy for DMD patients [13].…”

“…Another challenge associated with AAV-mediated delivery is the risk of AAV integration into DNA DSB sites [104]. Finally, AAV vectors may accumulate in the liver, posing the risk of deleterious side effects or dose-dependent toxicity [6].…”

“…Despite careful sgRNA design, the CRISPR/Cas9 system can tolerate a few mismatches and thus has the potential to generate DSBs at unintended sites. Despite this risk, off-target editing appears to occur at a low rate (<1%) in postmitotic skeletal muscle cells [6]. The risk of mutagenesis can be further minimized by several different strategies.…”

“…Symptoms begin to manifest during early childhood, progressively worsening throughout adolescence with loss of ambulation and a resultant lifespan of only 20-40 years [5]. Although corticosteroids can be administered to aid in symptom management, there is currently no completely curative treatment for DMD [6].…”