CRISPR Therapeutics for Duchenne Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder with a prevalence of approximately 1 in 3500-5000 males. DMD manifests as childhood-onset muscle degeneration, followed by loss of ambulation, cardiomyopathy, and death in early adulthood due to a lack of functional dystrophin protein. Out-of-frame mutations in the dystrophin gene are the most common underlying cause of DMD. Gene editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising therapeutic for DMD, as it can permanently correct DMD mutations and thus restore the reading frame, allowing for the production of functional dystrophin. The specific mechanism of gene editing can vary based on a variety of factors such as the number of cuts generated by CRISPR, the presence of an exogenous DNA template, or the current cell cycle stage. CRISPR-mediated gene editing for DMD has been tested both in vitro and in vivo, with many of these studies discussed herein. Additionally, novel modifications to the CRISPR system such as base or prime editors allow for more precise gene editing. Despite recent advances, limitations remain including delivery efficiency, off-target mutagenesis, and long-term maintenance of dystrophin. Further studies focusing on safety and accuracy of the CRISPR system are necessary prior to clinical translation.

Keywords: CRISPR; Duchenne muscular dystrophy (DMD); NHEJ; dystrophin; exon skipping; gene editing.

Figure 1

Dystrophin protein structure. Key functional domains, from left to right: N-terminal actin-binding domain, central rod domain with 24 repeats and 4 hinge regions, WW and cysteine-rich (CYS) domains (dystroglycan binding site), and carboxy-terminal (CT) domain (dystrobrevin and syntrophin binding sites). Since the rod domain is partially redundant, skipping or removing part of this region is typically well tolerated. Conversely, the N and C termini are essential for proper dystrophin function.

Figure 2

Timeline highlighting the major milestones in CRISPR gene editing for human diseases. Achievements in the field of DMD research are in red.

Figure 3

A schematic of DNA repair systems for gene editing with CRISPR/Cas9 in a hypothetical DMD patient harboring an exon 50 deletion mutation. Deleting exon 50 abolishes the reading frame and leads to a premature stop codon in exon 51, denoted by the “X”. Non-homologous end joining (NHEJ) or homology-directed repair (HDR) can restore the dystrophin reading frame. NHEJ exon skipping (single-cut) targets a splice site, thus skipping the adjacent exon. NHEJ exon deletion (double-cut) requires two sgRNAs flanking the out-of-frame exon(s) for removal. Exon reframing (single-cut) relies on indels generated by NHEJ to reframe the out-of-frame exon. Finally, HDR requires an exogenous DNA template to replace the missing or mutated exon in a precise manner. Scissors represent the sites targeted by CRISPR/Cas9.

Figure 4

Novel developments to the CRISPR/Cas9 system. (A) Precise base editing via dCas9 fused to a cytosine (C:G > T:A) or adenosine (A:T > G:C) deaminase. Base editing could either repair a nonsense mutation, or induce exon skipping by targeting the splice site. (B) Transcriptional activator targeted to the utrophin promoter (UTRN) in an attempt to compensate for the loss of functional dystrophin. (C) Homology-independent targeted integration to knock-in exon 50 in a hypothetical DMD patient lacking exon 50, leading to a frameshift and premature stop codon in exon 51 (denoted by X). A donor plasmid is delivered with the desired exon, flanked by CRISPR/Cas9 cut sites. Cas9 will cleave both the genomic DNA and the donor plasmid, followed by NHEJ. Scissors represent Cas9 cut sites. Abbreviations: dCas9, deactivated Cas9; CD, cytosine deaminase; AD, adenosine deaminase; TA, transcriptional activator; HITI, homology-independent targeted integration; NHEJ, non-homologous end joining.