Exon-Skipping in Duchenne Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a devastating, rare disease. While clinically described in the 19th century, the genetic foundation of DMD was not discovered until more than 100 years later. This genetic understanding opened the door to the development of genetic treatments for DMD. Over the course of the last 30 years, the research that supports this development has moved into the realm of clinical trials and regulatory drug approvals. Exon skipping to therapeutically restore the frame of an out-of-frame dystrophin mutation has taken center stage in drug development for DMD. The research reviewed here focuses on the clinical development of exon skipping for the treatment of DMD. In addition to the generation of clinical treatments that are being used for patient care, this research sets the stage for future therapeutic development with a focus on increasing efficacy while providing safety and addressing the multi-systemic aspects of DMD.

Fig. 1

Schematic of the DMD gene and exon skipping. Panel A: A schematic of the DMD gene from the Genome Browser (genome.ucsc.edu) at Xp21 with encoded mRNA transcripts is shown. Gene transcription is shown from left to right, with the three gene promoters driving expression of the full-length 427 kDa dystrophin (Dp427B, Dp427M, Dp427P), as well as down-stream gene promoters driving smaller molecular weight dystrophin proteins (Dp260, Dp140, Dp116, Dp71). Also shown is an expansion of exons 52, 53, and 54. The amino acids and encoding triplet codons are provided at the ends of each of these exons. Exon 52 ends in an incomplete codon for isoleucine (I-2554), requiring the last two bases from exon 53 to complete the codon. In contrast, exon 53 ends with a complete codon for lysine (K-2624), splicing to exon 54 that starts with a complete codon for glutamine (Q-2625). A gene mutation deleting exon 53 would then be out-of-frame, as an incomplete codon ending exon 52 would be fused to a complete codon on exon 54, leading to a frame shift in the resulting dystrophin mRNA. Panel B: This shows the consequence of drug-induced exon skipping by viltolarsen targeted to exon 53. A boys with DMD is shown as having a deletion mutation of exon 52, and when this patient’s dystrophin mRNA splices together the remaining exons (exon 51 to exon 52), this leads to a frame shift, mRNA out-of-frame, and no dystrophin protein. Viltolarsen binds to exon 53, and blocks its inclusion in the dystrophin mRNA. The drug-induced splicing of exon 51 to exon 54 results in an in-frame dystrophin mRNA, and Becker-like dystrophin protein.

Fig. 2

Lead candidate selection for exon 53 exon skipping. Panel A: Shown is a schematic of the 38 oligonucleotides tested for strength in blocking exon 53 splicing, and the experimental approach leading to lead compound selection (NS-065/NCNP-01; viltolarsen). Panel B: Dose-response analyses shows NS-065/NCNP-01 (viltolarsen) to achieve ∼70% exon skipping efficiency in cell cultures. From Watanabe et al. 2018 [62].

Fig. 3

Example of an RNA blot showing viltolarsen-induced exon skipping in DMD participant muscle. From Clemens et al. 2020 [40].