Skipping Multiple Exons to Treat DMD-Promises and Challenges

Abstract

Duchenne muscular dystrophy (DMD) is a lethal disorder caused by mutations in the DMD gene. Antisense-mediated exon-skipping is a promising therapeutic strategy that makes use of synthetic nucleic acids to skip frame-disrupting exon(s) and allows for short but functional protein expression by restoring the reading frame. In 2016, the U.S. Food and Drug Administration (FDA) approved eteplirsen, which skips DMD exon 51 and is applicable to approximately 13% of DMD patients. Multiple exon skipping, which is theoretically applicable to 80-90% of DMD patients in total, have been demonstrated in animal models, including dystrophic mice and dogs, using cocktail antisense oligonucleotides (AOs). Although promising, current drug approval systems pose challenges for the use of a cocktail AO. For example, both exons 6 and 8 need to be skipped to restore the reading frame in dystrophic dogs. Therefore, the cocktail of AOs targeting these exons has a combined therapeutic effect and each AO does not have a therapeutic effect by itself. The current drug approval system is not designed to evaluate such circumstances, which are completely different from cocktail drug approaches in other fields. Significant changes are needed in the drug approval process to promote the cocktail AO approach.

Keywords: Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9 (CRISPR/Cas9)-mediated genome editing; Duchenne/Becker muscular dystrophy (DMD/BMD); actin binding domain (ABD); antisense oligonucleotides (AOs); canine X-linked muscular dystrophy (CXMD); eteplirsen; golden retriever muscular dystrophy (GRMD); golodirsen; morpholino); multi-exon skipping; phosphorodiamidate morpholino oligomer (PMO.

Figure 1

Structures of PMOs and conjugates.

Figure 2

Mutation pattern of the CXMD/golden retriever muscular dystrophy (GRMD) dog models and exons 6–8 skipping strategy using AOs. (A) A point mutation in the acceptor splice site (ASS) in intron 6 in CXMD dogs leads to exon 7 being skipped from the dystrophic dog mRNA; (B) AOs are designed such that they bind to exons 6 and 8 causing them to be spliced out thereby correcting the reading frame. Exon 9 is spliced spontaneously along with exons 6 and 8.

Figure 3

CRISPR/Cas9-mediated gene editing for creating dystrophin mRNA with a corrected ORF by removing the premature stop codon due to C to T transition shown in red. The dual vector approach targets introns 51 and 53 to direct the excision of exons 52 and 53. The arrows depict the sgRNA target sites in the intronic region shown in 5′-3′ direction based on target strand.