Pre-clinical dose-escalation studies establish a therapeutic range for U7snRNA-mediated DMD exon 2 skipping

Tabatha R Simmons,Tatyana A Vetter,Nianyuan Huang,Adeline Vulin-Chaffiol,Nicolas Wein,Kevin M Flanigan

doi:10.1016/j.omtm.2021.03.014

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked progressive disease characterized by loss of dystrophin protein that typically results from truncating mutations in the DMD gene. Current exon-skipping therapies have sought to treat deletion mutations that abolish an open reading frame (ORF) by skipping an adjacent exon, in order to restore an ORF that allows translation of an internally deleted yet partially functional protein, as is seen with many patients with the milder Becker muscular dystrophy (BMD) phenotype. In contrast to that approach, skipping of one copy of a duplicated exon would be expected to result in a full-length transcript and production of a wild-type protein. We have developed an adeno-associated virus (AAV)-based U7snRNA exon-skipping approach directed toward exon 2, duplications of which represent 10% of all DMD duplication mutations. Deletion of exon 2 results in utilization of an exon 5 internal ribosome entry site (IRES) that allows translation beginning in exon 6 of a highly protective dystrophin protein, providing a wide therapeutic window for treatment. Both intramuscular and systemic administration of this vector in the Dup2 mouse model results in robust dystrophin expression and correction of muscle physiologic defects, allowing dose escalation to establish a putative minimal efficacious dose for a human clinical trial.

Highlights

The X-linked Duchenne muscular dystrophy (DMD) gene, which encodes the dystrophin protein, consists of 79 exons and at least 7 promoters
The difference between the Dup[2] and the deletion of exon 2 (D2)-associated phenotypes was explained by the identification of an internal ribosome entry site (IRES) within the DMD exon 5, which allows for cap-independent translational initiation in the case of an exon 2 deletion but is nonfunctional in the presence of an exon 2 duplication.[8]
Translation from this IRES results in the expression of a dystrophin isoform missing the calponin-homology 1 (CH1) domain within the N-terminal actin binding domain 1 (ABD1), but which is highly functional; expression of the same isoform is found in patients across North America carrying the DMD p.Trp3X founder allele, resulting in mildly symptomatic Becker muscular dystrophy (BMD) with ambulation into the seventh or eighth decade.[9]

Summary

INTRODUCTION

The X-linked DMD gene, which encodes the dystrophin protein, consists of 79 exons and at least 7 promoters. Differing from the case with skipping for deletion mutations, which results in creation of a BMD-like internally truncated transcript, skipping a single copy of exon 2 would restore an entirely normal mRNA transcript Skipping of both copies of exon 2 results in a stable transcript subject to translation, via the exon 5 IRES, of a dystrophin isoform that is highly stable, as demonstrated by multiple patients with an exon 1 nonsense mutation who express the same isoform with symptoms of only myalgias and hyperCKemia.[9] To test this as a potential therapeutic approach, we developed a mouse model with a duplication of exon 2 (Dup2).[10] In this mouse dystrophin expression is essentially absent, and pathologic features and physiologic defects are essentially identical to the common mdx mouse model, which carries a nonsense mutation in exon 23 (rendering it unsuitable for testing exon 2 skipping therapy). In contrast to exon skipping using a phosphorodiamidate morpholino oligomer (PMO),[6,11] we designed modified U7snRNAs with terminal antisense oligonucleotide sequences that target exon 2 definition

RESULTS

DISCUSSION

Findings

MATERIALS AND METHODS