Pre-clinical study of RM-101, a novel AAV-based gene therapy for USH2A-related Retinitis Pigmentosa

Abstract

Purpose : Mutations in the USH2A gene are the leading cause of autosomal recessive retinitis pigmentosa and Usher syndrome, with exon 13 mutations being the most common. Previous studies have demonstrated that the removal of exon 13 from the USH2A gene can lead to the production of a functional protein, suggesting that exon skipping could be a potential treatment for retinal degeneration associated with USH2A mutations. Here, we reported RM-101, a novel strategy based on adeno-associated virus (AAV) for exon skipping, aimed at treating retinal degeneration caused by USH2A exon 13 mutations.

Methods : We developed a U7 SmOPT snRNA that specifically targets USH2A exon 13 and encapsulated it into an AAV delivery system (RM-101). The safety and efficacy of RM-101 were evaluated in retinal organoids (ROs), USH2A humanized mice and cynomolgus macaques. The ROs used in our study contained a biallelic c.2299delG mutation in the USH2A gene, along with a flag tag inserted before the stop codon. The USH2A humanized mouse model was created by replacing the mouse Ush2a exon 12 with a human USH2A exon 13 carrying the c.2802T>G mutation.

Results : In wild-type ROs treated with RM-101, the skipping of exon 13 in USH2A transcripts was 52.8% ± 12.5%, while no exon 13 skipping was observed in control samples (Fig. 1A). RNA-Seq analysis confirmed that RM-101 specifically induced USH2A exon 13 skipping and no off-target splicing events were detected. Immunohistochemistry demonstrated that RM-101-treated ROs restored Usherin expression with correct subcellular localization (Fig. 1B). In mice treated with RM-101, there was a significant increase in USH2A exon 13 skipping in a dose-dependent manner (Fig. 1C) and sustained efficacy over time (Fig. 1D). A single subretinal administration of RM-101 in monkeys reasulted in 59.8% skipping of exon 12 within the cells of the retina area directly exposed to RM-101 and 24.3% in entire retina (Fig. 1E). Slit-lamp microscope and optical coherence tomography examinations did not reveal any significant abnormalities in these animals.

Conclusions : Our results demonstrate that RM-101 can effectively induce USH2A exon 13 skipping both in vitro and in vivo, and it has an acceptable safety profile. These preclinical studies provide support for RM-101 as a potential gene therapy for the treatment of USH2A-related retinal degeneration.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.