Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations

Abstract

Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G > T, both reside in exon 13. Skipping exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2a rmc1 mutants resulted in the production of usherinDexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in induced pluripotent stem cell (iPSC)-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection and induced a detectable level of exon skipping until at least 6 months post-injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13.

Molecular Inversion Probe-Based Sequencing of USH2A Exons and Splice Sites as a Cost-Effective Screening Tool in USH2 and arRP Cases

“…A phase 1/2 clinical trial is ongoing for evaluation of safety and tolerability of antisense oligonucleotides with the aim to induce skipping of exon 13 during pre-mRNA splicing (ClinGov ID: NCT03780257 (https://clinicaltrials.gov/ ct2/show/NCT03780257 accessed on 15 February 2021), consulted on 15 February 2021). This is promising for many subjects as the most common pathogenic USH2A variants affect exon 13; c.2299del (p.(Glu767Serfs*21)) and c.2276G>T(p.(Cys759Phe)) [15]. A similar strategy has been proposed to prevent the inclusion of a frame-disrupting pseudo-exon (PE40) [16,17].…”

Zebrafish as a Model to Evaluate a CRISPR/Cas9-Based Exon Excision Approach as a Future Treatment Option for EYS-Associated Retinitis Pigmentosa

“…The zebrafish is a pre-eminent model to study IRDs as they have a high fecundity, are amenable to genetic manipulation, and have a retinal structure comparable to humans [14]. As previously published for USH2A-associated retinal degeneration, proof of concept of exon-skipping therapy obtained in zebrafish has high translational value [3]. However, due to inter-species differences in the Eys protein and presence of translation regulatory elements in the deleted intronic regions, the effect of AON-induced skipping of exons 37-41 of human EYS on EYS expression and localization could be different.…”

“…Besides splice correction, AONs can also be used for the skipping of native exons that harbor disease-causing mutations, provided that the skipping of the respective exons does not affect the reading frame and that the exon does not encode a domain crucial for protein structure or function. AON-mediated exon skipping has already been shown to have a high therapeutic potential for large genes encoding (structural) proteins that are built up by a series of repetitive protein domains, including usherin and dystrophin [3,40]. The safety and therapeutic efficacy of AON-induced skipping of native exons has been explored most thoroughly for Duchenne muscular dystrophy (caused by mutations in the DMD gene), and two AON-based treatments have recently obtained market approval by the FDA [41][42][43][44][45].…”

“…The safety and therapeutic efficacy of AON-induced skipping of native exons has been explored most thoroughly for Duchenne muscular dystrophy (caused by mutations in the DMD gene), and two AON-based treatments have recently obtained market approval by the FDA [41][42][43][44][45]. In the same way, QR-421a-based skipping of USH2A exon 13 (642 bp) has already been successfully tested in a phase 1/2 clinical trial (#NCT03780257) and two pivotal phase 2/3 clinical trials are currently being planned [3]. In contrast to the previously mentioned DMDand USH2A-related therapeutic exon skipping strategies, the EYS region that we aimed to skip consists of multiple exons.…”

“…The protein-coding sequence exceeds the 4.7 kb packaging capacity of adeno-associated virus (AAV) vehicles, which are the currently preferred vehicles for retinal gene delivery [18]. An alternative therapeutic approach, which has already proven its potential in several (pre-)clinical studies for Usher syndrome, Duchenne muscular dystrophy, and CADASIL [3,19,20], is the antisense oligonucleotide (AON)-based skipping of native, in-frame exons harboring recurrent lossof-function mutations. In this study, we therefore investigated the therapeutic potential of exon skipping as a future treatment option for EYS-associated RP.…”