Investigation of Base-Editing-Mediated Exon Skipping as a Potential Gene Therapy for Usher Syndrome

Abstract

Purpose: Usher syndrome (USH) is an autosomal recessive form of retinitis pigmentosa (RP) and the majority of cases are caused by mutations in USH2A. Traditional gene replacement therapy cannot be applied to treat USH2A as the 15.6kb coding sequence of USH2A far exceeds the packaging capacity of commonly used adeno-associated virus (AAV) vectors. Previous studies have shown that in large proteins with repetitive domains, skipping of mutant exons that encode repetitive domains and splicing of the remaining adjacent exons that are in-frame lead to truncated proteins that retain full or partial functions. In this preliminary study, we investigated if a CRISPR base editing (BE) approach can be applied to edit USH2A splice sites in vitro to skip exons carrying disease-associated mutations to develop potential treatment for USH2A-associated retinal degeneration. Methods: Exons that harbor pathogenic USH2A mutations and whose neighboring exons are in-frame with each other were first selected. USH2A constructs that skip selected individual exon were cloned and the functions of the resulting truncated proteins were tested in in vitro culture of immortalized mouse organ of Corti-derived epithelia cell line, OC-k1. To investigate the application of CRISPR base editing approach to skip exons, human retinoblastoma cell line, WERi-Rb cells, were transfected with CRISPR base editor and gRNA plasmids targeting the splice sites upstream of the exons intended to be skipped. Transfected cells were FACS sorted, genomic DNAs were extracted, sequenced, and analyzed for base editing. RNA extraction and RT-PCR were performed to characterize exon skipping. Results: We cloned hUSH2A constructs that skip exon 24, 28, 56 or 62 and will assess their functionality using Ush2a null OC-k1 cells that exhibited severe impairment of ciliogenesis. We identified base editor and gRNA pairs that can edit splice acceptor sites of exon 24, 28, 56 and 62 of USH2A gene. Conclusions: Targeted exon-skipped hUSH2A constructs were generated for analysis of truncated USH2A protein function. CRISPR base editing approach could be applied to edit splice sites on USH2A. Further experiments will be needed to determine if alteration of USH2A splice sites lead to exon skipping and if skipping of exon 24, 28, 56 or 62 produces functional truncated protein products.