USS-USH2A

Rescuing the common deep intronic USH2A variant c.7595-2144A>G by single gRNA and AAV-packageable enhanced-deletion RNA-guided endonucleases

01.04.2023 bis 31.03.2024

Usher syndrome is an autosomal recessively inherited rare ciliopathy characterized by sensorineural hearing loss and subsequent retinal degeneration (i.e. Retinitis pigmentosa, RP). Among the three clinical Usher subclasses, mutations in the USH2A gene are responsible for ~50% of all Usher and 85% of Usher type 2 cases, respectively, and are also a common cause of isolated non-syndromic RP. Although many mutations in USH2A are private and therefore ultra-rare, important founder mutations have been identified. The deep-intronic variant (DIV) USH2A:c.7595-2144A>G is common and can account for up to ~ 4% of total USH2A alleles, depending on population. This DIV determines the activation of a cryptic splice site, leading to intron (pseudoexon) retention in the ma-ture mRNA transcript. The pseudoexon insertion in the mRNA results in a premature stop codon, leading to the
degradation of the mutant transcript via the nonsense mediated decay pathway. We want to rescue this missplicing phenotype by CRISPR/Cas9-mediated genome editing. We have bioengineered novel Enhanced-Deletion Cas variants – Cas9 variants fused to exonucleases: EDSpCas9 and EDsRGN, able to function by single gRNAs.