Abstract
Purpose :
Usher Syndrome is an autosomal recessive retinal degeneration associated with deafness. Usher type IIA is caused by mutations in the USH2A gene which encodes the protein usherin, which normally localises to the photoreceptor cilium and cochlear hair cells. CRISPR-mediated base editing is a potential treatment approach for 37.3% of USH2A mutations caused by single base transitions. USH2A c.11864G>A (p.W3955X) accounts for 3-5% of pathogenic alleles worldwide, and is potentially correctable with base editing. We developed an Ush2a knockout mouse model containing a premature termination codon in the homologous location of the murine Ush2a transcript (c.11840G>A; p.W3947X). We investigate the phenotype of this mouse as a model for testing of base editing therapeutics for Usher Syndrome.
Methods :
Ush2a-W3947X mice were generated using a homology directed repair knock-in strategy, with a single-stranded DNA template, gRNA and SpCas9 mRNA delivered to zygotes via pro-nuclear injection. Mice were bred on a C57BL/6J background with a repaired Cdh23 age-related hearing loss allele. Homozygous mice were evaluated at 3-, 6-, 9- and 12-months with OCT and ERG. Auditory brainstem response (ABR) threshold testing was performed at 9 weeks. Immunostaining of retinal and cochlear sections and western blot of retinal protein lysates were performed.
Results :
Heterozygous Ush2a-W3947X mice were successfully generated with successful insertion of a single copy of the donor confirmed by Sanger sequencing and a ddPCR copy counting assay. Usherin was not detected in immunostaining of retinal or cochlear sections of homozygous W3947X animals, or by western blot of retinal lysates. ABR thresholds were significantly elevated at higher frequencies (8-24kHz). No significant differences were seen in photoreceptor or total retinal thickness on OCT imaging relative to wildtype mice. No differences between dark-adapted, light adapted or flicker responses were seen with ERG testing between mutant and wildtype mice.
Conclusions :
The Ush2a-W3947X mouse lacks usherin expression in the retina and cochlea, and displays an early hearing loss phenotype. Ongoing analysis with age will determine if a slow retinal degenerative phenotype is present similar to other Ush2a-/-models. This model is useful for testing of base editing strategies for Usher syndrome.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.