Toward a gene-edited nonhuman primate model of Usher syndrome 1B

Abstract

Purpose : Better translational models are urgently needed for many retinal degenerative diseases to facilitate understanding pathogenetic processes and test potential therapies. Usher syndrome presents a particularly compelling need for such models, due to the devastating nature of the disease and the lack of rodent models showing retinal degeneration. Usher 1B is a primary target due to its rapid onset and prevalence among Usher subtypes. Nonhuman primates best mirror human retina anatomy and function by having a macula and fovea, as well as photoreceptor calyceal processes that are a major site of dysfunction in Usher syndrome but are absent in rodents.

Methods : We used CRISPR-Cas9 editing to create rhesus monkey embryos with mutations in exon 3 of MYO7A. Custom guide RNAs and Cas9 mRNA or protein were injected into zygotes 16 hours after in vitro insemination. Trophectoderm biopsies from day 8 blastocysts were genotyped, and those with desired mutations were selected for transfer to surrogate dams. Infant PBMCs, skin and cheek cells were sequenced to confirm infant genotype. Infant audition was assessed by auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE). Retinal structure and function were evaluated by multimodal retinal imaging and ERG.

Results : In the first live infant, genotyping of skin and buccal cells and single-cell sequencing of PBMCs showed a mosaic pattern with half of cells possessing a homozygous single base insertion in exon 3 resulting in a premature stop codon, while half were homozygous for wild type MYO7A. ABR and DPOAE responses were attenuated at one month of age but were normal in subsequent tests through 2 years of age, and normal retinal structure and function were present at all ages. A second infant born in November 2021 showed a compound heterozygote pattern with 1bp and 63bp deletions in exon 3. Auditory testing at 4 weeks of age showed no detectable ABR or DPOAE responses. Retinal imaging at 6 weeks detected no significant abnormalities. Development of the auditory and retinal phenotypes will be followed longitudinally.

Conclusions : This study showed the ability to induce mutations in the MYO7A gene in rhesus macaques, resulting in absence of auditory function in early infancy. Continued confirmation of an USH1B phenotype will set the stage for studies of gene therapy in this first gene-edited nonhuman primate model of Usher syndrome.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.