Abstract
Purpose :
To describe a novel no b-wave (nob) mouse with a naturally occurring Grm6 variant, leading to compromised visual transmission between photoreceptors and bipolar cells. Our objective was to enhance protein localization and function in this novel nob mouse by targeting specifically ON-bipolar cells or the entire retina.
Methods :
An initial discovery of a nob female mouse led to outbreeding with a male C57BL6J (wild type) mouse, and subsequent interbreeding of their offspring was conducted to regenerate the nob phenotype and facilitate gene discovery. Adeno-associated viruses encoding Grm6 under two different promoters (hGRM6-Grm6 and CMV-Grm6) were injected subretinally and intravitreally in our novel nob mice at P4 and P29. Electroretinogram (ERG) recordings were performed three months after gene therapy in Grm6 mutation mice. Correspondingly, immunolocalization and western blot studies were performed on retinal slices before and after treatment using antibodies against mGluR6, TRPM1, GRIK1, and PKCα.
Results :
The nob phenotypes from the founder mouse were successfully regenerated. Whole exome sequencing revealed a homozygous Grm6 missense variant. The historical appearance of the nob mice appeared normal without photoreceptor degeneration. There was no significant difference in the scotopic “a wave” amplitudes between wild type, Grm6 heterozygous, and Grm6 homozygous mice at P45, indicating that photoreceptor function was not affected. Flicker ERG confirmed the dysfunction of ON-bipolar cells, while the cone OFF-bipolar cells were not affected. Following gene therapy, in vivo fundus autofluorescence images of the retina displayed a well-distributed GFP signal, and retina cryosection demonstrated successful labeling of bipolar cells with GFP driven by hGRM6 promoter. The findings from functional ERG, immunolocalization, and western blot studies post-gene therapy will be presented at the conference.
Conclusions :
We identified a novel naturally occurring nob mouse with Grm6 variant, resembling in every respect examined the complete form of Congenital Stationary Night Blindness (CSNB) in humans. The outcomes of gene therapy are contingent on various factors that require careful consideration before advancing to human clinical trials.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.