Abstract
Purpose :
Usher syndrome Type 1C (USH1C) is an inherited deaf-blinding disease that affects Acadian populations in the United States and Canada. Specifically, the USH1C c.216G>A mutation encodes a truncated harmonin protein that disrupts photoreceptor and hair cell function. The goal of this study was to develop genetic approaches for long-term vision improvements in a murine model of USH1C. We hypothesized that AAV-mediated gene therapy and gene-editing approaches would increase full-length Ush1c expression and restore visual function in USH1C mice.
Methods :
An AAV vector co-expressing Ush1c-a1 and GFP was delivered via subretinal injection to USH1C mice at different postnatal (P) ages (P17, P22, P98). Transgene expression was determined by confocal scanning laser ophthalmoscopy (cSLO), immunohistochemistry (IHC), and polyacrylamide gel electrophoresis (PAGE). Changes in retinal structure, function, and visual behavior were assessed using optical coherence tomography (OCT), electroretinography (ERG), and a visual cliff assay, respectively. As an alternative approach, multiple gene-editing systems targeting the 216A mutation were developed and introduced into 293T cells alongside an USH1C minigene or into USH1C murine fibroblasts. Editing efficiency was determined by restriction fragment length polymorphism (RFLP) and sequencing analyses.
Results :
AAV-treated retinas showed increased full-length Ush1c mRNA and GFP fluorescence localized to the outer nuclear layer (ONL) and photoreceptor inner/outer segments (IS/OS) up to 6 months of age. Although cSLO showed GFP in approximately 20% of the fundus, these molecular changes did not correlate with improvements in retinal structure, function, or visual behavior. Multiple combinations of gene-editing plasmids yielded high rates of on-target editing (up to 46% in sorted 293T cells) with minimal off-target effects in the protospacer region.
Conclusions :
This study demonstrated successful AAV-mediated transgene expression in USH1C murine retina and successful gene editing of the human USH1C c.216G>A mutation, in vitro. Future studies will modify viral dose, Ush1c variant, gene promoter, and capsid structure to optimize long-term improvements in retinal structure, function, and visual behavior. We are currently optimizing the transfection protocol for murine fibroblasts to determine editing efficiency at the genomic 216A site.
This is a 2021 ARVO Annual Meeting abstract.