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Katelyn N Robillard, Sanford Boye, Shannon Boye, Nicolas G Bazan, Jennifer J Lentz; Genetic strategies to treat vision loss in a murine model of Acadian Usher syndrome. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2443.
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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 is to develop genetic strategies for long-term rescue of vision in a mouse model of USH1C. We hypothesize that AAV-mediated gene replacement and/or CRISPR gene-editing products will increase full-length wild type Ush1c expression and restore visual function to a mouse model of USH1C.
An AAV vector that co-expresses Ush1c and GFP reporter was used to treat USH1C mice at different ages (P16, P21, P90). Analysis of transgene expression was performed using confocal scanning laser ophthalmoscopy (cSLO), immunohistochemistry (IHC), and polyacrylamide gel electrophoresis (PAGE). As an alternative approach, CRISPR base editor and nickase systems were designed to target the 216A mutation. Candidate reagents were screened by co-transfection of 293T cells with an USH1C minigene. Editing efficiency was determined by restriction fragment length polymorphism (RFLP) and chromatogram analyses.
cSLO detected AAV-mediated GFP localized to the treatment bleb in USH1C mice at 4-weeks post-treatment. IHC revealed that GFP localized to the outer nuclear layer (ONL) and photoreceptor inner/outer segments (IS/OS). Full-length Ush1c transcripts were also detected by PAGE analysis of retinal tissues harvested 4-weeks post-treatment in USH1C mice. For CRISPR-mediated gene editing, RFLP of the targeted region produced additional bands resulting from 216A>G editing. Sequencing chromatograms also revealed a second G peak at the 216 location with up to 46% editing in sorted 293T cells.
This study demonstrates successful AAV-mediated transgene expression in murine retina and successful CRISPR-mediated gene editing of the human USH1C c.216G>A mutation in cells. Future studies will determine long-term effects of these approaches on retinal structure, function, and visual perception using optical coherence tomography, electroretinography, and a visual cliff assay, respectively.
This is a 2020 ARVO Annual Meeting abstract.
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