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Katelyn N Robillard, Marianne Hathaway, Nicolas G Bazan, Jennifer J Lentz; Genetic strategies to treat vision loss in a murine model of Usher syndrome Type 1C. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3407.
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© ARVO (1962-2015); The Authors (2016-present)
Usher syndrome is the leading inherited cause of deaf-blindness with 2.5% of cases attributed to mutations in the USH1C gene encoding harmonin. Antisense oligonucleotides targeting the 216A mutation transiently restore functional harmonin in the cochlea and retina, which correlates with short-term improvements in hearing, vestibular, and visual function in a mouse model of Usher syndrome Type 1C (USH1C). We now aim to develop genetic strategies that mediate long-term improvements in vision in USH1C mice.
Next generation sequencing RNA-Seq identified Ush1c-a1 as the most abundant variant in murine retina up to 1 year of age. Adeno-associated viral (AAV) vectors were designed to co-express the Ush1c-a1 variant with a fluorescent reporter and then validated, in vitro, for transgene expression. USH1C mice were treated at postnatal day 16 by subretinal injection of viral suspension, followed by analysis of transgene expression using fluorescent funduscopy, polyacrylamide gel electrophoresis (PAGE), and immunohistochemistry (IHC). As an alternative approach, plasmids were designed with modified clustered regularly interspaced short palindromic repeats (CRISPR) components and then transfected into cells. CRISPR systems were screened for editing efficiency of the 216A mutation using restriction fragment length polymorphism and sequencing analyses.
Funduscopic detection of viral fluorescence served as the inclusion criterion for prospective analyses. Following RNA isolation, RT-PCR, and cDNA amplification of Ush1c transcripts, PAGE produced multiple bands corresponding to existing mutant transcripts and viral full-length transcripts in treated eyes. IHC also revealed increased harmonin expression in the outer nuclear layer of treated eyes compared to contralateral controls. For genome editing, 3 modified CRISPR endonucleases and 12 corresponding gRNAs were successfully cloned into expression plasmids and tested in cell culture to determine candidate systems for future in vivo studies.
This study demonstrates successful AAV-mediated transgene expression in murine retina and describes the first CRISPR system designed to target the human USH1C c.216G>A mutation. Future studies will determine long-term effects of these approaches on retinal structure, function, and visual perception using electroretinography, optical coherence tomography, and a visual cliff assay.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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