Usher syndrome (Usher) is the leading inherited cause of combined deafness and blindness, affecting as many as 1 in 6000 in the general U.S. population. Type 1 Usher, the most severe form, is characterized by vestibular dysfunction and profound hearing impairment at birth followed by retinitis pigmentosa (RP) beginning in early adolescence. Worldwide, 2.5% of Usher cases are caused by mutations in the USH1C gene, which encodes harmonin. Previously, our laboratory created a knock-in mouse model containing the USH1C c.216G>A splicing mutation (216A) which is responsible for Usher Type 1C (USH1C) in the Acadian populations in the U.S. and Canada. Antisense oligonucleotides (ASOs) designed to target the 216A mutation block the aberrant splice site and improve Ush1c RNA splicing. Treatment of USH1C mice with ASOs improves the expression of full-length harmonin protein in the cochlea and retina with an increase in vestibular, auditory, and visual function comparable to wild type mice. These improvements are sustained for at least 1 year with additional treatments. Despite improvements, the underlying pathophysiological mechanisms by which the mutant Ush1c gene contributes to these clinical phenotypes, along with the mechanisms by which rescue of the mutant phenotype reverses visual impairment, remain largely unknown. Therefore, we hypothesize that RP functional impairments and underlying retinal defects associated with the USH1C c.216G>A mutation are caused by the loss of functional harmonin. This hypothesis will be tested in two Specific Aims: (1) test the prediction that Ush1c gene replacement therapy will restore harmonin levels in photoreceptor cells and correct RP in USH1C mice; and (2) test the prediction that genome editing of the 216A mutation via clustered regularly- interspaced short palindromic repeats (CRISPR)/Cas9 system will restore endogenous expression of harmonin in photoreceptor cells and correct RP in USH1C mice. Transgenic USH1C mice will be treated by subretinal injections of either Ush1c gene-expressing viral vectors or CRISPR/Cas9 genome-editing system at different ages. Ush1c transcript and harmonin protein expression will be measured using RT-PCR and western blot analysis, respectively. Optical coherence tomography (OCT) will be used to assess retinal integrity before and after treatments. Harmonin protein expression and localization in the retina will be measured by immunohistochemistry. Visual function will be assessed using electroretinography (ERG) analysis, and gross visual ability will be assessed using a visual cliff behavioral test. The research completed in both Aims will contribute to the development of a novel, one-time treatment regimen for correcting RP in USH1C. Molecular and structural analyses completed through the proposed research project will provide insight into loss or gain of function mechanisms caused by the USH1C c.216G>A mutation, which will help us to achieve our long-term goal of developing therapeutic strategies for translation into future clinical trials. Our proximity to the Acadian population gives us the unique advantage of developing treatments for local patients who are losing their vision.
Usher syndrome is the leading genetic cause of combined deafness and blindness in the United States, with a high prevalence in Acadian populations of South Louisiana in the United States and in Canada. Our work aims to develop a one-time gene therapy approach that will restore expression and function of harmonin, the primary mutated protein in Usher syndrome Type 1C, for a safe and effective treatment regimen to be used in future clinical trials.
