Abstract
Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken β-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder.
Highlights
Usher syndrome (USH) is an autosomal recessively inherited disorder responsible for more than half of combined deafness and blindness in humans [1, 2]
An AAV2 quad YF vector carrying a CLRN1-Venus transgene cDNA driven by a smCBA promoter was delivered to adult wild-type mouse eyes via either the subretinal or intravitreal route in order to examine the pattern of CLRN1 distribution by direct fluorescence imaging
CLRN1 was originally suggested to have a synaptic role based on its sequence homology with stargazin, a tetraspanin, which regulates the synaptic targeting of glutamate receptors in the brain [9]
Summary
Usher syndrome (USH) is an autosomal recessively inherited disorder responsible for more than half of combined deafness and blindness in humans [1, 2]. It represents a group of clinically and genetically heterogeneous disorders, classified into three major types, USH1, USH2, and USH3, depending on the onset and severity of the symptoms, and the presence or absence of vestibular function [3,4,5,6]. ES is the recipient of a grant from Macula Vision Research Foundation (MVRF), USA
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