Gene Therapy Studies for USH3: Characterization of the Knockout Mouse Retina and Search for an Efficient AAV Vector

Abstract

Purpose: : Usher syndrome type 3 (USH3) is an autosomal recessive disorder characterized by retinitis pigmentosa and progressive sensorineural deafness. USH3 is caused by mutations in the CLRN1 gene.The recently generated Clrn1 knockout (ko) mice have no histological or functional retinal abnormalities when studied by conventional methods, i.e. ERG, whereas they do have progressive hearing loss. The aims of this study were (1) to perform ERG on isolated Clrn1-ko-retina in order to detect possible subtle ERG abnormalities and (2) to search for suitable AAV vectors for delivery of wt Clrn1 into the Clrn1-ko-cochlea.

Methods: : Photoreceptor responses were measured with aspartate-isolated ERG from wt and Clrn1-ko mouse isolated retina. Transduction efficiencies of recombinant AAV2/1, 2/2, 2/5 and 2/8 vectors encoding GFP marker or HA-tagged CLRN1 were studied in vitro in HEK-293 cells and mouse cochlear tissue cultures. AAV2/1 and 2/2 encoding GFP were also studied in vivo in wt mice by microinjection of vector constructs to cochlea.

Results: : The ERGs of P80 Clrn1-ko mouse isolated retinas showed no abnormalities. AAV2/1 and 2/2 transduced HEK-293 cells more efficiently than AAV2/5 and 2/8. Transduction efficiency of self-complementary AAV2/2 was significantly higher than that of conventional AAV2/2. AAV2/1 and 2/2-mediated GFP expression was present in the organ of Corti of wt mouse 6 days after cochlear injection.

Conclusions: : Vector-based gene therapy is proven to be a suitable method for treating retinal dystrophies. We found no retinal phenotype in the Clrn1-ko mouse. Therefore we plan to test Clrn1-replacement in the Clrn1-ko mouse cochlea. Positive results may indicate that CLRN1 replacement in USH3 patients’ retina could be therapeutic.

Keywords: gene transfer/gene therapy