Subretinal injection in the USH1CR31∗ pig model leads to chorioretinal atrophy that limits evaluation of efficacy of an AAV-mediated gene therapy.

Usher syndrome (USH) is the most common form of hereditary deaf-blindness. There is no effective treatment for vision loss to date. Here we aimed to evaluate AAV-mediated ocular gene therapy for USH1C in pigs. USH1CR31∗ and wild type (WT) pigs were bilaterally subretinal injected with 200 μL of 1∗1011 vg/eye Anc80 AAV vectors expressing USH1C_a1 under the control of either a rod-specific human rhodopsin kinase (GRK1) promoter, a ubiquitous promoter (CAG), or vehicle control. Retinal structure and function were assessed by optical coherence tomography and electroretinography (ERG) at baseline and every six months post-injection. Decreased ERG amplitudes and reduction of total retinal thickness were observed in all groups following surgery. At 18 months post-injection, animals were sacrificed and treated and untreated retinal regions were analyzed by qRT-PCR, Western blots, pathohistological examinations, and immunohistochemistry. Both vectors resulted in high levels of USH1C_a1 transcript expression, but only slightly increased harmonin protein expression in USH1CR31∗ pigs. Across all treatment groups, chorioretinal atrophy (CRA) was observed, accompanied by reduced retinal thickness and increased GFAP expression. In conclusion, both vectors achieved efficient retinal transgene delivery in USH1CR31∗ pigs. However, CRA developed in all groups, even in vehicle-injected controls. The extensive retinal damage associated with CRA has likely masked a potentially beneficial therapeutic effect. For future translational development, optimization of dosing, and modifications in surgical technique will be of crucial importance.