<i>USH2A</i> High-Capacity Adenoviral Vector Restores usherin, whirlin, and ADGRV1 in Photoreceptor Cells of Retinal Organoids

Abstract Development of gene therapies for USH2A -associated diseases has been limited by the large size of the USH2A coding sequence, which exceeds the packaging capacity of adeno-associated viral vectors. Here, we evaluated high-capacity adenoviral vectors (HC-AdVs) as vehicles for a mutation-independent USH2A gene supplementation strategy in USH2A human retinal organoids. First, we generated human induced pluripotent stem cell (hiPSC) lines carrying a nonsense mutation in USH2A exon-61 (61KO). Similarly to our previously established exon-13 knockout model (13KO), 61KO hiPSC-derived retinal organoids showed reduced levels of usherin and its interacting proteins, adhesion G protein-coupled receptor V1 (ADGRV1), and whirlin, at the photoreceptor connecting cilium. Delivery of a HC-AdV5 encoding full-length USH2A under the control of the photoreceptor-specific human rhodopsin kinase (hGRK1) promoter restored usherin expression and promoted re-localization of ADGRV1 and whirlin to their physiological sites at the connecting cilium in 13KO and 61KO photoreceptors. Unexpectedly, usherin expression was also observed within mutant Müller glial cells. These findings provide proof-of-concept that HC-AdV5-mediated delivery of full-length USH2A can successfully promote the rescue of usherin and relocate its interacting partners in USH2A -defective retinal organoids, demonstrating its potential as a variant-independent therapeutic approach for inherited retinal disorders caused by USH2A mutations.