AAV-mediated gene augmentation therapy of CRB1-patient derived retinal organoids restores the histological and transcriptional retinal phenotype

Retinitis pigmentosa (RP) and Leber congenital amaurosis are inherited retinal dystrophies caused by mutations in, among others, the Crumbs homologue 1 (CRB1) gene. CRB1 is required for organizing apical-basal polarity and adhesion between photoreceptors and Müller glial cells. Using human CRB1 patient induced pluripotent stem cells from RP patients we derived CRB1 retinal organoids, with diminished expression of variant CRB1 protein with immunohistochemical analysis. Single cell RNA-sequencing revealed impact on, among others, the endosomal pathway and cell adhesion and migration in CRB1 patient derived retinal organoids compared to isogenic controls. Adeno-associated viral (AAV) vector-mediated hCRB2 or hCRB1 gene augmentation in Müller glial and photoreceptor cells partially restored the histological and differentially expressed genes phenotype. Altogether, we show proof-of-concept that AAV.hCRB1 or AAV.hCRB2 treatment improved the phenotype of CRB1 patient derived retinal organoids, providing essential information for future gene therapy approaches for patients with mutations in the CRB1 gene. CRB1 patient-derived retinal organoids were treated with AAV5.CMV.GFP and AAV5.CMV.hCRB1 or AAV5.CMV.hCRB2 were analyzed using scRNAseq at differentiation day 230. Samples were multiplexed using cell hashing oligonucleitode (HTO): each run contains 14 HTO with different organoids (multiple organoid lines, and tratments). In run 1, 2, and 3, CRB1 patient-derived retinal organoids P117, P128 and isogenic control of P128 (ISO-P128) with and without AAV.hCRB treatment were sequenced. In run 4 only P116 and ISO-P116 with and without AAV.hCRB treatment were sequenced.