Nonsense mutations in USH2A exon-13 activate the innate immune response in Müller glial cells

Pathological USH2A mutations cause Usher Syndrome type II, characterized by progressive retinitis pigmentosa and hearing and balance impairment. This study aims to investigate the cellular mechanisms underlying USH2A-related retinal degeneration using human induced pluripotent stem cell (hiPSC)-derived retinal organoids. The introduction of a homozygous nonsense mutation in the USH2A hotspot exon-13 resulted in normal photoreceptor development, but alterations in ciliary length, and loss of ciliary localization of usherin long form B and its interacting proteins, ADGRV1 and whirlin. Notably, single-cell RNA sequencing revealed unexpected significant changes in Müller glial cells (MGCs), with disruptions in the translation, innate immune response, and endolysosomal system. These findings suggest that, while photoreceptor cells are mildly affected by the exon-13 USH2A mutation, MGCs exhibit major dysfunction, potentially contributing to the disease progression and therefore shedding light on potential alternative therapeutic targets. Overall design: Isogenic control (ISO-CTRL) and USH2A KO (13KO) hiPSC-derived retinal organoids were analyzed by scRNAseq at differentiation day 225 in a single run. Samples were multiplexed using cell hashing oligonucleotide (HTO). The run contained 14 HTO. Number of organoids used: ISO-CTRL n=4, 13KO subclone E11 n=4, 13KO subclone G8 n=3, 13KO subclone B7 n=3.