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 loss of ciliary localization of usherin long form B and its interacting proteins, ADGRV1 and whirlin. Notably, single-cell RNA sequencing revealed unexpected significant transcriptional changes in Müller glial cells (MGCs), suggestive of 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 transcriptional changes, potentially contributing to the disease progression and therefore shedding light on potential alternative therapeutic targets.

Pathological USH2A mutations cause Usher Syndrome type II, characterized by progres-sive retinitis pigmentosa and hearing and balance impairment. This study aims to inves-tigate the cellular mechanisms underlying USH2A-related retinal degeneration using hu-man 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 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 there-fore shedding light on potential alternative therapeutic targets.

Loss-of-function mutations in the ABCA4 gene cause Stargardt disease (STGD1), the most common inherited macular dystrophy leading to progressive central vision loss. Here, we generated hiPSC-derived retinal organoids harboring a premature stop codon in exon-24 of ABCA4 to evaluate the impact of this mutation on mRNA and protein levels in a human model. Immunofluorescence analysis revealed the absence of ABCA4 protein in the mutant photoreceptor outer segment discs, while single-cell RNA sequencing detected no major transcriptional alterations in rods and cones. Unexpectedly, differential gene expression and pathway enrichment analyses of Müller glial cells (MGCs) and astrocytes highlighted disruption of neuronal development, microenvironment of glial cells, intercellular communication, and programmed cell death pathways. These findings suggest that ABCA4 might play a role in maintaining the retinal microenvironment homeostasis, and that the early transcriptomic response of MGCs and astrocytes preceding photoreceptor degeneration could contribute to Stargardt disease development. Significance Statement: Human induced pluripotent stem cell (hiPSC)-derived retinal organoids provide a powerful platform to investigate inherited retinal diseases. In this study, we generated ABCA4 -mutant hiPSC lines and differentiated them into retinal organoids to model Stargardt disease. Despite complete loss of ABCA4 protein from the photoreceptor outer segment discs, rods and cones exhibited minimal transcriptional alterations. In contrast, the ABCA4 variant triggered changes in the glial cell homeostasis, suggesting that Müller glial cells and astrocytes might exhibit an early response to photoreceptor dysfunction in the absence of ABCA4. Abstract Figure: Human induced pluripotent stem cells (hiPSCs) were engineered to generate ABCA4 -mutant cell lines, later differentiated into retinal organoids as a model of Stargardt disease. The organoids showed loss of ABCA4 from the outer segment discs of rod and cone photoreceptors, while the mutant Müller glial cells and astrocytes exhibited transcriptional changes in pathways involved in neuronal development, microenvironment, and programmed cell death.

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.