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Reactivating the phototransduction cascade with a mutation agnostic gene therapy preserves vision in Rod-Cone Dystrophies

ABSTRACT: Rod-cone dystrophy (RCD), or retinitis pigmentosa, involves genetic conditions where rod degeneration precedes cone degeneration, resulting in tunnel vision and eventual blindness. Previous studies attempted to restore cone activity and prolong vision by expressing microbial chloride pumps in degenerating cone photoreceptors. However, microbial opsins require high expression and intense light, limiting their effectiveness. In this work, we explored the phototransduction cascade in degenerating cones in two RCD mouse models. We observed that opsin and arrestin expression persists in the soma during outer segment degeneration. This led us to hypothesize that reactivating cones based on cone opsin signaling may be possible by expressing a target channel activated by G proteins recruited by cone opsin. Through adeno-associated viral (AAV) mediated expression of the G protein-coupled inwardly rectifying K (GIRK) channel, we achieved improvements in visual function in two RCD mouse models with mutations in different genes. Importantly, we validated the rationale of GIRK-mediated gene therapy in humans by confirming cone opsin and cone arrestin expression in cones of late-stage RCD patients. Our proposed approach involves GIRK channel expression in cones as a novel method to maintain high acuity, sensitivity, and color vision in RCD, independent of the underlying mutation.

ORGANISM(S): Mus musculus

PROVIDER: GSE286529 | GEO | 2025/01/13

REPOSITORIES: GEO

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Project description:The loss of cone photoreceptor cells, which are critical for optimal daylight vision, have the great impact on vision during retinal degenerations. Retinal differentiation of human induced pluripotent stem cell (hiPSC) sources could provide a renewable source of cone photoreceptors towards developing a cone cell replacement therapy to treat blindness. Demonstration of comparable gene expression profiles between human foetal and stem cell-derived cones at equivalent stages is required to progress the cell transplantation approach into the patient, as it is hypothesised stem cell-derived cones are required to show high levels of developmental recapitulation of the in vivo generated cones. In this study, the AAV2/9.pR2.1.GFP reporter was used to specifically label L/M-opsin cone photoreceptors in human foetal retinal samples, obtained from the MRC-Wellcome Trust Human Developmental Biology Resource, at a range of developmental stages. The L/M-opsin cone population represent the majority cone cell types in the adult human retina and are the only photoreceptors present within the fovea. Using fluorescence activated cell sorting, L/M-opsin GFP+ cones and GFP- retinal populations, alongside total foetal retinal samples containing all retinal cell tytpes, were isolated and processed for bulk RNA sequencing and downstream comparative analysis. Using DESeq2 differential gene expression analyses, statistically significant genes enriched within the GFP+ human foetal LM-opsin cone populations were determined which led to the identification of a cone enriched gene signature of human L/M-opsin cone photoreceptors. The AAV2/9.pR2.1.GFP reporter was applied to hiPSC-derived retinal cultures to isolate and process cone-like cell populations for RNA sequencing using the same strategy developed within the human foetal retina. Applying the cone enriched gene signature to the transcriptome of hiPSC-derived GFP+ samples at equivalent developmental stages revealed some expression similarities in genes found to be enriched within the late foetal L/M-opsin cone photoreceptors. This analysis overall revealed an intermediate stage of cone differentiation was achieved within the hiPSC-derived samples and the comparison to human foetal L/M-opsin gene express profiles suggesting further differentiation of hiPSC-derived sample is required.

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Reactivating the phototransduction cascade with a mutation agnostic gene therapy preserves vision in Rod-Cone Dystrophies

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