Generation of cell specific transcriptome atlases of Retinitis Pigmentosa

Abstract

Purpose : Retinitis Pigmentosa (RP) is a genetically heterogeneus condition that can be caused by mutations in more than 100 different genes. The final outcome is represented by degeneration of photoreceptor cells although the pathogenic events downstream of the causative mutations are still poorly characterized. To fill this gap of knowledge, we exploited the most recent advances in transcriptome analyses by carrying out single cell transcriptomics in two murine models of RP caused by mutations in different genes at three relevant stages of photoreceptor degeneration.

Methods : We collected optic cups, containing the retina and the retinal pigment epithelium (RPE) by dissecting the cornea and eliminating lens and vitreous. We pooled samples from at least three animals (for an average number of 30.000 cells/dataset). To generate single cell libraries, we used the Papain Dissociation System. Sequencing libraries were generated by using the Illumina 100 cycle sequencing kit and the samples were run on an Illumina NovaSeq 6000 sequencing system. A first pass bioinformatic analysis included normalization, clustering, multiplet filtering, and cell type assignment.

Results : We performed this analysis on two mouse models, namely the transgenic line RHO-P347S that corresponds to an autosomal dominant form of RP and the spontaneous mutant Pde6b^rd10 that corresponds to an autosomal recessive form of RP. We carried out the analysis at three different stages of disease progression, i.e., a preclinical stage, an acute stage (characterized by the peak of rod loss), and post-degeneration (characterized by complete rod loss and ongoing secondary cone degeneration). An initial analysis of the results suggested a decrease of rod photoreceptor viability already at early stages of disease progression.

Conclusions : This project will yield expression atlases for each retinal cell type at critical times of photoreceptor degeneration thus allowing a better dissection of the gene pathways involved in this pathological process. An improved knowledge of the pathways involved in photoreceptor degeneration progression could also be exploited to devise novel neuroprotective, gene/mutation-independent approaches that can potentially be applied to a broader population of RP patients as compared to gene-specific treatments.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.