RNA-seq analysis of USH2A -mutant organoids reveals distinct gene expression signatures in retinitis pigmentosa and Usher syndrome

Submission 43

RNA-seq analysis of USH2A-mutant organoids reveals distinct gene expression signatures in retinitis pigmentosa and Usher syndrome

PS2-34-Poster Presentation

Presented by: Suly Villa-Vasquez

Suly Villa-Vasquez ¹, Nejla Erkilic ^{1, 2}, Carla Sanjurjo-Soriano ¹, Carla Jimenez-Medina ¹, Isabelle Meunier ^{1, 2}, Jerome Roger ³, Vasiliki Kalatzis ^{1, 2}

¹ Institute of Neurosciences of Montpellier, Inserm U1298, University of Montpellier, France

² Maolya, National Reference Centre for Inherited Sensory Diseases, University of Montpellier, CHU, France

³ Paris-Saclay Institute of Neuroscience, CERTO-Retina France, CNRS, Université Paris-Saclay, Saclay, France

Objective:

Variants in USH2A account for up to 20% of cases of isolated autosomal recessive retinitis pigmentosa (RP) and about 85% of Usher syndrome (USH) type 2, with a prevalence of 1:4000 and 1:6000, respectively. However, the exact function of USH2A and the molecular mechanisms by which specific mutations lead to non-syndromic or syndromic phenotypes remain poorly understood. This study investigated the effects of USH2A mutations associated with either RP or USH, which associates RP and hearing loss, in patient-specific iPSC-derived retinal organoids, to identify transcriptomic differences between RP and USH-linked mutations, and to gain insight into the pathophysiological mechanisms underlying the development of differential phenotypes.

Methods:

Retinal organoids were differentiated from control iPSCs, patient-derived iPSCs harbouring mutations in USH2A associated with RP or USH, and their CRISPR-corrected isogenic controls. These organoids were subsequently analysed using bulk mRNA sequencing, differential gene expression and enrichment analysis.

Results:

Previous analyses showed inner and outer segment (IS/OS) defects in USH2A-mutant organoids, which were more severe in RP than in USH. These comprised reduced brush border lengths due to defective elongation of OS-like structures in RP, in comparison to shorter OS-like structures in USH. RP organoids also showed a rod defect, whereas USH organoids exhibited increased cones. These defects were notably improved upon CRISPR correction.

In RP, genes associated with cilia, photoreceptor outer segments, and innate immune response pathways were highly dysregulated. In USH, transcriptomic profiles revealed disruptions in pathways related to neuronal fate commitment, differentiation, visual perception, and vesicle transport. Additionally, cone-specific genes such as RXRG were upregulated in USH, potentially contributing to the previously observed cone enrichment, along with genes involved in inner ear development. CRISPR correction of the causative mutations rescued not only the disease phenotype but also restored partially the expression of genes involved mainly in visual system development and visual perception.

Conclusion:

This work revealed distinct transcriptomic signatures between RP and USH organoids USH2A-mutant retinal organoids, highlighting divergent disease mechanisms that correlate with their phenotypic differences, and demonstrating potential of CRISPR for molecular and phenotypic rescue.