Introduction

Retinitis Pigmentosa (RP) is the most frequent inherited retinal dystrophy, affecting around 2.5 million people and often leading to blindness in young adults. Among the causative genes, RHO, coding for rhodopsin, the light-absorbing protein that initiates the signal transmission cascade in rod photoreceptors, plays a central role. Several classifications of RHO mutations have been proposed based on clinical features and molecular effects. However, these classifications mainly rely on non-human models, such as overexpression in heterologous systems or rodent retinas, which do not fully replicate the human disease.

Objective

Human iPSC-derived retinal organoids offer a complementary model, enabling the study of RP in a human genetic and cellular context. However, due to inter-individual genetic variability, isogenic controls are required to validate the phenotype. Our first objective was to generate isogenic iPSC lines from patients carrying one of four RHO mutations: T17M, R135W, M216L, or P347L. Then, photoreceptor dystrophy mechanisms, associated with each specific mutation, can be investigated in retinal organoids obtained from mutated and corrected iPSC lines.

Methods

T17M, R135W, and P347L mutations are C>T transitions, amenable to correction using CRISPR-nCas9 base editing, which allows single-base changes without double-strand breaks. M216L (T>A) required classical CRISPR-mediated editing via homology-directed repair. Specific sgRNAs were designed for each mutation, and mRNA coding for the base editor was produced. iPSCs from patients were nucleofected with either base editor complexes or Cas9-sgRNA-template constructs.

Results

Edited clones were identified by Sanger sequencing. The four mutations were successfully corrected. Clones were validated for genome integrity (digital PCR), pluripotency (immunostaining), and absence of off-target effects. Mutated and corrected iPSC lines are differentiated into retinal organoids up to day 270, a late stage of retinal maturation, to assess photoreceptor degeneration by examining outer segment formation and performing immunofluorescence with specific photoreceptor markers.

Conclusion

The generation of reliable isogenic controls is a key step to allow RP physiopathology investigation using an appropriate and faithful model. This patient-derived system provides a robust platform to study the molecular mechanisms of RP associated with specific RHO mutations.