UNRAVELING THE IMPACT OF PRPF31-MUTATIONS IN EARLY RETINAL DEVELOPMENT USING RETINITIS PIGMENTOSA 11 PATIENT-DERIVED RETINAL ORGANOIDS

Abstract

Purpose : Retinitis pigmentosa type 11 (RP11) is a hereditary retinal disease due to mutations in one copy of the Pre-mRNA processing factor 31 (PRPF31) gene. RP11 symptoms usually manifest in early adulthood with impaired night vision, narrowing of the visual field, and in the final stage, loss of central vision. At cellular level, the disease is characterized by progressive cell death in the retinal layers, primarily affecting the retinal pigment epithelial layer and subsequently the photoreceptor layer. Induced pluripotent stem cells (iPSC) derived- human organoids are robust models that can reveal new mechanistic insight into the pathology of diseases and can be used as experimental platforms for drug screening and cell- and gene therapies. To investigate the impact of PRPF31 mutations in early- and late stages of retinal development and molecular mechanisms underlying RP11 disease progression, we are generating RP11 patient-derived retinal organoids (RO).

Methods : Patient-derived skin fibroblasts were reprogrammed into iPSC and further differentiated into early-stage retinal organoids. Prolonged cultivation in appropriated media and supplements is ongoing in our lab, aiming at the development of mature RO. Characterization of retinal markers throughout differentiation, from iPSC to early-stage RO, was performed via real-time polymerase chain reaction (qPCR), immunohistochemistry (IHC) and single-cell RNA-sequencing (scRNA). PRPF31 expression levels were assessed via qPCR, scRNA and targeted mass spectrometry.

Results : Characterization using cell markers confirmed the presence of all early-born neurons in RO at the early stage, both in patient- and control-derived RO. Moreover, lower levels of PRPF31 were detected in patient-derived iPSC and early-stage RO both at mRNA (about 50% lower) and protein (approx. 25% lower) compared to healthy control samples. In addition, the scRNA data revealed significantly lower PRPF31 expression in the patient's photoreceptor progenitors and early neurogenic precursors. The morphology of optic vesicles was not particularly affected.

Conclusions : These data indicate that PRPF31 mutations have a low impact on early retinal development. Subsequent characterization of mature RO, global proteome and scRNA analyses of iPSC, early- and late-stage RO will provide new insight into the pathomechanisms driving visual loss in RP11 patients.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.