Abstract
Purpose : Retinitis pigmentosa 11 (RP11) is an inherited degenerative retinal disease caused by heterozygous mutations in pre-mRNA processing factor 31 (PRPF31) for which there is currently no effective treatment available. RP11 features incomplete penetrance within affected families; the level of PRPF31 expression from the healthy allele determines whether mutation carriers develop symptoms. The CCR4-NOT transcription complex subunit 3 (CNOT3) is a major disease modifier that regulates PRPF31 levels via transcription inhibition. Higher CNOT3 levels are observed in RP11 cases. In this study, we aim to lower CNOT3 expression and function to indirectly upregulate functional PRPF31 from the healthy allele and rescue RP11 disease phenotypes.
Methods : Seventy-four antisense oligomers (ASO) were designed to target exonic splice enhancers to mediate exclusion of selected CNOT3 exons to (i) induce translational frameshift and mRNA decay or (ii) produce truncated low/non-functional CNOT3 isoform(s). PRPF31 expression and function were assessed in RP11 iPSC-derived retinal pigment epithelial (RPE) cells.
Results : We observed an inverse correlation between CNOT3 and PRPF31 mRNA levels in healthy fibroblasts (n=20). In iPSC-RPE cells, we found 10% higher expression of CNOT3 with 17% lower PRPF31 expression in a patient compared to an asymptomatic relative, both carrying a PRPF31 c.1205 C>A nonsense mutation. Fewer and shorter primary cilia were observed in the symptomatic patient RPE compared to asymptomatic, healthy RPE. Lowering CNOT3 levels and function with ASOs demonstrated a 1.7-fold increase in PRPF31 expression in patient RPE and significantly improved cilia number and length. Healthy cilia play an integral role in phagocytosis and are crucial for normal retinal function.
Conclusions : Subtle changes in CNOT3 and PRPF31 levels in retinal cells determine disease penetrance in PRPF31 mutation carriers within an affected family. Modulating expression levels of these proteins can reverse the cellular disease phenotype in RP11. ASOs are effective modulators of CNOT3 expression and function with the ability to increase PRPF31 transcription from the unaffected allele to an expected therapeutic level. Future studies will assess the restoration of transcriptional profiles linked to improved PRPF31 levels in patient derived iPSC-retinal organoids and RPE compared to healthy controls.