DESCRIPTION (provided by applicant): The long-term goals of the proposed research are to improve understanding of the pathogenesis of RNA splicing factor retinitis pigmentosa (RP) so that therapies can be developed for these blinding disorders. Mutations in genes that encode RNA splicing factors are the second most common cause of the dominant form of RP, and thus are an important cause of vision loss. The splicing factors affected, pre-mRNA processing factor (PRPF) 3, PRPF4, PRPF6, PRPF8, PRPF31, and SNRNP200 are highly conserved components of the spliceosome, the complex which excises introns from nascent RNA transcripts to generate mature mRNAs. Since RNA splicing is required in all cells, it is not clear how mutations in these ubiquitous proteins lead to retina-specific disease. Since all of the RNA splicing factors identified to harbor mutations that cause RP are associated with the U4/U6/U5 tri-snRNP complex of the spliceosome, it has been hypothesized that defects in RNA splicing underlie the RNA splicing factor forms of RP. What remains unclear is if specific aberrantly spliced transcripts cause the retinal disease, or if generalized alterations in splicing which occu globally affect the retina more than other organs. To investigate the mechanisms by which mutations in RNA splicing factors cause retinal degeneration, gene targeted Prpf3, Prpf8 and Prpf31 mutant mice were generated, and are being used to test the hypothesis that aberrant splicing underlies the retinal disease. In the past funding period cell autonomous defects in retinal pigment epithelial (RPE) cell function were identified in the gene targeted mice. These are recapitulated in human ARPE-19 cells following knockdown PRPF31, suggesting that RPE cells are the primary target cell type in RNA splicing factor RP. RNA-seq analyses of the RPE from the three gene targeted mouse lines suggest that altered splicing of a limited number of transcripts underlies the defects in RPE function, and thus the retinal degenerative disease. In Aim 1 of the proposed research the effects of the identified altered transcripts on RPE cell function will be tested in cultured RPE cells and in the RPE in vivo to determine which of the altered transcripts could be responsible for disease. In Aim 2, these studies will be extended to human cells, since it is now possible to generate RPE cells from human induced pluripotent stem cells (hiPSCs) and to introduce specific mutations into hiPSCs using genome editing tools. In Aim 3, the hypothesis that gene augmentation therapy can provide therapeutic benefit for PRPF31 disease will be tested. This is a logical extension of prior work, since mutations in PRPF31 have been shown to cause disease via haploinsufficiency, and RPE cells have been identified as the target cell type in RNA splicing factor RP. Viewed together, these Aims provide a logical pathway toward defining the pathogenesis of RNA splicing factor RP, and initiating work towards developing therapies for these forms of RP.

PUBLIC HEALTH RELEVANCE: Inherited retinal degenerations are important causes of blindness. The goals of the proposed research are to investigate how mutations in a class of genes that encode RNA splicing factors cause retinal degeneration, and to work toward developing gene therapy for one genetic subtype of disease.