The broad, long-term objectives of the proposed research are to improve our understanding of the pathogenesis of the RNA splicing factor forms of retinitis pigmentosa (RP) so that therapies for these blinding disorders can be developed. 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, PRPF8, PRPF31, RP9 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. Given the function of all 5 of these proteins in spliceosome, it has been hypothesized that defects in RNA splicing underlie these forms of RP. But, lack of suitable animal model systems for the RNA splicing factor forms of RP, and inability to study the transcriptome in sufficient detail have limited the ability to study these disorders and test this hypothesis. The animal models and research tools needed to successfully investigate the pathogenesis of these disorders have been developed by the research team. Gene targeted mouse models for PRPF3, PRPF8, PRPF31 forms of RNA splicing factor RP have been generated. All three of these mutant Prpf mice develop degenerative changes in the RPE, and RPE cells isolated from these mice are dysfunctional, suggesting RPE may be primary cell type affected in these disorders. In addition, the use of next generation sequencing - based transcriptome analyses (RNA-seq) to study these diseases has been developed, providing the opportunity to determine if defects in RNA splicing underlie these disorders. These tools will be used in the proposed research to answer several key questions about the PRPF3, 8 and 31 forms of RNA splicing factor RP.
In Aim 1, the hypothesis that RPE dysfunction leads to the retinal degeneration observed in the RNA splicing factor forms of RP will be tested.
In Aim 2, RNA-seq transcriptome analyses of tissues from the three Prpf gene targeted mouse models will be performed to determine if abnormal splicing underlies these disorders.
In Aim 3, the potential pathogenicity of the aberrant mRNAs detected in Aim 2 will be tested on RPE cells in culture and in vivo to identify the ones that are pathogenic. The answers to these questions will elucidate the mechanisms of disease, and provide the information needed to develop therapies for these forms of blindness. For example, if the specific splicing alterations that cause RP in patients with these disorders can be identified, it is possible that anti-sense oligonucleotides could be employed to correct the identified splicing errors. This approach has shown promise for treating muscular dystrophy in vivo, including a report of efficacy in a small clinical trial.

Public Health Relevance

Mutations in genes that encode the RNA splicing factors PRPF3, PRPF8, PRPF31 are among the most common causes of the inherited blinding disorder retinitis pigmentosa (RP). The goal of the proposed research is to investigate how mutations in these genes cause retinal degeneration. This knowledge will facilitate the development of therapies to prevent vision loss from these blinding disorders.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Biology and Diseases of the Posterior Eye Study Section (BDPE)
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Shen, Grace L
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University of Pennsylvania
Schools of Medicine
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Buskin, Adriana; Zhu, Lili; Chichagova, Valeria et al. (2018) Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa. Nat Commun 9:4234
Hafler, Brian P; Comander, Jason; Weigel DiFranco, Carol et al. (2016) Course of Ocular Function in PRPF31 Retinitis Pigmentosa. Semin Ophthalmol 31:49-52
Farkas, Michael H; Au, Elizabeth D; Sousa, Maria E et al. (2015) RNA-Seq: Improving Our Understanding of Retinal Biology and Disease. Cold Spring Harb Perspect Med 5:a017152
Farkas, Michael H; Lew, Deborah S; Sousa, Maria E et al. (2014) Mutations in pre-mRNA processing factors 3, 8, and 31 cause dysfunction of the retinal pigment epithelium. Am J Pathol 184:2641-52
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Grant, Gregory R; Farkas, Michael H; Pizarro, Angel D et al. (2011) Comparative analysis of RNA-Seq alignment algorithms and the RNA-Seq unified mapper (RUM). Bioinformatics 27:2518-28

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