Retinitis pigmentosa (RP) is a form of inherited retinal degeneration that is characterized by the progressive loss of photoreceptor cells (rods and cones) of the retina. While mutations in many genes have been implicated in the disease, we propose to investigate how mutations in genes required for RNA splicing cause dominant RP. RNA splicing is an essential function that occurs in every cell of the human body where the spliceosome processes pre-mRNA transcripts by removing introns (non-coding regions) and splicing the exons (coding regions) together to form a mature mRNA transcript. Although this is a general process, mutations in genes encoding four components of the spliceosome have been found to contain mutations that cause dominant RP. The four genes are called Pre-RNA Processing Factors 3, 8, and 31 (PRPF3, PRPF8, and PRPF31), and PAP-1. Mutations in these genes are the second most common cause of dominant RP. Despite their prevalence, the mechanism by which mutation in these genes cause vision loss is not understood. Specifically, it is not understood if the mutations cause splicing defects ubiquitously or locally in the retina. We believe that if we can increase our understanding of the pathogenesis of these mutations, then we will be able to develop treatments to prevent vision loss from these forms of RP. To study how mutations in PRPF3 and PRPF8 cause blindness, we generated mice with mutations in their Prpf3 and Prpf8 genes that mimic mutations found in people with RP. These mice are called Prpf3- T494M and Prpf8-H2309M knockin mice to indicate the specific mutations that we introduced into the genes of these mice. By studying the retinas of the Prpf3-T494M and Prpf8-H2309M knockin mice, we have found that mutations in PRPF3 and PRPF8 may cause vision loss by damaging the retinal pigment epithelium (RPE) cells of the retina. The RPE cells maintain the health of the photoreceptor cells. If the RPE cells are not healthy, then the photoreceptor cells will become unhealthy as well.
In Specific Aim 1 of the proposed research, we plan to continue these studies to determine if the RPE is the primary site of the retinal defect caused by mutations in the RNA splicing factors. This will include experiments to compare the function of the RPE cells in the Prpf3-T494M and Prpf8-H2309M knockin mice to those in normal control mice. From preliminary studies of RNA splicing in the retinas of the Prpf3-T494M and Prpf8-H2309M knockin mice, we believe that RNA splicing is generally disrupted in these animals. This data is consistent with results from studies of another form of disease caused by mutations in an RNA splicing factor. In this case, the neurologic disorder spinal muscular atrophy (SMA), which is caused by mutations in the survival motor neurons (SMN) gene.
In Specific Aim 2 of the proposed research, we plan to expand our studies of RNA splicing in the Prpf3-T494M and Prpf8-H2309M knockin mice in order to 1. Determine if defects in RNA splicing are widespread in these animals, and 2. Identify the specific RNA splicing defects that occur in the retina of these mice, and thus may be leading to sickness and death of retinal cells in these diseases. We expect that these studies will answer several important questions. First, is the RPE the primary site of the retinal defect caused by mutations in RNA splicing factors? Second, are RNA splicing defects widespread in the Prpf3-T494M and Prpf8-H2309M knockin mice? And third, what splicing defects occur in the retina that could lead to the sickness and death of retinal cells in these diseases? These are important questions, because the answers to them are critically important parts of our long-term effort to develop therapies for the RNA splicing factor forms of RP. There are several examples of treatments directed at correcting defects in RNA splicing being successfully applied in animal models of disease. In addition, a small clinical trial of such a therapy has recently been completed. We believe that if we can identify the specific splicing alterations that cause RP in patients with these disorders, it is possible that similar therapeutic approaches could be developed for treating these diseases.

Public Health Relevance

Retinitis Pigmentosa is a disorder that affects at least 1:4000 people worldwide and is characterized by the progressive loss of vision that begins as early as childhood with complete vision loss decades later. Mutations in proteins associated with the splicosome are the second leading cause of this disease and we aim to understand how these ubiquitously expressed proteins cause pathogenesis specifically in the retina.

National Institute of Health (NIH)
National Eye Institute (NEI)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F05-C (20))
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Shen, Grace L
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University of Pennsylvania
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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
Farkas, Michael H; Grant, Gregory R; White, Joseph A et al. (2013) Transcriptome analyses of the human retina identify unprecedented transcript diversity and 3.5 Mb of novel transcribed sequence via significant alternative splicing and novel genes. BMC Genomics 14:486
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