The Role of RNA Splicing Factors in Retinal Degeneration

描述（由申请人提供）：色素性视网膜炎（RP）是一种遗传性视网膜变性，其特征是视网膜感光细胞（视杆细胞和视锥细胞）的逐渐丧失。虽然许多基因的突变与该病有关，但我们建议研究RNA剪接所需基因的突变如何导致显性RP。RNA剪接是发生在人体每个细胞中的基本功能，剪接体通过去除内含子（非编码区）和将外显子（编码区）剪接在一起形成成熟的mRNA转录物来处理前mRNA转录物。虽然这是一个普遍的过程，但编码剪接体四个组分的基因突变已被发现包含导致显性RP的突变。这四个基因被称为前rna加工因子3、8和31 （PRPF3、PRPF8和PRPF31）和PAP-1。这些基因的突变是显性RP的第二大常见原因。尽管它们普遍存在，但这些基因突变导致视力丧失的机制尚不清楚。具体来说，目前尚不清楚这些突变是否会导致视网膜中普遍或局部的剪接缺陷。我们相信，如果我们能够增加对这些突变发病机制的了解，那么我们将能够开发出防止这些形式的RP导致视力丧失的治疗方法。为了研究PRPF3和PRPF8基因突变是如何导致失明的，我们制造了PRPF3和PRPF8基因突变的小鼠，这些突变与RP患者的基因突变相似。这些小鼠被称为Prpf3- T494M和Prpf8-H2309M敲入小鼠，以表明我们在这些小鼠的基因中引入的特定突变。通过对PRPF3 - t494m和PRPF8 - h2309m敲入小鼠视网膜的研究，我们发现PRPF3和PRPF8的突变可能通过损伤视网膜的视网膜色素上皮（RPE）细胞而导致视力丧失。RPE细胞维持感光细胞的健康。如果RPE细胞不健康，那么光感受器细胞也会不健康。在本研究的Specific Aim 1中，我们计划继续这些研究，以确定RPE是否是由RNA剪接因子突变引起的视网膜缺陷的主要部位。这将包括比较Prpf3-T494M和Prpf8-H2309M敲入小鼠与正常对照小鼠RPE细胞功能的实验。通过对Prpf3-T494M和Prpf8-H2309M敲入小鼠视网膜中RNA剪接的初步研究，我们认为RNA剪接在这些动物中普遍被破坏。这一数据与另一种由RNA剪接因子突变引起的疾病的研究结果一致。在这种情况下，神经系统疾病脊髓性肌萎缩症（SMA），这是由存活运动神经元（SMN）基因突变引起的。在拟进行的研究的Specific Aim 2中，我们计划扩大我们在Prpf3-T494M和Prpf8-H2309M敲入小鼠中RNA剪接的研究，以达到1。2.确定RNA剪接缺陷是否在这些动物中普遍存在；确定这些小鼠视网膜中发生的特异性RNA剪接缺陷，从而可能导致这些疾病中视网膜细胞的疾病和死亡。我们期望这些研究将回答几个重要的问题。首先，RPE是由RNA剪接因子突变引起的视网膜缺损的原发部位吗？第二，RNA剪接缺陷在Prpf3-T494M和Prpf8-H2309M敲入小鼠中普遍存在吗？第三，视网膜上发生了哪些剪接缺陷，可能导致这些疾病中视网膜细胞的生病和死亡？这些都是重要的问题，因为它们的答案是我们长期努力开发RP的RNA剪接因子形式的治疗方法的关键部分。有几个针对纠正RNA剪接缺陷的治疗方法成功应用于动物疾病模型的例子。此外，这种疗法的一个小型临床试验最近已经完成。我们相信，如果我们能够确定导致这些疾病患者RP的特定剪接改变，就有可能开发出类似的治疗方法来治疗这些疾病。

DESCRIPTION (provided by applicant): 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.