The Role of RNA Splicing Factors in Retinal Degeneration

RNA剪接因子在视网膜变性中的作用

• 批准号: 7914613
• 负责人: Michael Farkas
• 金额: $ 4.76万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7914613
• 关键词: Affect; Animal Disease Models; Animals; Blindness; Cells; Cessation of life; Childhood; Clinical Trials; Code; Data; Data Analyses; Defect; Disease; Exons; Functional RNA; Genes; Health; Human body; Introns; Lead; Messenger RNA; Mus; Mutation; Pathogenesis; Patients; Photoreceptors; Prevalence; Process; Proteins; RNA Processing; RNA Splicing; Research; Retina; Retinal; Retinal Cone; Retinal Defect; Retinal Degeneration; Retinitis Pigmentosa; Role; Site; Spinal Muscular Atrophy; Spliceosomes; Structure of retinal pigment epithelium; Therapeutic; Transcript; Vertebrate Photoreceptors; disease-causing mutation; inherited retinal degeneration; mRNA Precursor; nervous system disorder; prevent; public health relevance; research study; survival motor neuron gene; therapy development

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.

描述（由申请人提供）：视网膜色素变性（RP）是一种遗传性视网膜变性，其特征在于视网膜感光细胞（视杆细胞和视锥细胞）的进行性丧失。虽然许多基因的突变与这种疾病有关，但我们建议研究RNA剪接所需的基因突变如何导致显性RP。RNA剪接是发生在人体每个细胞中的基本功能，其中剪接体通过去除内含子（非编码区）并将外显子（编码区）剪接在一起以形成成熟mRNA转录物来加工前mRNA转录物。虽然这是一个普遍的过程，但已发现编码剪接体四种组分的基因突变含有导致显性RP的突变。这四个基因被称为前RNA加工因子3，8和31（PRPF 3，PRPF 8和PRPF 31）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。具体地说，目前还不清楚这些突变是否在视网膜中普遍或局部引起剪接缺陷。我们相信，如果我们能够增加我们对这些突变的发病机制的理解，那么我们将能够开发治疗方法来预防这些形式的RP导致的视力丧失。为了研究PRPF 3和PRPF 8的突变如何导致失明，我们产生了Prpf 3和Prpf 8基因突变的小鼠，这些突变模拟了RP患者中发现的突变。这些小鼠被称为Prpf 3-T494 M和Prpf 8-H2309 M敲入小鼠，以表明我们引入这些小鼠基因的特定突变。通过研究Prpf 3-T494 M和Prpf 8-H2309 M基因敲入小鼠的视网膜，我们发现PRPF 3和PRPF 8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，那么感光细胞也会变得不健康。在拟议研究的具体目标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Prpf 3-T494 M和Prpf 8-H2309 M敲入小鼠中的RPE细胞与正常对照小鼠中的RPE细胞的功能的实验。从Prpf 3-T494 M和Prpf 8-H2309 M敲入小鼠视网膜中RNA剪接的初步研究中，我们认为RNA剪接在这些动物中通常被破坏。这一数据与RNA剪接因子突变引起的另一种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），这是由运动神经元生存（SMN）基因突变引起的。在拟议研究的具体目标2中，我们计划扩展我们在Prpf 3-T494 M和Prpf 8-H2309 M敲入小鼠中的RNA剪接研究，以1.确定RNA剪接缺陷是否在这些动物中广泛存在，以及2.确定这些小鼠视网膜中发生的特定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。我们希望这些研究能够回答几个重要的问题。首先，视网膜色素上皮是由RNA剪接因子突变引起的视网膜缺陷的主要部位吗？第二，RNA剪接缺陷是否在Prpf 3-T494 M和Prpf 8-H2309 M敲入小鼠中广泛存在？第三，视网膜中发生了什么样的剪接缺陷，可能导致这些疾病中视网膜细胞的疾病和死亡？这些都是重要的问题，因为它们的答案是我们为RP的RNA剪接因子形式开发疗法的长期努力的至关重要的部分。有几个例子的治疗，旨在纠正缺陷的RNA剪接被成功地应用于动物模型的疾病。此外，最近还完成了这种疗法的小型临床试验。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。 公共卫生关系： 色素性视网膜炎是一种影响全球至少1：4000人的疾病，其特征是早在儿童时期就开始的视力逐渐丧失，数十年后完全丧失视力。与剪接体相关的蛋白质突变是这种疾病的第二大原因，我们的目标是了解这些普遍表达的蛋白质如何在视网膜中引起发病机制。