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: 8055973
• 负责人: Michael Farkas
• 金额: $ 2.06万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8055973
• 关键词: Affect; Alternative Splicing; Animal Disease Models; Animals; Biological Assay; Blindness; Cells; Cessation of life; Characteristics; Childhood; Clinical Trials; Code; Complex; Data; Data Analyses; Defect; Deposition; Disease; Excision; Exons; Functional RNA; Functional disorder; Gene Expression Profile; Genes; Genetic; Goals; Health; Human body; Inherited; Introns; Lead; Light; Messenger RNA; Molecular; Mus; Mutant Strains Mice; Mutation; Nature; Neural Retina; Pathogenesis; Patients; Phagocytosis; Photoreceptors; Prevalence; Primary Cell Cultures; Process; Proteins; RNA; RNA Processing; RNA Splicing; Research; Retina; Retinal; Retinal Cone; Retinal Defect; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Ribonucleoproteins; Role; Site; Spinal Muscular Atrophy; Spliceosomes; Structure of retinal pigment epithelium; Technology; Testing; Therapeutic; Tissues; Transcript; Vertebrate Photoreceptors; Vision; base; disease-causing mutation; inherited retinal degeneration; insight; mRNA Precursor; nervous system disorder; next generation; photoreceptor degeneration; 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）：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）逐渐丧失。虽然许多基因的突变与该疾病有关，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 剪接是人体每个细胞中发生的一项重要功能，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的 mRNA 转录物来处理前 mRNA 转录物。尽管这是一个一般过程，但已发现编码剪接体四种成分的基因突变包含导致显性 RP 的突变。这四个基因称为 Pre-RNA 处理因子 3、8 和 31（PRPF3、PRPF8 和 PRPF31）以及 PAP-1。这些基因突变是显性 RP 的第二常见原因。尽管它们很普遍，但这些基因突变导致视力丧失的机制尚不清楚。具体来说，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，如果我们能够加深对这些突变发病机制的了解，那么我们将能够开发出治疗方法来预防这些形式的 RP 导致的视力丧失。为了研究 PRPF3 和 PRPF8 突变如何导致失明，我们培育了 Prpf3 和 Prpf8 基因突变的小鼠，这些突变模仿了 RP 患者中发现的突变。这些小鼠被称为 Prpf3-T494M 和 Prpf8-H2309M 敲入小鼠，以表明我们引入这些小鼠基因中的特定突变。通过研究Prpf3-T494M和Prpf8-H2309M敲入小鼠的视网膜，我们发现PRPF3和PRPF8的突变可能通过损害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损的主要部位。这将包括将 Prpf3-T494M 和 Prpf8-H2309M 敲入小鼠中 RPE 细胞的功能与正常对照小鼠中的 RPE 细胞功能进行比较的实验。根据对 Prpf3-T494M 和 Prpf8-H2309M 敲入小鼠视网膜中 RNA 剪接的初步研究，我们认为这些动物的 RNA 剪接普遍被破坏。该数据与 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）是由运动神经元存活基因（SMN）突变引起的。在拟议研究的具体目标 2 中，我们计划扩大对 Prpf3-T494M 和 Prpf8-H2309M 敲入小鼠中 RNA 剪接的研究，以便 1. 确定 RNA 剪接缺陷是否在这些动物中普遍存在，以及 2. 识别这些小鼠视网膜中发生的特定RNA剪接缺陷，因此可能导致这些疾病中视网膜细胞患病和死亡。我们期望这些研究能够回答几个重要问题。首先，RPE是RNA剪接因子突变引起的视网膜缺损的原发部位吗？其次，RNA剪接缺陷在Prpf3-T494M和Prpf8-H2309M敲入小鼠中是否普遍存在？第三，视网膜中出现哪些剪接缺陷可能导致这些疾病中视网膜细胞患病和死亡？这些都是重要的问题，因为这些问题的答案是我们长期努力开发 RNA 剪接因子形式的 RP 疗法的至关重要的部分。有几个针对纠正 RNA 剪接缺陷的治疗方法已成功应用于疾病动物模型的例子。此外，这种疗法的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出现 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。 公共卫生相关性： 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视力逐渐丧失，数十年后视力完全丧失。与剪接体相关的蛋白质突变是这种疾病的第二个主要原因，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。