The Pathogenesis of RNA Splicing Factor RP

RNA剪接因子RP的发病机制

• 批准号: 8108021
• 负责人: Eric A Pierce
• 金额: $ 21.88万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8108021
• 关键词: Affect; Alleles; Amyotrophic Lateral Sclerosis; Animal Experimentation; Animal Model; Antisense Oligonucleotides; Atrophic; Base Sequence; Biological Models; Blindness; Cells; Clinical Trials; Complex; Defect; Disease; Functional disorder; Gene Expression Profile; Gene Targeting; Generations; Genes; Goals; Health; Human; Individual; Inherited; Introns; Knowledge; Lead; Messenger RNA; Molecular; Motor; Mus; Muscular Dystrophies; Mutant Strains Mice; Mutation; Neurodegenerative Disorders; Pathogenesis; Pathogenicity; Patients; Phenotype; Process; Proteins; RNA; RNA Splicing; Reporting; Research; Retina; Retinal; Retinal Degeneration; Retinitis Pigmentosa; Small Nuclear Ribonucleoproteins; Spinal; Spliceosomes; Testing; Tissues; Transcript; cell type; disease-causing mutation; improved; in vivo; mRNA Precursor; mouse model; mutant; next generation; prevent; protein expression; therapy development; tool

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：拟议研究的广泛的长期目标是提高我们对色素性视网膜炎的RNA剪接因子形式（RP）的发病机理的理解，以便可以开发出这些盲目疾病的疗法。编码RNA剪接因子的基因突变是RP主要形式的第二大原因，因此是视力丧失的重要原因。受影响的剪接因子，前MRNA加工因子（PRPF）3，PRPF8，PRPF31，RP9和SNRNP200是剪接体的高度保守成分，该组件是从新生的RNA转录物中激发内含子以产生成熟的mRNA的复合物。由于在所有细胞中都需要RNA剪接，因此尚不清楚这些无处不在的蛋白质中突变如何导致视网膜特异性疾病。鉴于所有这些蛋白质中的所有5种在剪接体中的功能，已经假设RNA剪接中的缺陷是这些形式的RP。但是，缺乏用于RP的RNA剪接因子形式的合适动物模型系统，并且无法详细研究转录组的研究能力限制了研究这些疾病的能力和检验该假设。研究团队已经开发了成功研究这些疾病发病机理所需的动物模型和研究工具。已经生成了用于PRPF3，PRPF8，PRPF31 RNA剪接因子RP的基因靶向小鼠模型。所有这三个突变的PRPF小鼠都会在RPE中发生退化性变化，并且从这些小鼠中分离出的RPE细胞均功能障碍，这表明RPE可能是这些疾病中影响的主要细胞类型。此外，已经开发了基于下一代测序的基于基于测序的转录组分析（RNA -SEQ）来研究这些疾病，从而提供了确定RNA剪接中缺陷的机会。这些工具将在拟议的研究中使用，以回答有关RNA剪接因子RP的几个关键问题。在AIM 1中，将测试RPE功能障碍导致在RP的RNA剪接因子形式中观察到的视网膜变性的假设。在AIM 2中，将执行来自三个PRPF基因靶向小鼠模型的组织的RNA-SEQ转录组分析，以确定这些疾病是否是这些疾病的基础。在AIM 3中，在AIM 2中检测到的异常mRNA的潜在致病性将在培养物和体内的RPE细胞上进行测试，以鉴定那些具有致病性的细胞。这些问题的答案将阐明疾病的机制，并提供开发这些形式失明疗法所需的信息。例如，如果可以鉴定出引起这些疾病患者RP的特定剪接改变，则可以使用抗敏感性寡核苷酸来纠正已识别的剪接误差。这种方法显示了治疗体内肌营养不良症的希望，包括在一项小型临床试验中的疗效报告。