The Pathogenesis of RNA Splicing Factor Retinitis Pigmentosa

RNA剪接因子视网膜色素变性的发病机制

• 批准号: 9254547
• 负责人: Eric A Pierce
• 金额: $ 59.68万
• 依托单位: MASSACHUSETTS EYE AND EAR INFIRMARY
• 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 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9254547
• 关键词: Affect; Alternative Splicing; Amyotrophic Lateral Sclerosis; Atrophic; B-Lymphocytes; Blindness; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Defect; Disease; Epithelial; Event; Functional disorder; Funding; Gene Targeting; Generations; Genes; Genetic; Goals; Human; Introns; Lead; Mediating; Messenger RNA; Motor; Mus; Mutant Strains Mice; Mutation; Neural Retina; Neurodegenerative Disorders; Organ; Pathogenesis; Pathway interactions; Patients; Phenotype; Proteins; RNA; RNA Splicing; Reporting; Research; Retina; Retinal Degeneration; Retinal Diseases; Retinal Pigments; Retinitis Pigmentosa; Small Nuclear Ribonucleoproteins; Spinal; Spliceosomes; Structure of retinal pigment epithelium; Testing; Therapeutic; Transcript; Work; cell type; disease-causing mutation; disorder subtype; gene augmentation therapy; gene therapy; genome editing; improved; in vivo; induced pluripotent stem cell; inherited retinal degeneration; knock-down; lymphoblast; mRNA Precursor; mutant; public health relevance; therapy development; tool; transcriptome; transcriptome sequencing

 DESCRIPTION (provided by applicant): The long-term goals of the proposed research are to improve understanding of the pathogenesis of RNA splicing factor retinitis pigmentosa (RP) so that therapies can be developed for these blinding disorders. 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, PRPF4, PRPF6, PRPF8, PRPF31, 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. Since all of the RNA splicing factors identified to harbor mutations that cause RP are associated with the U4/U6/U5 tri-snRNP complex of the spliceosome, it has been hypothesized that defects in RNA splicing underlie the RNA splicing factor forms of RP. What remains unclear is if specific aberrantly spliced transcripts cause the retinal disease, or if generalized alterations in splicing which occu globally affect the retina more than other organs. To investigate the mechanisms by which mutations in RNA splicing factors cause retinal degeneration, gene targeted Prpf3, Prpf8 and Prpf31 mutant mice were generated, and are being used to test the hypothesis that aberrant splicing underlies the retinal disease. In the past funding period cell autonomous defects in retinal pigment epithelial (RPE) cell function were identified in the gene targeted mice. These are recapitulated in human ARPE-19 cells following knockdown PRPF31, suggesting that RPE cells are the primary target cell type in RNA splicing factor RP. RNA-seq analyses of the RPE from the three gene targeted mouse lines suggest that altered splicing of a limited number of transcripts underlies the defects in RPE function, and thus the retinal degenerative disease. In Aim 1 of the proposed research the effects of the identified altered transcripts on RPE cell function will be tested in cultured RPE cells and in the RPE in vivo to determine which of the altered transcripts could be responsible for disease. In Aim 2, these studies will be extended to human cells, since it is now possible to generate RPE cells from human induced pluripotent stem cells (hiPSCs) and to introduce specific mutations into hiPSCs using genome editing tools. In Aim 3, the hypothesis that gene augmentation therapy can provide therapeutic benefit for PRPF31 disease will be tested. This is a logical extension of prior work, since mutations in PRPF31 have been shown to cause disease via haploinsufficiency, and RPE cells have been identified as the target cell type in RNA splicing factor RP. Viewed together, these Aims provide a logical pathway toward defining the pathogenesis of RNA splicing factor RP, and initiating work towards developing therapies for these forms of RP.

 描述（由适用提供）：拟议研究的长期目标是提高对RNA剪接因子色素性视网膜炎（RP）的发病机理的理解，以便可以针对这些盲目疾病开发治疗。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。影响的剪接因子，前MRNA处理因子（PRPF）3，PRPF4，PRPF6，PRPF8，PRPF8，PRPF31和SNRNP200是剪接体的高配置成分，该组件是从新生的RNA转录中对内含子进行激发的复合物，以产生成熟的MATURE MRNA。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。由于鉴定出引起RP的携带突变的所有RNA剪接因子都与剪接体的U4/U6/U5 Tri-SnRNP复合物相关，因此已假设RNA剪接中的缺陷是RP的RPN的RNA拼接因子形式的基础。尚不清楚的是，特定的异常剪接的转录本是否引起残留疾病，或者在全球影响视网膜上比其他器官更大的剪接变化。为了研究RNA剪接因子中的突变导致永久性变性的机制，基因靶向PRPF3，PRPF8和PRPF31突变小鼠，并用于检验以下假说，即异常剪接是残基疾病的基础。在过去的资金期间，在基因靶向小鼠中鉴定出视网膜色素上皮（RPE）细胞功能的细胞自主缺陷。敲除PRPF31后，这些在人ARPE-19细胞中被概括，这表明RPE细胞是RNA剪接因子RP中的主要靶细胞类型。来自三个基因靶向小鼠系的RPE的RNA-seq分析表明，有限数量的转录本的剪接改变了RPE功能中缺陷的基础，因此改变了视网膜退行性疾病。在拟议的研究的目标1中，将在培养的RPE细胞和RPE IN VIVO中测试所鉴定的转录物对RPE细胞功能的影响，以确定哪些改变的转录本可能导致疾病。在AIM 2中，这些研究在AIM 3中，将测试基因增强疗法可以为PRPF31疾病提供治疗益处的假设。这是先前工作的逻辑扩展，因为已证明PRPF31中的突变通过单倍弥补引起疾病，并且RPE细胞已被确定为RNA剪接因子RP中的靶细胞类型。共同考虑，这些目标为定义RNA剪接因子RP的发病机理提供了逻辑途径，并开始为这些形式的RP开发疗法而开始工作。