Elucidating splicing factor function and retinal splicing programmes: developing new therapeutic strategies for splicing factor retinitis pigmentosa

阐明剪接因子功能和视网膜剪接方案：开发剪接因子色素性视网膜炎的新治疗策略

基本信息

批准号：
MR/T017503/1
负责人：
Majlinda Lako
金额：
$ 168.12万
依托单位：
Newcastle University
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2020
资助国家：
英国
起止时间：
2020 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=MR%2FT017503%2F1
关键词：
Elucidating splicing factor function retinal

项目摘要

Retinitis pigmentosa (RP) is a common form of hereditary, progressive sight loss: it has a prevalence of 1 in 2500 and more than 1 million people affected worldwide. A major form of RP is caused by defects ("mutations") in genes that encode protein components ("splicing factors") of the "spliceosome". The spliceosome is a complex of proteins that ensure the new RNA transcripts formed ("transcribed") from genes are then correctly spliced to form the final messenger or mRNA. The cell then uses the final mRNA to encode the production of proteins. Splicing removes non-coding RNA ("introns") from the essential coding regions ("exons") that encode proteins. An analogy is the editing of unwanted or nonsensical passages out of a set of instructions, so that only intelligible words and sentences remain in the final text. The spliceosome is the cellular apparatus that performs the editing and ensures the fidelity and specificity of splicing.RP caused by mutations in splicing factors is a perplexing condition because splicing is ubiquitous in cells, but the condition only causes the degeneration of retinal cells. Previous work has suggested that mis-splicing of genes that encode retinal proteins may be important. However, our recently published work suggests that defective splicing of components of the splicing apparatus itself is the fundamental molecular defect. This positive feedback loop appears to only occur in retinal cells, suggesting that the targeting of this process might be particularly effective as a possible treatment. This work developed experimental methods and applied them to understand the function of PRPF31 in RP. In the present proposal, we now wish to broaden our investigations to include PRPF8, since this is a key structural component at the heart of the spliceosome and is essential for correct splicing. Mutations in PRPF8 are also a major cause of splicing factor RP.In order to understand this mechanism of disease in greater depth and to assess potential treatments, we will use special cell systems that closely model human retinal tissue. We will use patient-specific human stem cells differentiated into retinal cells, allowing us to study cellular structures and functions in retinal tissue derived from patients with splicing factor RP. These investigations would be impossible if we were to rely on the very limited clinical resources of patient tissue, inappropriate cell models such as skin fibroblasts, or the available mouse mutants that do not recapitulate the human disease. We will use biochemical methods to understand the effect of PRPF8 mutations on the structure and function of the spliceosome. We will combine these studies with "next generation" or clonal sequencing to determine the nucleotide sequences of RNA from patient-derived retinal tissue. This will determine which tissue is first affected during retinal degeneration, what types of splicing defects occur and which genes are affected. These studies will then inform the design of pre-clinical studies into potential treatments of PRPF8-related RP, for example by specific ablation ("knock-down") of the mutant form of the protein in retinal cells.The outcome of this proposed research will establish the disease mechanisms for RP caused by mutations in PRPFs, specifically PRPF8 and PRPF31, enabling the development of future therapeutic strategies to treat splicing factor RP. Current clinical trials for ocular gene therapies have focused on severe, early-onset disorders such as retinal dystrophies. However, there remains a large and unmet clinical need for the treatment of adult-onset RP, a large proportion of which are due to defects in PRFPs. These conditions present a particular challenge because patients can have useful residual vision into their fifth decade. A clear understanding of disease mechanism and greater requirement to demonstrate safety is therefore required before proceeding to clinical trials.

色素性视网膜炎 (RP) 是一种常见的遗传性进行性视力丧失：患病率为 2500 分之一，全世界有超过 100 万人受到影响。 RP的主要形式是由编码“剪接体”的蛋白质成分（“剪接因子”）的基因缺陷（“突变”）引起的。剪接体是一种蛋白质复合物，可确保从基因形成（“转录”）的新 RNA 转录本正确剪接以形成最终的信使或 mRNA。然后细胞使用最终的 mRNA 来编码蛋白质的产生。剪接从编码蛋白质的基本编码区（“外显子”）中去除非编码 RNA（“内含子”）。打个比方，就是从一组指令中编辑出不需要的或无意义的段落，以便最终文本中只保留可理解的单词和句子。剪接体是执行编辑并确保剪接的保真度和特异性的细胞装置。剪接因子突变引起的RP是一种令人费解的情况，因为剪接在细胞中普遍存在，但这种情况只会导致视网膜细胞的变性。先前的研究表明，编码视网膜蛋白的基因的错误剪接可能很重要。然而，我们最近发表的工作表明，拼接装置本身组件的拼接缺陷是基本的分子缺陷。这种正反馈循环似乎只发生在视网膜细胞中，这表明该过程的靶向作为一种可能的治疗方法可能特别有效。这项工作开发了实验方法并应用它们来了解 PRPF31 在 RP 中的功能。在目前的提案中，我们现在希望扩大我们的研究范围以包括 PRPF8，因为这是剪接体核心的关键结构成分，对于正确剪接至关重要。 PRPF8的突变也是剪接因子RP的主要原因。为了更深入地了解这种疾病机制并评估潜在的治疗方法，我们将使用密切模拟人类视网膜组织的特殊细胞系统。我们将使用分化为视网膜细胞的患者特异性人类干细胞，使我们能够研究源自具有剪接因子 RP 的患者的视网膜组织的细胞结构和功能。如果我们依赖于非常有限的患者组织临床资源、不适当的细胞模型（例如皮肤成纤维细胞）或不能重现人类疾病的可用小鼠突变体，那么这些研究将是不可能的。我们将利用生化方法来了解PRPF8突变对剪接体结构和功能的影响。我们将把这些研究与“下一代”或克隆测序相结合，以确定来自患者来源的视网膜组织的 RNA 核苷酸序列。这将确定在视网膜变性期间哪些组织首先受到影响、发生什么类型的剪接缺陷以及哪些基因受到影响。这些研究将为 PRPF8 相关 RP 潜在治疗的临床前研究设计提供信息，例如通过视网膜细胞中蛋白质突变形式的特异性消融（“敲低”）。这项拟议研究的结果将建立由 PRPF（特别是 PRPF8 和 PRPF31）突变引起的 RP 疾病机制，从而能够开发未来治疗剪接因子 RP 的治疗策略。目前眼部基因疗法的临床试验主要集中在严重的早发性疾病，例如视网膜营养不良。然而，对于成人发病的 RP 的治疗仍然存在大量且未得到满足的临床需求，其中很大一部分是由于 PRFP 的缺陷造成的。这些情况提出了特殊的挑战，因为患者在五十几岁的时候仍能保持有用的残余视力。因此，在进行临床试验之前，需要清楚地了解疾病机制并提高证明安全性的要求。