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

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1 / 2500，全世界有超过100万人受到影响。RP的一种主要形式是由编码“剪接体”蛋白质成分（“剪接因子”）的基因中的缺陷（“突变”）引起的。剪接体是一种蛋白质复合物，它确保从基因中形成的新RNA转录物（“转录”）被正确剪接，形成最终的信使RNA。然后，细胞使用最终的mRNA编码蛋白质的产生。剪接将非编码RNA（内含子）从编码蛋白质的基本编码区（外显子）中移除。打个比方，从一组说明中删去不需要的或无意义的段落，这样在最终文本中只留下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是一种令人费解的疾病，因为剪接在细胞中普遍存在，但这种疾病只引起视网膜细胞的变性。先前的研究表明，编码视网膜蛋白的基因的错误剪接可能是重要的。然而，我们最近发表的工作表明，剪接装置本身的组件剪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，这表明靶向这一过程可能是一种特别有效的治疗方法。本工作开发了实验方法，并应用于了解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，包括PRPF8，因为这是剪接体中心的关键结构成分，对正确剪接至关重要。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的机制并评估潜在的治疗方法，我们将使用特殊的细胞系统来密切模拟人类视网膜组织。我们将使用分化为视网膜细胞的患者特异性人类干细胞，使我们能够研究来自剪接因子RP患者的视网膜组织中的细胞结构和功能。如果我们依赖于非常有限的患者组织临床资源，不合适的细胞模型，如皮肤成纤维细胞，或现有的小鼠突变体，不可能重现人类疾病，这些研究是不可能的。我们将使用生化方法来了解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”或克隆测序结合起来，以确定来自患者视网膜组织的RNA的核苷酸序列。这将决定在视网膜变性期间哪个组织首先受到影响，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的潜在治疗方法的临床前研究的设计提供信息，例如，通过特异性消融（“敲除”）视网膜细胞中蛋白质的突变形式。本研究的结果将建立由PRPF8和PRPF31突变引起的RP的发病机制，为未来治疗剪接因子RP的治疗策略的发展提供支持。目前眼部基因治疗的临床试验主要集中在严重的早发性疾病，如视网膜营养不良。然而，成人起病RP的治疗仍有很大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。这些情况提出了一个特别的挑战，因为患者在第五个十年中可能还有有用的残余视力。因此，在进行临床试验之前，需要对疾病机制有一个清晰的认识，并对安全性有更大的要求。

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.