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

• 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

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）是遗传性，渐进视力丧失的一种常见形式：在2500中，它的患病率为1500分，全世界有超过100万人。 RP的一种主要形式是由编码“剪接体”的蛋白质成分（“剪接因子”）的基因中的缺陷（“突变”）引起的。剪接体是蛋白质的复合体，可确保从基因中形成（转录的）新的RNA转录本被正确剪接以形成最终的信使或mRNA。然后，细胞使用最终mRNA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”）。一个类比是从一组说明中编辑了不必要的或荒谬的段落，因此最终文本中只保留可理解的单词和句子。剪接体是执行编辑的细胞设备，并确保由剪接因子突变引起的剪接和特异性。RP是一种令人困惑的条件，因为剪接在细胞中无处不在，但该条件仅引起视网膜细胞的变性。先前的工作表明，编码视网膜蛋白的基因的错误分解可能很重要。但是，我们最近发表的工作表明，剪接设备本身的组件分量有缺陷是基本的分子缺陷。这种正反馈回路似乎仅发生在视网膜细胞中，这表明该过程的靶向可能特别有效地作为一种可能的治疗方法。这项工作开发了实验方法，并将其应用于理解RP中PRPF31的功能。在本提案中，我们现在希望扩大调查以包括PRPF8，因为这是剪接体核心的关键结构成分，对于正确的剪接至关重要。 PRPF8中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，我们将使用特殊的细胞系统，这些细胞系统对人类视网膜组织进行密切模拟。我们将使用分化为视网膜细胞的患者特异性人干细胞，从而使我们能够研究源自具有剪接因子RP患者的视网膜组织中的细胞结构和功能。如果我们依靠患者组织的临床资源非常有限，不适当的细胞模型，例如皮肤成纤维细胞或不概括人类疾病的可用小鼠突变体，那么这些研究将是不可能的。我们将使用生化方法来了解PRPF8突变对剪接体的结构和功能的影响。我们将将这些研究与“下一代”或克隆测序结合起来，以确定来自患者衍生的视网膜组织RNA的核苷酸序列。这将确定在视网膜变性期间首先影响哪种组织，发生了哪种类型的剪接缺陷以及哪些基因受到影响。然后，这些研究将告知临床前研究对与PRPF8相关的RP的潜在治疗方法的设计，例如，通过视网膜细胞中蛋白质突变体形式的特定消融（“敲除”）。这项拟议的研究的结果将建立疾病的疾病机制，用于在PRPF和PRPF8和PRPF31的策略中引起的RP引起的疾病机制，并建立prpf31的未来效果。 RP。目前针对眼基因疗法的临床试验集中于严重的早期发作性疾病，例如视网膜营养不良。但是，对于成人发作的RP的治疗仍然存在很大且未满足的临床需求，其中很大一部分是由于PRFP中的缺陷。这些条件带来了一个特殊的挑战，因为患者可以在第五个十年中具有有用的残留视力。因此，在进行临床试验之前，需要清楚地了解疾病机制和更大的要求证明安全性。

项目成果

Progressive accumulation of cytoplasmic aggregates in PRPF31 retinal pigment epithelium cells interferes with cell survival

PRPF31视网膜色素上皮细胞中细胞质聚集物的逐渐积累干扰细胞存活

• DOI: 10.1002/ctd2.89
• 发表时间: 2022
• 期刊: Clinical and Translational Discovery
• 作者: Georgiou M

Molecular diagnoses in the congenital malformations caused by ciliopathies cohort of the 100,000 Genomes Project.

• DOI: 10.1136/jmedgenet-2021-108065
• 发表时间: 2022-08
• 期刊: Journal of medical genetics
• 影响因子: 4

Unlocking the potential of the UK 100,000 Genomes Project-lessons learned from analysis of the "Congenital Malformations caused by Ciliopathies" cohort.

• DOI: 10.1002/ajmg.c.31965
• 发表时间: 2022-03
• 期刊: AMERICAN JOURNAL OF MEDICAL GENETICS PART C-SEMINARS IN MEDICAL GENETICS
• 影响因子: 3.1
• 作者: Best, Sunayna;Inglehearn, Chris F.;Watson, Christopher M.;Toomes, Carmel;Wheway, Gabrielle;Johnson, Colin A.
• 通讯作者: Johnson, Colin A.

Incorporating microglia-like cells in human induced pluripotent stem cell-derived retinal organoids.

• DOI: 10.1111/jcmm.17670
• 发表时间: 2023-02
• 期刊: Journal of cellular and molecular medicine
• 影响因子: 5.3

Progressive protein aggregation in PRPF31 patient retinal pigment epithelium cells: the mechanism and its reversal through activation of autophagy

• DOI: 10.1101/2021.10.11.463925
• 发表时间: 2021-10
• 期刊: bioRxiv
• 作者: Maria Georgiou;Chunbo Yang;R. Atkinson;K. Pan;A. Buskin;Marina Moya Molina;J. Collin;J. Al-Aama-J.-Al-Aam