Towards Gene Editing for Inherited Immunodeficiencies: Functional modelling & correction of novel disease-causing STAT1 variants

针对遗传性免疫缺陷的基因编辑：功能建模

• 批准号: 2075769
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2075769
• 关键词: Towards; Gene; Editing; Inherited; Immunodeficiencies

Signal transducer and activator of transcription 1 (STAT1) is a vital component of the Janus kinase (JAK)/STAT signalling cascade which transduces signals from diverse cell surface receptors to initiate gene transcription. STAT1 plays a particularly key role in immunity, as demonstrated by the insufficient immune responses generated by STAT1-deficient mice and by patients with STAT1 loss-of-function mutations. In 2011, autosomal dominant STAT1 gain-of-function (GOF) mutations were first identified in humans by van de Veerdonk et al. and Liu et al. They are now known to be the main cause of chronic mucocutaneous candidiasis and are further associated with an enhanced susceptibility to bacterial and viral infections, autoimmunity, aneurysms and cancer. These outcomes owe mainly to impaired Th 17 immunity, defective natural killer cell maturation and function, B cell lymphopenia and exaggerated responsiveness to interferons. The mechanism behind STAT1 GOF disease was previously thought to be hinderance of STAT1 dephosphorylation, leading to a build-up of phosphorylated STAT1 (pSTAT1) in the nucleus and thus overexpression of STAT1-dependent genes. However, this has been complex to decipher, and recent data suggests STAT1 GOF mutations have no effect on the dephosphorylation of STAT1, and the elevated pSTAT1 levels are explained through higher levels of total STAT1 protein. At present, JAK inhibitors such as Ruxolitinib and Jakinibs are being used to treat STAT1 GOF manifestations but, for unknown reasons, this method proves only effective for some. Alternative approaches such as allo-HSCT have been trialled but remain difficult, with only 40% overall survival reported in 20 cases to date. With this in mind, this project aims to utilise the novel gene-editing platform of CRISPR technology to a) correct the specific missense defects and restore wild-type STAT1 (wt-STAT1) sequence or b) knock out the overactive allele to cause haploinsufficiency as a therapeutic for STAT1 GOF disease. Initially, we will work to model the disease utilising the U3A cell line that lack endogenous STAT1 and transduce these with lentiviral constructs encoding wt-STAT1 or STAT1 GOF mutations. We will then design and screen multiple synthetic guide RNAs (sgRNAs) for their ability and specificity to guide Cas9 protein to the overactive allele for its cleavage. Having then selected the optimal sgRNAs, they will be taken forward for assessment in T cells, and used to initiate the homology-directed repair of GOF mutations via an exogenous DNA encoding wt-STAT1. If one/both of the approaches are successful, we will then explore the use of this technology in haematopoietic stem cells and potentially the practicality of the approach in vivo. Furthermore, in conjunction with the gene-editing aspect of the project, we will look to further deconstruct the pathogenesis of GOF STAT1 disease using our panel of U3A cell lines and patient primary cells, with a particular interest in GOF STAT1 disease in myeloid cells which appear to be neglected in the literature thus far.

信号转导和转录激活因子1（STAT 1）是Janus激酶（JAK）/STAT信号级联的重要组成部分，其从不同的细胞表面受体转导信号以启动基因转录。STAT 1在免疫中起着特别关键的作用，正如STAT 1缺陷小鼠和STAT 1功能丧失突变患者产生的免疫应答不足所证明的那样。2011年，货车de Veerdonk et al.和Liu et al.首次在人类中发现了常染色体显性STAT 1功能获得性（GOF）突变。现在已知它们是慢性粘膜皮肤念珠菌病的主要原因，并进一步与细菌和病毒感染、自身免疫、动脉瘤和癌症的易感性增强相关。这些结果主要归因于受损的Th 17免疫、缺陷的自然杀伤细胞成熟和功能、B细胞淋巴细胞减少和对干扰素的过度反应。STAT 1 GOF疾病背后的机制以前被认为是STAT 1去磷酸化的阻碍，导致磷酸化STAT 1（pSTAT 1）在细胞核中的积累，从而导致STAT 1依赖性基因的过表达。然而，这是复杂的破译，最近的数据表明STAT 1 GOF突变对STAT 1的去磷酸化没有影响，并且通过更高水平的总STAT 1蛋白解释了升高的pSTAT 1水平。目前，JAK抑制剂如Ruxolitinib和Jakinibs被用于治疗STAT 1 GOF的表现，但由于未知的原因，这种方法仅对某些人有效。替代方法，如allo-HSCT已经过试验，但仍然很困难，到目前为止，在20例病例中报告的总生存率仅为40%。考虑到这一点，该项目旨在利用CRISPR技术的新型基因编辑平台来a）纠正特定的错义缺陷并恢复野生型STAT 1（wt-STAT 1）序列或B）敲除过度活跃的等位基因以引起单倍不足，作为STAT 1 GOF疾病的治疗方法。最初，我们将利用缺乏内源性STAT 1的U3 A细胞系建立疾病模型，并用编码wt-STAT 1或STAT 1 GOF突变的慢病毒构建体将其转染。然后，我们将设计和筛选多个合成的指导RNA（sgRNA），以确定它们将Cas9蛋白引导到过度活跃的等位基因进行切割的能力和特异性。然后选择了最佳的sgRNA，它们将在T细胞中进行评估，并用于通过编码wt-STAT 1的外源DNA启动GOF突变的同源性定向修复。如果一种/两种方法都成功，我们将探索这种技术在造血干细胞中的应用，并可能探索这种方法在体内的实用性。此外，结合该项目的基因编辑方面，我们将使用我们的U3 A细胞系和患者原代细胞进一步解构GOF STAT 1疾病的发病机制，特别关注骨髓细胞中的GOF STAT 1疾病，迄今为止，这似乎在文献中被忽视了。