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

信号转导和转录激活因子1(STAT1)是Janus Kinase(JAK)/STAT信号转导通路的重要组成部分，通过转导多种细胞表面受体的信号来启动基因转录。STAT1在免疫中发挥着特别关键的作用，STAT1缺陷小鼠和STAT1功能缺失突变患者产生的免疫反应不足就证明了这一点。2011年，van de Veerdonk等人首次在人类中发现了常染色体显性显性STAT1功能增益(GOF)突变。和Liu等人。它们现在被认为是慢性皮肤粘膜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加有关。这些结果主要是由于Th17免疫功能受损，自然杀伤细胞成熟和功能缺陷，B细胞淋巴细胞减少和对干扰素的过度反应性。STAT1 GOF病的发病机制以前被认为是抑制STAT1去磷酸化，导致核内磷酸化的STAT1(PSTAT1)积聚，从而导致STAT1依赖基因的过度表达。然而，这一点很难破译，最近的数据表明，STAT1 GOF突变对STAT1的去磷酸化没有影响，pSTAT1水平的升高是通过高水平的总STAT1蛋白来解释的。目前，JAK抑制剂如Ruxolitinib和Jakinibs正被用于治疗STAT1 GOF症状，但由于未知的原因，这种方法仅对某些患者有效。替代方法，如异基因造血干细胞移植，已经进行了试验，但仍然很困难，到目前为止，在20例病例中报告的总存活率只有40%。有鉴于此，本项目旨在利用CRISPR技术的新型基因编辑平台a)纠正特定的错义缺陷并恢复野生型STAT1(wt-STAT1)序列或b)敲除过度活跃的等位基因导致单倍体不足，作为治疗STAT1 GOF疾病的方法。最初，我们将利用缺乏内源性STAT1的U3A细胞系来建立疾病模型，并用编码wt-STAT1或STAT1 GOF突变的慢病毒构建体转导这些细胞。然后，我们将设计和筛选多个合成引导RNA(SgRNAs)，以了解它们引导Cas9蛋白切割过度活跃的等位基因的能力和特异性。然后，在选择了最佳的sgRNAs后，它们将被带到T细胞中进行评估，并通过编码wt-STAT1的外源DNA启动GOF突变的同源定向修复。如果这两种方法中的一种/两种都成功了，我们将探索这项技术在造血干细胞中的应用，并潜在地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U3A细胞系和患者原代细胞小组进一步解构GOF STAT1疾病的发病机制，尤其是在髓系细胞中的GOF STAT1疾病，到目前为止文献中似乎忽略了这一点。

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.