The Development of Novel, Therapeutic Gene Editing Strategies for STAT1 gain-of-function Immunodeficiency.

造血干细胞基因疗法（HSC-GT）正在迅速成为许多单基因原发性免疫缺陷（PID）的可行治疗选择，迄今已有几项成功的应用。然而，到目前为止，临床HSC-GT仅具有靶向功能丧失（LoF）PID，其中使用病毒载体基因添加或CRISPR/Cas介导的同源定向修复（HDR）的简单基因添加方法就足够了。这种方法不适合杂合功能获得性（GoF）突变，这是一个日益认识到的严重PID的原因。一种这样的GoF PID是STAT 1 GoF疾病，其由信号转导子和转录激活子1（STAT 1）中的种系GoF突变引起，并且特征在于Janus相关激酶（JAK）-STAT信号传导途径的过度激活。患者患有广泛的临床表型，包括慢性粘膜皮肤念珠菌病、严重的机会性感染、自身免疫和恶性肿瘤等。治疗选择有限，抗生素预防和感染的及时治疗以及针对自身免疫的全身免疫抑制构成了保守治疗的支柱。一小部分患者可能会接受异基因造血干细胞移植（HSCT），尽管这与相对较差的结局相关（约40-50%的存活率）。最近，少数患者接受了JAK抑制剂治疗，但没有关于这种方法的耐受性，有效性或持久性的数据。HSC-GT为STAT 1 GoF疾病提供了一种有吸引力的替代治疗选择，并将在本博士项目中进行探索。将研究几种不同的基因编辑方法，包括但不限于：- 突变体GoF STAT 1等位基因的靶向破坏-使用HDR和/或碱基编辑策略靶向修复STAT 1 GoF突变-在内源性STAT 1起始密码子的控制下插入野生型STAT 1 cDNA体外和体内（鼠模型）功能测定。该项目不仅与STAT 1 GoF疾病的治疗相关，而且将成为GoF PID治疗性基因编辑的首例案例研究。此外，这些基因编辑策略也可能更广泛地适用于患有其他先天性STAT 1缺陷的患者，包括那些患有STAT 1缺陷的患者。

Haematopoietic stem cell gene therapies (HSC-GT) are fast becoming a viable treatment option for many monogenic primary immunodeficiencies (PID), with several successful applications to date. However, thus far clinical HSC-GT has only targeted loss-of-function (LoF) PID where simple gene addition approaches using viral vector gene addition or CRISPR/Cas-mediated homology directed repair (HDR) suffice. Such approaches are not appropriate for heterozygous gain-of-function (GoF) mutations, which are an increasingly recognized cause of severe PID. One such GoF PID is STAT1 GoF disease, caused by germline GoF mutations in signal transducer and activator of transcription 1 (STAT1), and characterized by overactivation of the Janus associated kinase (JAK)-STAT signaling pathway. Patients suffer from a broad clinical phenotype including Chronic Mucocutaneous Candidiasis, severe opportunistic infections, autoimmunity and malignancy amongst others. Treatment options are limited, with antimicrobial prophylaxis and prompt treatment of infections together with systemic immunosuppression for autoimmunity constituting the mainstay of conservative management. A small group of patients may undergo allogeneic haematopoietic stem cell transplantation (HSCT), although this has been associated with relatively poor outcomes (~40-50% survival rate). Very recently a small number of patients have been treated with JAK inhibitors, but no data exists on tolerability, efficacy or durability of this approach.HSC-GT offers an attractive alternative treatment option for STAT1 GoF disease and will be explored in this PhD project. Several different gene editing approaches will be investigated including but not limited to: - Targeted disruption of the mutant GoF STAT1 allele- Targeted repair of STAT1 GoF mutations using HDR and/or base editing strategies - Insertion of wild-type STAT1 cDNA under control of the endogenous STAT1 start codon The efficacy of the above gene editing strategies in correcting immune cell function will be tested using various in-vitro and in-vivo (murine models) functional assays. This project is relevant not only for the treatment of STAT1 GoF disease, but will be a first-of-its-kind case study for therapeutic gene editing of GoF PID. Furthermore, these gene editing strategies may also be more widely applicable to patients with other inborn errors in STAT1, including those with STAT1 deficiency.