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免疫缺陷(PID)的可行治疗选择，迄今已有几个成功的应用。然而，到目前为止，临床HSC-GT只有靶向功能丧失(LoF)的PID，而简单的基因添加方法使用病毒载体基因添加或CRISPR/Cas介导的同源定向修复(HDR)就足够了。这种方法不适合杂合性功能增益(GOF)突变，这是越来越多人认识到的严重PID的原因。信号转导和转录激活因子1(STAT1)基因突变引起的STAT1GOF病就是其中之一，其特征是Janus相关蛋白-STAT信号通路的过度激活。患者患有广泛的临床表型，包括慢性皮肤粘膜念珠菌病、严重的机会性感染、自身免疫和恶性肿瘤等。治疗选择有限，抗菌素预防和及时治疗感染，以及全身免疫抑制自身免疫构成保守治疗的主要内容。一小部分患者可以接受异基因造血干细胞移植(HSCT)，尽管这与相对较差的结果(约40-50%的存活率)有关。最近，少数患者接受了JAK抑制剂的治疗，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STAT1 Gof病提供了一种有吸引力的替代治疗方案，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：-靶向中断突变的GOF STAT1等位基因-使用HDR和/或碱基编辑策略靶向修复STAT1突变-在内源STAT1起始密码子的控制下插入野生型STAT1 cDNA上述基因编辑策略在纠正免疫细胞功能方面的有效性将通过各种体外和体内(小鼠模型)功能分析来测试。该项目不仅与STAT1戈夫病的治疗相关，而且将是首个针对戈夫-彼德治疗性基因编辑的案例研究。此外，这些基因编辑策略也可能更广泛地适用于STAT1中其他先天错误的患者，包括那些STAT1缺乏症患者。

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.