Development of a lentiviral gene therapy vector to modify effector T and NK cells to control disease activity in perforin deficient hemophagocytic lymphohistiocytosis (HLH) patients

噬血细胞性淋巴组织细胞增生症（HLH）是一种免疫失调性疾病，其定义为极端炎症的症状，导致包括骨髓、肝脏和中枢神经系统在内的各种器官的致命损伤。主要特征为发热、脾肿大、眼睑下垂、呼吸困难和血细胞减少引起的感染、高胆红素血症、低纤维蛋白原血症、噬血细胞作用、NK细胞活性降低、铁蛋白和sCD 25升高。几种原发性免疫缺陷易于发生HLH，称之为噬血细胞综合征。在大多数这些疾病中，基因受到影响，这些基因是导致穿孔素和颗粒酶诱导靶细胞凋亡的细胞溶解分泌途径的一部分。在实施结构化方案之前，1年生存率非常低。如今，随着造血干细胞移植（HSCT）的引入，在最佳情况下存活率达到> 90%，尽管接受HSCT的患者仍有发生HSCT的任何常见并发症的风险。对于缺乏匹配供体的患者，需要其他治疗选择。在少数非恶性遗传疾病中，自体干细胞的离体校正似乎是一个有吸引力的选择。在过去的二十年中，通过病毒转导操作自体干细胞的技术已经使基因治疗方案在有限数量的候选diseases.In家族性HLH（FHL）II型-这应该是构成最大的组FHL患者-在PRF 1基因（编码穿孔素）突变导致升高的抗原呈递树突状细胞与连续的T细胞超活化。伦敦大学学院儿童健康研究所的分子免疫学单位目前开发了一种安全有效的编码穿孔素基因的慢病毒基因治疗载体，可用于修饰自体造血干细胞，用于治疗穿孔素缺乏的HLH。最初的实验表明，将穿孔素基因的正确拷贝导入造血干细胞可以恢复穿孔素缺陷小鼠中观察到的T和NK细胞缺陷。这些重建的小鼠在病毒攻击后可免受HLH的发展。类似地，将基因转移到自体外周T或NK细胞中可能控制患有HLH的患者的疾病活动。这项任务，这构成了研究员的项目，将通过开发最好的病毒载体来促进小鼠细胞中T和NK细胞中穿孔素的表达来实现。在选择最佳效应细胞群（其提供HLH小鼠模型中细胞毒性缺陷的校正）之后，我们将评估穿孔素重构效应细胞在鼠模型中控制病毒诱导的疾病的能力。如果基因转移成功，进一步的临床试验应该是这些临床前研究的主要目标。

Hemophagocytic lymphohistiocytosis (HLH) is an immune dysregulatory disorder, which is defined by symptoms of extreme inflammation leading to fatal damage in various organs including bone marrow, liver and central nervous system. Key features are fever, splenomegaly, bleedings, dyspnea and infections due to cytopenia, hypertriglyceridemia, hypofibrinogenemia, hemophagocytosis, decreased NK cell activity, elevated ferritin and sCD25. Several primary immunodeficiencies are prone to HLH, terming these diseases hemophagocytic syndromes. In most of these disorders, genes are affected, which are part of the cytolytic secretory pathway that cause perforin and granzymes to induce apoptosis in target cells. Before the implementation of a structured protocol the 1-year survival had been very low. Nowadays with the introduction of hematopoetic stem cell transplantation (HSCT) the survival reaches > 90% in an optimal setting, though patients receiving HSCT stay at risk of developing any of the usual complications of HSCT. In patients, who lack of a well-matched donor, other curative options are warranted. In few non-malignant genetic disorders ex-vivo correction of autologous stem cells seems to be an attractive option. In the past two decades techniques for manipulations of autologous stem cells by viral transduction has enabled gene therapy protocols in a limited number of candidate diseases.In familial HLH (FHL) type II - which is supposed to constitute the largest group among patients with FHL - mutations in the PRF1 gene (coding perforin) lead to elevated antigen presentation by dendritic cells with consecutive T cell hyperactivation. The Molecular Immunology Unit at the UCL Institute of Child Health currently develops a safe and effective lentiviral gene therapy vector encoding the perforin gene, that can be used to modify autologous hematopoietic stem cells for treatment of perforin deficient HLH. Initial experiments have shown that introduction of the correct copy of the perforin gene into hematopoietic stem cells can restore the T and NK cell defects seen in the perforin deficient mice. These reconstituted mice are protected against development of HLH after viral challenge. Similarly, gene transfer into autologous peripheral T or NK cells might control disease activity in patients suffering from HLH. This task, which constitutes the project of the research fellow, will be achieved by the development of the best viral vector to promote perforin expression in T and NK cells in mouse cells. After selection of the best effector cell population, which provides correction of cytotoxic defects in the mouse model of HLH, we will assess the ability of perforin reconstituted effector cells in controlling virus induced disease in the murine model. In case of a successful gene transfer further clinical trials are supposed to be a main goal of these pre-clinical studies.