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

噬血细胞淋巴组织细胞增多症是一种免疫调节障碍，其特征是严重的炎症症状，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害。主要特征是发热、脾肿大、出血、呼吸困难和因细胞减少、高甘油三酯血症、低纤维蛋白原血症、吞噬血细胞、NK细胞活性降低、铁蛋白和sCD25升高而引起的感染。几种原发免疫缺陷容易发生HLH，这些疾病称为噬血细胞综合征。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。在实施结构化议定书之前，1年存活率很低。如今，随着造血干细胞移植(HSCT)的引入，在最佳的环境下，存活率可达90%，尽管接受HSCT的患者仍有发生HSCT常见并发症的风险。对于缺乏匹配捐赠者的患者，有必要选择其他治疗方法。在少数非恶性遗传病中，体外矫正自体干细胞似乎是一个有吸引力的选择。在过去的20年里，通过病毒转导操纵自体干细胞的技术已经使基因治疗方案能够在有限数量的候选疾病中实现。在家族性HLH(FHL)中，II型被认为是FHL患者中最大的群体，PRF1基因(编码穿孔素)的突变导致树突状细胞递送抗原增加，并连续T细胞激活。伦敦大学学院儿童健康研究所的分子免疫学研究所目前正在开发一种安全有效的慢病毒基因治疗载体，编码穿孔素基因，可用于修饰自体造血干细胞，用于治疗穿孔素缺乏症。初步实验表明，将正确拷贝的穿孔素基因导入造血干细胞可以修复穿孔素缺陷小鼠的T和NK细胞缺陷。这些重组的小鼠在病毒攻击后被保护以防止HLH的发展。同样，将基因转移到自体外周T细胞或NK细胞可能会控制HLH患者的疾病活动性。这项任务是这位研究员的项目，将通过开发最好的病毒载体来促进穿孔素在小鼠T细胞和NK细胞中的表达来实现。在选择了最佳效应细胞群后，我们将评估穿孔素重组效应细胞在小鼠模型中控制病毒诱导疾病的能力。在基因转移成功的情况下，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.