Neonatal Chemoselection Following Ex Vivo Gene Transfer For Hereditary Disorders

描述(由申请人提供)：同种异体移植是治疗各种遗传性疾病的标准，由于缺乏相容的供者，移植的造血干细胞(HSC)数量少，必要的准备方案和移植后免疫抑制的毒性，以及移植物抗宿主病(GVHD)，异基因移植受到限制。遗传性疾病基因治疗应用的障碍包括基因表达变化、HSC转导效率低下以及对载体和治疗性基因的免疫反应。我们建议通过独特地将新生儿时期的移植与体内扩增耐药供体细胞的阳性选择策略相结合来绕过干细胞移植和基因治疗的这些障碍。假设1：新生儿期是移植转基因异基因HSC以实现长期植入的唯一免疫个体发育窗口。新生儿移植为探索诱导对新抗原的耐受机制和异基因干细胞植入提供了一个模型系统。慢病毒载体介导的P140K-甲基鸟嘌呤甲基转移酶(MGMT)基因可在体外转导胎肝和骨髓来源的HSC。这种变异的DNA修复酶对内源性MGMT的抑制剂苯基鸟嘌呤和氯乙基化试剂(如BCNU)具有抵抗力，使供体细胞能够通过体内化学选择在干细胞水平上进行浓缩。假设2：基于MGMT的阳性选择策略将既扩大供体干细胞，又耗尽供体和宿主来源的同种异体反应细胞，消除了毒性消融或免疫抑制治疗的需要。移植这种转基因同种异体造血干细胞的潜在风险包括移植物抗宿主病和插入突变，导致不受控制的克隆性增殖。假设3：携带MGMT和胸苷激酶(TK)的阳性/阴性慢病毒载体转导HSC将使增殖的同种异体反应T细胞的阴性选择成为可能。更昔洛韦的阴性选择也使恶性克隆种群得以消除。具体目的：目的1：非清髓性、同基因MGMT-GFP慢病毒转导的HSC在新生儿体内的移植和体内化学选择将被用来确定是否诱导对GFP新抗原的耐受。目的：将MGMT-TK慢病毒转导的HSC用于半异基因模型半相合移植。我们将确定稳定供体嵌合体的潜在机制，并使用更昔洛韦控制GVHD或自主克隆群体，如果它们出现的话。目的：在地中海贫血小鼠模型上测试非清髓性、半异基因新生儿移植/化学选择的治疗效果。我们在这些新生儿移植模型中提出的研究将产生明确的数据，对遗传和其他疾病的基因治疗和移植具有广泛的意义。 公共卫生相关性：尽管造血干细胞移植是许多血液相关疾病的唯一治愈方法，但相关风险往往限制了这种治疗的应用。我们的目标是减少与移植相关的发病率和死亡率，并使更多受影响的个体能够及早纠正遗传性疾病。我们开发了一种结合基因治疗和干细胞移植的新方法，减少或消除了目前移植方案和程序中使用的有毒、大剂量化疗和免疫抑制的需要。

DESCRIPTION (provided by applicant): Allogeneic transplantation, the standard-of-care for a variety of inherited disorders, is limited by a shortage of compatible donors, low numbers of engrafting hematopoietic stem cells (HSC), the toxicity of necessary preparative regimens and post-transplant immunosuppression, and graft vs. host disease (GVHD). Hurdles to the application of gene therapy for hereditary disorders include variable gene expression, inefficient transduction of HSC, and immune responses to vectors and therapeutic genes. We propose to circumvent these hurdles to HSC transplantation and gene therapy by uniquely combining transplantation during the neonatal period, when tolerance may be more readily achieved, with a positive selection strategy for in vivo amplification of drug-resistant donor cells. HYPOTHESIS 1: The neonatal period is a unique window during immune ontogeny for transplantation of genetically modified allogeneic HSC to achieve long-term engraftment. Neonatal transplantation provides a model system for exploring mechanisms of tolerance induction to neoantigens, and allogeneic stem cell engraftment. Fetal liver and bone marrow-derived HSC will be transduced ex vivo by lentivirus-mediated gene transfer of P140K-methylguanine methytransferase (MGMT). This variant DNA repair enzyme confers resistance to benzylguanine, an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU, enabling enrichment of donor cells at the stem cell level with in vivo chemoselection. HYPOTHESIS 2: An MGMT-based positive selection strategy will both expand donor stem cells and deplete allo-reactive cells of donor and host origin, eliminating the need for toxic ablative or immunosuppressive treatment. Potential risks of transplantation of such genetically modified allogeneic HSC include GVHD and insertional mutagenesis resulting in uncontrolled clonal proliferation. HYPOTHESIS 3: HSC transduction with a positive/negative lentiviral vector incorporating MGMT and thymidine kinase (TK) will enable negative selection of proliferating alloreactive T cells. Negative selection with ganciclovir also enables elimination of malignant clonal populations. Specific Aims: AIM 1: Non-myeloablative, transplantation of syngeneic MGMT-GFP lentivirus-transduced HSC in neonates and in vivo chemoselection will be used to determine whether tolerance to the GFP neoantigen is induced. AIM 2: Neonatal transplantation and in vivo selection of MGMT-TK lentivirus-transduced HSC will be applied in a semi-allogeneic model recapitulating haploidentical transplantation. We will define mechanisms underlying stable donor chimersim and use ganciclovir to control GVHD or autonomous clonal populations, should they arise. AIM 3: The therapeutic efficacy of non-myeloablative, semi-allogeneic neonatal transplantation/chemoselection will be tested in a murine model of ¿ thalassemia. The studies we propose in these neonatal transplantation models will generate definitive data with broad implications for gene therapy and transplantation for genetic and other disorders. Public Health Relevance: Although hematopoietic stem cell transplantation is the only curative therapy for many blood-related disorders, associated risks often limit the application of this treatment. Our goal is to reduce the morbidity and mortality associated with transplantation and to enable early correction of inherited disorders in larger numbers of affected individuals. We have developed a new approach combining gene therapy and stem cell transplantation that reduces or eliminates the need for toxic, high dose chemotherapy and immunosuppression used in current transplant protocols and procedures.