Neonatal Chemoselection Following Ex Vivo Gene Transfer For Hereditary Disorders

抽象的。同种异体移植是各种遗传性疾病的标准治疗方法， 缺乏相容的供体，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，以及移植物抗宿主病（GVHD）。 遗传性疾病基因治疗应用的障碍包括可变基因表达，低效的基因治疗， HSC的转导以及对载体和治疗基因的免疫应答。我们建议规避 这些障碍，HSC移植和基因治疗的独特组合移植期间， 新生儿期，当耐受性可能更容易实现，与体内的积极选择策略 耐药供体细胞的扩增。假设1：新生儿期是一个独特的窗口， 免疫个体发生用于移植遗传修饰的同种异体HSC以实现长期植入。 新生儿移植为探索新生儿移植耐受诱导机制提供了一个模型系统。 抗原和同种异体干细胞移植。将转导胎肝和骨髓来源的HSC 通过慢病毒介导的P140 K-甲基鸟嘌呤甲基转移酶（MGMT）的基因转移离体。该变型 DNA修复酶赋予对内源性MGMT的抑制剂苄基鸟嘌呤和 氯乙基化剂如BCNU，能够在干细胞水平上富集供体细胞， 化学选择假设2：基于MGMT的阳性选择策略将扩增供体干细胞 细胞和消耗供体和宿主来源的同种异体反应性细胞，消除了对毒性消融或 免疫抑制治疗。这种基因修饰的同种异体HSC移植的潜在风险 包括GVHD和插入诱变，导致不受控制克隆增殖。假设3：HSC 用掺入MGMT和胸苷激酶（TK）的阳性/阴性慢病毒载体转导将 能够负选择增殖的同种异体反应性T细胞。更昔洛韦的阴性选择也使 消除恶性克隆种群。 特定目的：AIM 1：同基因MGMT-GFP慢病毒转导的非清髓性移植 将使用新生儿中的HSC和体内化学选择来确定是否对GFP neo. 抗原被诱导。目的2：新生儿移植和MGMT-TK慢病毒转导的体内筛选 HSC将应用于重现单倍体相合移植的半同种异体模型。我们将定义 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 人口，如果他们出现。目的3：探讨非清髓性半异基因新生儿骨髓移植的疗效 移植/化学选择将在小鼠模型测试？地中海贫血我们建议的研究 这些新生儿移植模型将产生明确的数据，对基因治疗具有广泛的意义。 以及遗传和其他疾病的移植。项目叙述 虽然造血干细胞移植是许多血液相关疾病的唯一治愈性疗法， 相关的风险往往限制了这种治疗的应用。我们的目标是降低发病率和死亡率 与移植相关，并使大量的遗传性疾病的早期纠正成为可能。 受影响的个人。我们已经开发出一种新的方法结合基因治疗和干细胞 减少或消除对毒性、高剂量化疗和免疫抑制的需要的移植 用于当前的移植方案和程序。

Abstract. 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 neo- antigens, 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 neo- antigen 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. Project Narrative 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.