Neonatal Chemoselection Following Ex Vivo Gene Transfer For Hereditary Disorders

遗传性疾病离体基因转移后的新生儿化学选择

• 批准号: 8259431
• 负责人: KARIN L GAENSLER
• 金额: $ 38.24万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-12 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8259431
• 关键词: Affect; Alloantigen; Allogenic; Allografting; Anemia; Animal Model; Animals; Antigens; Autologous; Biological Models; Blood; Bone Marrow; Caring; Carmustine; Cells; Chimerism; Clinical Trials; Confounding Factors (Epidemiology); DNA Repair Enzymes; Data; Disease; Dissociation; Dose; Drug resistance; Engraftment; Experimental Designs; Fetal Liver; Frequencies; Ganciclovir; Gene Expression; Gene Transfer; Genes; Genetic; Genomics; Goals; Graft Rejection; Growth; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hereditary Disease; High Dose Chemotherapy; Homologous Transplantation; Human; Immune; Immune response; Immunosuppression; Immunosuppressive Agents; Inborn Genetic Diseases; Inbred BALB C Mice; Individual; Inherited; Insertional Mutagenesis; Isogenic transplantation; Laboratories; Lentivirus Vector; Malignant - descriptor; Mediating; Medical; Modeling; Modification; Morbidity - disease rate; Mus; Mutagenesis; Neonatal; Other Genetics; Outcome; Population; Procedures; Proliferating; Proto-Oncogene Protein c-kit; Protocols documentation; Regimen; Regulatory Element; Resistance; Risk; Safety; Site; Stem cell transplant; Stem cells; Subfamily lentivirinae; T cell response; T-Lymphocyte; Testing; Thalassemia; Thymidine Kinase; Time; Tissues; Toxic effect; Transgenes; Transplantation; Treatment Efficacy; Variant; abstracting; base; cellular transduction; conditioning; cytotoxic; dosage; gene function; gene therapy; graft vs host disease; improved; in vivo; in vivo Model; inhibitor/antagonist; lentivirally transduced; mortality; mouse model; neonatal exposure; neonate; novel strategies; reconstitution; stem cell population; therapeutic gene; transduction efficiency; vector

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.

抽象的。同种异体移植是一种治疗各种遗传性疾病的标准疗法，它受到 相容供者短缺，移植造血干细胞(HSC)数量较少， 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基因表达可变、效率低下 HSC的转导，以及对载体和治疗性基因的免疫反应。我们建议绕过 造血干细胞移植和基因治疗的这些障碍通过在移植过程中独特地结合移植来实现 新生儿期，在体内采用积极的选择策略，可能更容易获得耐受性 耐药供体细胞的扩增。假设1：新生儿期是一个独特的窗口 免疫个体发生学为移植转基因异基因造血干细胞实现长期植入。 新生儿移植为探索诱导耐受机制提供了一个模型系统。 抗原和同种异体干细胞植入。胎儿肝脏和骨髓来源的HSC将被转导 慢病毒介导的P140K-甲基鸟嘌呤甲基转移酶(MGMT)基因的体外转移。这个变种 DNA修复酶使人对内源性MGMT的抑制剂--苯基鸟嘌呤产生抵抗力，并对 氯乙基化试剂，如BCNU，使供体细胞能够在体内干细胞水平上浓缩 化学选择。假设2：基于MGMT的积极选择策略将扩大供者干细胞 细胞和耗尽供体和宿主来源的同种异体反应细胞，消除了有毒的消融或 免疫抑制治疗。这种转基因异基因造血干细胞移植的潜在风险 包括GVHD和插入突变，导致不受控制的克隆性增殖。假设3：HSC 携带MGMT和TK的阳性/阴性慢病毒载体的转导将 启用对增殖的同种异体反应T细胞的阴性选择。更昔洛韦的阴性选择也使 消除恶性克隆种群。 具体目标：目标1：同基因MGMT-GFP慢病毒转导的非清髓性移植 新生儿体内的HSC和体内的化学选择将被用来确定是否对GFP neo- 抗原被诱导出来。目的2：转导MGMT-TK慢病毒的新生儿移植及体内筛选 HSC将应用于半异基因模型的半相合移植。我们将定义 稳定供体嵌合体和使用更昔洛韦控制GVHD或自主克隆的潜在机制 人口，如果它们出现的话。目的3：非清髓半异基因新生儿的治疗效果 移植/化学选择将在小鼠模型中进行测试？地中海贫血。我们在报告中提出的研究 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。项目叙事 虽然造血干细胞移植是治疗许多血液相关疾病的唯一疗法， 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，并使更多的人能够及早纠正遗传性疾病 受影响的个体。我们已经开发出一种结合基因治疗和干细胞的新方法。 减少或消除有毒的大剂量化疗和免疫抑制的需要的移植 在当前的移植方案和程序中使用。