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: 7621030
• 负责人: KARIN L GAENSLER
• 金额: $ 38.63万
• 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/7621030
• 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; Regulatory Element; Resistance; Risk; Safety; Site; Stem cell transplant; Stem cells; Subfamily lentivirinae; T-Lymphocyte; Testing; Thalassemia; Thymidine Kinase; Time; Tissues; Toxic effect; Transgenes; Transplantation; Treatment Efficacy; Treatment Protocols; Variant; 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; public health relevance; reconstitution; response; stem cell population; therapeutic gene; transduction efficiency; vector

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.

描述（由申请人提供）：同种异体移植是多种遗传性疾病的标准治疗方法，但受到相容性供体短缺、移植造血干细胞（HSC）数量少、必要准备方案的毒性和移植后免疫抑制以及移植物抗宿主病（GVHD）的限制。基因治疗遗传性疾病的障碍包括基因表达的变化、HSC转导的低效以及对载体和治疗基因的免疫反应。为了规避这些障碍，我们建议在新生儿期进行HSC移植和基因治疗，因为在新生儿期更容易获得耐受性，同时采用积极的选择策略，在体内扩增耐药供体细胞。假设1：新生儿期是转基因异体造血干细胞移植实现长期移植的免疫个体发育的独特窗口期。新生儿移植为探索对新抗原的耐受诱导机制和异体干细胞移植提供了一个模型系统。胎儿肝脏和骨髓源性HSC将通过慢病毒介导的p140k -甲基鸟嘌呤甲基转移酶（MGMT）基因转移在体外转导。这种变异的DNA修复酶能够抵抗苯鸟嘌呤（一种内源性MGMT抑制剂）和氯乙基化剂（如BCNU），从而通过体内化学选择在干细胞水平上富集供体细胞。假设2：基于mgmt的阳性选择策略既能扩增供体干细胞，又能消耗供体和宿主来源的同种异体反应细胞，从而消除毒性消融或免疫抑制治疗的需要。这种转基因异体造血干细胞移植的潜在风险包括GVHD和插入性突变，导致克隆增殖失控。假设3：用含有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性T细胞的阴性选择成为可能。更昔洛韦的阴性选择也能够消除恶性克隆群体。目的1：新生儿非清髓移植同源MGMT-GFP慢病毒转导的HSC和体内化学选择将用于确定是否诱导对GFP新抗原的耐受。目的2：新生儿移植和体内选择MGMT-TK慢病毒转导的HSC将应用于半同种异体模型，重现单倍体移植。我们将确定稳定供体嵌合的机制，并使用更昔洛韦来控制GVHD或自主克隆群体，如果它们出现的话。目的3：将在地中海贫血小鼠模型中测试非清髓性、半同种异体新生儿移植/化疗的治疗效果。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。