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）：同种异体移植是各种遗传性疾病的标准治疗，但受到相容供体短缺、移植造血干细胞（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）的限制。遗传性疾病的基因治疗应用的障碍包括可变的基因表达，HSC的低效转导，以及对载体和治疗基因的免疫应答。我们建议绕过这些障碍，HSC移植和基因治疗的独特结合移植在新生儿时期，当耐受性可能更容易实现，在体内扩增的耐药供体细胞的积极选择策略。假设1：新生儿期是免疫个体发育过程中的一个独特窗口，用于移植基因修饰的同种异体HSC以实现长期植入。新生儿移植为探索对新抗原的耐受诱导机制和同种异体干细胞移植提供了模型系统。胎儿肝脏和骨髓来源的HSC将通过慢病毒介导的P140 K-甲基鸟嘌呤甲基转移酶（MGMT）基因转移进行离体转导。这种变体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的抗性，使得能够在干细胞水平上用体内化学选择富集供体细胞。假设2：基于MGMT的阳性选择策略将扩增供体干细胞并耗尽供体和宿主来源的同种异体反应性细胞，从而消除对毒性消融或免疫抑制治疗的需要。这种遗传修饰的同种异体HSC移植的潜在风险包括GVHD和插入诱变，导致不受控制的克隆增殖。假设3：用掺入MGMT和胸苷激酶（TK）的阳性/阴性慢病毒载体转导HSC将能够阴性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能够消除恶性克隆群体。具体目标：目标1：将使用新生儿中的同基因MGMT-GFP慢病毒转导的HSC的非清髓性移植和体内化学选择来确定是否诱导对GFP新抗原的耐受性。目标2：新生儿移植和MGMT-TK慢病毒转导的HSC的体内选择将应用于重现单倍体相合移植的半同种异体模型中。我们将定义稳定供体嵌合体的潜在机制，并使用更昔洛韦来控制GVHD或自主克隆群体，如果他们出现。目标3：将在地中海贫血的鼠模型中测试非清髓性、半同种异体新生儿移植/化学选择的治疗功效。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影响。 公共卫生相关性：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，但相关风险往往限制了这种治疗的应用。我们的目标是降低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早期纠正遗传性疾病。我们开发了一种结合基因治疗和干细胞移植的新方法，可以减少或消除目前移植方案和程序中使用的毒性，高剂量化疗和免疫抑制剂的需求。