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: 7463365
• 负责人: KARIN L GAENSLER
• 金额: $ 38.6万
• 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/7463365
• 关键词: Affect; Alloantigen; Allogenic; Allografting; Anemia; Animal Model; Animals; Antigens; Autologous; Biological Models; Blood; Bone Marrow; Caring; Carmustine; Carmustine/O6-Benzylguanine; Cells; Chimerism; Clinical Trials; Compatible; Condition; 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; Green Fluorescent Proteins; 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; Numbers; Other Genetics; Outcome; Population; Procedures; Proliferating; Proto-Oncogene Protein c-kit; Protocols documentation; Public Health; Regulatory Element; Resistance; Risk; Safety; Site; Standards of Weights and Measures; Stem cell transplant; Stem cells; Subfamily lentivirinae; T-Lymphocyte; Testing; Thalassemia; Therapeutic immunosuppression; Thymidine Kinase; Time; Tissues; Toxic effect; Transgenes; Transplantation; Treatment Efficacy; Treatment Protocols; Variant; base; cellular transduction; 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; response; 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护理标准，但受到相容供体短缺、移植造血干细胞 (HSC) 数量低、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。应用基因疗法治疗遗传性疾病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反应。我们建议通过独特地将新生儿期移植（此时更容易实现耐受）与耐药供体细胞体内扩增的正向选择策略相结合，来规避 HSC 移植和基因治疗的这些障碍。假设1：新生儿期是免疫个体发育过程中转基因同种异体HSC移植实现长期植入的独特窗口。新生儿移植为探索新抗原耐受诱导和同种异体干细胞植入机制提供了一个模型系统。胎儿肝脏和骨髓来源的 HSC 将通过慢病毒介导的 P140K-甲基鸟嘌呤甲基转移酶 (MGMT) 基因转移进行离体转导。这种变体 DNA 修复酶赋予对苄基鸟嘌呤（内源性 MGMT 抑制剂）和氯乙基化剂（如 BCNU）的抗性，从而能够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 的正选策略将既扩增供体干细胞，又消除供体和宿主来源的同种异体反应细胞，从而消除对毒性消融或免疫抑制治疗的需要。这种转基因同种异体 HSC 移植的潜在风险包括 GVHD 和插入突变，导致不受控制的克隆增殖。假设 3：使用包含 MGMT 和胸苷激酶 (TK) 的正/负慢病毒载体转导 HSC 将能够对增殖的同种反应性 T 细胞进行阴性选择。更昔洛韦的负选择也能够消除恶性克隆群体。具体目标： 目标 1：在新生儿中非清髓性移植同基因 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。目标2：MGMT-TK慢病毒转导的HSC的新生儿移植和体内选择将应用于再现半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，并使用更昔洛韦来控制 GVHD 或自主克隆群体（如果出现）。目标 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对基因治疗和遗传及其他疾病的移植具有广泛影响的明确数据。 公共健康相关性：虽然造血干细胞移植是许多血液相关疾病的唯一治疗方法，但相关风险往往限制了这种治疗的应用。我们的目标是降低与移植相关的发病率和死亡率，并能够及早纠正大量受影响个体的遗传性疾病。我们开发了一种将基因治疗和干细胞移植相结合的新方法，可以减少或消除当前移植方案和程序中使用的有毒、高剂量化疗和免疫抑制的需要。