Enhancing immune regulation in gene therapy for hemophilia

项目总结/摘要 腺相关病毒（AAV）载体广泛用于人类基因治疗：例如，两种产品已被FDA批准。 批准和三种载体，通过肝脏基因转移纠正X连锁出血性疾病血友病， 正在进行III期临床试验。对载体或转基因的不期望的和有时意想不到的免疫应答 产品可能阻碍长期治疗，包括CD 8 + T细胞和抗体对转基因产品的反应 和病毒衣壳，其在高全身性载体剂量下偶尔诱导严重的免疫毒性。 因此，短期免疫抑制，例如通过皮质类固醇，现在已经被纳入到治疗中。 正在探索许多方案和B细胞耗竭。这项建议旨在阐明分子和细胞 这些免疫应答是对AAV转导的小鼠和人肝细胞的先天性和适应性免疫应答的基础。 这些实验旨在验证中心假设，即先天免疫的靶向阻断 反应和特定的细胞因子信号传导途径与增强的免疫调节相结合， 这是成功的肝脏AAV定向基因转移所必需的。这一概念是基于 研究表明：1。肝脏是转基因诱导免疫耐受的良好靶点 由于其微环境最终使调节性T细胞（Treg）能够控制抗体， 和T细胞反应。2.使用雷帕霉素本身或与其他药物组合的免疫调节方案 药物导致所需的免疫调节。3. captain特异性CD 8 + T细胞的交叉致敏需要 树突状细胞的不同亚群之间的合作，其中关键涉及TLR 9-MyD 88信号转导和IFN I. 4.对于肝脏中转基因产物特异性CD 8 + T细胞的TLR 9非依赖性活化，IL-1 R是必需的。 5.最先进的体外培养技术和在体内扩增人肝细胞和人肝细胞的能力， 免疫缺陷小鼠中的先天免疫细胞已经被开发出来。6.中和抗体的形成 可以预防肝AAV基因转移时针对凝血因子VIII和衣壳的抗体， 通过最佳的免疫调节实现对血友病A小鼠的持续治疗。根据这些发现， 该提案寻求i）定义AAV中人肝细胞和肝环境的先天免疫应答 ii）确定IL-1 R-MyD 88信号通路促进CD 8 + T细胞增殖的机制; iii）开发最佳的瞬时免疫调节方案， 防止B和T细胞对转基因产物和载体的应答，从而确保持续的治疗。

PROJECT SUMMARY/ABSTRACT Adeno-associated viral (AAV) vectors are widely used in human gene therapy: e.g. two products have been FDA approved and three vectors, which correct the X-linked bleeding disorder hemophilia by hepatic gene transfer, are in Phase III clinical trials. Undesired and sometimes unexpected immune responses to vector or transgene product can impede long-lasting therapy, including CD8+ T cell and antibody responses to transgene product and viral capsid, which at high systemic vector doses occasionally induce severe immune toxicities. Consequently, short-term immune suppression, for example by corticosteroids has now been incorporated into many protocols and B cell depletion is being explored. This proposal seeks to elucidate the molecular and cellular underpinnings of innate and adaptive immune responses to AAV-transduced mouse and human hepatocytes. The experiments are designed to test the central hypothesis that a targeted blockade of innate immune responses and specific cytokine signaling pathways combined with an empowered immune regulation are requisite for a successful hepatic AAV-directed gene transfer. This concept is based upon the outcomes of studies, which establish that: 1. The liver is an excellent target for immune tolerance induction to transgene products owing to its microenvironment that ultimately empowers regulatory T cells (Treg) to control antibody and T cell responses. 2. Immune modulatory protocols using rapamycin by itself or in combination with other drugs result in the desired immune regulation. 3. Cross-priming of capsid-specific CD8+ T cells requires cooperation between different subsets of dendritic cells, which critically involves TLR9-MyD88 signaling and IFN I. 4. For TLR9-independent activation of transgene product-specific CD8+ T cells in the liver, IL-1R is requisite. 5. State-of-the art in vitro culture techniques and ability to in vivo expand both human hepatocytes and human innate immune cells in immunodeficient mice have been developed. 6. Formation of neutralizing antibodies directed against coagulation factor VIII and capsid upon hepatic AAV gene transfer can be prevented and sustained therapy is achieved hemophilia A mice by optimal immune modulation. Based on these findings, the proposal seeks to i) define the innate immune response of human hepatocytes and liver environment in AAV gene transfer; ii) define the mechanism by which the IL-1R-MyD88 signaling pathway promotes CD8+ T cell responses in hepatic AAV gene transfer; and iii) develop optimal transient immune modulatory regimens that prevent B and T cell responses to the transgene product and the vector and thereby assure sustained therapy.