Enhancing immune regulation in gene therapy for hemophilia

项目摘要/摘要 腺相关病毒(AAV)载体广泛用于人类基因治疗：例如，两种产品已通过FDA 批准和三个载体，通过肝脏基因转移纠正X连锁出血性疾病血友病， 正在进行第三阶段的临床试验。对载体或转基因的不良免疫反应，有时甚至是意想不到的免疫反应 产品会阻碍长期治疗，包括CD8+T细胞和对转基因产品的抗体反应 以及病毒衣壳，在高全身媒介剂量下，偶尔会引起严重的免疫毒性。 因此，短期免疫抑制，例如由皮质类固醇引起的，现在已被纳入 许多方案和B细胞耗尽正在被探索。这一提议试图阐明分子和细胞 对AAV转导的小鼠和人肝细胞的先天和获得性免疫反应的基础。 这些实验旨在检验中心假设，即有针对性地阻断先天免疫 反应和特定的细胞因子信号通路与增强的免疫调节相结合 是成功的肝脏AAV导向的基因转移所必需的。这一概念是基于 研究表明：1.肝脏是诱导免疫耐受转基因的极佳靶点 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.单独使用雷帕霉素或与其他药物联合使用的免疫调节方案 药物可以起到理想的免疫调节作用。3.衣壳特异性CD8+T细胞的交叉激发需要 树突状细胞不同亚群之间的合作，其中关键涉及TLR9-MyD88信号和干扰素 对于肝脏中转基因产物特异性CD8+T细胞的TLR9非依赖性激活，IL-1R是必需的。 5.最新的体外培养技术和体内扩增人肝细胞和人的能力 免疫缺陷小鼠的先天免疫细胞已经被开发出来。6.中和抗体的形成 针对凝血因子VIII和衣壳蛋白对肝脏AAV基因转移的预防和治疗 持续治疗是血友病A小鼠通过优化免疫调节实现的。基于这些发现， 该提案旨在i)确定AAV中人肝细胞和肝环境的先天免疫反应 基因转移；ii)确定IL-1R-MyD88信号通路促进CD8+T细胞的机制 肝脏AAV基因转移的反应；以及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反应，从而确保持续治疗。

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.