Immunology of Factor IX Gene Transfer to Liver

描述（由申请人提供）： 近年来，X连锁出血性疾病血友病（由编码凝血因子VIII，血友病A或因子IX，血友病B的基因突变引起）的基因治疗已在几个I/II期临床试验中进行了测试。临床方案包括肌肉和肝脏定向因子IX（F.IX）基因转移与腺相关病毒（AAV）载体。来自这些试验和动物模型临床前研究的数据表明，免疫应答可能是这种安全、有效和非常有前途的治疗方法成功治疗的最大障碍。同样重要的是，凝血因子抑制性抗体（抑制剂）的形成是血友病治疗的一个普遍问题和严重并发症。维持免疫系统对转基因产物的无反应性是成功的基因治疗的关键要求。在基因替代疗法中，治疗性基因产物可代表可经受适应性免疫应答的新抗原。对F.IX的抑制剂形成是这种反应的一个例子，其通常依赖于CD 4 + T辅助。通过基因转移治疗的最理想的结果是诱导对由转基因表达介导的治疗性蛋白质的免疫耐受性，而不需要额外操纵免疫系统。在最近的研究中，我们证明了在用AAV-2载体进行肝基因转移后，最低水平的表达可靠地在几种但不是所有品系的小鼠中诱导了对F.IX的稳健的免疫耐受。本申请的目标是建立通过肝基因转移诱导对F.IX的免疫耐受的最佳方案，研究肝抗原呈递如何导致T细胞对转基因产物耐受的机制，并开发用于体内产生F. IX特异性调节性T细胞的方案。我们假设，一个最佳的肝脏基因转移协议可以实现高成功率的耐受性诱导，无论接受基因转移的动物的遗传背景。免疫耐受将被定义为持续的肝脏和全身F.IX表达（没有抗体形成）和对随后用F.IX蛋白免疫的应答失败。防止对转基因产物的有害免疫应答的替代策略是在基因转移之前产生F. IX特异性调节性T细胞。基于调节性T细胞的有效活化在随后的治疗期间阻止了F. IX特异性T和B细胞的假设，将评估两种替代策略：粘膜（鼻）抗原施用和设计为在诱导CD 4 + CD 25+调节性T细胞的同时删除效应T细胞的新方案。将比较所有策略对F. IX无反应的持续时间和稳健性。 公共卫生相关性声明： 该项目将开发新的策略，用于预防治疗遗传性出血性疾病血友病时的不良免疫反应。对凝血因子的免疫应答代表血友病患者治疗的严重并发症，增加发病率和死亡率，并显著增加治疗成本。因此，非常需要上级免疫耐受方案。

DESCRIPTION (provided by applicant): Gene therapy for the X-linked bleeding disorder hemophilia (which is caused by mutations in the gene encoding blood coagulation factor VIII, hemophilia A, or factor IX, hemophilia B) has been tested in several Phase I/II clinical trials in recent years. Clinical protocols included muscle- and liver-directed factor IX (F.IX) gene transfer with adeno-associated viral (AAV) vectors. Data from these trials and from pre-clinical studies in animal models indicate that immunological responses likely represent the largest hurdle for successful therapy by this otherwise safe, efficacious, and highly promising treatment approach. Equally important, formation of inhibitory antibodies (inhibitors) to coagulation factors is a general concern and a serious complication of any treatment of hemophilia. Maintaining unresponsiveness of the immune system to the transgene product is a key requirement for successful gene therapy. In gene replacement therapy, the therapeutic gene product may represent a neo-antigen that can be subject to an adaptive immune response. Inhibitor formation to F.IX is an example of such a response, which is typically dependent on CD4+ T help. The most desirable outcome of treatment by gene transfer is induction of immune tolerance to the therapeutic protein mediated by transgene expression without a need for additional manipulation of the immune system. In recent studies, we demonstrated that a minimal level of expression upon hepatic gene transfer with AAV-2 vectors reliably induced robust immune tolerance to F.IX in several but not all strains of mice. The goals of this application are to establish an optimal protocol for induction of immune tolerance to F.IX by hepatic gene transfer, to investigate the mechanism of how hepatic antigen presentation leads to T cell tolerance to the transgene product, and to develop protocols for in vivo generation of F.IX-specific regulatory T cells. We hypothesize that an optimal hepatic gene transfer protocol can achieve a high success rate of tolerance induction regardless of the genetic background of the animal receiving gene transfer. Immune tolerance will be defined as sustained hepatic and systemic F.IX expression (without antibody formation) and failure to respond to subsequent immunization with F.IX protein. An alternative strategy to prevent deleterious immune responses to the transgene product is the generation of F.IX-specific regulatory T cells prior to gene transfer. Based on the hypothesis that efficient activation of regulatory T cells prevents F.IX-specific T and B cells during subsequent therapy, two alternative strategies will be evaluated: mucosal (nasal) antigen administration and a novel protocol designed to delete effector T cells concomitant with induction of CD4+CD25+ regulatory T cells. All strategies will be compared for duration and robustness of unresponsiveness to F.IX. Public Health Relevance Statement: This project will develop novel strategies for prevention of undesired immune responses in treatment of the inherited bleeding disorder hemophilia. Immune responses to coagulation factors represent a severe complication of treatment of patients with hemophilia, increase morbidity and mortality, and substantially increase cost of treatment. Therefore, superior immune tolerance protocols are highly desirable.