Factor IX tolerance in hemophilia gene therapy

描述（由申请人提供）：血友病B是一种由IX因子（FIX）缺乏引起的x连锁出血性疾病。B型血友病患者反复出现自发性出血，严重的患者因主要关节反复出血而致残。目前对B型血友病的治疗包括输注FIX浓缩液以应对出血发作。这种方法是非预防性的，高达5%的B型血友病患者在接触FIX后产生抗FIX抗体。持续输注FIX浓缩液可诱导小鼠对FIX的耐受，但机制尚不清楚。然而，持续输注疗法的巨大成本使得大多数血友病患者难以接受这种治疗，即使在发达的西方国家也是如此。基于肌内直接注射重组腺相关病毒血清型2 （AAV2）的基因治疗可以提供FIX的持续表达，但在免疫能力强的动物中产生抗FIX抑制抗体是复杂的。申请人最近报道了直接肌内注射AAV血清型载体（AAV1）在免疫功能正常的血友病B小鼠中持续和完全的血友病B表型纠正而不形成抗fix抑制抗体。了解FIX免疫耐受的机制对于血友病治疗的成功发展至关重要。我们假设，骨骼肌中AAV1表达的高水平FIX通过辅助性T淋巴细胞能量或克隆性缺失和/或调节性T淋巴细胞的产生的机制诱导FIX耐受。该假设将通过完成两个特定目标来验证：1)研究持续高水平的FIX表达对诱导FIX耐受的影响；2)在基于AAV1的基因转移方案中研究抗原呈递细胞的成熟状态和辅助T细胞在抗原呈递后的反应。申请人的长期目标是开发一种成功的血友病B基因治疗方法。本提案的目的是阐明直接肌内注射AAV1后FIX耐受的机制，开发一种新的方法，不仅可以防止血友病B患者产生抑制性FIX抗体，还可以诱导对预先存在的抑制性FIX抗体产生耐受。

DESCRIPTION (provided by applicant): Hemophilia B is an X-linked hemorrhagic disorder due to factor IX (FIX) deficiency. Hemophilia B patients suffer from recurrent spontaneous bleeding episodes and the severely affected patients become crippled resulting from recurrent bleeding in the major joints. Current treatment for hemophilia B consists of infusion of FIX concentrates in response to the bleeding episode. This approach is non-prophylactic and up to 5 percent of hemophilia B patients develop anti-FIX antibody after exposure to FIX. Continuous infusion of FIX concentrates can induce FIX tolerance, although the underlying mechanism is not clear. However, the huge cost of continuous infusion therapy makes it prohibitive to apply this treatment to the majority of hemophilia patients, even in developed western countries. Gene therapy based on direct intramuscular injection of recombinant adeno-associated virus serotype 2 (AAV2) can provide sustained expression of FIX, but is complicated by generation of anti-FIX inhibitory antibodies in immune competent animals. The applicants recently reported that direct intramuscular injections of AAV serotype one vectors (AAV1) effected sustained and complete hemophilia B phenotype correction without formation of anti-FIX inhibitory antibody in immunocompetent hemophilia B mice. Understanding the mechanism accounting for immune tolerance to FIX is crucial for the development of successful hemophilia therapy. We hypothesize that sustained expression of high levels of FIX expressed from AAV1 in skeletal muscle induces FIX tolerance by mechanism(s) of helper T lymphocyte energy or clonal deletion, and/or generation of regulatory T lymphocytes. The hypothesis will be tested by completing two specific aims: 1) investigating the effects of sustained high level FIX expression on induction of FIX tolerance, and 2) investigating the state of maturation of antigen presenting cells and helper T cell responses after antigen presentation in an AAV1- based gene transfer protocol. The applicant's long-term goal is to develop a successful gene therapy approach for hemophilia B. The objective of this proposal is to elucidate the mechanism(s) accounting for FIX tolerance after direct intramuscular injection of AAV1, developing a novel approach not only to prevent generation of inhibitory FIX antibody but also to induce tolerance to pre-existing inhibitory FIX antibody in hemophilia B patients.