FIX tolerance in hemophilia gene therapy

描述（由申请方提供）：血友病B是一种由于因子IX（FIX）缺乏引起的X连锁出血性疾病。血友病B患者患有复发性自发性出血发作，并且严重受累的患者由于主要关节中的复发性出血而致残。血友病B的当前治疗包括输注FIX浓缩物以应对出血事件。这种方法是非预防性的，高达5%的血友病B患者在暴露于FIX后产生抗FIX抗体。FIX浓缩物的连续输注可诱导FIX耐受性，但其潜在机制尚不清楚。然而，持续输注治疗的巨大成本使得即使在西方发达国家，也禁止将这种治疗应用于大多数血友病患者。基于直接肌内注射重组腺相关病毒血清型2（AAV 2）的基因治疗可提供FIX的持续表达，但由于在免疫活性动物中产生抗FIX抑制性抗体而变得复杂。申请人最近报道了直接肌内注射AAV血清型1载体（AAV 1）实现了持续和完全的血友病B表型校正，而在免疫活性血友病B小鼠中不形成抗FIX抑制性抗体。了解FIX免疫耐受的机制对于开发成功的血友病治疗至关重要。我们假设骨骼肌中AAV 1表达的FIX持续高水平表达通过辅助性T淋巴细胞能量或克隆缺失和/或调节性T淋巴细胞的产生机制诱导FIX耐受。将通过完成两个特定目标来检验该假设：1）研究持续高水平FIX表达对诱导FIX耐受性的影响，以及2）研究在基于AAV 1的基因转移方案中抗原呈递后抗原呈递细胞和辅助T细胞应答的成熟状态。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AAV 1直接肌内注射后FIX耐受性的机制，开发一种新方法，不仅可防止产生抑制性FIX抗体，还可诱导血友病B患者对既存抑制性FIX抗体的耐受性。

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.