FIX tolerance in hemophilia gene therapy

描述(申请人提供)：血友病B是一种X连锁出血性疾病，由凝血因子IX(FIX)缺乏引起。血友病B患者反复自发性出血，受严重影响的患者由于主要关节反复出血而变得残废。目前对血友病B的治疗包括输注FIX浓缩液以应对出血事件。这种方法是非预防性的，高达5%的血友病B患者在接触FIX后会产生抗FIX抗体。持续输注FIX浓缩液可以诱导FIX耐受性，尽管潜在的机制尚不清楚。然而，持续输液疗法的巨大成本使其无法应用于大多数血友病患者，即使在西方发达国家也是如此。基于直接肌肉注射重组腺相关病毒2型(AAV2)的基因治疗可以提供FIX的持续表达，但在具有免疫能力的动物中产生抗FIX抑制抗体而变得复杂。申请人最近报道，直接肌肉注射AAV血清1型载体(AAV1)在免疫活性血友病B小鼠中实现了持续和完全的血友病B表型纠正，而没有形成抗FIX抑制抗体。了解免疫耐受修复的机制对于血友病治疗的成功发展至关重要。我们推测，AAV1FIX基因在骨骼肌中持续高水平表达可通过辅助性T淋巴细胞能量机制(S)或克隆性缺失和/或调节性T淋巴细胞的产生来诱导FIX耐受。这一假设将通过完成两个特定的目标来验证：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，研究抗原提呈细胞和辅助T细胞在抗原提呈后的成熟状态。申请人的长期目标是开发一种成功的血友病B的基因治疗方法。本建议的目的是阐明肌肉注射AAV1后导致FIX耐受的机制(S)，开发一种新的方法，不仅可以防止抑制性FIX抗体的产生，而且可以诱导血友病B患者对原有抑制性FIX抗体的耐受。

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.