Immune tolerance induction by AAV-FVIII gene therapy for canine hemophilia A with inhibitors

血友病A（HA）是一种X-连锁出血性疾病，由突变引起的因子VIII（FVIII）缺乏引起 F8基因该疾病影响全世界1：5，000的男性出生。FVIII蛋白的替代治疗是 有效预防/控制出血，但约30%的患者产生FVIII抑制剂（中和 同种抗体），使FVIII无效;因此，增加发病率和死亡率。免疫耐受 诱导（ITI）是消除抑制剂/恢复对FVIII的免疫耐受性的唯一成功策略。ITI 治疗方案基于FVIII蛋白注射数月/年，在约60%的病例中有效，但高剂量的FVIII蛋白注射对患者的生存率无显著影响。 成本（约100万美元/年）阻止了它在发达国家以外的使用。因此，抑制剂根除的新方法 是迫切需要的。正在进行的无抑制剂的HA的AAV肝脏基因治疗临床试验导致 治疗性FVIII。这些研究使用在HEK-293或Sf 9细胞中生产的载体，这些细胞在其生物学特性上不同。 基础生物学和临床结果。我们的中心假设是AAV-FVIII肝基因治疗是一种理想的治疗方法。 ITI方案基于我们在抑制剂HA犬中的概念验证报告（Finn et al Blood，2010）和新的 初步数据。我们将使用2种新的具有犬F8基因的不同高反应抑制剂HA犬模型 与可能受益于AAV ITI的最具挑战性的抑制剂患者组相关的突变。的 该建议的基本原理是单次注射AAV肝基因治疗提供了（A）有效的根除 高滴度抑制剂，（B）恢复和维持高应答HA犬对FVIII的免疫耐受性 和（C）在抑制剂根除后改善出血表型的连续FVIII表达。的 具体目的是目的1：确定AAV基因治疗在诱导免疫耐受中的功效。 我们将继续努力在狗模型中测试AAV基因疗法， 几个不同的品种，以确定与抑制剂根除动力学相关的因素（转基因 水平/持续时间）。目的2：确定通过rAAV-Sf 9衍生的AAV-cFVIII基因治疗ITI在肿瘤中的潜力。 高反应HA犬模型。在AAV-Sf 9-TdR治疗后3年，FVIII表达最近意外下降。 HA患者的FVIII引起了持久性的担忧。到目前为止，根本的机制是未知的。我们 假设这可能是Sf 9衍生载体和/或FVIII的组合。我们将在狗身上确定， AAV-Sf 9随时间推移影响FVIII表达水平及其根除抑制剂和诱导耐受性的能力 到FVIII。目的3：定义AAV介导的ITI的潜在机制。在这些新颖的狗模型中，我们将 表征特定B细胞，尤其是调节性T细胞池和在AAV-ITI后的功能， 确定两种细胞系来源的载体系统中免疫耐受的潜在机制。成功 该提案的完成将支持针对抑制剂HA患者的AAV肝脏基因治疗临床试验。的能力 基因治疗诱导免疫耐受的可能性与用酶治疗的其他遗传性疾病有关 替代治疗并伴有抗药中和抗体。

Hemophilia A (HA) is an X-linked bleeding disorder caused by a deficiency in factor VIII (FVIII) due to mutations in the F8 gene. The disease affects 1:5,000 male born worldwide. Replacement therapy with FVIII protein is effective in preventing/controlling bleeding but ~30% of patients develop inhibitors to FVIII (neutralizing alloantibodies) that renders FVIII ineffective; thus, increasing morbidity and mortality. Immune tolerance induction (ITI) is the only successful strategy for eradication of inhibitors/restore immune tolerance to FVIII. ITI regimens are based on FVIII protein injections for months/years and is efficacious in ~60% of cases, but the high cost (~1million/year) prevents its use outside the developed word. Thus, new approaches for inhibitor eradication are urgently needed. Ongoing AAV liver gene therapy clinical trials for HA without inhibitors resulted in therapeutic FVIII. These studies are using vectors manufactured in either HEK-293 or Sf9 cells that differ in their basic biology and clinical outcomes. Our central hypothesis is that AAV-FVIII liver gene therapy is an ideal ITI regimen based on our proof-of-concept report in inhibitor HA dogs (Finn et al Blood, 2010) and novel preliminary data. We will use 2 novel distinct high-responding inhibitor HA dog models with the canine F8 gene mutations associated with the most challenging inhibitor patient group that likely would benefit from AAV ITI. The rationale of this proposal is that a single injection of AAV liver gene therapy provides (A) efficient eradication of high titer inhibitors, (B) restoration and maintenance of immune tolerance to FVIII in high responding HA dogs and (C) continuous FVIII expression that improves the bleeding phenotype after inhibitor eradication. The specific aims are Aim 1: Determine the efficacy of AAV gene therapy in inducing immune tolerance in high-responding HA dog models.We will advance our efforts testing AAV gene therapy in dog models across several distinct breeds to define the factors associated with kinetics of inhibitor eradication (transgene levels/duration). Aim 2: Determine the potential of ITI by rAAV-Sf9-derived AAV-cFVIII gene therapy in high-responding HA dog models. Recent unexpected decline of FVIII expression after 3 years post- AAV-Sf9- FVIII in HA patients raised concerns of durability. To date, the underlying mechanism is unknown. We hypothesized that this could be a combination of Sf9-derived vector and/or FVIII. We will determine in dogs if AAV-Sf9 impacts FVIII expression levels over time and its ability to eradicate inhibitors and to induce tolerance to FVIII. Aim 3: Define the mechanism(s) underlying AAV-mediated ITI. In these novel dog models, we will characterize specific B cells and especially T regulatory cells pool and function following AAV-ITI and to determine the underlying mechanism of immune tolerance in both cell line-derived vector systems. Successful completion of this proposal would support AAV liver gene therapy clinical trial for inhibitor HA patients. The ability of gene therapy to induce immune tolerance is likely to be relevant to other genetic diseases treated with enzyme replacement therapy and complicated by antidrug neutralizing antibodies.