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

血友病A(HA)是一种X连锁的出血性疾病，由突变引起的凝血因子VIII(FVIII)缺乏引起 在F8基因中。这种疾病影响到全世界出生的1：5000的男性。FVIII蛋白的替代疗法是 有效预防/控制出血，但约30%的患者会产生FVIII的抑制剂(中和 同种抗体)，使FVIII无效；因此，增加了发病率和死亡率。免疫耐受 诱导(ITI)是根除抑制物/恢复FVIII免疫耐受的唯一成功策略。ITI 方案是基于FVIII蛋白注射数月/数年，在约60%的病例中有效，但高 成本(约100万/年)阻止了它在发达世界以外的地方使用。因此，根除抑制剂的新方法 都是迫切需要的。正在进行的AAV肝脏基因治疗HA的临床试验结果为 治疗性FVIII。这些研究使用了在HEK-293或Sf9细胞中制造的载体，这两种细胞的 基础生物学和临床结果。我们的中心假设是AAV-FVIII肝脏基因治疗是理想的 基于我们在抑制HA犬中的概念验证报告的ITI方案(Finn等人血液，2010)和新的 初步数据。我们将使用两种不同的新的高反应抑制物狗模型，带有犬F8基因 与最具挑战性的抑制剂患者组相关的突变可能会从AAV ITI中受益。这个 这项建议的基本原理是，一次注射AAV肝脏基因疗法可提供(A)有效的根除 高效价抑制剂，(B)高应答HA犬对FVIII免疫耐受的恢复和维持 和(C)持续的FVIII表达，改善了抑制剂根除后的出血表型。这个 目的1：确定AAV基因治疗诱导小鼠免疫耐受的效果。 高响应性HA犬模型。我们将推进在狗模型中测试AAV基因疗法的努力 几个不同的品种来确定与抑制物根除(转基因)动力学相关的因素 级别/持续时间)。目的2：通过rAAV-Sf9衍生的AAV-cFVIII基因治疗，确定ITI的潜能 高响应性HA犬模型。AAV-Sf9术后3年后FVIII表达最近意外下降 HA患者的FVIII引起了人们对耐久性的担忧。到目前为止，潜在的机制尚不清楚。我们 假设这可能是Sf9衍生载体和/或FVIII的组合。我们将在狗身上确定是否 AAV-Sf9随时间影响FVIII的表达水平及其清除抑制物和诱导耐受的能力 致FVIII。目的3：明确腺病毒介导的ITI的机制(S)。在这些新奇的狗模型中，我们将 AAV-ITI和TO后特异性B细胞，特别是T调节细胞池和功能的特征 确定两种细胞系衍生载体系统中免疫耐受的潜在机制。成功 这项建议的完成将支持AAV肝脏基因治疗针对抑制剂HA患者的临床试验。一种能力 诱导免疫耐受的基因治疗的研究可能与其他用酶治疗的遗传病有关。 替代疗法，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.