Mechanisms of Innate and Adaptive Immune Responses to AAV-FVIII Gene Transfer

项目摘要/摘要 腺相关病毒载体肝内基因转移治疗X连锁出血 混乱的血友病已经进入了多个I/II期和4个III期临床试验，所有这些试验都在寻求稳定恢复 肝细胞中长期表达的止血作用。与其他治疗方式不同，基因 治疗有可能治愈这种疾病，从而消除了频繁注射凝血的需要。 因子或其他药物。然而，实现持续的纠正一直是一个重大挑战。 血友病A(凝血因子VIIII，FVIII缺乏，约占血友病患者的80%)。FVIII是 很难在高水平上表达。因此，需要较高的媒介剂量。尽管如此，FVIII活动在正常情况下 在临床试验中达到了超生理范围。虽然这一结果在2017年得到了广泛的庆祝，但它 很明显，FVIII在这些高水平的表达是不稳定的。所有患者的发病率都在逐渐下降 在2-4年观察，一直到治疗范围的低端，引起了对耐用性的严重担忧。 其他研究开始显示类似的结果，而较低的FVIII水平，导致较温和的载体 剂量，似乎是持续的。肝毒性和类固醇治疗是第一个阶段的典型特征。 大剂量基因转移之年。现在越来越多的人意识到基础科学研究对 达到安全有效的效果。我们的初步数据有力地支持了这样一个假设，即 对FVIII和载体(实质上受载体影响)的细胞应激和先天免疫反应 设计)限制了治疗表达的稳定性，也促进了获得性免疫。众所周知， FVIII的高水平表达导致内质网中未折叠蛋白的积累，细胞应激和 毒性、聚集和未折叠蛋白反应(UPR)的诱导。我们的数据显示，肝脏AAV 基因转移，虽然有可能诱导对转基因产品的免疫耐受，但也激活了先天的 可导致细胞免疫反应的免疫途径和细胞因子反应。AAV载体引入 病原体相关分子模式(PAMP)。细胞应激和先天免疫是相互关联的， 损伤相关的分子模式(抑制)驱动免疫力。事实上，我们看到FVIII的表达逐渐丧失 在血友病A小鼠模型中，尽管缺乏抗体形成。该项目特别提出：i) 确定“亚基因组颗粒”引起对AAV的先天和获得性免疫反应的机制- 原代人肝细胞/天然免疫细胞和小鼠模型中的FVIII；ii)定义免疫反应 载体和转基因设计在肝脏AAV基因转移中对抗FVIII的作用机制； 明确导致FVIII表达逐渐丧失的机制，并制定持续治疗方案 和免疫耐受性。

PROJECT SUMMARY/ABSTRACT Hepatic in vivo gene transfer with adeno-associated viral (AAV) vectors for treatment of the X-linked bleeding disorder hemophilia has advanced to multiple Phase I/II and 4 Phase III clinical trials, all seeking to stably restore hemostasis through long-term expression in hepatocytes. As opposed to other treatment modalities, gene therapy has the potential to cure the disease, thus eliminating the need for frequent injections of coagulation factors or other medicines. However, it has been a major challenge to accomplish sustained correction of hemophilia A (deficiency in coagulation factor VIIII, FVIII, representing ~80% of hemophilic patients). FVIII is difficult to express at high levels. Hence, high vector doses are required. Nonetheless, FVIII activity in the normal to super-physiological range was achieved in clinical trial. While this result was widely celebrated in 2017, it has since become clear that FVIII expression at these high levels is not stable. A gradual decline in all patients was observed in years 2-4, down to the lower end of the therapeutic range, raising serious concerns about durability. Other studies begin to show similar outcome, whereas lower FVIII levels, resulting from more modest vector doses, appear to be sustained. Hepatotoxicity and treatment with steroids were typical features during the first year of high-dose gene transfer. There is now a growing realization that basic science studies are paramount to achieve safety and efficacy. Our preliminary data strongly support the hypothesis that the closely interlinked cellular stress and innate immune responses to FVIII and the vector (which are substantially impacted by vector design) limit stability of therapeutic expression and also drive adaptive immunity. It is well established that expression of FVIII at high levels results in the accumulation of unfolded protein in the ER, cellular stress and toxicity, aggregation, und induction of the unfolded protein response (UPR). Our data show that hepatic AAV gene transfer, while having the potential to induce immune tolerance to transgene products, also activates innate immune pathways and cytokine responses that can lead to cellular immune responses. AAV vectors introduce pathogen-associated molecular patterns (PAMPs). Cellular stress and innate immunity are interlinked, and damage-associated molecular patters (DAMPs) drive immunity. In fact, we see gradual loss of FVIII expression in a hemophilia A mouse models despite a lack of antibody formation. This project specifically proposes to: i) Define the mechanisms by which “subgenomic particles” cause innate and adaptive immune responses to AAV- FVIII in primary human hepatocytes/innate immune cells and in murine models; ii) define immune response mechanisms against FVIII in hepatic AAV gene transfer as a function of vector and transgene design; and iii) define the mechanisms that lead to gradual loss of FVIII expression and develop protocols for sustained therapy and immune tolerance.