Toward Safer Gene Therapy for Hemophilia A

项目总结摘要 X连锁出血性疾病血友病的基因治疗很有希望实现持久的治愈。 将腺相关病毒(AAV)基因转移到男性重型肝炎患者肝脏的四项临床试验 血友病目前正在进行多个第三阶段临床试验。血友病A(因子缺乏 )是一种更常见的疾病(约80%的患者)，传统上更难治疗 通过基因治疗，因为FVIII是一个大分子，不能有效地表达和分泌。尽管如此， 初步结果显示该病已完全治愈。然而，FVIII水平大幅下降超过 时间，引发了耐受性的令人担忧的问题，患者也经历了长期的轻度肝毒性 尽管在基因治疗的第一年期间进行了类固醇药物治疗。最近的多个观察结果引发了严重的 关于肝脏基因治疗血友病A的安全性的问题这些迫切需要解决 这种有希望的方法可以安全地应用于患者，并实现持续矫正。例如, 肝毒性和FVIII表达下降的原因尚不清楚，这突显了我们在 了解载体和肝细胞之间的相互作用以及FVIII表达和 肝细胞，以及免疫系统在长期结果中的作用。人们也再次对此感到担忧 关于插入突变。我们将解决这些与生物的生物学有关的基本和机械问题 AAV和FVIII。这一建议的中心假设是AAV和FVIII的多个相互关联的特征 生物学限制了治疗性表达的持久性，并造成了严重的安全问题。此外，我们假设 解开这些机制将允许设计将这些问题降至最低的载体和协议，因此 从而获得持久的治疗和更高的安全性。该项目结合了FVIII生物学、细胞应激 反应，免疫学和AAV媒介生物学，并被组织成3个科学项目，一个行政 核心和2个科学核心。项目1(考夫曼)寻求克服FVIII蛋白错误折叠和细胞毒性。 项目2(肖)将揭示导致亚基因组AAV载体颗粒形成的机制 在载体生产过程中通过核酸酶和重组活动形成。项目3(Herzog)将定义 AAV-FVIII基因转移的先天和获得性免疫反应机制。三个方面的目标 项目将得到一个行政核心(核心A)的支持，这是一个为体外培养提供人类肝细胞的核心 和体内研究(核心B)，以及执行AAV载体的开发和分子分析的核心(核心 c)。总体而言，该项目将个别调查人员的专门知识应用于解决主要 FVIII生物学、血友病的基因治疗、肝脏导向的基因转移和分子生物学中的未解问题 和AAV载体的免疫生物学。

PROJECT SUMMARY ABSTRACT Gene therapy for the X-linked bleeding disorder hemophilia holds much promise to accomplish a lasting cure. Four clinical trials utilizing adeno-associated viral (AAV) gene transfer to the livers of males with severe hemophilia are currently being investigated in multiple Phase III clinical trials. Hemophilia A (deficiency in factor VIII, FVIII), the more common form of the disease (~80% of patients), has traditionally been more difficult to treat by gene therapy because FVIII is a large molecule and not efficiently expressed and secreted. Nonetheless, initial results demonstrated complete correction of the disease. However, FVIII levels declined substantially over time, raising worrying questions about durability, and patients also experienced prolonged mild hepatotoxicity despite steroid drug treatment during the first year of gene therapy. Multiple recent observations raise serious questions about the safety of hepatic gene therapy for hemophilia A. These urgently need to be addressed so that this promising approach can be safely applied to patients and to achieve sustained correction. For instance, the reasons for hepatotoxicity and for the decline in FVIII expression are unclear, highlighting critical gaps in our knowledge of the interactions between the vector and hepatocytes and between the FVIII expression and hepatocytes, as well as the role of the immune system in long-term outcome. There is also renewed concern about insertional mutagenesis. We will address these basic and mechanistic questions related to the biology of AAV and FVIII. The central hypothesis of this proposal is that multiple interconnected features of AAV and FVIII biology limit durability of therapeutic expression and pose serious safety concerns. Further, we postulate that unraveling these mechanisms will allow for design of vectors and protocols that minimize these problems, thus resulting in lasting therapy and enhanced safety. The program combines expertise in FVIII biology, cellular stress responses, immunology, and AAV vector biology and is structured into 3 scientific Projects, an administrative Core and 2 scientific Cores. Project 1 (Kaufman) seeks to overcome FVIII protein misfolding and cell toxicity. Project 2 (Xiao) will uncover the mechanisms that lead to formation of subgenomic AAV vector particles that form during vector production through nuclease and recombination activities. Project 3 (Herzog) will define the mechanisms of innate and adaptive immune responses to AAV-FVIII gene transfer. The objectives of the three projects will be supported by an administrative core (Core A), a core that provides human hepatocytes for in vitro and in vivo studies (Core B), and a core that performs development and molecular analysis of AAV vectors (Core C). Overall, this project applies the expertise of the individual investigators towards addressing major unanswered questions in FVIII biology, gene therapy for hemophilia, liver-directed gene transfer, and molecular and immunobiology of AAV vectors.