Toward Safer Gene Therapy for Hemophilia A

项目摘要 X连锁出血性疾病血友病的基因治疗有望实现持久治愈。 四项临床试验利用腺相关病毒（AAV）基因转移到男性肝脏的严重 血友病目前正在多项III期临床试验中进行研究。血友病A（因子缺乏） VIII，FVIII），更常见的疾病形式（约80%的患者），传统上更难以治疗 因为FVIII是一种大分子，不能有效地表达和分泌。尽管如此， 初步结果显示疾病已完全治愈。然而，FVIII水平大幅下降， 时间，提出了令人担忧的持久性问题，患者还经历了长期的轻度肝毒性 尽管在基因治疗的第一年进行了类固醇药物治疗。最近的多项观察发现， 关于血友病A肝脏基因治疗安全性的问题。这些问题迫切需要解决， 这种有前途的方法可以安全地应用于患者，并实现持续的矫正。比如说， 肝毒性和FVIII表达下降的原因尚不清楚，这突出了我们研究中的关键空白。 了解载体与肝细胞之间的相互作用以及FVIII表达与 肝细胞，以及免疫系统在长期结果中的作用。也有新的关注 关于插入突变我们将解决这些基本的和机械的问题，有关的生物学， AAV和FVIII。该提议的中心假设是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引起严重的安全性问题。此外，我们假设， 解开这些机制将允许设计载体和协议，最大限度地减少这些问题， 导致持久的治疗和增强的安全性。该计划结合了FVIII生物学，细胞应激 反应，免疫学和AAV载体生物学，并分为3个科学项目，一个行政项目， 核心和2个科学核心。项目1（考夫曼）旨在克服FVIII蛋白错误折叠和细胞毒性。 项目2（Xiao）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性形成。项目3（赫尔佐格）将定义 对AAV-FVIII基因转移的先天性和适应性免疫应答的机制。三个目标 这些项目将得到一个管理核心（核心A）的支持，该核心为体外培养提供人肝细胞。 和体内研究（核心B），以及进行AAV载体的开发和分子分析的核心（核心 C）。总的来说，该项目利用调查员个人的专门知识解决重大问题， FVIII生物学、血友病基因治疗、肝脏定向基因转移和分子生物学中尚未回答的问题 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.