Develop humanized AAV vectors for liver targeting and neutralizing antibody evasion

腺相关病毒载体已成功应用于临床试验 血友病。FDA最近批准了两种基于AAV的基因治疗药物。卢克图尔纳一直是 一种罕见失明的一次性治疗价值85万美元，Zolgensma价值210万美元 脊椎肌肉萎缩。利用AAV载体进行基因治疗显示了一个潜在的巨大市场。虽然 在临床研究中取得成功，两个问题限制了AAV载体在慢性病患者中的更广泛应用 需要全身注射AAV载体以有效肝靶向治疗的血友病患者：低人肝细胞 转导和中和抗体(NAB)介导的阻断AAV转导。有几种方法 已被探索用于增强AAV转导或中和抗体逃避。AAV的工程化 Capsid代表了一种非常强大和流行的技术，并被广泛研究以开发新的 AAV载体在临床前动物模型或NAB体外逃逸中的转导增强。一直以来 证明了小鼠实验的结果不能概括大型动物的结果，如 对于灵长类动物和狗，在动物细胞和器官中产生的AAV变体的数据可能不会转化为 人类应用程序。最近，一种与人肝细胞异种移植的小鼠模型被用于开发 以人肝为靶向基因治疗的AAV载体。我们已经从AAV中分离出几个AAV突变体 人中和抗体存在下人肝细胞异种移植小鼠的肝脏 (IVIG)使用AAV改组文库方法。在这个应用中，我们将研究这些突变体的能力 转导人肝细胞，躲避NAB。在鉴定出最好的高效突变体之后 人肝细胞转导和对NAB的高逃避能力，我们将研究其安全性和长期疗效 在一次注射后，这些突变体用于血友病基因治疗与AAVNAb的小鼠模型。 这些突变体在血友病小鼠中的成功验证将为翻译成 更大的血友病模型，希望用AAV NAB安全有效地治疗血友病患者 只用一次剂量。

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials in patients with hemophilia. Two AAV based gene therapy drugs have been recently approved by the FDA. Luxturna has been valued at $850,000 for a one-time treatment for a rare form of blindness and Zolgensma at $2,100,000 for spinal muscle atrophy. Gene therapy with AAV vectors has shown a potentially huge market. Although successful in clinical studies, two concerns restrict a broader AAV vector application for patients with hemophilia who need systemic administration of AAV vector for effective liver targeting: low human hepatocyte transduction and neutralizing antibody (Nab)-mediated blocking of AAV transduction. Several approaches have been explored for AAV transduction enhancement or neutralizing antibody evasion. Engineering of the AAV capsid represents a very powerful and popular technology, and has been extensively studied to develop novel AAV vectors for transduction enhancement in pre-clinical animal models or Nab escape in vitro. It has been demonstrated that the results from mouse experiments do not recapitulate those of large animals such as primates and dogs, the data for AAV variants generated in animal cells and organs may not translate into human application. Recently, a mouse model xenografted with human hepatocytes has been used to develop AAV vectors for targeting gene therapy relative to human liver. We have isolated several AAV mutants from the liver of chimeric mice xenografted with human hepatocytes in the presence of human neutralizing antibodies (IVIG) using AAV shuffling library approach. In this application, we will study the ability of these mutants to transduce human hepatocytes and evade Nabs. Following the identification of the best mutants with efficient human hepatocyte transduction and high ability for Nab evasion, we will study the safety and long-term efficacy of these mutants for hemophilia gene therapy in a mouse model with AAV Nabs after a single injection. Successful validation of these mutants in hemophilic mice will provide a proof-of-concept for translation to larger models of hemophilia in hopes of safely and effectively treating hemophilia patients with AAV Nabs using a single dose.