Develop humanized AAV vectors for liver targeting and neutralizing antibody evasion

腺相关病毒（AAV）载体已成功应用于临床试验中的患者， 血友病两种基于AAV的基因治疗药物最近已被FDA批准。Luxturna一直是 价值850 000美元的一次性治疗一种罕见的失明，价值2 100 000美元的Zolgensma， 脊髓性肌肉萎缩利用AAV载体进行基因治疗已显示出潜在的巨大市场。虽然 尽管在临床研究中取得了成功，但两个问题限制了AAV载体在患有 需要全身施用AAV载体用于有效肝靶向血友病：低人肝细胞 转导和中和抗体（Nab）介导的AAV转导阻断。有几种方法 已经探索了AAV转导增强或中和抗体逃避。AAV工程 衣壳代表了一种非常强大和流行的技术，并且已经被广泛研究以开发新的衣壳。 用于临床前动物模型中的转导增强或体外Nab逃逸的AAV载体。已经 表明小鼠实验的结果并不能概括大型动物的结果， 在灵长类动物和狗中，动物细胞和器官中产生的AAV变体的数据可能无法转化为 人类应用最近，已经使用异种移植人肝细胞的小鼠模型来开发 用于相对于人肝脏的靶向基因治疗的AAV载体。我们已经分离出几个AAV突变体从 在存在人中和抗体的情况下异种移植人肝细胞的嵌合小鼠的肝脏 使用AAV改组文库方法在IVIG中表达。在本申请中，我们将研究这些突变体的能力， 培养人肝细胞和逃避Nabs。在鉴定出具有高效的 人肝细胞转导和Nab逃避的高能力，我们将研究其安全性和长期疗效 在单次注射后，在具有AAV Nabs的小鼠模型中使用这些突变体进行血友病基因治疗。 这些突变体在血友病小鼠中的成功验证将为翻译提供概念验证， 更大的血友病模型，希望用AAV Nabs安全有效地治疗血友病患者 使用单一剂量。

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.