Rational design of AAV vectors with human hepatocyte tropism and neutralizing antibody evasion

腺相关病毒(AAV)载体已成功地应用于不同疾病患者的临床试验 精神错乱。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850,000美元，Zolgensma定价2,100,000美元 治疗脊椎肌肉萎缩。因此，基于AAV载体的基因治疗是一个越来越有吸引力的市场。 尽管在临床研究中取得了成功，但有两个问题限制了AAV载体在患者中的更广泛应用 全身给药后需要肝靶向AAV基因治疗：低人肝细胞 转导和中和抗体(NAB)介导的AAV转导抑制。几种方法 已探索用于AAV转导增强或衣壳NAB逃避。AAV的工程化 Capsid提供了一种非常强大和流行的技术，已被广泛研究以开发新的 AAV载体在动物模型中增强转导或在体外NAB逃逸。然而，它一直是 证明了小鼠实验的结果不能概括大型动物的结果，如 灵长类动物和狗。因此，在动物细胞和器官中产生的AAV变体的数据可能无法翻译 转化为成功的人类应用。最近，建立了小鼠人肝细胞异种移植模型。 用于研制用于基因治疗的人肝靶向AAV载体。在我们之前的研究中，我们有 从人肝细胞异种移植嵌合小鼠的肝脏中成功分离到几株AAV突变体 使用AAV改组衣壳文库方法的人NAB(IVIG)的存在。具体来说，BDRK001 (AAV突变体LP2-10)表现出比任何其他AAV血清型或 变种人。然而，与BDRK001相比，BDRK001在人肝细胞中的转导没有增强 最佳自然血清型。在这个应用中，我们将使用合理的设计策略来生成新的AAV衣壳，通过 BDRK001的可变区I(VRI)结构域交换使用天然血清型或高人肝突变株 向心性。然后，该小组将在嵌合小鼠身上进行人类肝细胞转导的评估(AIM 1)和 NAB规避(目标2)。Bedock这种方法的长期目标是低剂量AAV基因治疗 成功治疗多种肝病，与患者的NAB患病率无关。

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials in patients with diverse disorders. 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 priced at $2,100,000 for spinal muscle atrophy. As such, AAV vector based gene therapy is an increasingly attractive market. Although successful in clinical studies, two concerns restrict broader AAV vector applications for patients requiring liver targeted AAV gene therapy following systemic administration: low human hepatocyte transduction and neutralizing antibody (Nab)-mediated inhibition of AAV transduction. Several approaches have been explored for AAV transduction enhancement or capsid Nab evasion. Engineering of the AAV capsid presents a very powerful and popular technology that has been extensively studied to develop novel AAV vectors for enhanced transduction in animal models or Nab escape in vitro. However, it has been demonstrated that the results from mouse experiments do not recapitulate those of large animals such as primates and dogs. Thus, the data for AAV variants generated in animal cells and organs may not translate into successful human applications. Recently, a mouse xenograft model with human hepatocytes has been used to develop human liver targeted AAV vectors for gene therapy. In our previous studies, we have successfully isolated several AAV mutants from the liver of chimeric mice with human hepatocyte xenografts in the presence of human Nabs (IVIG) using the AAV shuffled capsid library approach. Specifically, BDRK001 (AAV mutant LP2-10) demonstrated a much higher ability to evade Nabs than any other AAV serotypes or mutants. However, BDRK001 was not enhanced for transduction in human hepatocytes when compared to the best natural serotype. In this application, we will use rational design strategy to generate novel AAV capsids by variable region I (VRI) domain swapping of BDRK001 using natural serotypes or mutants with high human liver tropism. This panel will then be evaluated in chimeric mice for human hepatocyte transduction (Aim 1) and Nab evasion (Aim 2). Bedrock's long-term goal of this approach is low dose AAV gene therapy for the successful treatment of a variety of liver diseases, independent of the patient's Nab prevalence.