Directed evolution of AAV vectors for hemophilia to evade neutralization

描述：腺相关病毒(AAV)载体已经成功地用于血友病B患者的I期临床试验。然而，在血友病基因治疗的临床试验中遇到的障碍之一是对AAV的体液免疫，这是因为AAV载体是从自然感染人类的野生型AAV改造而来的。设计中和抗体(NAB)的载体有多种方法，包括化学修饰、衣壳的合理设计和组合诱变以及NAB滴度的生物耗尽。这些方法只在细胞系或动物模型上进行了测试，已经证明小鼠实验的结果不能代表灵长类和狗等大型动物的结果，因此在小鼠组织中产生的AAVNAB逃逸突变的数据可能并不总是翻译成人类的数据。此外，由于缺乏关于AAV在人肝脏中转导的信息，阻碍了AAV载体在血友病基因治疗中的应用。近年来，人肝细胞异种移植小鼠模型已被用于构建人肝靶向基因治疗的AAV载体。然而，该模型产生的结果是否会直接转化为人体临床试验也是未知的。为了解决这些突出问题(发展具有NAB逃逸活性的人嗜肝AAV)，我们将建立犬肝细胞异种移植小鼠模型，并检测犬肝细胞移植小鼠和正常犬的AAV血清型的转导效率，以验证该模型的可行性(Aim1a)。接下来，我们将应用AAV定向进化策略来筛选能够避开在狗身上产生的AAV NAB并具有犬肝趋向性的AAV变体(目标1b和1c)。接下来，我们将使用这些犬NAB规避突变体将优化的犬FIX注射到预先免疫AAV的血友病B犬体内，并检查血友病表型的纠正情况(目标2)。最后，我们将利用人IVIG作为NAB的来源，在人肝细胞异种移植的小鼠模型中，开发具有躲避AAV NAB能力的临床人嗜肝AAV突变体(目标3)。

DESCRIPTION: Adeno-associated virus (AAV) vectors have been successfully used in phase I clinical trials in patients with hemophilia B. However, one of the obstacles encountered in clinica trial for hemophilia gene therapy is humoral immunity to AAV, a consequence of the fact that AAV vectors have been engineered from a wild-type AAV naturally infecting humans. Several approaches have been considered to design neutralizing antibody (Nab)-evading AAV vectors, including chemical modification, rational design and combinatorial mutagenesis of the capsid as well as biological depletion of Nab titer. These approaches have only been tested on cell lines or in animal models, it has been demonstrated that the result from mouse experiments does not represent that from big animals such as primates and dogs, so the data for AAV Nab escape mutants generated in mice tissues may not always translated into that in human. Further, the dearth of information on AAV transduction in human liver hinders the application of AAV vector in hemophilia gene therapy. Recently mouse model xenografted with human hepatocytes has been used to develop AAV vector for human liver targeting gene therapy. However, it is also unknown whether the result generated from this model will be directly translated into human clinical trial. To address these outstanding concerns (development of human liver tropic AAV with Nab escape activity), we will establish a mouse xenografted model with canine hepatocytes and examine the transduction efficiency of AAV serotypes in canine hepatocytes in both canine hepatocyte xenografted mice and normal dogs to validate the model feasibility (Aim1a). Next we will apply the AAV directed evolution strategy to select AAV variants which can evade AAV Nab generated in dogs and have canine liver tropism (Aim 1b and 1c). Next we will use these canine Nab evasion mutants to deliver optimized canine FIX into hemophilia B dogs pre-immunized with AAV and to examine the correction of hemophilia phenotype (Aim 2). Finally, we will utilize human IVIG as the source of Nabs to develop clinical human liver-tropic AAV mutants with the capacity of evade AAV Nabs in mouse model xenografted with human hepatocytes (Aim 3).