Directed evolution of AAV vectors for hemophilia to evade neutralization

描述：腺相关病毒（AAV）载体已成功用于血友病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AAV载体已经从天然感染人类的野生型AAV工程化的事实的结果。已经考虑了几种方法来设计逃避中和抗体（Nab）的AAV载体，包括衣壳的化学修饰、合理设计和组合诱变以及Nab滴度的生物消耗。这些方法仅在细胞系或动物模型中进行了测试，已经证明来自小鼠实验的结果并不代表来自大型动物如灵长类动物和狗的结果，因此小鼠组织中产生的AAV Nab逃逸突变体的数据可能并不总是转化为人类的数据。此外，缺乏关于AAV在人类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，人肝细胞异种移植的小鼠模型已被用于开发人肝靶向基因治疗的AAV载体。然而，目前还不清楚该模型产生的结果是否会直接转化为人体临床试验。为了解决这些突出问题（开发具有Nab逃逸活性的人肝嗜性AAV），我们将建立犬肝细胞异种移植模型，并在犬肝细胞异种移植小鼠和正常犬中检查犬肝细胞中AAV血清型的转导效率，以验证模型可行性（Aim1a）。接下来，我们将应用AAV定向进化策略来选择可以逃避在狗中产生的AAV Nab并具有犬肝向性的AAV变体（Aim 1b和1c）。接下来，我们将使用这些犬Na B逃避突变体将优化的犬FIX递送到用AAV预免疫的血友病B犬中，并检查血友病表型的校正（目的2）。最后，我们将利用人IVIG作为Nab的来源来开发临床人嗜肝性AAV突变体，其具有在异种移植有人肝细胞的小鼠模型中逃避AAV Nab的能力（目的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).