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b型血友病患者的I期临床试验。然而，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载体是由自然感染人类的野生型AAV改造而来的结果。目前，人们已经考虑了多种方法来设计逃避中和抗体（Nab）的AAV载体，包括化学修饰、衣壳的合理设计和组合诱变以及Nab滴度的生物耗损。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并不代表灵长类动物和狗等大型动物的结果，因此小鼠组织中产生的AAV Nab逃逸突变的数据可能并不总是适用于人类。此外，关于AAV在人肝脏中的转导信息的缺乏阻碍了AAV载体在血友病基因治疗中的应用。近年来，利用人肝细胞异种移植小鼠模型，建立了用于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。为了解决这些突出的问题（开发具有Nab逃逸活性的人嗜肝性AAV），我们将建立犬肝细胞的小鼠异种移植模型，并在移植犬肝细胞的小鼠和正常犬中检测犬肝细胞中AAV血清型的转导效率，以验证模型的可行性（Aim1a）。接下来，我们将应用AAV定向进化策略来选择能够逃避狗体内产生的AAV Nab并具有犬肝亲性（Aim 1b和1c）的AAV变体。接下来，我们将使用这些犬Nab逃避突变体将优化的犬FIX传递到预先接种AAV的血友病B犬中，并检查血友病表型的纠正（目的2）。最后，我们将利用人IVIG作为nab的来源，在移植人肝细胞的小鼠模型中开发具有逃避AAV nab能力的临床人嗜肝AAV突变体（Aim 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).