Molecular Evolution Strategies to Derive New Recombinant AAV Vectors

描述（由申请人提供）：AAV介导的基因转移为基因治疗提供了巨大的希望。然而，早期基于肝脏的基因治疗试验的成功由于预先存在的体液免疫而受到阻碍，并且转基因表达是短暂的，因为免疫应答针对含有衣壳的肝细胞。该项目的目标是开发一类基于腺相关病毒（AAV）的新型人类基因转移载体，其结合靶组织的高效体内转导，并具有规避或克服人类宿主免疫应答的能力。我们已经展示了在DNA改组（或分子进化）反应中使用8种不同的天然分离的衣壳来制备衣壳基因文库的原理验证研究。文库中的每个衣壳含有来源于起始衣壳的不同片段的组合的独特序列。我们已经选择了在人肝细胞中促进AAV感染并且对汇集的人免疫血清具有抗性的衣壳。第一个被载体化并被广泛研究的衣壳具有重要的生物学特性，这些特性导致了基因转移模式的一些改进，并提供了与衣壳生物学相关的新信息。然而，这些最初的研究使我们相信，衣壳的体内选择可能与我们与基于人类肝脏的基因治疗相关的最终目标更相关。为了测试这一前提，我们计划制作额外的衣壳文库，以在选择之前增加输入衣壳的多样性。然后将在培养物中的原代细胞上（如前所述）以及在含有由至少50%人肝细胞组成的嵌合肝的新小鼠模型中的体内选择新的AAV衣壳。用于分离对人免疫应答具有抗性的AAV衣壳的选择压力将使用一系列抗体策略在体外和体内进行。将在小鼠和犬血友病B动物模型中测试具有新型AAV衣壳的AAV载体。重要的是，我们还可以通过使用这些载体测量人嵌合小鼠模型中的人肝细胞转导来确定这些动物模型在体内预测人细胞的AAV转导中的价值。将测试显示有希望的载体对人T细胞应答的免疫原性潜力。希望这种形式的选择性压力将使我们能够分离AAV衣壳序列，从而允许有效的体内转导，同时避免来自预先存在的免疫的中和，以及可能的细胞介导的免疫应答，这些免疫应答困扰我们先前针对血友病的AAV-2肝脏临床试验。 公共卫生相关性：我们建议的目标是利用分子进化来创建新的AAV基因转移载体，具有上级特性，并克服目前基于自然界中发现的病毒的载体的局限性。一旦新的AAV载体被创建，它们将在人类疾病的动物模型中进行测试。

DESCRIPTION (provided by applicant): AAV-mediated gene transfer offers great promise for gene therapy. However, the success of early liver based gene therapy trials has been hampered due to pre-existing humoral immunity and transgene expression is short-lived because of an immune response directed against the capsid containing hepatocytes. The goal of this project is to develop a novel class of human gene transfer vectors based on adeno-associated virus (AAV) that combines high efficiency in vivo transduction of target tissues, with the capability to circumvent or overcome host immunologic responses in humans. We have shown proof-of-principle studies using 8 diverse naturally isolated capsids in DNA shuffling (or molecular evolution) reactions to make a library of capsid genes. Each capsid in the library contains a unique sequence derived from a combination of different pieces of the starting capsids. We have selected for capsids that promote AAV infection in human hepatocytes and that are resistant to pooled human immune sera. The first capsid that was vectorized and extensively studied has important biological properties that have resulted in some improvemenet in gene transfer paradigms as well as provided new information related to capsid biology. However, these first studies have led us to believe that in vivo selection of capsids may be more relevant to our ultimate goal related to human liver-based gene therapy. To test this premise, we plan to make additional capsid libraries that will increase the diversity of input capsids prior to selection. Novel AAV capsids will then be selected for on primary cells in culture (as before) as well as in vivo in a new mouse model containing a chimeric liver that is made up of at least 50% human hepatocytes. Selection pressure for isolating AAV capsids resistant to the human immune response will be carried out using a battery of antibody strategies both in vitro and in vivo. AAV vectors with the novel AAV capsids will be tested both in mouse and canine hemophilia B animal models. Importantly, we can also determine the value of these animal models in predicting AAV transduction of human cells in vivo by using these vectors to measure human hepatocyte transduction in the human- chimeric mouse model. Vectors showing promise will be tested for their immunogenic potential for human T cell responses. It is hoped that this form of selective pressure will enable us to isolate AAV capsid sequences allowing efficient in vivo transduction while evading both neutralization from pre-existing immunity, and possibly cell-mediated immune responses that plagued our previous AAV-2 liver clinical trial for hemophilia. PUBLIC HEALTH RELEVANCE: The goal of our proposal is to use molecular evolution to create new AAV gene transfer vectors that have superior properties and overcome the limitations of the current vectors based on viruses found in nature. Once the new AAV vectors are created, they will be tested in animal models of human disease.