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受到了阻碍，因为预先存在的体液免疫和转基因表达是短暂的，因为免疫反应直接针对含有衣壳的肝细胞。本项目的目标是开发一种基于腺相关病毒（AAV）的新型人类基因转移载体，该载体结合了高效的靶组织体内转导，以及规避或克服人体宿主免疫反应的能力。我们已经展示了在DNA洗牌（或分子进化）反应中使用8种不同的自然分离衣壳的原理证明研究，以建立衣壳基因文库。文库中的每个衣壳都包含一个独特的序列，该序列来自于起始衣壳的不同片段的组合。我们选择的衣壳能够促进AAV在人肝细胞中的感染，并且能够抵抗汇集的人免疫血清。第一个被载体化和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范式的一些改进，并提供了与衣壳生物学相关的新信息。然而，这些初步研究使我们相信，衣壳的体内选择可能与我们的最终目标更相关，即基于人类肝脏的基因治疗。为了验证这个前提，我们计划制作额外的衣壳库，以增加选择之前输入衣壳的多样性。然后，新的AAV衣壳将被选择用于培养的原代细胞（和以前一样）以及含有至少50%人类肝细胞组成的嵌合肝脏的新小鼠模型的体内。分离抗人类免疫反应的AAV衣壳的选择压力将在体外和体内使用一系列抗体策略进行。携带新型AAV衣壳的AAV载体将在小鼠和犬血友病B动物模型中进行测试。重要的是，我们还可以通过使用这些载体在人-嵌合小鼠模型中测量人肝细胞转导来确定这些动物模型在预测人体内AAV细胞转导方面的价值。将测试有希望的载体对人类T细胞反应的免疫原性潜力。希望这种形式的选择压力将使我们能够分离AAV衣壳序列，从而在体内有效转导，同时避免预先存在的免疫中和，以及可能的细胞介导的免疫反应，这些免疫反应困扰着我们之前的AAV-2血友病肝脏临床试验。

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.