Molecular Evolution Strategies to Derive New Recombinant AAV Vectors

衍生新重组 AAV 载体的分子进化策略

基本信息

批准号：
8230691
负责人：
Mark A Kay
金额：
$ 55.33万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-01 至 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8230691
关键词：
Animal Model Animals Antibodies Biological Biology Canis familiaris Capsid Cells Clinical Trials DNA DNA Shuffling Data Dependovirus Development Directed Molecular Evolution Disease Future Gene Transfer Genes Genetic Genome Goals Hemophilia A Hemophilia B Heparin Binding Hepatocyte Hereditary Disease Human Humoral Immunities Immune Sera Immune response Immunity Immunologics In Vitro Libraries Life Liver Measures Mediating Modification Molecular Evolution Mus Nature Peptides Plague Population Primary carcinoma of the liver cells Primates Property Reaction Recombinant adeno-associated virus (rAAV)Reporting Resistance Role Scheme Serotyping Serum T cell response Testing Tissues Toxic effect Variant Viral hepatitis Virion Virus Virus Diseases adeno-associated viral vector base cell mediated immune response gene therapy gene transfer vector human disease immunogenic improved in vivo interest mouse model neutralizing antibody novel pre-clinical pressure public health relevance response success transduction efficiency transgene expression vector

项目摘要

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.

描述（由申请人提供）：AAV 介导的基因转移为基因治疗提供了广阔的前景。然而，由于预先存在的体液免疫，早期基于肝脏的基因治疗试验的成功受到阻碍，并且由于针对含有衣壳的肝细胞的免疫反应，转基因表达是短暂的。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，该载体将目标组织的高效体内转导与规避或克服人类宿主免疫反应的能力相结合。我们已经展示了在 DNA 改组（或分子进化）反应中使用 8 种不同的自然分离的衣壳来构建衣壳基因库的原理验证研究。文库中的每个衣壳都包含源自起始衣壳不同片段组合的独特序列。我们选择了能够促进人肝细胞中 AAV 感染并且对混合的人免疫血清具有抗性的衣壳。第一个被矢量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。为了测试这个前提，我们计划制作额外的衣壳库，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（如前所述）以及包含嵌合肝脏的新小鼠模型体内选择新型 AAV 衣壳，该嵌合肝脏由至少 50% 的人类肝细胞组成。将使用一系列体外和体内抗体策略来分离对人类免疫反应具有抗性的 AAV 衣壳的选择压力。带有新型 AAV 衣壳的 AAV 载体将在小鼠和犬 B 型血友病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预测人细胞体内AAV转导方面的价值。将测试显示出前景的载体对人类 T 细胞反应的免疫原性潜力。希望这种形式的选择性压力将使我们能够分离 AAV 衣壳序列，从而实现有效的体内转导，同时避免先前存在的免疫的中和，以及可能困扰我们之前的 AAV-2 肝脏临床试验的细胞介导的免疫反应。血友病。公共健康相关性：我们提案的目标是利用分子进化来创建新的 AAV 基因转移载体，该载体具有优越的特性，并克服现有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。