Molecular Evolution Strategies to Derive New Recombinant AAV Vectors

衍生新重组 AAV 载体的分子进化策略

• 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
• 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

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疗法提供了巨大的希望。然而，由于先前存在的体液免疫和转基因表达，基于早期肝的基因治疗试验的成功受到了阻碍，因为针对含有含CapsID的肝细胞的免疫反应。该项目的目的是基于腺相关病毒（AAV）开发一类新型的人类基因转移载体，该病毒（AAV）结合了靶组织的体内转导高效率，并能够规避或克服人类的宿主免疫反应。我们已经在DNA洗牌（或分子进化）反应中使用8种不同自然分离的衣壳进行了原理证明研究，以形成一个衣壳基因库。库中的每个衣壳都包含一个独特的序列，该序列源自不同片段的起始衣壳的组合。我们选择了促进人类肝细胞中AAV感染的衣壳，并且对合并的人类免疫血清具有抵抗力。对矢量化和广泛研究的第一个衣壳具有重要的生物学特性，从而导致基因转移范式的一些改进，并提供了与Capsid生物学有关的新信息。但是，这些第一批研究使我们相信，在体内选择衣壳可能与我们与基于人类肝的基因疗法有关的最终目标更相关。为了测试此前提，我们计划制作其他CAPSID库，以在选择之前增加输入CAPSID的多样性。然后，将在培养物中的原代细胞上选择新颖的AAV衣壳（如前所述），以及在含有嵌合肝脏的新小鼠模型中的体内，该模型由至少50％的人类肝细胞组成。隔离AAV CAPSID的选择压力将使用体外和体内的一系列抗体策略来实现对人免疫反应的抗性。与新型AAV衣壳的AAV载体将在小鼠和犬类血友病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类小鼠模型中测量人类肝细胞转导，确定这些动物模型在预测人体体内AAV转导的价值。表现出希望的向量将被测试，以证明其对人T细胞反应的免疫原性潜力。希望这种选择性压力能够使我们能够隔离AAV CAPSID序列，从而可以有效地体内转导，同时避免通过预先存在的免疫力进行中和，并可能困扰我们先前的Hemophilia的AAV-2肝临床试验。 公共卫生相关性：我们的提案的目的是使用分子进化来创建具有卓越特性的新AAV基因转移向量，并基于自然界中的病毒克服当前向量的局限性。一旦创建了新的AAV矢量，它们将在人类疾病的动物模型中进行测试。