Selection of New rAAV Vectors Using Replicating Viral Capsids Libraries

使用复制病毒衣壳文库选择新的 rAAV 载体

基本信息

批准号：
8861132
负责人：
Mark A Kay
金额：
$ 59.31万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-03-01 至 2020-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8861132
关键词：
Adenoviruses Affect Aliquot Amino Acid Sequence Animal Model Antibodies Bioinformatics Biological Assay Capsid Cells Clinical Clinical Trials Cloning Communities Complex Cystic Fibrosis DNA DNA Packaging DNA Shuffling Disease Disease susceptibility Dose Duchenne muscular dystrophy Equilibrium Evolution Family Genes Genome Goals Guidelines Helper Viruses Hemophilia A Hemophilia B Hemorrhage Hepatitis C Hepatitis C virus Hepatocyte Homologous Gene Human Immune Immunoglobulin G In Vitro Infection Infusion procedures Intravenous Immunoglobulins Intravenous infusion procedures Libraries Liver Modeling Molecular Mus Outcome Output Patients Peptide Library Plasmids Property Proteins Protocols documentation Recombinants Relative (related person)Research Serial Passage Signal Transduction Site Specificity Structure T cell response Tissues Trees Variant Viral Viral Genome Virion Virus Western Blotting Work adeno-associated viral vector base co-infection cost field study gene therapy gene transfer vector hereditary blindness high risk high throughput screening in vivo lipoprotein lipase nonhuman primate novel particle pathogen pre-clinical preclinical study pressure public health relevance screening single molecule success therapeutic gene transduction efficiency transgene expression vector

项目摘要

DESCRIPTION: Recombinant adeno-associated viral vectors (rAAV) are showing promising clinical results in a few diseases including hemophilia B, hereditary blindness and lipoprotein lipase deficiency. Even with the early limited success in treating "simple" disease entities, there are a number of limitations with the current vectors. For example, in the hemophilia B trials, AAV8-hFIX transduced patient livers after IV infusion and an improvement in the bleeding diathesis was demonstrated, yet the following difficulties remain: 1) some patients develop a transient transaminitis thought to be due to a T-cell response directed against AAV8 capsids; 2) transgene expression based on vector dose per body mass is far less than predicted from mouse studies (~10x) and non- human primate studies (~3-5x), suggesting transduction in human hepatocytes is not optimal; 3) pre-existing neutralizing anti-AAV8 antibodies are found in about 1/3 of patients; 4) re-administration of a different vector maybe required; 5) a limitation i the size of AAV genome packaging makes it difficult or even impossible to treat other similar diseases (e.g. hemophilia A, the more common form of hemophilia). Due to the fact that significant differences in rAAV transduction are dictated by small variations in capsid amino acid sequences, we have pursued molecular shuffling of capsid genes to create AAV libraries with extensive sequence variants (~10e7). These novel capsids replace the wild-type capsid in a viral plasmid and are used to produce infectious virus in the presence of a helper virus. Replicating AAV is grown under selective conditions that we vary according to the desired outcome, and those with a selective advantage are isolated and their sequences determined. These capsids can then be used to package therapeutic genes and their transduction determined. We and others have selected AAV capsids with new properties, many of which have become very useful to the gene therapy community. However, we feel the full utility of replicating capsid libraries has yet to be reached. We plan to assemble new more complex libraries and select for enriched capsids after serial passage in primary human hepatocytes maintained in a chimeric mouse-human liver model under different selection pressures. We will make vectors from the most selected capsids and study their transduction in vitro and in vivo. Finally, we will use these libraries in two high-risk, high-payoff screens: 1) select capsids with novel cell specificity (e.g. an AAV that only transduces hepatocytes infected with hepatitis- C virus); 2) select capsids that can package larger genomes, thus expanding the utility of AAV vectors for diseases whose expression cassette just exceeds current packaging limits (e.g. cystic fibrosis, Duchenne muscular dystrophy and hemophilia A,). We believe this work will not only provide new AAV vectors for expanded use in research and clinical gene therapy, but will also provide important quantitative guidelines to make AAV capsid shuffling more efficient for all groups pursuing this line of research.

描述：重组腺相关病毒载体 (rAAV) 在治疗 B 型血友病、遗传性失明和脂蛋白脂肪酶缺乏症等少数疾病方面显示出有希望的临床效果，尽管早期在治疗“简单”疾病方面取得了有限的成功。目前的载体存在许多局限性，例如，在血友病 B 试验中，AAV8-hFIX 在静脉输注后转导患者肝脏，并证明出血素质有所改善，但仍然存在以下困难：1）一些患者出现了短暂性转氨炎被认为是由于针对 AAV8 衣壳的 T 细胞反应所致；2) 基于单位体重载体剂量的转基因表达远低于小鼠研究的预测 (~10x)非人类灵长类动物研究 (~3-5x)，表明人类肝细胞中的转导并非最佳；3) 在约 1/3 的患者中发现了预先存在的中和性抗 AAV8 抗体；4) 重新施用不同的载体5) AAV 基因组包装的大小的限制使得治疗其他类似疾病变得困难甚至不可能（例如血友病 A，血友病的更常见形式）。 rAAV 转导的差异是由衣壳氨基酸序列的微小变化决定的，我们对衣壳基因进行分子改组，以创建具有广泛序列变异的 AAV 文库 (~10e7)，这些新型衣壳取代了病毒质粒中的野生型衣壳，并且用于在辅助病毒存在的情况下产生传染性病毒，我们根据所需结果在选择性条件下生长，并分离出具有选择性优势的病毒并确定其序列。然后，衣壳可用于包装治疗基因并确定其转导。我们和其他人选择了具有新特性的 AAV 衣壳，其中许多对基因治疗界非常有用。我们计划组装新的更复杂的文库，并在不同选择压力下在嵌合小鼠-人肝脏模型中维持的原代人肝细胞中连续传代后选择富集的衣壳。最后，我们将在两个高风险、高回报的筛选中使用这些文库：1）选择具有新颖细胞特异性的衣壳（例如仅转导感染丙型肝炎病毒的肝细胞的 AAV）。）；2）选择可以包装更大基因组的衣壳，从而扩大AAV载体对于表达盒刚刚超出当前包装限制的疾病（例如囊性纤维化， Duchenne 肌营养不良症和血友病 A，）我们相信这项工作不仅会提供新的 AAV 载体以扩大在研究和临床基因治疗中的应用，而且还将提供重要的定量指导，使 AAV 衣壳改组对于所有追求这一路线的团体更加有效。研究。