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：
9022412
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/9022412
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 DNA cassette Disease Disease susceptibility Dose Duchenne muscular dystrophy Equilibrium Evolution Family Genes Genome Goals Guidelines Health 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 Research Serial Passage Signal Transduction Site Specificity Structure T cell response Tissues Trees Variant Viral Viral Genome Virion Virus Virus Replication 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 humanized mouse in vivo lipoprotein lipase nonhuman primate novel particle pathogen pre-clinical preclinical study pressure 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）在几种疾病中显示出有望的临床结果，包括血友病B，遗传性盲和脂蛋白脂肪酶缺乏症。即使早期在治疗“简单”疾病实体方面取得了有限的成功，那里是当前向量的许多局限性。例如，在血友病B试验中，AAV8-HFIX在静脉输注后转导患者的生命和出血性核糖的改善，但仍存在以下困难：1）有些患者会出现瞬时跨动脉炎，认为是由于针对AAV8 Capsids的T细胞反应引起的； 2）基于每个体重的载体剂量的转基因表达远远低于小鼠研究（〜10X）和非人类灵长类动物研究（〜3-5X）所预测的，这表明人肝细胞的转导并不最佳。 3）在大约1/3的患者中发现了预先存在的中和抗AAV8抗体； 4）可能需要重新管理其他矢量； 5）限制AAV基因组包装的大小使得治疗其他类似疾病的困难甚至不可能（例如血友病A，是血友病的更常见形式）。由于RAAV转导的显着差异取决于衣壳氨基酸序列的较小变化决定了capsid基因的分子洗牌，以创建具有广泛序列变体的AAV库（〜10E7）。这些新型的衣壳取代了病毒质粒中的野生型衣壳，并用于在存在辅助病毒的情况下产生传染性病毒。复制AAV是在我们根据所需结果变化的选择性条件下生长的，并且具有选择性优势的人是隔离的，并确定其序列。然后，这些衣壳可用于包装治疗基因及其翻译。我们和其他人选择了具有新特性的AAV Capsids，其中许多对基因治疗社区非常有用。但是，我们认为复制Capsid库的全部用途尚未到达。我们计划在不同选择压力下保持在嵌合小鼠 - 人类肝模型中的原代人肝细胞中的串行通道后，组装新的更复杂的文库，并选择富集的衣壳。我们将从最选择的衣壳中制作矢量，并在体外和体内研究它们的翻译。最后，我们将在两个高风险的高付费屏幕中使用这些文库：1）选择具有新型细胞特异性的衣壳（例如，仅转导肝炎病毒感染的肝细胞的AAV）； 2）选择可以包装较大基因组的衣壳，从而扩展了AAV载体的疾病的效用，其表达盒的疾病刚刚超过了当前的包装极限（例如囊性纤维化，Duchenne肌肉营养不良和血友病A）。我们认为，这项工作不仅将为研究和临床基因疗法提供新的AAV媒介，而且还将提供重要的定量指南，以使AAV Capsid改组为所有追求这一研究的小组更有效。