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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.