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Molecular Biology of Recombinant AAV Genomes

重组 AAV 基因组的分子生物学

基本信息

批准号：
7569346
负责人：
WEIDONG XIAO
金额：
$ 35.94万
依托单位：
CHILDREN'S HOSP OF PHILADELPHIA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-02-15 至 2009-02-06
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7569346
关键词：
Address Adopted Affect Apoptosis Appearance Binding Biological Assay Biology Bromodeoxyuridine Capsid Capsid Proteins Cell Count Cells Chromosomes Clinical Clinical Research Clinical Trials Complication Cultured Cells DNA DNA Damage DNA annealing DNA biosynthesis Development Dose Double Stranded DNA Virus Episome Frequencies Gene Expression Gene Transfer Genome Genome Stability Genomic Instability Hepatocyte Human Immune response In Situ Infection Kinetics Knowledge Label LacZ Genes Liver Measures Methods Molecular Molecular Biology Molecular Profiling Mus Nature Non-Viral Vector Nuclear Pathogenicity Pathway interactions Patients Play Procedures Process Production Recombinant adeno-associated virus (rAAV)Recombinants Reporter Reporter Genes Research Activity Research Personnel Role Safety Sampling Serotyping Signal Transduction Single-Stranded DNA Southern Blotting System Techniques Temperature Tern Testing Time Transgenic Mice Transgenic Model Uncertainty Viral Viral Tumor Antigens Virion adeno-associated viral vector cell mediated immune response cellular transduction chromosome replication cost effective design ds-DNA expression vector gene therapy human subject improved in vivo mouse model particle plasmid DNA pre-clinical pressure prevent programs repaired sialosyl-T antigen tissue tropism transduction efficiency transgene expression vector vector genome

项目摘要

Recent advances in AAV vectorology have allowed recombinant AAV (rAAV) vectors to be used for human clinical trials. However, the basic biology of AAV vectors is still not well understood. This has been a major limitation for the full exploitation of the usefulness of rAAV vectors. Despite numerous research activities focusing on the tissue tropism and applications for rAAV vectors, the fate of rAAV genomes in vivo and the mechanisms of AAV genome conversion have not been well characterized. Preclinical clinical studies suggest that a disturbing dose of IxlO14 vector genomes may be necessary for a human subject. Our hypothesis is that rAAV genome instability seriously reduces its efficiency. The single stranded (ss). AAV genome can be recognized by host cells as DNA damage signal which leads to a cascade of AAV genome loss/degradation immediately after uncoating. Such mechanism will not allow substantial free ss AAV to exist in the host for an extended period. The limiting step for efficient rAAV transduction is therefore more likely to be intracellular processing of AAV virions and both ss and double stranded AAV genome loss/degradation. In addition, the random integration of AAV genomes is a major concern for AAV vectors. To address this issue, we plan to quantify rAAV integration frequency. Hence, our specific aims are 1). To study the conversion of AAV genome from single stranded DNA to double stranded form. 2). To study the stability of double stranded AAV genomes 3). To quantify the frequency of rAAV integration in vivo. The successful execution of these specific aims will help identify new strategies for improving rAAV transductionefficiency and utilizingrAAV vectors safely.

AAV 载体学的最新进展使得重组 AAV (rAAV) 载体可用于人类临床试验。然而，AAV 载体的基本生物学仍不清楚。这已经是一个重大的充分利用 rAAV 载体的有效性的限制。尽管进行了大量的研究活动重点关注rAAV载体的组织趋向性和应用、rAAV基因组在体内的命运和 AAV 基因组转换的机制尚未得到很好的表征。临床前临床研究表明人类受试者可能需要令人不安的1x1014载体基因组剂量。我们的假设是 rAAV 基因组的不稳定性严重降低了其效率。单链（ss）。 AAV基因组可以是被宿主细胞识别为 DNA 损伤信号，导致 AAV 基因组丢失/降解级联脱漆后立即。这种机制不会允许大量免费的 ss AAV 在主机中存在延长期限。因此，有效 rAAV 转导的限制步骤更有可能是在细胞内 AAV 病毒粒子的处理以及 ss 和双链 AAV 基因组丢失/降解。此外， AAV 基因组的随机整合是 AAV 载体的一个主要问题。为了解决这个问题，我们计划量化 rAAV 整合频率。因此，我们的具体目标是 1)。研究AAV的转化基因组从单链DNA转变为双链形式。 2）。研究双链AAV的稳定性基因组3）。量化体内 rAAV 整合的频率。这些具体措施的成功执行目标将有助于确定提高 rAAV 转导效率和利用 rAAV 载体的新策略安全。