Gene Targeting and Integration by Adeno-Associated Virus Vectors

腺相关病毒载体的基因靶向和整合

• 批准号: 8249899
• 负责人: David W Russell
• 金额: $ 42.27万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-15 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8249899
• 关键词: Adult; Area; COL1A2 gene; Cell Differentiation process; Cell Proliferation; Cell model; Cells; Chromosomes; Clinical; Collagen Gene; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytomegalovirus; DNA Sequence; Dependovirus; Development; Disease; Duchenne muscular dystrophy; Dystrophin; Enhancers; Episome; Fibroblasts; Frequencies; Funding; Gene Targeting; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genome; Genomics; Grant; Hepatocyte; Hereditary Disease; Human; Hypoxanthine Phosphoribosyltransferase; Individual; Insertional Mutagenesis; Interleukin 2 Receptor Gamma; Lead; Lesch-Nyhan Syndrome; Liver neoplasms; Location; Malignant neoplasm of liver; Maps; Mediating; Methods; Mitotic; Modeling; Modification; Mus; Mutate; Mutation; Neomycin resistance gene; Newborn Infant; Osteogenesis Imperfecta; Patients; Phase; Plasmids; Pluripotent Stem Cells; Positioning Attribute; Primary carcinoma of the liver cells; Proliferating; Proviruses; Recovery; Regulator Genes; Relative (related person); Research; Risk; Role; Site; Staging; Stem cells; System; Technology; Testing; Therapeutic; Therapeutic Human Experimentation; Time; Transcript; Transplantation; X-Linked Severe Combined Immunodeficiency; adeno-associated viral vector; base; cell type; density; disease-causing mutation; embryonic stem cell; fetal; follow-up; genetic manipulation; genome-wide; genotoxicity; homologous recombination; human disease; human embryonic stem cell; human stem cells; improved; induced pluripotent stem cell; mammalian genome; novel strategies; promoter; public health relevance; recombinational repair; therapy development; transgene expression; tumor; tumorigenesis; vector; vector genome

DESCRIPTION (provided by applicant): Gene Targeting and Integration by Adeno-Associated Virus Vectors Adeno-associated virus (AAV) vectors can efficiently deliver genes to many cell types and produce long- term transgene expression. Although AAV vector genomes often persist without altering a cell's chromosomes, they can also permanently modify the genome through gene targeting or integration at non- homologous sites. In some cases integration is essential for therapeutic or experimental efficacy. In the previous funding period this grant supported our research on AAV-mediated gene targeting and non- homologous integration. Gene targeting refers to the genetic modification of a specific site on a chromosome that shares DNA sequence with the vector. Non-homologous integration refers to the introduction of the vector genome at unrelated, non-specific chromosomal sites. In this proposal we will follow up on our prior results in three areas. In each case we will use AAV vectors to manipulate and probe the mammalian genome, further developing this important vector system. First, we will take advantage of the high frequency and accuracy of AAV-mediated gene targeting to correct several disease-causing mutations in human stem cells. The corrected and uncorrected stem cells produced will serve as ideal cellular models of human disease, and the targeting vectors employed can be used therapeutically when transplantation therapies are developed for patient-derived pluripotent stem cells. Second, using modern sequencing technologies we will determine where AAV-mediated gene targeting and non-homologous vector integration occur on a genome-wide basis. Based on our prior results, this should identify human chromosomal regions prone to breakage, and map recombination frequencies in cultured human cells for the first time. Third, we will follow up on our unexpected observation that AAV vector integration at a specific mouse locus can lead to liver tumors. Given the potential importance of these findings, it is essential that we establish whether this was a unique example unlikely to be encountered in other settings, or a real concern for the clinical use of AAV. This research has significant potential to develop new therapies for genetic disease and establish the risks of AAV vectors. PUBLIC HEALTH RELEVANCE: Relevance The proposed research will improve adeno-associated virus (AAV) vectors, which are increasingly used in research and therapeutic applications. New methods for correcting disease-causing mutations will be developed, which may ultimately be used in cellular therapies. The possible risks of AAV vectors will also be studied, with relevance for their clinical use and the study of liver cancer.

描述（由申请人提供）： 腺相关病毒载体腺相关病毒（AAV）载体的基因靶向和整合可以有效地将基因传递给许多细胞类型，并产生长期的转基因表达。尽管AAV载体基因组通常在不改变细胞的染色体的情况下持续存在，但它们也可以通过非同源部位的基因靶向或整合来永久修改基因组。在某些情况下，整合对于治疗或实验功效至关重要。在上一个资金期间，该赠款支持我们对AAV介导的基因靶向和非同源整合的研究。靶向基因是指与载体共享DNA序列的染色体上特定位点的遗传修饰。非同源整合是指在无关的非特异性染色体位点引入载体基因组。在此提案中，我们将在三个领域进行以前的结果。在每种情况下，我们都会使用AAV矢量来操纵和探测哺乳动物的基因组，从而进一步开发这种重要的向量系统。首先，我们将利用AAV介导的基因靶向的高频和准确性来纠正人类干细胞中的几种引起疾病的突变。所产生的校正和未经校正的干细胞将用作人类疾病的理想细胞模型，并且在为患者衍生的多能干细胞开发移植疗法时，可以使用治疗的靶向载体进行治疗。其次，使用现代测序技术，我们将确定AAV介导的基因靶向和非同源载体整合的位置。基于我们先前的结果，这应该确定容易破裂的人类染色体区域，并首次在培养的人类细胞中绘制重组频率。第三，我们将跟进我们意外的观察结果，即在特定小鼠基因座处的AAV矢量积分会导致肝肿瘤。鉴于这些发现的潜在重要性，我们必须确定在其他环境中不太可能遇到这是一个独特的例子，还是临床使用AAV的真正关注点。这项研究具有开发遗传疾病的新疗法并确定AAV载体的风险的巨大潜力。 公共卫生相关性： 相关性拟议的研究将改善与腺相关的病毒（AAV）载体，这些病毒媒介越来越多地用于研究和治疗应用。将开发用于纠正引起疾病突变的新方法，最终可以用于细胞疗法。还将研究AAV载体的可能风险，并与其临床使用和研究肝癌有关。