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）： 腺相关病毒载体的基因靶向和整合腺相关病毒（AAV）载体可以有效地将基因递送至多种细胞类型并产生长期转基因表达。尽管AAV载体基因组通常在不改变细胞染色体的情况下持续存在，但它们也可以通过基因靶向或非同源位点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效果至关重要。在之前的资助期间，这笔赠款支持了我们对 AAV 介导的基因靶向和非同源整合的研究。基因打靶是指对与载体共享DNA序列的染色体上特定位点进行遗传修饰。非同源整合是指在不相关的、非特异性的染色体位点引入载体基因组。在本提案中，我们将在三个领域跟进我们之前的成果。在每种情况下，我们都将使用 AAV 载体来操纵和探测哺乳动物基因组，进一步开发这一重要的载体系统。首先，我们将利用 AAV 介导的基因靶向的高频率和准确性来纠正人类干细胞中的几种致病突变。所产生的校正和未校正的干细胞将作为人类疾病的理想细胞模型，并且当为患者来源的多能干细胞开发移植疗法时，所使用的靶向载体可用于治疗。其次，利用现代测序技术，我们将确定 AAV 介导的基因靶向和非同源载体整合在全基因组基础上发生的位置。根据我们之前的结果，这应该可以识别容易断裂的人类染色体区域，并首次绘制培养的人类细胞中的重组频率。第三，我们将跟进我们意外的观察结果，即 AAV 载体在特定小鼠位点的整合可导致肝脏肿瘤。考虑到这些发现的潜在重要性，我们有必要确定这是否是在其他情况下不太可能遇到的独特例子，或者是 AAV 临床使用的真正问题。这项研究在开发遗传病新疗法和确定 AAV 载体的风险方面具有巨大潜力。 公共卫生相关性： 相关性拟议的研究将改进腺相关病毒（AAV）载体，该载体越来越多地用于研究和治疗应用。将开发纠正致病突变的新方法，最终可能用于细胞疗法。还将研究 AAV 载体的可能风险，及其临床应用和肝癌研究的相关性。