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AAV-mediated targeted DNA integration in mouse ES cells

AAV 介导的小鼠 ES 细胞中的靶向 DNA 整合

基本信息

批准号：
7491818
负责人：
RALPH MICHAEL LINDEN
金额：
$ 30.56万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-09-20 至 2009-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7491818
关键词：
19q13.4 Acute Address Adenovirus Infections Adult Affect Animal Model Attention Biological Assay Biological Models Biology Birth Cardiac Cell Therapy Cell Transplantation Cell Transplants Cells Cellular biology Characteristics Chromosomal Stability Chromosomes, Human, Pair 19 Clinical Trials Cloning Complex Condition Cultured Cells DNA DNA Integration DNA Sequence Rearrangement DNA biosynthesis Dependovirus Derivation procedure Development Diabetes Mellitus Disease Disruption EPS8L1 gene Embryo Environment Experimental Designs Fetal Development Frequencies Future Gene Delivery Gene Targeting Gene Transfer Generations Genes Genome Genomics Germ Lines Heart failure Human Human Genome Human Virus In Vitro Infection Insertional Mutagenesis Knock-out Knockout Mice Lead Life Cycle Stages Link Maintenance Mediating Monitor Mus Parkinson Disease Phenotype Physiology Population Principal Investigator Proteins Protocols documentation Range Research Research Personnel Retroviridae Risk Signal Transduction Site Source Stem cells Structure System Technology Testing Tilia Tissues Transformed Cell Line Transgenes Transgenic Animals Transgenic Mice Transplantation Troponin I Viral Virus Integration Virus Replication base blastocyst body system cell killing cell type cellular transduction day embryonic stem cell gene therapy in utero in vivo leukemia mouse genome programs promoter research study site-specific integration stem success therapeutic gene therapeutic transgene transcription factor transgene expression vector viral DNA

项目摘要

DESCRIPTION (provided by applicant): Adeno-associated virus (AAV) is unique in that it establishes latency by integrating its genome into human chromosome 19 at 19q13.4, termed AAVS1. While this feature has been contemplated as useful for AAV- based strategies for targeted gene delivery, little is known about AAV and its integration mechanism in vivo. Most of the viral life cycle characteristics are concluded from observations in cell culture, as no suitable animal model has been available yet. Recently, we demonstrated that the chromosomal signals required for human site-specific integration are conserved in the mouse genome in a region corresponding to the human target site. Based on this finding we propose to study AAV-mediated transgene targeting in mouse ES cells (Aim 1). This discovery has also opened the opportunity to investigate the biology of wt AAV integration in an as yet unexplored model system. Once ES gene targeting has been established, it will be possible to extend those studies to wild type AAV integration (Aim 1). The ES system will allow us to ask a range of new questions. Does wild type AAV integration affect the differentiation of ES cells into any or all of the lineages? This question is significant since it has been proposed that AAV can infect in utero. Furthermore, introduction of ES cells, that contain integrated AAV, into the blastocyst and subsequent derivation of transgenic mice (in effect knock-out mice for the integration locus) will tell us unambiguously, what - if any - effects can be expected from heterozygous disruption of the integration locus (Aim 2). The need for safe and efficient gene targeting of ES cells has grown since recent developments in stem cell biology have focused considerable attention on the use of cell-based therapies for the treatment of complex diseases. The success of such an approach, however, will require the ability to genetically modify stem cells ex vivo. For example, an obstacle to cell based therapies is the likelihood for the destruction of the newly transplanted cells by immunoreactive cells and/or allo-rejection of the transplanted cells. Therefore, we deem it necessary to investigate whether AAV-based targeted insertion of a transgene into mouse ES cells diminishes concerns about insertional mutagenesis. While in differentiated cells the potential consequences of insertional mutagenesis are apparently negligible, in fast-dividing ES cells, lacking a G1 checkpoint, this concern needs to be addressed. Differentiation assays of ES cells, both in vitro (embryoid body system) and in vivo (transgenic mice), offer the possibility to investigate whether AAVS1 represents a safe targeting site. In addition, these assays will allow us to study if this chromosomal context permits optimal transgene expression in various lineages and tissues. Once established, this system will allow us to test a variety of lineage specific as well as regulated promoters for their activities within this particular chromosomal environment (Aim 3).

描述（由申请人提供）：腺相关病毒（AAV）的独特之处在于，它通过将其基因组整合到人类19号染色体19q13.4处建立潜伏期，称为AAVS1。虽然这一特征被认为对基于AAV的靶向基因递送策略有用，但对AAV及其在体内的整合机制知之甚少。由于没有合适的动物模型，大多数病毒的生命周期特征是通过细胞培养观察得出的。最近，我们证明了人类位点特异性整合所需的染色体信号在小鼠基因组中与人类靶位点对应的区域中是保守的。基于这一发现，我们建议研究aav介导的转基因靶向小鼠胚胎干细胞（Aim 1）。这一发现也为在一个尚未探索的模型系统中研究wt - AAV整合的生物学提供了机会。一旦确定了ES基因的靶向性，将有可能将这些研究扩展到野生型AAV整合（Aim 1）。ES系统将允许我们提出一系列新问题。野生型AAV整合是否会影响胚胎干细胞向任何或所有谱系的分化？这个问题很重要，因为有人提出AAV可以在子宫内感染。此外，将含有整合AAV的胚胎干细胞引入囊胚和随后衍生的转基因小鼠（实际上是敲除整合位点的小鼠）将明确地告诉我们，整合位点的杂合破坏（如果有的话）会产生什么影响（目标2）。由于干细胞生物学的最新进展将相当多的注意力集中在使用基于细胞的疗法治疗复杂疾病上，因此对安全有效的胚胎干细胞基因靶向的需求日益增长。然而，这种方法的成功将需要在体外对干细胞进行基因修饰的能力。例如，基于细胞的治疗的一个障碍是新移植的细胞可能被免疫反应性细胞破坏和/或移植细胞的同种异体排斥反应。因此，我们认为有必要研究基于aav的转基因靶向插入小鼠胚胎干细胞是否减少了对插入突变的担忧。虽然在分化细胞中插入突变的潜在后果显然可以忽略不计，但在缺乏G1检查点的快速分裂胚胎干细胞中，这一问题需要得到解决。胚胎干细胞在体外（胚状体系统）和体内（转基因小鼠）的分化试验，为研究AAVS1是否代表一个安全的靶向位点提供了可能性。此外，这些分析将使我们能够研究这种染色体背景是否允许在各种谱系和组织中进行最佳的转基因表达。一旦建立，该系统将允许我们测试各种谱系特异性以及调控启动子在这种特定染色体环境中的活动（目的3）。