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

• 批准号: 7032149
• 负责人: RALPH MICHAEL LINDEN
• 金额: $ 32.23万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 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/7032149
• 关键词: adeno associated virus group; cell cycle; cell differentiation; embryonic stem cell; gene delivery system; gene expression; gene targeting; genetic promoter element; genetically modified animals; host organism interaction; laboratory mouse; transfection; virus integration

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号染色体的19q13.4处来建立潜伏期，称为AAVS1。虽然这一特征被认为可用于基于 AAV 的靶向基因传递策略，但人们对 AAV 及其体内整合机制知之甚少。大多数病毒生命周期特征是通过细胞培养观察得出的，因为目前还没有合适的动物模型。最近，我们证明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相对应的区域中是保守的。基于这一发现，我们建议研究 AAV 介导的小鼠 ES 细胞转基因靶向（目标 1）。这一发现也为在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 ES 基因靶向确定，就有可能将这些研究扩展到野生型 AAV 整合（目标 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 ES 细胞分化为任何或所有谱系？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 ES 细胞引入囊胚并随后衍生出转基因小鼠（实际上是整合位点敲除小鼠）将明确告诉我们，整合位点的杂合破坏可能会产生什么影响（如果有的话）（目标 2）。自从干细胞生物学的最新发展将相当多的注意力集中在使用基于细胞的疗法来治疗复杂疾病以来，对 ES 细胞的安全有效的基因靶向的需求不断增长。然而，这种方法的成功需要能够对干细胞进行离体基因修饰。例如，基于细胞的疗法的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。因此，我们认为有必要研究基于 AAV 的转基因靶向插入小鼠 ES 细胞是否可以减少对插入突变的担忧。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，但在快速分裂的 ES 细胞中，由于缺乏 G1 检查点，这一问题需要得到解决。 ES 细胞的体外（胚体系统）和体内（转基因小鼠）分化分析为研究 AAVS1 是否代表安全靶向位点提供了可能性。此外，这些测定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实现最佳转基因表达。一旦建立，该系统将使我们能够测试各种谱系特异性以及受调控的启动子在该特定染色体环境中的活性（目标 3）。