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19q13.4处（称为AAVS 1）来建立潜伏期。虽然该特征被认为可用于靶向基因递送的基于AAV的策略，但关于AAV及其体内整合机制知之甚少。大多数病毒的生命周期特征是从细胞培养中的观察得出的，因为还没有合适的动物模型。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对应于人类靶位点的区域中是保守的。基于这一发现，我们提出在小鼠ES细胞中研究AAV介导的转基因靶向（Aim 1）。这一发现也开启了在尚未探索的模型系统中研究wt AAV整合的生物学的机会。一旦建立了ES基因靶向，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整合是否影响ES细胞分化为任何或所有谱系？这个问题是重要的，因为已经提出AAV可以在子宫内感染。此外，将含有整合的AAV的ES细胞引入胚泡中并随后衍生出转基因小鼠（实际上敲除整合基因座的小鼠）将明确地告诉我们，从整合基因座的杂合破坏可以预期什么效果（如果有的话）（目的2）。由于干细胞生物学的最新发展已经将相当大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ES细胞的安全和有效基因靶向的需求已经增长。然而，这种方法的成功将需要离体遗传修饰干细胞的能力。例如，基于细胞的疗法的障碍是免疫反应性细胞破坏新移植的细胞和/或移植细胞的同种异体排斥的可能性。因此，我们认为有必要研究是否基于AAV的转基因靶向插入到小鼠ES细胞减少插入突变的关注。虽然在分化的细胞中，插入诱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，缺乏G1检查点，需要解决这个问题。ES细胞的分化测定，在体外（胚状体系统）和体内（转基因小鼠），提供了可能性，以调查是否AAVS 1代表一个安全的靶向位点。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的最佳转基因表达。一旦建立，该系统将允许我们测试各种谱系特异性以及调节启动子在该特定染色体环境中的活性（目的3）。