DEVELOPING ZFN'S AS MOLECULAR TOOLS FOR TARGETED INTEGRATION

开发 ZFN 作为靶向整合的分子工具

• 批准号: 7646236
• 负责人: SRINIVASAN CHANDRASEGARAN
• 金额: $ 31.16万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7646236
• 关键词: AIDS prevention; Address; Affect; Affinity; Alleles; Applications Grants; Arabidopsis; Binding; Biological; Biological Assay; Biology; CCR5 gene; CD34 gene; Cell Fraction; Cell Line; Cells; Chemokine (C-C Motif) Receptor 5; Chromosomes; Cleaved cell; Clinical; DNA; DNA Sequence; Development; Dimerization; Double Strand Break Repair; Drosophila genus; Fingers; Future; Gene Conversion; Gene Mutation; Gene Targeting; Gene-Modified; Generations; Genes; Genetic Medicine; Genome; Genomics; Goals; Hematopoietic; Hematopoietic stem cells; Human; Human Genome; Hybrids; K562 Cells; Laboratories; Laboratory Research; Ligation; Location; Mammalian Cell; Mammals; Mediating; Modeling; Modification; Molecular; Monophenol Monooxygenase; Mus; Mutate; Mutation; N Domain; Nonhomologous DNA End Joining; Paste substance; Plants; Plasmids; Population; Process; Proteins; Publishing; Reporter; Reporting; Site; Specificity; Stem cells; System; T-Lymphocyte; Techniques; Technology; Testing; To specify; Toxic effect; Transgenes; Translations; Work; X-Linked Severe Combined Immunodeficiency; Zinc Fingers; albino mouse; cell injury; cell type; cellular transduction; cytotoxic; design; efficacy testing; embryonic stem cell; endonuclease; gamma-Chemokines; gene correction; gene therapy; homologous recombination; human embryonic stem cell; improved; in vivo; macrophage; mammalian genome; melanocyte; monomer; novel; nuclease; receptor; recombinational repair; repaired; research study; response; stem; stem cell biology; therapeutic target; tool

DESCRIPTION (provided by applicant): DEVELOPING ZFNs AS MOLECULAR TOOLS FOR TARGETED INTEGRATION A sought after goal of molecular biologists has been the ability to manipulate or modify plant and mammalian genomes including the human genome at specific sites. Gene targeting - the process of replacing a gene by homologous recombination (HR) - uses an extra-chromosomal fragment of template DNA and invokes the cell's own repair machinery for gene conversion. Gene targeting is not a very efficient process in mammalian cells; about only 1 in a million treated cells undergo the desired gene modification. Zinc finger nucleases (ZFNs) that combine the non-specific cleavage domain (N) of Fokl endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver site-specific DSB to the genome, and stimulate local HR to repair the DSB. Several labs have shown that ZFNs find and cleave their chromosomal target in cells and stimulate local HR to repair the DSB. In this proposal, we address 3 barriers to wider implementation of ZFN-mediated gene targeting in Genetic Medicine. First, existing strategies for selecting ZFPs with high affinity and strict sequence-specificity are laborious and cumbersome. We have developed bacterial one-hybrid, single-reporter systems to rapidly select and refine sequence-specificity of ZFNs, which we propose to further test. Second, even though our 3-finger ZFNs cleave specific sites in vivo, they may be toxic to cells due to cleavage at secondary (non-targeted) sites. Imparting greater sequence-specificity to the ZFNs, we hypothesize, will make them less toxic. We propose to examine the efficacy of ZFN-mediated gene targeting in cells by comparing pairs of 3- and 4-finger ZFNs, recognizing 18 bp and 24 bp DNA sequence, respectively. Third, precisely targeted site-specific modification of 2 important human cell types, namely primary CD34+ haematopoietic stem-progenitor cells (HSPCs) and embryonic stem cells (HESCs), cannot be done effectively by routine technologies. This limits laboratory research in human cells and potential translation to clinical therapies. We propose to test the efficacy and toxicity of ZFN-mediated gene targeting in these key cell types. We have chosen the chemokine (C-C motif) receptor 5 (CCR5) locus of the human genome as a model locus for ZFN-mediated targeted disruption in primary human HSPCs and HESCs.

描述（由申请人提供）：开发ZFN作为靶向整合的分子工具，是分子生物学家寻求的目标的能力，它可以操纵或修改植物和哺乳动物基因组在特定地点，包括人类基因组。基因靶向 - 通过同源重组（HR）代替基因的过程 - 使用模板DNA的染色体外片段，并调用细胞自身的修复机器以进行基因转换。基因靶向不是哺乳动物细胞中非常有效的过程。只有一百万个经过处理的细胞中只有1个经历了所需的基因修饰。将FOKL核酸内切酶的非特异性切割结构域（N）与锌指蛋白（ZFPS）相结合的锌指核酸酶（ZFN）提供了一种将特异性DSB传递到基因组的一般方法，并刺激局部HR来维修DSB。几个实验室表明，ZFN在细胞中发现并裂解其染色体靶标，并刺激局部HR修复DSB。在此提案中，我们解决了在遗传医学中更广泛实施ZFN介导的基因靶向的3个障碍。首先，选择具有高亲和力和严格序列特异性的ZFP的现有策略是费力且繁琐的。我们已经开发了细菌的单杂交，单重孢子系统来快速选择和完善ZFN的序列特异性，我们建议这进一步测试。其次，即使我们的3指ZFN在体内切割特定位点，它们也可能因次级（非靶向）位点的切割而对细胞有毒。我们假设将更大的序列特异性赋予ZFN会使它们的毒性较小。我们建议通过比较3和4指ZFN对ZFN介导的基因靶向在细胞中的疗效，分别识别18 bp和24 bp DNA序列。第三，准确靶向的位点特异性修饰对2种重要的人类细胞类型，即原发性CD34+造血干型干细胞（HSPC）和胚胎干细胞（HESC），无法通过常规技术有效地进行。这限制了人类细胞中的实验室研究，并可能转化为临床疗法。我们建议在这些关键细胞类型中测试ZFN介导的基因靶向的疗效和毒性。我们选择了人类基因组的趋化因子（C-C基序）受体5（CCR5）基因座作为ZFN介导的原代人HSPC和HESC中ZFN介导的靶向破坏的模型基因座。