DEVELOPING ZFN'S AS MOLECULAR TOOLS FOR TARGETED INTEGRATION

开发 ZFN 作为靶向整合的分子工具

基本信息

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
项目类别：
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

项目摘要

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分子生物学家追求的目标是能够在特定位点操纵或修饰植物和哺乳动物基因组（包括人类基因组）。基因打靶——通过同源重组 (HR) 替换基因的过程——使用模板 DNA 的染色体外片段，并调用细胞自身的修复机制进行基因转换。在哺乳动物细胞中，基因打靶并不是一个非常有效的过程；大约只有百万分之一的处理细胞经历了所需的基因修饰。锌指核酸酶 (ZFN) 将 Fokl 核酸内切酶的非特异性切割结构域 (N) 与锌指蛋白 (ZFP) 结合在一起，提供了一种将位点特异性 DSB 传递到基因组并刺激局部 HR 修复 DSB 的通用方法。多个实验室已经表明，ZFN 可以找到并切割细胞中的染色体靶标，并刺激局部 HR 修复 DSB。在本提案中，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，现有的选择具有高亲和力和严格序列特异性的 ZFP 的策略既费力又麻烦。我们开发了细菌单杂交、单报告系统来快速选择和完善 ZFN 的序列特异性，我们建议对其进行进一步测试。其次，尽管我们的 3 指 ZFN 在体内切割特定位点，但由于在次要（非目标）位点进行切割，它们可能对细胞有毒。我们推测，赋予 ZFN 更大的序列特异性将降低它们的毒性。我们建议通过比较分别识别 18 bp 和 24 bp DNA 序列的 3 指和 4 指 ZFN 对来检查 ZFN 介导的基因靶向在细胞中的功效。第三，常规技术无法有效地对两种重要的人类细胞类型，即原代CD34+造血干祖细胞（HSPC）和胚胎干细胞（HESC）进行精确靶向位点特异性修饰。这限制了人类细胞的实验室研究和临床治疗的潜在转化。我们建议测试 ZFN 介导的基因靶向在这些关键细胞类型中的功效和毒性。我们选择人类基因组的趋化因子（C-C 基序）受体 5 (CCR5) 位点作为 ZFN 介导的原代人类 HSPC 和 HESC 靶向破坏的模型位点。