RHODOSPIN GENE CORRECTION BY OLIGONUCLEOTIDE TARGETING

通过寡核苷酸靶向进行视紫红质基因校正

• 批准号: 6314816
• 负责人: JOHN H WILSON
• 金额: $ 25万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6314816
• 关键词: CHO cells; autosomal dominant trait; electroporation; embryonic stem cell; flow cytometry; fluorescent dye /probe; fusion gene; gene mutation; gene targeting; gene therapy; genetic techniques; genetic transcription; green fluorescent proteins; human tissue; laboratory mouse; nucleic acid sequence; oligonucleotides; polymerase chain reaction; retinitis pigmentosa; rhodopsin; tissue /cell culture

DESCRIPTION (Adapted from the applicant's abstract): Triplex-directed and other oligonucleotide-based strategies will be tested for feasibility as gene-specific therapies for autosomal dominant retinitis pigmentosa (ADRP) caused by defects in the rhodopsin gene. In the course of this work the investigator will develop technologies that can be used for testing a variety of therapeutic approaches to ADRP and other autosomal dominant genetic disorders. As any therapy for ADRP must ultimately be tested and optimized in animals, the investigator has chosen mice because of the ease with which their genomes can be modified. The investigator proposes to create a mouse ES cell line selectable segment of DNA that can be efficiently targeted by site-specific recombination. These modified ES cells will permit efficient, selectable 'knock-in' of any form of rhodopsin, cDNA or genomic, wild-type or mutant, from any species, for a broad range of physiological studies. The investigator will "knock-in" modified human rhodopsin genes, designed to serve as sensitive cellular detectors of the effects of oligonucleotide treatments on rhodopsin gene expression, correction and knockout. For whole mouse studies, the investigator will use fusions of rhodopsin with green fluorescent protein (GFP). Oligonucleotide effects on transcription will be measured as a decrease in fluorescence intensity; oligonucleotide effects on gene correction and mutation will be measured by the appearance of GFP fluorescence starting with rhodopsin-GFP fusion constructs that are not expressed (GFP-). Cellular studies will precede whole animal experiments in order to test various treatment parameters. Although oligonucleotide-mediated effects on transcription can be measured readily, detection of recombination-based correction and mutational knockout requires specialized constructs, which the investigator will initially test at the selectable adenosine phosphoribosyl transferase (APRT) gene in CHO cells. A sensitive detection system will allow us to detect weak signals that can then be improved and optimized. Using CHO oligonucleotides (TFOs), RNA/DNA chimeric oligonucleotides and oligonucleotide analogues such as peptide nucleic acids (PNAs), for their ability to inhibit transcription, correct gene defects, and introduce mutations. These results will be used as the basis for developing treatment protocols in mice.

描述（改编自申请人的摘要）：三重指导和其他 将测试基于寡核苷酸的策略，以实现可行性 常染色体显性视网膜炎色素（ADRP）的基因特异性疗法 由视紫红质基因缺陷引起。在这项工作中 研究人员将开发可用于测试多样性的技术 ADRP和其他常染色体显性遗传的治疗方法 疾病。由于对ADRP的任何疗法最终都必须在 动物，研究人员选择了小鼠，因为他们的轻松 基因组可以修改。研究人员建议创建鼠标ES单元 可以有效针对的DNA的线路可选段 特定地点重组。这些修饰的ES细胞将允许有效， 任何形式的视紫红质，cDNA或基因组，野生型或野生型或 来自任何物种的突变体进行广泛的生理研究。这 研究者将“敲入”改良的人类视紫红质基因，旨在服务 作为寡核苷酸治疗作用的敏感细胞检测器 视紫红质基因表达，校正和敲除。对于整个小鼠研究， 研究者将使用绿色荧光蛋白的融合蛋白融合 （GFP）。寡核苷酸对转录的影响将被测量为减少 在荧光强度；寡核苷酸对基因校正和 突变将通过GFP荧光的出现开始测量 Rhodopsin-GFP融合构建体未表达（GFP-）。细胞研究 将在整个动物实验之前进行测试 参数。尽管寡核苷酸介导的对转录的影响可能是 容易测量，基于重组的校正和突变的检测 淘汰赛需要专门的结构，调查员最初将 在CHO中选择的可选腺苷磷酸糖基转移酶（APRT）基因的测试 细胞。敏感检测系统将使我们能够检测到弱信号 然后可以改进和优化。使用CHO寡核苷酸（TFOS），RNA/DNA 嵌合寡核苷酸和寡核苷酸类似物（例如肽核） 酸（PNA），因为它们抑制转录，正确基因缺陷的能力， 并引入突变。这些结果将被用作发展的基础 小鼠的治疗方案。