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）：Triplex-directed 和 other 基于寡核苷酸的策略将进行可行性测试 常染色体显性遗传性色素性视网膜炎 (ADRP) 的基因特异性疗法 由视紫红质基因缺陷引起。在这项工作的过程中 研究人员将开发可用于测试各种 ADRP 和其他常染色体显性遗传的治疗方法 失调。由于 ADRP 的任何疗法最终都必须在以下方面进行测试和优化： 动物，研究人员选择了老鼠，因为它们很容易 基因组可以被修改。研究人员提议创建小鼠 ES 细胞 DNA 的线选择片段，可以有效地靶向 位点特异性重组。这些改良的 ES 细胞将允许高效、 任何形式的视紫红质、cDNA 或基因组、野生型或 来自任何物种的突变体，用于广泛的生理学研究。这 研究人员将“敲入”经过修饰的人类视紫红质基因，旨在服务 作为寡核苷酸治疗效果的敏感细胞检测器 视紫红质基因表达、校正和敲除。对于整个小鼠研究， 研究人员将使用视紫红质与绿色荧光蛋白的融合物 （绿色荧光蛋白）。寡核苷酸对转录的影响将通过减少来测量 荧光强度；寡核苷酸对基因校正的影响和 突变将通过 GFP 荧光的出现来测量，起始为 未表达的视紫红质-GFP 融合构建体 (GFP-)。细胞研究 将在整个动物实验之前进行，以测试各种治疗方法 参数。尽管寡核苷酸介导的转录效应可以 易于测量，检测基于重组的校正和突变 敲除需要专门的构建体，研究者首先会 CHO 中选择性腺苷磷酸核糖转移酶 (APRT) 基因的测试 细胞。灵敏的检测系统将使我们能够检测到微弱的信号 然后可以进行改进和优化。使用 CHO 寡核苷酸 (TFO)、RNA/DNA 嵌合寡核苷酸和寡核苷酸类似物，例如肽核酸 酸（PNA），具有抑制转录、纠正基因缺陷的能力， 并引入突变。这些结果将作为开发的基础 小鼠的治疗方案。