MECHANISMS OF GLOBIN GENE SILENCING

球蛋白基因沉默的机制

• 批准号: 6030924
• 负责人: Mark C Walters
• 金额: $ 12.87万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-10 至 1999-09-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6030924
• 关键词: Retroviridae; chromatin; erythroid stem cell; gene expression; gene induction /repression; gene therapy; genetic enhancer element; genetic promoter element; genetic recombination; genetic regulatory element; genetic transcription; globin; hemoglobin F; hemoglobinopathy; phenotype; plasmids; point mutation; recombinase; reporter genes; tissue /cell culture; transfection /expression vector

The human globin genes have long been the focus of intense study, because they are altered by many mutations that cause hereditary anemias with wide-ranging and often devastating effects. The well-characterized molecular basis for clinically significant hemoglobinopathies like sickle cell disease and beta-thalassemia major has made these disorders natural targets for replacement gene therapy. A major obstacle to gene therapy for these and other disorders is the silencing of transcription that occurs after genomic integration of replacement gene vectors. We have developed and utilized novel methods, termed Recombinase Mediated Cassette Exchange (RMCE), to study transcriptional silencing. These methods are based on the Lox/Cre system of site-specific recombination and permit the efficient exchange of one plasmid construct for another in the same chromosomal site, thereby eliminating the influence of integration position on subsequent studies of transcriptional silencing. The broad aims of this proposal are to test the hypothesis that retroviral elements are targeted for silencing by chromatin, and find means of overcoming such silencing. The experimental system we have developed makes it possible to study a wide range of cis-acting elements that may contribute to stable expression of erythroid genes. These studies have particular relevance to replacement gene therapy for sickle cell anemia and other clinically significant hemoglobinopathies, as identification of cis-acting elements sufficient to overcome transcriptional silencing will promote the development of improved gene therapy vectors.

人类球基因长期以来一直是激烈研究的重点， 因为它们被许多引起遗传性贫血的突变所改变 具有广泛且经常具有毁灭性的影响。 特殊的 临床上重要的血红蛋白病的分子基础 镰状细胞疾病和β地中海贫血专业使这些疾病 替代基因治疗的天然靶标。 基因的主要障碍 这些和其他疾病的治疗是转录的沉默 这是在基因组整合替代基因载体后发生的。 我们 已经开发和利用了新方法，称为重组酶介导 盒式交换（RMCE），研究转录沉默。这些 方法基于位点特异性重组的LOX/CRE系统 并允许有效交换一种质粒构建体 在同一染色体部位，从而消除了 在随后的转录沉默研究中的整合位置。 该提议的广泛目的是检验以下假设。 逆转录病毒元素是针对染色质沉默的目标，并找到 克服这种沉默的手段。 我们拥有的实验系统 开发的可以研究广泛的顺式作用元素 这可能有助于红细胞基因的稳定表达。 这些 研究与镰刀的替代基因疗法特别相关 细胞贫血和其他临床意义的血红蛋白病，AS 识别足以克服的顺式作用元素 转录沉默将促进改善基因的发展 治疗载体。