MECHANISMS OF GLOBIN GENE SILENCING

长期以来，人类珠蛋白基因一直是密集研究的重点， 因为它们会被许多导致遗传性贫血的突变所改变 其影响范围很广，而且往往是毁灭性的。性格特点很好的 临床上有重要意义的血红蛋白疾病的分子基础 镰状细胞病和严重的β-地中海贫血造成了这些疾病 替代基因治疗的天然靶点。基因的主要障碍 这些疾病和其他疾病的治疗方法是沉默转录 这发生在替换基因载体的基因组整合之后。我们 开发和利用了新的方法，称为重组酶中介 磁带交换(RMCE)，以研究转录沉默。这些 方法基于位点特异性重组的Lox/Cre系统 并允许一种质粒构造与另一种质粒构造的有效交换 在相同的染色体位置，从而消除了 关于转录沉默后续研究的整合立场。 这项提议的主要目的是检验这一假设 逆转录病毒元件被染色质沉默，并发现 克服这种沉默的方法。我们拥有的实验系统 开发使研究广泛的顺式作用元素成为可能 这可能有助于红系基因的稳定表达。这些 研究与镰刀的替代基因疗法特别相关 细胞性贫血和其他临床上显著的血红蛋白病，如 识别足以克服的顺式作用元件 转录沉默将促进改良基因的发展 治疗载体。

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.