MECHANISMS OF GLOBIN GENE SILENCING

人类珠蛋白基因长期以来一直是研究的焦点， 因为它们被许多突变所改变，这些突变会导致遗传性贫血， 影响范围广，往往是毁灭性的。 充分表征的 具有临床意义的血红蛋白病的分子基础 镰状细胞病和β-地中海贫血使这些疾病 替代基因疗法的天然靶点。 基因的主要障碍 治疗这些和其他疾病的方法是沉默转录 发生在替换基因载体的基因组整合之后。 我们 已经开发和利用了新的方法，称为酶介导的 盒交换（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 并允许一种质粒构建体与另一种质粒构建体的有效交换 在相同的染色体位点，从而消除了 整合位置对转录沉默的后续研究。 这项建议的主要目的是检验以下假设： 逆转录病毒元件被染色质靶向沉默，并发现 克服这种沉默的方法。 我们的实验系统 开发了一种可以研究广泛的顺式作用元件 这可能有助于红细胞基因的稳定表达。 这些 研究与镰状细胞替代基因疗法特别相关， 细胞性贫血和其他临床显著的血红蛋白病，如 顺式作用元件的鉴定足以克服 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.