PNA-based strategies to reverse gamma-globin gene silencing*

描述(由申请人提供)： 血红蛋白病(如镰状细胞病)和地中海贫血是红细胞功能缺陷，表现为中度至危及生命的贫血。胎儿珠蛋白的重新激活通过补偿缺失的β-珠蛋白链(在β-地中海贫血中)或通过干扰突变的血红蛋白聚合(在镰状细胞疾病中)，为这些成年患者提供了治疗上的好处。 关于控制类β珠蛋白合成的研究已经成功地集中在破译簇中每个基因最大限度表达的机制上。然而，对这一提议感兴趣的是，观察表明，主动沉默在产生正确的组织和发育特异性表达方面也发挥着重要作用。特别是，位于伽马珠蛋白启动子内并抑制其活性的CCTTG序列提供了增加伽马珠蛋白表达的新靶点，在这种情况下，通过逆转沉默或去抑制。 除了与通过鉴定相关的伽马抑制蛋白来阐明珠蛋白发育调控的机制细节有内在联系外，产生合适的伽马去阻遏蛋白还增加了它可以用来在转录上重新激活成年红系细胞内的胎儿珠蛋白基因的可能性。以这种方式改善镰状细胞疾病和β-地中海贫血对生命的威胁为通过以下具体目标实现这一目标提供了相当好的临床基础：1.将合成多肽核酸(PNA)，并测试其通过干扰CCTTG结合抑制物来重新激活胎儿珠蛋白启动子的能力；2.这些PNA分子的设计将被修改，以实现有效的细胞和核进入；3.将从生化角度分离、鉴定和鉴定与β-珠蛋白CCTTG元件结合的假定抑制物。 这些目标的最终结果将是提供一种转录试剂，将测试其在成人红系环境中重新激活胎儿珠蛋白基因的能力，并确定导致该基因正常沉默的阻遏因子。

DESCRIPTION (provided by applicant): Hemoglobinopathies (such as sickle cell disease) and thalassemias are defects of red blood cell function that are manifested as moderate to life-threatening anemias. Reactivation of fetal globin provides a therapeutic benefit to these adult patients by compensating for absent beta-globin chains (in beta-thalassemia) or by interfering with polymerization of mutant hemoglobin (in sickle cell disease). Studies on the control of beta-like globin synthesis have successfully focused on deciphering the mechanisms by which each gene within the cluster is maximally expressed. However, of interest for this proposal are observations indicating that active silencing also plays an important role in generating correct tissue- and developmental specificity of expression. In particular, CCTTG sequences that reside within the gamma-globin promoter and inhibit its activity provide a novel target with which to increase gamma-globin expression, in this case by reversal of silencing or derepression. Apart from its intrinsic relevance to illuminating mechanistic details of globin developmental regulation by identification of the relevant gamma-repressor protein, generating a suitable gamma-derepressor raises the possibility that it can be used to transcriptionally reactivate the fetal globin gene within the adult erythroid cell. Ameliorating the life-threatening effects of sickle cell disease and beta-thalassemias in this way provides a considerable clinical rationale for pursuing this goal by the following specific aims: 1. Peptide nucleic acids (PNAs) will be synthesized and tested for their ability to reactivate the fetal globin promoter via interference with the CCTTG-binding repressor; 2. The design of these PNA molecules will be modified to enable efficient cell and nuclear entry; 3. The putative repressor that binds to the ?-globin CCTTG element will be biochemically isolated, identified, and characterized. The end results of these aims will be to make available a transcriptional reagent that will be tested for its ability to reactivate the fetal globin gene within the adult erythroid environment, and to have identified the repressor responsible for normal silencing of this gene.