PNA-based strategies to reverse gamma-globin gene silenc

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的缺陷，表现为中度至危及生命的贫血。胎儿珠蛋白的再活化通过补偿缺失的β-珠蛋白链（在β-地中海贫血中）或通过干扰突变血红蛋白的聚合（在镰状细胞病中）为这些成年患者提供治疗益处。 对β-样珠蛋白合成控制的研究已经成功地集中在破译簇内每个基因最大表达的机制上。然而，对于该提议感兴趣的是表明主动沉默在产生正确的表达的组织和发育特异性中也起重要作用的观察结果。特别是，位于γ-珠蛋白启动子内并抑制其活性的CCTTG序列提供了一种新的靶点，在这种情况下，通过逆转沉默或去阻遏来增加γ-珠蛋白表达。 除了通过鉴定相关的γ-阻遏蛋白来阐明珠蛋白发育调控的机制细节的内在相关性之外，产生合适的γ-去阻遏蛋白提高了它可用于转录再激活成人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病和β-地中海贫血的危及生命的影响为通过以下具体目标追求这一目标提供了相当大的临床理由：1.将合成肽核酸（PNA）并测试其通过干扰CCTTG结合阻遏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，以使有效的细胞和核进入; 3.假定的阻遏物结合到？-将对珠蛋白CCTTG元件进行生化分离、鉴定和表征。 这些目标的最终结果将是提供一种转录试剂，该试剂将被测试其在成人红细胞环境中重新激活胎儿珠蛋白基因的能力，并鉴定负责该基因正常沉默的阻遏物。

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.