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

Mount Sinai School Of Medicine Of Nyu, New York NY

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Abstract

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.