Activation of human gamma-globin gene expression via triplex-forming oligonucleotide (TFO)-directed mutations in the gamma-globin gene 5' flanking region.

Human beta-globin disorders, such as sickle cell anemia and beta-thalassemia, are relatively common genetic diseases cause by mutations in the beta-globin gene. Increasing gamma-globin gene expression has been found to greatly reduce the disease symptom. However, the gamma-globin gene is developmentally regulated and normally expressed at high levels only during the fetal stage of human development. We have explored the possibility of activating the gamma-globin gene expression by triplex-forming oligonucleotide (TFO)-directed targeted mutagenesis. Using a psoralen-conjugated TFO designed to bind to a site overlapping with an Oct-1 binding site at the -280 region of the gamma-globin gene, targeted mutagenesis of the Oct-1 binding site has been achieved by transfecting the in-vitro-formed plasmid-oligo complex into human normal fibroblast (NF) cells. The mutation frequency at the target site was estimated to be 20% by direct DNA sequencing analysis. In-vitro protein binding assays indicated that these mutations reduced Oct-1 binding to the target site. In-vivo gene expression assays demonstrated activation of gamma-globin gene expression from these mutations in mouse erythroleukemia (MEL) cells. The levels of the gamma-globin gene expression increased by as much as fourfold in mutants with single base changes. These results suggest that the -280 region of the Agamma-globin gene negatively regulates the gamma-globin gene expression, and mutations at the Oct-1 binding site can lead to activation of the gamma-globin gene and generate the hereditary persistence of fetal hemoglobin (HPFH) condition. This study may provide a novel approach for gene therapy of sickle cell disease. The data may also have implications in gene therapy for other diseases including genetic diseases and cancers by introducing mutations into transcription factor binding sites to alter the levels of target gene expression.