Document Type

Article

Publication Date

11-15-1996

Abstract

Hemoglobin A2 (HbA2; α2δ2) is a powerful inhibitor of HbS (α2β2/(S) polymerization. However, HbA2 levels are normally low in sickle cell patients. We show that a major reason for low δ-globin gene expression is the defective CACCC box at -90 in the δ-globin promoter. When the CACCC box defect in δ is corrected, expression of an HS2 δ/Luciferase reporter is equivalent to HS2 β/Luciferase. Erythroid Krupple-like factor (EKLF), which binds to the CACCC box of the β-globin gene and activates high-level expression, does not bind to the normal δ-globin promoter. Our goal is to design a modified EKLF that binds to the defective δ-globin promoter and enhances δ-globin gene expression. To test the feasibility of this strategy, we inserted the β-globin CACCC box at -90 of the δ-globin gene promoter to produce an HS2 δ(CAC)-β construct and quantitated human δ- and β-globin mRNA in stably transformed murine erythroleukemia (MEL) cells. δ-Globin mRNA in these cells was 22.0% ± 9.0% of total human globin mRNA (δ/δ + β) as compared with 3.0% ± 1.3% in the HS2 δ-β control. In a second set of experiments a GAL4 DNA-binding site was inserted at -90 of the δ-globin gene to produce an HS2 δ(GAL4)-β construct. This construct and a GAL4((1-147))/EKLF expression vector were stably transfected into MEL cells. δ-Globin mRNA in these cells was 27.8% ± 7.1% of total human globin mRNA as compared with 9.9% ± 2.5% in the HS2 δ(GAL4)-β plus GAL4((1-147)) control. These results show that δ-globin gene expression can be significantly increased by a modified EKLF. Based on these results, we suggest that modified EKLFs, which contain zinc fingers designed to bind specifically to the defective δ-globin CACCC box, may be useful in gene therapy approaches to increase HbA2 levels and inhibit HbS polymerization.

Publication Source (Journal or Book title)

Blood

First Page

4051

Last Page

4057

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