KLF1 drives the expression of fetal hemoglobin in British HPFH

Abstract

β-Hemoglobinopathies are among the most common single-locus inherited diseases. In this condition, high fetal hemoglobin (HbF) levels have been found to be beneficial, and boosting HbF expression is seen as an attractive therapy. Naturally occurring mutations in the fetal promoter can result in high HbF persisting into adulthood in a benign condition known as hereditary persistence of fetal hemoglobin (HPFH). Individuals with one form of HPFH, British HPFH, carry a T to C substitution at position -198 of the fetal gene promoter. These individuals exhibit HbF levels of up to 20%, enough to ameliorate the symptoms of β-hemoglobinopathies. Here, we use clustered regularly interspaced short palindromic repeat-mediated genome editing to introduce the -198 substitution into human erythroid HUDEP-2 cells and show that this mutation is sufficient to substantially elevate expression of HbF. We also examined the molecular mechanism underlying the increase in fetal expression. Through a combination of in vitro and in vivo studies, we demonstrate that the mutation creates a de novo binding site for the important erythroid gene activator Krüppel-like factor 1 (KLF1/erythroid KLF). Our results indicate that introducing this single naturally occurring mutation leads to significantly boosted HbF levels.

Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

“…We designed an sgRNA that programs ABE to simultaneously mutate -198T to C in the promoter driving HBG1 expression, and -198T to C in the promoter driving HBG2 expression, by placing the target A•T base pair at protospacer position 7. These mutations are known to confer British-type HPFH and enable fetal hemoglobin production in adults 37 . ABE7.10 installed the desired T•A to C•G mutations in the HBG1 and HBG2 promoters with 29% and 30% efficiency, respectively, in HEK293T cells (Fig.…”

Disruption of the BCL11A Erythroid Enhancer Reactivates Fetal Hemoglobin in Erythroid Cells of Patients with β-Thalassemia Major

“…Attempts to faithfully recreate HPFH γ-globin promoter mutations through gene editing have been associated with low efficiency in normal adult CD34+ cells ex vivo because less efficient homology- or microhomology-mediated repair is required 6, 7. By contrast, attempts to induce nuclease-mediated inactivation of HbF “silencers” (i.e BCL11A,12, 13, 14 LRF, 8 or KLF1 15 ) can be more efficient because they require repair of DSBs with non-homologous end joining (NHEJ). One of several approaches targeting HbF reawakening is suppression of an important developmental silencer of γ-globin, BCL11A.…”

Recent progress in understanding and manipulating haemoglobin switching for the haemoglobinopathies

“…Recreation of the ‐175T>C ( HBG2 :g.‐175T>C) HPFH mutation in K562 cells by gene editing using TALENs resulted in elevated fetal haemoglobin expression and de novo binding of TAL1 to the mutant HBG2 promoter (Wienert et al , ). Similarly the British HPFH mutation ‐198T>C ( HBG1 :g.‐198T>C) results in de novo binding of KLF1, increased physical interaction with the LCR, and elevated γ‐globin expression in HUDEP‐2 cells to a level similar to that found in individuals heterozygous for the mutation (Wood et al , ; Wienert et al , ). These studies indicate that gain of binding sites for transcriptional activators at the fetal globin gene promoters could be a potent strategy for HbF reactivation.…”