Correction of sickle cell disease in adult mice by interference with fetal hemoglobin silencing

Abstract

Persistence of human fetal hemoglobin (HbF, α(2)γ(2)) in adults lessens the severity of sickle cell disease (SCD) and the β-thalassemias. Here, we show that the repressor BCL11A is required in vivo for silencing of γ-globin expression in adult animals, yet dispensable for red cell production. BCL11A serves as a barrier to HbF reactivation by known HbF inducing agents. In a proof-of-principle test of BCL11A as a potential therapeutic target, we demonstrate that inactivation of BCL11A in SCD transgenic mice corrects the hematologic and pathologic defects associated with SCD through high-level pancellular HbF induction. Thus, interference with HbF silencing by manipulation of a single target protein is sufficient to reverse SCD.

Fig. 1

BCL11A loss in adult mice reverses γ-silencing. (A) Expression of BCL11A protein in CD71⁺Ter119⁺ fetal liver (FL) and bone marrow (BM) cells of control (EpoR-Cre-) and BCL11A knockout (EpoR-Cre+) β-YAC mice. Glyceraldehyde phosphate dehydrogenase (GAPDH) was analyzed as a loading control. (B) Expression of human fetal (γ) globin genes was monitored by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in FL cells (E12.5 to E18.5) or peripheral blood of postnatal animals (1 to 30 weeks old). Data are plotted as percentage of γ-globin over total β-like human globin gene levels in control (EpoR-Cre-) and BCL11A knockout (EpoR-Cre+) β-YAC mice (N ≥ 4 per genotype at each time point). Results are means ± SD. All γ-globin levels for the different genotypes are significantly different (P < 1 × 10⁻⁵, two-tailed t test). (C) Immunohistochemistry for HbF was performed on E16.5 FLs from EpoR-Cre- and EpoR-Cre+ animals. (D) Transcriptional profiling of control (Bcl11a^+/+) and Bcl11a^–/– (EpoR-Cre+) CD71⁺Ter119⁺ erythroid cells (N = 3 per genotype). Probes corresponding to mouse α- and β-globin genes are indicated by arrows. Hba-x, ζ-globin; Hba-a1/a2, α-globin; Hbb-y, εy-globin; Hbb-bh1, βh1-globin; Hbb-b1/b2, β-globin. (E) Expression of human γ-globin genes was monitored by qRT-PCR in control (Mx1-Cre-) and BCL11A knockout (Mx1-Cre+) mice before and after polyinosinepolycytidine (pIpC) (N ≥ 4 per genotype at each time point; *P < 1 × 10⁻⁵).

Fig. 2

DNA demethylation and HDAC inhibition enhance residual γ-globin expression. (A) CpG methylation within the Gγ promoter was measured by bisulfite sequencing analysis in CD71⁺Ter119⁺ erythroid progenitors in control (Bcl11a^+/+) and Bcl11a^–/– (EpoR-Cre+) β-YAC mice at various development stages. Human primary FL and BM proerythroblasts (Pro-E) were analyzed as controls. The percentage of methylated CpG dinucleotides is shown for each sample. A diagram of the human β-globin cluster is shown on the top. (B) Expression of human γ-globin genes was monitored by qRT-PCR in control (Bcl11a^+/+) and Bcl11a^–/– (EpoR-Cre+) β-YAC mice before and after 5-azaD treatment (N = 6 per genotype per treatment). (C) In vivo chromatin occupancy of BCL11A, acetyl-H3 lysine 9 (H3K9ac), HDAC1, trimethyl-H3 lysine 27 (H3K27me3), and RNA Pol II was determined by ChIP-chip in primary human erythroid progenitors. A genome browser representation of binding patterns at the human β-globin cluster is shown. (D) Expression of human γ-globin genes was monitored by qRT-PCR in control (Bcl11a^+/+, N = 8) and Bcl11a^–/– (EpoR-Cre+, N = 12) β-YAC mice before and after SAHA treatment.

Fig. 3

Inactivation of BCL11A rescues sickle cell defects in humanized SCD mice. (A) Representative blood smears of control, SCD, and SCD/Bcl11a^–/– mice are shown at 1000× magnification. (B) RBC life span is significantly extended in SCD/Bcl11a^–/– mice at every time point compared with SCD mice (N ≥ 4; P < 0.01). Results are means ± SEM. (C) Correction of splenomegaly in SCD/Bcl11a^–/– mice (N ≥ 3 per genotype). Results are means ± SEM. (D) Expression of fetal (γ) and sickle adult (β^s) globin genes was monitored by qRT-PCR in the peripheral blood of control, SCD, and SCD/Bcl11a^–/– animals (8 to 10 weeks old; N = 5, 6, and 4, respectively). Results are means ± SEM. (E) Distribution of HbF in red cells. Representative graphs for control, SCD, and SCD/Bcl11a^–/– animals are shown. The same scale is used in all three graphs, and the mean percentage of F cells (HbF/HbA double-positive) is shown (N = 5, 6, and 4, respectively).