Correction of Murine Sickle Cell Disease Through Interference with Fetal Hemoglobin Silencing

Abstract 351

The switch from fetal (HbF, α₂γ₂) to adult (HbA, α₂β₂) hemoglobin is critical to the pathogenesis of the major hemoglobin disorders, sickle cell disease (SCD) and β-thalassemias. Increased HbF expression ameliorates the clinical severity of these conditions, forming the basis for the goal of elucidating mechanisms of globin switching and developing target-based therapies to enhance HbF production. The rationale for this therapeutic approach rests on the hypothesis that manipulation of a single target would lead to sufficient HbF reactivation for clinical benefit. Despite recent advances in understanding the complex processes of developmental globin switching and HbF silencing, this hypothesis is untested. The transcription factor BCL11A is a critical mediator of HbF switching and silencing. Previously we showed that BCL11A controls globin switching in mice and silencing of HbF expressed from human β-globin locus transgenes in mouse fetal liver, and contributes to HbF silencing in primary human erythroid cells. Furthermore, we demonstrated that BCL11A is required in vivo for HbF silencing in adult animals, yet dispensable for red cell production. These findings illustrate favorable features of BCL11A as a potential target for reactivation of HbF in the β-hemoglobinopathies.

Here, we test whether impairment of BCL11A is sufficient to ameliorate disease symptoms in a relevant preclinical model of a major β-hemoglobin disorder, the humanized SCD mouse model (“Berkeley” SCD model, Paszty et al. Science 278:876–878, 1997). These mice express exclusively human sickle hemoglobin and faithfully recapitulate the principal hematologic and histopathologic phenotypes of patients with SCD. We introduced BCL11A-null alleles into the “Berkeley” SCD model (SCD Bcl11a^fl/fl EpoR-Cre+, hereafter called SCD/Bcl11a^−/− mice). Loss of BCL11A in SCD animals restored the hematologic deficits to normal values. Compared with sickle animals, SCD/Bcl11a^−/− mice have marked increases in RBC counts (6.4 ± 0.5 to 9.8 ± 0.4 × 10¹²/L; non-sickle control 10.1 ± 0.2 × 10¹²/L ) and hemoglobin level (7.8 ± 0.6 to 13.6 ± 0.7 g/L; non-sickle control 13.1 ± 0.3 g/L), accompanied by a significant reduction in reticulocyte counts (38.2% ± 3.9% to 7.0% ± 0.3%; non-sickle control 3.1% ± 0.6%). Consistent with correction of hematologic parameters, sickled erythrocytes were absent in the peripheral blood of SCD/Bcl11a^−/− mice and red cell lifespan was substantially normalized. In addition, tissue damage in multiple organs was prevented in SCD/Bcl11a^−/− mice. Urine osmolality of SCD/Bcl11a^−/− mice was restored (from 1037 ± 82 mOsm in SCD mice to 2133 ± 333 mOsm in SCD/Bcl11a^−/− mice), indicating improved renal function.

To assess the extent to which these phenotypic findings were related to HbF induction, we determined the level of γ-globin expression and cellular distribution of HbF in SCD/Bcl11a^−/− mice. Compared with SCD mice, expression of γ-globin genes was markedly elevated in adult SCD/Bcl11a^−/− mice (1.3% to 28.3% of total β-like human globins; P = 0.00017). Peripheral blood of control and SCD mice contained few F cells (3.6% and 7.2%, respectively). In contrast, peripheral blood of SCD/Bcl11a^−/− mice displayed strong pancellular staining of HbF, and F cells contributed to 85.1% of total RBCs (P = 3.9 × 10⁻⁵). The level of HbF expression achieved in the absence of BCL11A is well beyond that needed to impede HbS polymerization.

In conclusion, we demonstrate that inactivation of a single component involved in HbF regulation, the transcription factor BCL11A, provides phenotypic correction of SCD in a mouse model. Our findings provide a crucial “proof of principle” for targeted reactivation of HbF as a realistic therapeutic goal.