Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype
Christian Brendel, Swaroopa Guda, Raffaele Renella, Daniel E. Bauer, Matthew C. Canver, Young-Jo Kim, Matthew M. Heeney, Denise Klatt, Jonathan Fogel, Michael D. Milsom, Stuart H. Orkin, Richard I. Gregory, David A. Williams
Christian Brendel, Swaroopa Guda, Raffaele Renella, Daniel E. Bauer, Matthew C. Canver, Young-Jo Kim, Matthew M. Heeney, Denise Klatt, Jonathan Fogel, Michael D. Milsom, Stuart H. Orkin, Richard I. Gregory, David A. Williams
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Research Article Hematology

Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype

Abstract

Reducing expression of the fetal hemoglobin (HbF) repressor BCL11A leads to a simultaneous increase in γ-globin expression and reduction in β-globin expression. Thus, there is interest in targeting BCL11A as a treatment for β-hemoglobinopathies, including sickle cell disease (SCD) and β-thalassemia. Here, we found that using optimized shRNAs embedded within an miRNA (shRNAmiR) architecture to achieve ubiquitous knockdown of BCL11A profoundly impaired long-term engraftment of both human and mouse hematopoietic stem cells (HSCs) despite a reduction in nonspecific cellular toxicities. BCL11A knockdown was associated with a substantial increase in S/G2-phase human HSCs after engraftment into immunodeficient (NSG) mice, a phenotype that is associated with HSC exhaustion. Lineage-specific, shRNAmiR-mediated suppression of BCL11A in erythroid cells led to stable long-term engraftment of gene-modified cells. Transduced primary normal or SCD human HSCs expressing the lineage-specific BCL11A shRNAmiR gave rise to erythroid cells with up to 90% reduction of BCL11A protein. These erythrocytes demonstrated 60%–70% γ-chain expression (vs. < 10% for negative control) and a corresponding increase in HbF. Transplantation of gene-modified murine HSCs from Berkeley sickle cell mice led to a substantial improvement of sickle-associated hemolytic anemia and reticulocytosis, key pathophysiological biomarkers of SCD. These data form the basis for a clinical trial application for treating sickle cell disease.

Authors

Christian Brendel, Swaroopa Guda, Raffaele Renella, Daniel E. Bauer, Matthew C. Canver, Young-Jo Kim, Matthew M. Heeney, Denise Klatt, Jonathan Fogel, Michael D. Milsom, Stuart H. Orkin, Richard I. Gregory, David A. Williams

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Figure 5

Amelioration of SCD-specific disease parameters in vivo using the Berkeley-SCD mouse model.

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Amelioration of SCD-specific disease parameters in vivo using the Berkel...
(A) Lineage-negative bone marrow cells from Berkeley-SCD mice were transduced with the LCR-shRNAmiR5 vector and transplanted into lethally irradiated BoyJ recipients. As controls, SFFV-EGFP–transduced SCD or untransduced WT cells were transplanted and peripheral blood hematologic parameters of transplanted animals were obtained. Published reference values in the nontransplant setting are indicated by lines (dotted line, Berkeley-SCD; continuous line, WT mice). Control groups are shown in light gray, the LCR-shRNAmiR5–transplanted group in dark gray. *P < 0.05; **P < 0.01. (B) Representative FACS plots of peripheral blood cells stained for the erythroid differentiation markers CD71 and Ter119. Percentages in plots indicate the fraction of CD71+Ter119+ high erythroid precursor cells. Data are the mean ± SD, n = 3. HCT, hematocrit; HGB, hemoglobin; LCR, β-globin locus control region and β-globin proximal promoter; Retics, reticulocytes; SFFV, spleen focus-forming virus promoter.