Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype
Christian Brendel, … , Richard I. Gregory, David A. Williams
Christian Brendel, … , Richard I. Gregory, David A. Williams
Published September 6, 2016
Citation Information: J Clin Invest. 2016;126(10):3868-3878. https://doi.org/10.1172/JCI87885.
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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 2

Erythroid-specific expression of the shRNAmiR prevents engraftment defect of hematopoietic stem cells.

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Erythroid-specific expression of the shRNAmiR prevents engraftment defec...
(A) Configuration of the erythroid-specific LCR-shRNAmiR vector. (B) Competitive repopulation assay between LCR-shRNAmiR– and SFFV-shRNAmiR–transduced cells. The relative contribution of gene-modified cells in peripheral blood erythroid cells (defined by CD71 and Ter119 positivity) of transplanted animals was analyzed at 4, 8, and 12 weeks after transplantation. The dashed line indicates the predicted balanced hematopoietic output. Data points above or below this line indicate an overrepresentation of gene-modified cells derived from the LCR vector– (above the line) or the SFFV-shRNAmiR–transduced (below the line) population. Each data point represents an individual recipient mouse. (C) Competitive repopulation between LCR-shRNAmiR– and SFFV-BFP– (empty vector) transduced cells. Analysis as described in panel B. (D) In vivo expression profile of the LCR-shRNAmiR vector in mice. The gating strategy is shown at the top and expression of Venus in different cell lineages is shown below. Numbers in plots indicate percentage Venus+ cells and the mean fluorescence intensity. (E) Summary of the data shown in D normalized to the percentage Venus+ cells in the CD71+Ter119+ population. Data are the mean ± SD, n = 3. HS2 and HS3, DNAse hypersensitive sites 2 and 3 from the β-globin locus control region; LCR, β-globin locus control region and β-globin proximal promoter; SFFV, spleen focus-forming virus promoter; SSC, side scatter.