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 1

Knockdown of BCL11A reduces engraftment of murine and human hematopoietic stem cells.

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Knockdown of BCL11A reduces engraftment of murine and human hematopoieti...
(A) Lentiviral vectors used in competitive transplantation assays: the SFFV-shRNAmiR vector is shown above and the control vectors are shown below. The dark vertical lines represent the shRNA targeting BCL11A. (B) Murine competitive repopulation assays. The contribution of gene-modified cells in repopulated mice was analyzed in peripheral blood (PB), bone marrow (BM), and spleen (Spl) (week 12). The dashed line indicates the predicted hematopoietic output based on the input of cells. Data points above or below this line indicate an overrepresentation of gene-modified cells derived from the control vector (above the line) or the SFFV-shRNAmiR transduced population (below the line). (C) Reconstituted murine bone marrow B cell compartment (left panel) derived from HSCs transduced with SFFV-shRNAmiR versus control vectors and the LSK content (right panel). Each data point in B and C represents an individual recipient. P values are indicated in the figure. (D) Reconstitution of NSG mice with transduced human cells. Human CD34+ cells were transduced with SFFV-shRNAmiR or SFFV-EGFP and transplanted into NSG mice. The percentage of gene-modified human B cells, monocytes, and granulocyte fractions at 14 weeks relative to the initial transduction rate is shown. (E) NSG mice were transplanted with SFFV-shRNAmiR– or SFFV-EGFP–transduced hCD34+ cells. The cell cycle status of human gene-modified cells in the bone marrow at 2 weeks was assessed by DNA/Ki76 staining. Representative flow diagrams are shown. (F) The cell cycle distribution of gene-modified cells in vivo (as in panel E) was quantified. Each data point represents an individual mouse. ***P < 0.001; *P < 0.05. Error bars in all figures = SD. Statistical analysis: 2-tailed t test. Ψ, packaging signal; cPPT, central polypurine tract; Gran, granulocytes; Mono, monocytes; NS, not significant; RRE, rev-responsive element; SA, splice acceptor; SD, splice donor; SFFV, spleen focus-forming virus promoter; sinLTR, self-inactivating long terminal repeat; SSC, side scatter; wPRE, woodchuck hepatitis virus posttranscriptional regulatory element.