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.
View: Text | PDF
Research Article Hematology Article has an altmetric score of 75

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

×

Figure 4

The LCR-shRNAmiR vector does not inhibit human hematopoietic stem cell engraftment in NSG mice and induces γ-globin in erythroid cells derived from engrafted normal and SCD patient CD34+ cells.

Options: View larger image (or click on image) Download as PowerPoint
The LCR-shRNAmiR vector does not inhibit human hematopoietic stem cell e...
(A) Healthy donor–derived CD34+ cells were transduced and transplanted into NSG-mice. Fourteen to 16 weeks later, CD34+ cells were isolated from the bone marrow and subjected to erythroid in vitro differentiation followed by FACS sorting and RT-qPCR analysis. Each data point represents the result from an individual animal. (B) Specificity of the LCR-shRNAmiR vector in human cells isolated from the bone marrow (BM) of NSG mice. Shown is the percentage of Venus+ cells in freshly isolated CD34+ cells and in 3 different erythroid (Ery) maturation stages 1 week after start of erythroid in vitro differentiation. (C) Sickle cell patient CD34+ cells were transduced and differentiated in vitro. RT-qPCR analysis was performed on FACS sorted transduced cells after 18 days of culture. Symbols indicate different patients and each data point represents an independent replicate. (D) Western blot quantification of BCL11A in transduced erythroid cells derived from a single SCD patient. Data are the mean ± SD, n = 3. **P ≤ 0.01; ***P ≤ 0.001. Statistics: 1-way ANOVA. FSC, forward scatter; HU, hydroxyurea; LCR, β-globin locus control region and β-globin proximal promoter; NT, nontargeting shRNAmiR; ntd, nontransduced; SFFV, spleen focus-forming virus promoter.