Go to JCI Insight
  • About
  • Editors
  • Consulting Editors
  • For authors
  • Publication ethics
  • Publication alerts by email
  • Advertising
  • Job board
  • Contact
  • Clinical Research and Public Health
  • Current issue
  • Past issues
  • By specialty
    • COVID-19
    • Cardiology
    • Gastroenterology
    • Immunology
    • Metabolism
    • Nephrology
    • Neuroscience
    • Oncology
    • Pulmonology
    • Vascular biology
    • All ...
  • Videos
    • Conversations with Giants in Medicine
    • Video Abstracts
  • Reviews
    • View all reviews ...
    • Complement Biology and Therapeutics (May 2025)
    • Evolving insights into MASLD and MASH pathogenesis and treatment (Apr 2025)
    • Microbiome in Health and Disease (Feb 2025)
    • Substance Use Disorders (Oct 2024)
    • Clonal Hematopoiesis (Oct 2024)
    • Sex Differences in Medicine (Sep 2024)
    • Vascular Malformations (Apr 2024)
    • View all review series ...
  • Viewpoint
  • Collections
    • In-Press Preview
    • Clinical Research and Public Health
    • Research Letters
    • Letters to the Editor
    • Editorials
    • Commentaries
    • Editor's notes
    • Reviews
    • Viewpoints
    • 100th anniversary
    • Top read articles

  • Current issue
  • Past issues
  • Specialties
  • Reviews
  • Review series
  • Conversations with Giants in Medicine
  • Video Abstracts
  • In-Press Preview
  • Clinical Research and Public Health
  • Research Letters
  • Letters to the Editor
  • Editorials
  • Commentaries
  • Editor's notes
  • Reviews
  • Viewpoints
  • 100th anniversary
  • Top read articles
  • About
  • Editors
  • Consulting Editors
  • For authors
  • Publication ethics
  • Publication alerts by email
  • Advertising
  • Job board
  • Contact
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

Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype

  • Text
  • PDF
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 3

Efficient knockdown of BCL11A leads to high γ-globin and HBF induction in human primary cells in vitro.

Options: View larger image (or click on image) Download as PowerPoint
Efficient knockdown of BCL11A leads to high γ-globin and HBF induction i...
(A) Representative Western blot showing a side by side comparison of BCL11A knockdown efficiency mediated by SFFV or LCR-shRNAmiR vectors in erythroid cells differentiated in vitro from transduced hCD34+ cells. The top panels show BCL11A immunoblot (XL and L isoforms) and the bottom panels show actin as loading control. Different lanes represent different shRNA targeting sequences (see methods). (B) Quantification of Western blots derived from 3 independent experiments. Data represent the mean ± SD of densitometric analysis of immunoblots. (C) Induction of γ-globin (by RT-qPCR) and HbF induction (by HPLC) of erythroid cells differentiated in vitro from transduced hCD34+ cells. Data are the mean ± SD, n = 3. Vertical arrows indicate the shRNAmiRs chosen for further experiments. Average vector copy numbers (VCNs) are indicated below. (D) Representative HPLC plots showing HbF (black) and HbA (white) peaks for the 3 selected candidates. (E) Differentiation state of controls (mean of NT and SFFV-EGFP) and LCR-shRNAmiR (mean of 3, 5, and 8) vector-transduced CD34+ cells after 18 days of erythroid culture. n = 3. (F) Correlation between the degree of knockdown of BCL11A and induction of HbF in transduced hCD34+ cells after erythroid in vitro differentiation. Each data point represents an individual sample. LCR, β-globin locus control region and β-globin proximal promoter; NT, nontargeting shRNAmiR; ntd, nontransduced; SFFV, spleen focus-forming virus promoter.

Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

Sign up for email alerts