Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype

• Published in: The Journal of clinical investigation 2016-10, Vol.126 (10), p.3868-3878
• Main Authors: Brendel, Christian, Guda, Swaroopa, Renella, Raffaele, Bauer, Daniel E, Canver, Matthew C, Kim, Young-Jo, Heeney, Matthew M, Klatt, Denise, Fogel, Jonathan, Milsom, Michael D, Orkin, Stuart H, Gregory, Richard I, Williams, David A
• Format: Article
• Language: English
• Subjects: Anemia, Sickle Cell - therapy; Animals; Antigens, CD34 - metabolism; Apoptosis; Biomedical research; Blood diseases; Bone marrow; Care and treatment; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell cycle; Cell Lineage; Cells, Cultured; Charitable foundations; Cloning; Disease; Experiments; Gene expression; Gene Knockdown Techniques; Gene therapy; Genetic aspects; Genetic Therapy; Genotype & phenotype; Graft vs Host Disease - prevention & control; Health aspects; Hematopoiesis; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells - physiology; Humans; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, SCID; MicroRNA; Mortality; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Phenotype; Polymerization; Properties; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; Software; Stem cells; Transcription factors; Transplants & implants; Vectors (Biology)
• ISSN: 0021-9738; 1558-8238
• DOI: 10.1172/JCI87885

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.