Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype.
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.
Christian
C
Brendel
Swaroopa
S
Guda
Raffaele
R
Renella
Daniel E
DE
Bauer
Matthew C
MC
Canver
Young-Jo
YJ
Kim
Matthew M
MM
Heeney
Denise
D
Klatt
Jonathan
J
Fogel
Michael D
MD
Milsom
Stuart H
SH
Orkin
Richard I
RI
Gregory
David A
DA
Williams
D000755:Anemia, Sickle Cell / Q000628:therapy*; D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D019070:Cell Lineage; D002478:Cells, Cultured; D055785:Gene Knockdown Techniques; D015316:Genetic Therapy; D006086:Graft vs Host Disease / Q000517:prevention & control; D006410:Hematopoiesis; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000502:physiology; D006801:Humans; D008810:Mice, Inbred C57BL; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins
D018952:Antigens, CD34; C486563:BCL11A protein, human; D002352:Carrier Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins