Highly efficient therapeutic gene editing of human hematopoietic stem cells

Wu Y, Zeng J, Roscoe BP, Liu P, Yao Q, Lazzarotto CR, Clement K, Cole MA, Luk K, Baricordi C, Shen AH, Ren C, Esrick EB, Manis JP, Dorfman DM, Williams DA, Biffi A, Brugnara C, Biasco L, Brendel C, Pinello L, Tsai SQ, Wolfe SA, Bauer DE.
Source: Nat Med
Publication Date: (2019)
Issue: 25(5): 776-783
Research Area:
Immunotherapy / Hematology
Stem Cells
Gene Expression
Cells used in publication:
CD34+ cell, human
Species: human
Tissue Origin: blood
Culture Media:
4D-Nucleofector™ X-Unit

In this publication Wu et al. are using Nucleofection to obtain highly efficient and selection-free method for genome editing in human hematopoietic stem cells. CD34+ HSPCs isolated from mobilized peripheral blood were thawed into X-VIVO 15 (Lonza, 04–418Q) supplemented with 100ng/ml human stem cell factor (SCF), 100ng/ml human thrombopoietin (TPO), and 100ng/ml recombinant human Flt3-ligand (Flt3-L).

HSPCs were electroporated with Cas9 RNP 24h afer thawing and maintained in X-VIVO media with cytokines. Electroporation was performed using Lonza P3 Primary Cell 4D Nucleofector Kit (V4XP-3032 for 20µl Nucleocuvette Strips or V4XP-3024 for 100µl Nucleocuvettes) using the program EO-100.

For 20µl Nucleocuvette Strips, the RNP complex was prepared by mixing 200 pmol Cas9 and 200 pmol sgRNA and incubating for 15min at room temperature immediately before electroporation. For indicated experiments in which glycerol was supplemented, 30% glycerol solution was added to Cas9 protein before addition of sgRNA. HSPCs (50000 ) resuspended in 20µl P3 solution were mixed with RNP and transferred to a cuvette for electroporation.


Re-expression of the paralogous gamma-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe ß-globin disorders sickle cell disease (SCD) and ß-thalassemia by induction of fetal hemoglobin (HbF, a2?2)1. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells2-6. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal ?-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with ß-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.