Therapeutic adenine base editing of human hematopoietic stem cells

Liao J, Chen S, Hsiao S, Jiang Y, Yang Y, Zhang Y, Wang X, Lai Y, Bauer DE, Wu Y
Source: Nat Commun.
Publication Date: (2023)
Issue: 14(1): 207
Research Area:
Immunotherapy / Hematology
Cells used in publication:
CD34+ cell, human
Species: human
Tissue Origin: blood
Culture Media:
4D-Nucleofector® X-Unit

RNP and mRNA electroporation

Electroporation was performed using Lonza 4D Nucleofector (V4XP-3032 for 20 µl Nucleocuvette Strips or V4XP-3024 for 100 µl Nucleocuvette Strips) following the manufacturer’s instructions. The chemical MS-sgRNA (2'-O-methyl 3' phosphorothioate modifications in the first and last three nucleotides) was ordered from GenScript. CD34+ HSPCs were thawed and maintained in X-VIVO medium supplemented with cytokines 24 h before electroporation. For 20-µl Nucleocuvette Strips, the RNP complex was prepared by mixing protein (100 pmol) and sgRNA (300 pmol, full-length product reporting method) and incubating for 15 min at room temperature immediately before electroporation. Fifty thousand HSPCs resuspended in 20µl of P3 solution were mixed with RNP and transferred to a cuvette for electroporation with program EO-100. For mRNA 20-µl Nucleocuvette Strips electroporation, the mRNA complex was prepared by mixing ABE8e mRNA (1 µg) and sgRNA (300 pmol). For 100-µl cuvette electroporation, the RNP complex was made by mixing 500 pmol ABE protein and 1500 pmol sgRNA. Next, 5M HSPCs were resuspended in 100 µl of P3 solution for RNP electroporation as described above. The electroporated cells were resuspended with X-VIVO media with cytokines and changed into EDM 24 h later for in vitro differentiation. For mouse transplantation experiments, cells were maintained in X-VIVO 15 with SCF, TPO, and Flt3-L for 0–1 day as indicated before infusion. Single guide RNA sequences, PAM and related deep-seq primers are listed in Supplementary Data 1.


In ß-thalassemia, either -globin induction to form fetal hemoglobin (a22) or ß-globin repair to restore adult hemoglobin (a2ß2) could be therapeutic. ABE8e, a recently evolved adenine base editor variant, can achieve efficient adenine conversion, yet its application in patient-derived hematopoietic stem cells needs further exploration. Here, we purified ABE8e for ribonucleoprotein electroporation of ß-thalassemia patient CD34+ hematopoietic stem and progenitor cells to introduce nucleotide substitutions that upregulate -globin expression in the BCL11A enhancer or in the HBG promoter. We observed highly efficient on-target adenine base edits at these two regulatory regions, resulting in robust ?-globin induction. Moreover, we developed ABE8e-SpRY, a near-PAMless ABE variant, and successfully applied ABE8e-SpRY RNP to directly correct HbE and IVS II-654 mutations in patient-derived CD34+ HSPCs. Finally, durable therapeutic editing was produced in self-renewing repopulating human HSCs as assayed in primary and secondary recipients. Together, these results support the potential of ABE-mediated base editing in HSCs to treat inherited monogenic blood disorders.