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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: Nat Med. 2021 Mar 18;27(4):677–687. doi: 10.1038/s41591-021-01284-y

Fig. 2 ∣. WGR of α-globin using a promoterless reporter.

Fig. 2 ∣

a, AAV6 donor design for integration of HBB-T2A-YFP at the HBA1 locus. b, Percentage of CD34−/CD45− HSPCs acquiring RBC surface markers—GPA and CD71—as determined by flow cytometry. Bars represent median ± interquartile range. Values represent biologically independent HSPC donors: n = 6 for mock; n = 3 for RNP only, GFP WGR and HBB UTRs; n = 4 for AAV only and HBB UTRs without intron 2 (HBB-i2); and n = 5 for HBA1 UTRs and HBA2 UTRs. c, Percentage of GFP+ cells as determined by flow cytometry. Bars represent median ± interquartile range. Values represent biologically independent HSPC donors: n = 9 for mock; n = 4 for RNP only and HBB UTRs without intron 2; n = 11 for AAV only; n = 12 for GFP WGR; n = 3 for HBB UTRs; and n = 5 for HBA1 and HBA2 UTRs. ***P = 0.0035 by unpaired two-tailed t-test. d, Targeted allele frequency in bulk edited population as determined by ddPCR. Bars represent median ± interquartile range. Values represent biologically independent HSPC donors: n = 1 for mock; n = 5 for RNP only; n = 2 for AAV only; n = 7 for GFP WGR; n = 3 for HBB, HBA1 and HBB UTRs without intron 2; and n = 5 for HBA2 UTRs. ***P = 0.00023 by unpaired two-tailed t-test. e, MFI of GFP+ cells for each treatment as determined by the BD FACS Aria II platform. Values represent biologically independent HSPC donors: n = 6 for mock and AAV only; n = 4 for RNP only and HBB UTRs without intron 2; n = 3 for HBB UTRs; and n = 5 for HBA1 and HBA2 UTRs. Bars represent median ± interquartile range. *P= 0.037, ***P = 0.012 by unpaired two-tailed t-test without adjustment for multiple comparisons. f, Representative flow cytometry staining (FCS) and gating scheme for human HSPCs targeted at HBA1 with HBA1 UTRs donor and differentiated into RBCs. This indicates that only RBCs (CD34−/CD45−/CD71+/GPA+) are able to express the integrated T2A-YFP marker. E1–3, exons 1–3. K, 1,000.