Fig. 2: Modulation of synEPOR effect by addition of signal peptide and EPOR truncation.

A Schematic of second-generation synEPORs integrated at CCR5 locus. Red boxes represent the FKBP domain; yellow and green triangles indicate EPOR and IL6 SPs, respectively; dashed line represents EPOR truncation. Expected functional ligands are displayed above each construct. B Percentage of edited HSPCs that acquired erythroid markers (CD34-APC^-/CD45-V450^-/CD71-PE-Cy7⁺/GPA-PE⁺) +/−BB normalized to unedited cells +EPO at d14 of differentiation. synEPOR 1.5 data from Fig. 1C shown for comparison. Bars represent median +/-SEM; *p = 0.0052 and **p = 0.0006 by unpaired two-tailed t test across distinct samples. C Fold change of edited allele frequencies over the course of differentiation +/−BB and +/−EPO. Bars represent median +/−SEM; *p = 0.0178 and **p = 0.000048 comparing d0 vs. d14 within treatment by unpaired two-tailed t test across distinct samples. N = 4 biological replicates for all 1.5.IL6SP samples as well as −BB/−EPO and +BB/−EPO conditions edited with t1.5.IL6SP; N = 6 for −BB/+EPO and +BB/+EPO conditions edited with t1.5.IL6SP. D Representative flow cytometry staining and gating scheme for synEPOR-edited HSPCs at d14 of differentiation −EPO and +/−BB. Arrows indicate that only gated cells are displayed on the subsequent plot. E Representative hemoglobin tetramer HPLC plots at d14 of erythroid differentiation. +BB and −BB/−EPO conditions were from cells edited with synEPOR; +EPO condition was from unedited cells. All plots normalized to 1e6 cells. Source data are provided as a Source Data file. F AlphaFold2-based structure prediction of truncated EPOR and synEPOR. SP was removed since this sequence will be cleaved following translocation to the membrane. TMD labeled with an arrow as a reference point.