Fig 4. Correction of the E6V mutation in SCD patient-derived HSPCs.

a) gDNA from HBB RNP-treated SCD-HSPCs was harvested and INDELs were analyzed via TIDE software (N=4 different SCD patient donors). b) HBB-targeted SCD-HSPCs were analyzed for GFP expression by flow cytometry (N=4 different SCD patient donors) c) SCD HSPCs were targeted with rAAV6 corrective SNP donor. HBB allele types were analyzed by sequencing of TOPO-cloned PCR fragments derived from In-Out PCR. 50-100 TOPO clones were analyzed from each of three different HSPC donors (N=3 different SCD patient donors). d) SCD HSPCs were targeted with anti-sickling HBB cDNA-EF1-tNGFR correction donor. Frequencies of tNGFR⁺ cells were analyzed by flow cytometry (N=3 different SCD patient donors). e) SCD Mock HSPCs and sorted SCD tNGFR^high HSPCs were differentiated into erythrocytes in vitro. Representative FACS plots from Day 21 of differentiation show cell surface markers associated with erythrocytes (GPA⁺/CD45^-/ CD71⁺/CD34^-) f) HbS, HbA, and HbAS3 mRNA expression was quantified by RT-qPCR in erythrocytes differentiated from HBB-edited or Mock SCD HSPCs. All mRNA transcript levels were normalized to the RPLP0 input control (N=2-3 different SCD patient donors).