Figure 2.

Homology-Directed Repair at the HBB Nuclease Target Site Using rAAV6 Donor Template

(A) Schematic representation of rAAV6 cassettes designed to drive either a GTC (E6V) introducing a sickle mutation or a GAA (E6optE) introducing a codon-optimized change at codon 6 by HDR. (B) Experimental timeline for testing gene editing with RNP and rAAV6 delivery followed by erythroid differentiation in mPBSCs. (C) WT (%) and HDR (%) measured by ddPCR and NHEJ (%) measured by ICE sequencing, respectively, following electroporation with RNP alone, transduction with rAAV6 donor template alone, or co-delivery of RNP and GTC (E6V) rAAV6 donor template, at the indicated concentrations (donor n = 4). (D) RP-HPLC analysis of erythroid cells to measure β-globin expression in cells treated with RNP only, rAAV6 only, or RNP plus GTC (E6V) rAAV6 (donor n = 7). (E) WT (%) and HDR (%) measured by ddPCR and NHEJ (%) measured by ICE sequencing, respectively, following electroporation with RNP alone, transduction with rAAV6 donor template alone, or co-delivery of RNP and GAA (E6optE) rAAV6 donor template, at the indicated concentrations (1% rAAV6; donor n = 3). (F) RP-HPLC analysis of erythroid cells to measure β-globin expression in cells treated with RNP only, rAAV6 only, or RNP plus GAA (E6optE) rAAV6 (donor n = 3). β^A, adult globin; β^S, sickle globin; γ^G, gamma 2; γ^A, gamma 1. All bar graphs show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. p value was calculated by comparing each sample mean of NHEJ (%), HDR (%), WT (%), or globin sub-type (%) with the respective NHEJ (%), HDR (%), WT (%), or globin sub-type (%) of the mock sample by two-way ANOVA with Dunnett’s multiple comparison. Asterisks are color matched to the respective mock sample. See also Figures S2–S4.