Figure 2.

The bifunctional βAS3m/miR7m vector efficiently and safely corrects the RBC sickling phenotype

(A) Structure of the βAS3m/miR7m LV. The sequence of miR7m aligned to the βAS3 transgene is shown in the top panel. The βAS3 transgene (βAS3 original) was modified to avoid its targeting by miR7m, thus generating the βAS3 modified transgene (βAS3m) harboring mismatches (green) with the βAS3 original transgene corresponding to silent mutations, as shown by the amino acid sequence (black boxes). (B) Infectivity (TU/ng p24) was calculated based on infectious (TU/mL) and physical (p24 antigen ng/mL) titers (Figure S5). Data are shown as mean ± SD. (n = 3 independent LV productions per LV). (C) Mobilized (2 donors) or non-mobilized (1 donor) peripheral blood HSPCs from SCD donors were either mock transduced (mock; n = 1 per donor) or transduced with the βAS3 or βAS3m/miRnt (n = 5–6 independent biological replicates per donor) or βAS3m/miR7m (n = 2–5 independent biological replicates per donor) LV at different MOIs for 24 h. After transduction, cells were differentiated in liquid culture toward the erythroid lineage. VCN/cell was measured 14 days after transduction by ddPCR in erythroblasts. Globin expression (qRT-PCR, HPLC, and flow cytometry) and RBC differentiation markers and properties (sickling assay) were evaluated in erythroblasts along differentiation or in mature RBCs (see also Figure S5). (D–G) βAS3- and βAS3m/miRnt-transduced samples were pooled in all analyses. VCN/cell is indicated below each graph. (F and G) 2 SCD donors. (D) HbAS3 and HbS expression in mature RBCs measured by CE HPLC. Linear regression, ∗∗∗p = 0.0005 (n = 3, 17, and 10 independent biological replicates for mock-, βAS3- or βAS3/miRnt-, and βAS3/miRBCL11A-transduced samples, respectively; 3 SCD donors). (E) Frequency of sickling RBCs after 1-h incubation at low oxygen tension (0% O₂). Unpaired t test, ∗∗p < 0.01 (n = 2, 11, and 5 independent biological replicates for mock-, βAS3- or βAS3/miRnt-, and βAS3/miRBCL11A-transduced samples, respectively; 2 SCD donors). (F) Frequency of enucleated RBCs measured by flow cytometry on days 13 and 20 of erythroid differentiation. (G) Frequencies of (left) CD36⁺, (center) CD71⁺, and (right) CD235a⁺ erythroid cells measured by flow cytometry along differentiation. (H) Distribution of βAS3-LV or βAS3m/miR7m-LV integration sites in exonic, intronic, and intergenic genomic regions in HD HSPCs (n = 1). (I) mRNA-seq-based analysis comparing mRNA expression between two sample groups: βAS3 and mock (left), βAS3m/miRnt and βAS3 (center), and βAS3m/miR7m and βAS3m/miRnt (right) (2 SCD donors were used; n = 2 and 3 independent biological replicates for mock- and LV-transduced samples, respectively). Differentially expressed genes (DEGs) with an FDR of less than 0.05 and an absolute log2 fold change (logFC) greater than 1 are highlighted in red (upregulated [UP]) or blue (downregulated [DOWN]). Genes that are not differentially expressed are represented in gray (NotSig).