Figure 3.

Testing of the bifunctional LV AS3m.C in SCD HSPCs

(A) Schematic representation of the protocol used to transduce and transfect SCD HSPCs. 24 h after thawing, HSPCs were transduced with LV AS3m.C and after 48 h cells were transfected with the Cas9-GFP protein and differentiated into mature RBCs following a 21-day differentiation protocol. (B) Correlation between VCN and indel frequency in cells treated with LV AS3m.C (two mobilized SCD donors). R² and line-of-best-fit equation are indicated. (C) Indel frequency in LV AS3m.C-treated SCD HSPCs measured by TIDE analysis after PCR amplification and Sanger sequencing of the target region in HBB exon 1 and the potential off-target region in the transgene (AS3m). (D) Evaluation of off-target activity. GUIDE-seq analysis of gR-C in 293T cells (left panel). The protospacer targeted by gR-C and the protospacer adjacent motifs (PAMs) are shown in the first line. Off-targets and their mismatches with the on-target (highlighted in color), sequencing read counts, and chromosomal position are reported. Deep-sequencing analysis of off-target editing events in mature erythroblasts derived from SCD HSPCs treated with LV AS3m.C (right panel). Transduced and mock-transfected SCD cells (Ctr) are indicated in red and edited samples (AS3m.C) in black. (E) Correlation between VCN (left panel) or indel frequency (right panel) and percentage of HbS and HbAS3 (determined by CE-HPLC) in SCD RBCs derived from mock-transfected (orange and light blue) and Cas9-transfected (red and dark blue) SCD HSPCs transduced with LV AS3m.C (two mobilized SCD donors). R² and line-of-best-fit equation are indicated. Dashed lines indicate the VCN required to achieve equal amounts of HbS and HbAS3. (F) Relative expression of HBB mRNA normalized to α-globin in untreated SCD cells (Ctr) (n = 6) and in cells treated with LV AS3m.C (n = 8, 2 mobilized SCD donors). ∗∗∗∗p < 0.0001 (unpaired t test). Horizontal lines indicate median and first and third quartiles. (G) Representative CE-HPLC chromatograms showing the Hb profile of in vitro-differentiated mature erythroblasts. From top to bottom: β-thalassemic cells (thal), healthy donor cells (HD), HD cells treated with gR-C plasmid (HD+gR-C), SCD cells (SCD), SCD cells transduced with LV AS3m.C and mock transfected, and SCD cells transduced with LV AS3m.C and transfected with Cas9-GFP protein.