Figure 2.

Combination of lentiviral and genome editing technologies is efficient in HUDEP-2 cells

(A) Schematic representation of the LVs carrying either the AS3 globin gene (LV AS3, strategy #1) or the AS3m globin gene and a sgRNA-expressing cassette (LV AS3m.gR). Both AS3- and AS3m-globin genes are under the control of a short β-globin promoter (β-p) and a mini-βLCR containing HS2 and HS3. The anti-sickling amino acid substitutions are reported in red. The modified nucleotides in LV AS3m, avoiding its targeting by the gR-C, are reported in green. We indicated the amino acids below the nucleotide sequence. sgRNA expression is under the control of the human U6 promoter (U6p). gR-C targets a region in exon 1 (Ex1) of HBB (strategy #2), gR-BCL11A targets the +58-kb erythroid-specific enhancer of BCL11A (strategy #3), and gR-13bpdel targets the BCL11A binding site within the HBG promoters (strategy #4). (B) Schematic representation of the LV transduction and plasmid transfection protocol used in HUDEP-2 cells. Cells were transduced with LV AS3m carrying gR-C, BCL11A, and 13bpdel sgRNAs and transfected after 10 days with a Cas9-GFP plasmid. Transfection efficiency was 30%–60%. FACS-sorted Cas9-GFP⁺ cells were differentiated into mature erythroblasts. (C) Relative expression of HBB and AS3m mRNAs normalized to α-globin, as detected by qRT-PCR. VCN values are reported in blue and indels in black below the graph. Ctr UTs are untreated HUDEP-2 cells and Ctr TEs are HUDEP-2 cells transfected only with TE buffer. Transduced samples that were mock transfected are indicated with “–.” The percentage of HBB decrease upon LV AS3m.C treatment is reported above the histogram bars. Data are expressed as mean ± SEM. (D) RP-HPLC quantification of globin chains in Cas9-treated and control (mock-transfected) LV AS3m.C-transduced cells. β-Like globin chains were normalized to α-globin chains. VCN values are reported in blue and indels in black below the graph. Ctr UTs are untreated HUDEP-2 cells and Ctr TEs are HUDEP-2 cells transfected only with TE buffer. Transduced samples that were mock transfected are indicated with “–” and harbored no indels. The percentage of β-globin decrease upon LV AS3m.C treatment and the α-globin/non-α-globin ratio are reported above the histogram bars. (E) CE-HPLC chromatograms (left panel) and quantification (right panel) of Hb tetramers in Cas9-treated and control mock-transfected LV AS3m.C-transduced cells. We calculated the percentage of each Hb type over the total Hb tetramers. VCN values are reported in blue and indels in black below the graph. Ctr UTs are untreated HUDEP-2 cells and Ctr TEs are HUDEP-2 cells transfected only with TE buffer. The decrease in HbA expression upon LV AS3m.C treatment is reported as percentage above the histogram bars. (F) Editing efficiency (indel frequency) in LV AS3m.C-treated HUDEP-2 cells 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) in Cas9-GFP⁺ HUDEP-2 erythroid cells. (G) Relative HBG expression normalized to α-globin, as detected by qRT-PCR in Cas9-treated and mock-transfected cells transduced with LV AS3m.BCL11A or LV AS3m.13bpdel. VCN values are reported in blue and indels in black below the graph. Ctr UTs are untreated HUDEP-2 cells and Ctr TEs are HUDEP-2 cells transfected only with TE buffer. Data are expressed as mean ± SEM. (H) RP-HPLC quantification of globin chains in Cas9-treated and mock-transfected cells transduced with LV AS3m.BCL11A or LV AS3m.13bpdel. β-Like globin chains are normalized over α-globin. VCN values are reported in blue and indels in black below the graph. α-Globin/non-α-globin ratios are reported above the histogram bars. Ctr UTs are untreated HUDEP-2 cells and Ctr TEs are HUDEP-2 cells transfected only with TE buffer. (I) CE-HPLC chromatograms (left panel) and quantification (right panel) of Hb tetramers in Cas9-treated and mock-transfected LV AS3m.BCL11A- and LV AS3m.13bpdel-transduced cells. We plotted the percentage of each Hb type over the total Hb tetramers. VCN values are reported in blue and indels in black below the graph. The percentage of HbA decrease is reported above the histogram bars. Ctr UTs are untreated HUDEP-2 cells and Ctr TEs are HUDEP-2 cells transfected only with TE buffer.