Figure 1
From: Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII
Construction and validation of transgene vectors for expression of human B domain deleted FVIII. (A) Schematic diagrams of pCDNA4 plasmid containing human wild-type (WT) full length F8 cDNA (7065 bp) or modified BDD-F8 cDNA (4425 bp), both driven by a CMV promoter. BDD-F8 cDNA contained an amino acid substitution, R1645H (red), and a short B domain sequence (66 bp nucleotides/22 amino acid residues encoded). (B)The donor vector pX602-AAV-BDD-F8 for CRISPR/Cas9-mediated insertion contains an inverted terminal repeat (ITR), gRNA-PAM sequence, 3′-splicing sequence of Alb intron 13, and Alb exon 14 codon sequence, followed by P2A, BDD-F8 cDNA, a second gRNA-PAM sequence and another ITR. The entire BDD-F8 cassette was 4642 bp in length. (C–E) FVIII expression and functional activity tests in HEK-293 cells transfected without (control) or with pCDNA4-WT-F8 or pCDNA4-BDD-F8 plasmids. At 4 days after transfection, (C) mRNA and (D) protein expression of WT-F8 or BDD-F8 were assessed by real-time RT-PCR and immunofluorescence, respectively. Scale bars, 10 µm. (E) Content and activity of FVIII or BDD-FVIII protein in the cell culture medium, released from HEK-293 cells, were measured by ELISA and Chromogenix FVIII activity assay, respectively. Data are means ± SE from 3 independent experiments, except that ELISA measurements were performed in only one of these 3 experiments.
