Evaluation of modified lentiviral vectors for FVIII expression in cBOECs. (A) Schematic representation of the third-generation, self-inactivating lentiviral vector containing the cFVIII transgene under the control of the human EF1α promoter into which 1 to 3 copies of the 164-bp endothelial-specific enhancer element (–5.5HSCR were inserted. Unique AgeI (A), XhoI (X), and FseI (F) restriction sites in the promoter were used to insert the endothelial enhancer element into either the AgeI, or AgeI and XhoI, or AgeI and XhoI and FseI sites for constructs containing 1, 2, or 3 copies, respectively. (B) Confocal analysis of cultured BOECs representative of those isolated from hemophilia A dogs. BOECs were immunostained with vWF (green) and were used only if >90% of the cells stained positive for vWF. (C-F) Confocal microscopic analysis of BOECs transduced with Lenti-EF1α-(–5.5HSCR)-cFVIII and immunostained with vWF (green), 4,6 diamidino-2-phenylindole (DAPI) nuclear counterstain (blue), and FVIII (red). (E-F) Merged images of FVIII and vWF staining. (G) cBOECs (1 × 105 cells) cultured in 6-well plates were transduced with Lenti-EF1α-cFVIII, Lenti-EF1α(–5.5HSCR)-cFVIII, Lenti-EF1α-2(–5.5HSCR)-cFVIII, or Lenti-EF1α-3(–5.5HSCR)-cFVIII containing 0, 1, 2, or 3 copies, respectively, of the endothelial enhancer element. Culture media was collected 3 days later and FVIII activity (FVIII:C) was measured. Experiments were repeated at least 3 times.
