Lentiviral Vector for Hemophilia B Treatment

We have designed a LV vector containing the human FAH gene under control of a liver-specific promoter (HCR-AAT; hepatic control region enhancer and alpha-1 antitrypsin promoter), currently being trialed in humans for the treatment of hemophilia B^{40 (link)}. A schematic representation of this vector is provided in Fig. 1a. In order to generate viral vectors, the LV-SFFV-eGFP or LV-AAT-huFAH expression construct, together with the packaging plasmid p8.91 and the vesicular stomatitis virus glycoprotein G-encoding plasmid pVSV-G, was transfected into 293 T/17 cells (CRL-11268, ATCC, Manassas, VA) using 1 mg/ml polyethylenimine (Polysciences, Warrington, PA). Viral supernatant was harvested 48 and 72 h after transfection, filtered through a 0.45-μm filter, and concentrated by ultracentrifugation (70,000 g, 1.5 h at 4 °C). After resuspension in serum-free media (DMEM, Thermo Fisher Scientific, Waltham, MA), LV vectors were aliquoted and stored at −80 °C. Vector titers were determined by p24 enzyme-linked immunosorbent assay and qPCR using the Lenti-X Provirus Quantitation Kit (Clontech, Mountain View, CA).