2.2. AAV-HDV Production

HDV genome containing AAV vector production was based on transient transfections and performed as described [17]. Cells were harvested by pelleting at 1000 g for 15 min 72 h after transfection. Cells were then washed with PBS and resuspended in 7.4 mL AAV lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM MgCl₂ in H₂O). Cell lysate was exposed to three freeze–thaw cycles and treated with 50 U/mL benzonase for 30 min at 37 °C. Purification of the AAV-HDV stock was performed via an iodixanol gradient ranging from 60% to 15% iodixanol. Centrifugation was carried out in a SW55Ti rotor (Beckmann Coulter, Brea, CA, USA) for 2 h at 50,000 rpm at 4 °C. After centrifugation, the AAV-HDV vector was collected from the 40% iodixanol phase. Viral DNA was isolated by incubating 5 µL virus stock with 5 µL TE Buffer (from the Plasmid Gigaprep Kit) and 10 µL NaOH (2M) for 30 min at 56 °C and adding 480 µL HCl (40 mM) thereafter. Viral genome titers were determined by qPCR using the AAVpro^® Titration Kit (Takara Bio, Kusatsu, Japan) following manufacturer’s instructions. AAV stocks were stored at −80 °C.