FIGURE 1.

Overview of AAV-PhP.B capsids and in vitro testing. (A) Alignment of AAV5, AAV5-PhP.B, AAV9, and AAV9-PhP.B at PhP.B insertion. (B) Binding by AAV9 of CHO Lec-2 cells due to the modification of the cell surface in glycan receptor exposing galactose, whereas AAV-5 transduces Huh-7 cells through binding to sialic acid. Receptors on the cell surface are depicted in this figure. (C) Schematic overview of the expression cassette containing the cytomegalovirus (CMV) early enhancer element and chicken beta-actin promoter (CAG), green fluorescent protein (GFP), and poly(A) tail. CHO Lec-2 cells were inoculated in triplicate with GFP encoding constructs of each capsid. All capsids are capable of inducing GFP expression at 7 days postinoculation. (D) Graphical representation of expression cassette used containing CMV-promoter-luciferase gene and poly(A) tail. A dose–response of each capsid in CHO Lec-2 cells measured as relative luminescence (RLU). A total of 2 × 10⁴ cells/well were infected in a serial dilution starting at a multiplicity of infection (MOI) of 4 × 10⁶ cells/well in a 96-well plate. (E) Inoculation of HuH-7 cells with AAV vectors. The addition of PhP.B to AAV-5 resulted in a decreased transduction in both cell types. Experiments were performed in triplicate in a 24-well plate, seeding 1 × 10⁵ cells/well for HuH-7 cells. AAV5, orange bar; AAV5-PhP.B, pink bar; AAV9, light green; and AAV9-PhP.B, dark green. Asterisks indicate a statistical significance of p ≤ 0.005.