752. Effect of 127 Mutations at 72 Positions on the Surface of Adeno-Associated Virus Type-2

Our goal is to identify mutations on the external surface of AAV-2 capsid that reduce neutralization by antibodies, but have minimal effects on biological properties. 127 mutations were made at 72 positions (55 % of surface area) deemed most likely to be accessible to antibody binding based on manual docking of IgG and AAV-2 structures. Single alanine substitutions (57), single non-alanine substitutions (41), multiple mutations (27), and insertions (2) were made. Mutant rAAV-2 lacZ was generated by triple transfection of 293 cells. Freeze-thaw lysates of the transfected cells were prepared and assayed for total capsid synthesis (Western blotting), DNA packaging (Q-PCR), heparin-binding (ceramic beads), and in vitro transduction (Hep G2 cells/etoposide). CsCl gradient-purified viruses were made for antibody binding and in vitro neutralization assays.