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.All mutants make capsid proteins and package DNA at levels within 10-fold of wild type. All mutants bind heparin as well as wild type, except for 7 which are close to or within the heparin binding site. 42 of 98 single mutants transduced at least as well as wild type. Several mutants had increased transducing activity. One, a S to T mutant, had 9-fold greater transducing activity than wild type. Combination (up or down) mutants usually transduced at the same level as that of the single mutant with the lowest level of transduction.13 of 15 single alanine substitution mutants with