AAV vectors and injection

Recombinant AAV (rAAV)2/2. Control and Knockout vectors were generated and purified in accordance with the method described previously (Fujita et al, 2015). Briefly, the Control or Knockout plasmid, AAV2 serotype–specific packaging plasmid, and helper plasmid, in a ratio of 1:1:3, were mixed with polyethyleneimine (PEI; Polysciences Inc.) and incubated for 10 min to form complexes. The transfection complexes were added to HEK293T cells and left for 72 h. The cells were harvested and lysed by freeze and thaw (3×) in PBS. AAV2 was bound to an AVB Sepharose column (GE Healthcare) and eluted with 50 mM glycine, pH 2.7, into 1M Tris, pH 8.8. AAV2 were washed with PBS and concentrated to a volume 100–150 μl using Vivaspin 4 concentrators. The viral vector was reconstituted at 1.0 × 10⁹ genome copy/ml and used for intravitreal injection (2.0 μl/injection) into mice aged 5 wk old.