AAV vector production, purification, and quantification.

Recombinant AAV vectors were produced by transfecting HEK293 cells at 70 to 80% confluence with polyethylenimine using the triple-plasmid transfection protocol (34). Recombinant vectors packaging single-stranded genomes encoding firefly luciferase driven by the chicken β-actin promoter (ssCBA-Luc) or self-complementary green fluorescence protein driven by a hybrid chicken β-actin promoter (scCBh-GFP) were generated using this method. Subsequent steps involving the harvesting of recombinant AAV vectors and downstream purification were carried out as described earlier (35). Recombinant AAV vector titers were determined by quantitative PCR with primers amplifying AAV2 inverted terminal repeat regions (ITRs) 5′-AACATGCTACGCAGAGAGGGAGTGG-3′ and 5′-CATGAGACAAGGAACCCCTAGTGATGGAG-3′.