Recombinant AAV production

This study utilized rAAV carrying Firefly Luciferase with a Cytomegalovirus (CMV) promoter. rAAV luciferase (rAAV-Luc) was generated by the standard triple transfection method using the XX6-80 Ad helper plasmid with packaging plasmid (pXR1, 2, 4–9, 3.1, SASTG) and the terminal repeat plasmid carrying Firefly Luciferase transgene. The plasmids were transfected into HF1 cells with polyethylenimine. rAAVs were harvested from both cells and media 72 h posttransfection and then purified by two cesium chloride gradients.^16,17 The purified vector was desalted using Amicon Ultra-4 50kDa Centrifugal Columns (MilliporeSigma, Burlington, MA), eluted in sterile phosphate-buffered saline (PBS), and sterilized with polyethersulfone syringe filters (VWR, Radnor, PA). The genome containing particle titer was evaluated by quantitative polymerase chain reaction (qPCR).