Aav293 cells

The three rAds, namely rAd-amiRSn, rAd-amiRCD163 and rAd-amiRcon, were generated by transfecting AAV-293 cells (Stratagene, USA) with the rAd vectors according to the instruction manual for AdEasy™ Adenoviral Vector System (Agilent Technologies, USA). The rAds were amplified on AAV-293 cells, purified using ViraBind™ Adenovirus Miniprep Kit (CELL BIOLABS, USA) by following the manufacturer’s protocol, and titrated on AAV-293 cells as fluorescent formation units (FFU)/ml. For transduction of PK-15 cells, the cells were seeded in triplicates at 5 × 10⁴ cells/well on 24-well plates, and grown to 80% confluent growth. After two time wash with PBS, the cells were transduced (37°C for 2 h) with different doses (MOI) of rAds. The transduction of primary PAMs was carried out as previously described [33 (link)]. Briefly, the cells were cultured for 7 days in the growth medium conditioned with 20% of L-929 cell culture medium. The cells were trypsinized, diluted to 10⁵cells/ml with the same medium, mixed with different doses of rAds and centrifuged at 2000 × g for 1 h at 37°C. After wash again with PBS, the cells were grown for additional 48 h in the fresh medium and assayed for GFP-positive cells on BD FACSAia III or by fluorescent microscopy.

Three plasmids, an AAV vector, pAAV-DJ (Cell Biolabs, San Diego, CA, USA) and pHelper were transfected into 80–90% confluent AAV293 cells (Cell Biolabs). After 3 days of incubation, cells were harvested. AAV vectors produced in AAV293 cells were purified by CsCl (Nacalai Tesque) gradient ultracentrifugation using a SW41Ti rotor (Beckman-Coulter Life Science, Indianapolis, IN) at 35,000 rpm for 72 h at 16 °C. The formed gradients were fractionated, and the AAV titer of each fraction was evaluated by qPCR (TOYOBO, Osaka, Japan). AAV-rich fractions were combined and dialyzed using a dialysis cassette Slide-A-Lyzer (Thermo Fisher Scientific) to remove CsCl. The final AAV titer was determined by qPCR.