The 10-amber 13-amber hoc-del soc-del T4 heads were purified according to previously described protocols (6). To produce rAAV-DJ, the triple-plasmid transfection method (Cell Biolabs, CA) was used according to the manufacturer’s instructions. Briefly, plasmids, including an adenovirus helper plasmid (pHelper), a rep/cap plasmid expressing rep and cap (pAAV-DJ or pAAV-DJ-H75A-D76N), and a transgene plasmid carrying the AAV transgene cassette (AAV genome) (pAAV-GFP, pAAV-mCherry, pAAV-luciferase, pAAV-HA4900, or pAAV-VRCO1) (1:1:1) were cotransfected into HEK293 cells expressing adenovirus E1a and E1b proteins using polyethyleneimine (Polysciences, PA). After 72 hours, cells were harvested by centrifugation at 1140g for 10 min. The cell pellet was resuspended in lysis buffer [50 mM tris-HCl (pH 8.5) and 0.15 M NaCl] and lysed by three freeze/thaw cycles in dry ice–ethanol and a 37°C water bath. Benzonase was added to the mixture (50 U/ml final concentration), and the lysate was incubated for 30 min at 37°C. The lysate was clarified by centrifugation at 3700g for 20 min, and the virus-containing supernatant was considered to be the crude lysate. Viruses were then purified by discontinuous iodixanol density gradient centrifugation at 500,000 rpm for 16 hours at 4°C (type 55 Ti rotor; Beckman, CA) and using HiTrap AVB Sepharose HP according to the manufacturer’s instructions (GE Healthcare, IL). The peak fractions were dialyzed against PBS-MK buffer [PBS (pH 7.4), 2.5 mM KCl, and 1 mM MgCl2] in Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific, MA). The dialyzed AAV particles were concentrated and stored at −80°C. AAV titers were determined using a QuickTiter AAV Quantitation kit (Cell Biolabs, CA).

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