rAAV-HNF4α

Low-passage HEK293T were maintained at 37°C with 5% CO₂ in Dulbecco's modified Eagle medium supplemented with 10% FBS.

To produce rAAV8, a triple cotransfection procedure was used to introduce an rAAV vector plasmid (pAAV-CMV-mHNF4α or pAAV-CMV-GFP) together with pXR8, AAV8 helper plasmid carrying AAV rep and cap genes and pXX6-80, and Ad helper plasmid at a 1:1:1 molar ratio (51).

Briefly, HEK293T cells were transfected using poly-ethylenimine (PEI; linear; molecular weight, 25,000; Polysciences, Inc.), and the medium was replaced at 18 hours after transfection. Cells were harvested 72 hours after transfection, subjected to 3 rounds of freeze–thawing, and then digested with 100 U/mL Benzonase (EMD Millipore) at 37^°C for 1 hour. Viral vectors were purified by iodixanol (Serumwerk Bernburg AG) gradient ultracentrifugation (52), followed by further concentration using Amicon ultra-15 100K (100,000-molecular-weight cutoff, Merck Millipore) and washed with PBS (−/−). The final concentration of rAAV8 particles was 2.78E+10 vg per microliter (AAV-CMV-mHNF4α) and 2.35E+10 vg per microliter (pAAV- CMV-GFP). Mice were injected via tail vein with 5E11 vg 48 hours following inoculation with cancer cells.