AAV production

AAV293 cells (Agilent Technologies) were cultured in DMEM (Mediatech) supplemented with 10% FBS and 1× antibiotic antimycotic solution (Mediatech). Cells were cultured in a humidified incubator with 5% CO₂ and 95% air at 37°C, confirmed to be mycoplasma free, and authenticated by short tandem repeat DNA profiling. The cells were transfected with three plasmids [AAV transgene, pHelper (Agilent Technologies), and AAV2/9] for viral production. Viral particles were purified using a discontinuous iodixanol (Sigma) gradient and resuspended in PBS with 10% glycerol and stored at −80°C. Viral titer or vector genome number was determined by quantitative PCR using custom TaqMan assays specific for woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequences. Standard curves for WPRE were obtained from serial dilutions over a 6 log range of the corresponding plasmids. AAV-mediated gene delivery provides a means to achieve long-lasting transgene expression without the inflammatory responses that are commonly associated with other viral vectors. When introduced into adult mice, sustained expression of up to 1 year has been observed. The major site of transgene expression is the hepatocytes.