Generation and Purification of AAV Particles

AAVs were generated in HEK 293T cells (ATCC) using Polyethylenimine (PEI)^{61 (link)}. 72 hours post transfection, viral particles were harvested from the media and after 120 hours from cells and the media. Viral particles from the media were precipitated with 40% polyethylene glycol (Sigma, 89510-1KG-F) in 500 mM NaCl and combined with cell pellets for processing. The cell pellets were suspended in 500 mM NaCl, 40 mM Tris, 2.5 mM MgCl₂, pH 8, and 100 U/mL of salt-activate nuclease (Arcticzymes) at 37°C for 30 minutes. Afterwards, the cells were clarified by centrifugation at 2,000 × g and then purified over iodixanol (Optiprep, Sigma; D1556) step gradients (15%, 25%, 40%, and 60%)^{62 (link)}. Viruses were concentrated using Amicon filters (EMD, UFC910024), and formulated in sterile phosphate buffer saline (PBS). Virus titers were measured by determining the number of DNAse I-resistant vg using qPCR using a linearized genome plasmid as a standard^{61 (link)}.