Production and Purification of AAV Vectors

AAV was produced in AAVpro(R) 293T cells (Takara, Kyoto, Japan; #632273) by co-transfection of the genome pAAV, an AAV helper plasmid, and pUCmini-iCAP-PHP.eB. pUCmini-iCAP-PHP.eB was a gift from Viviana Gradinaru (http://n2t.net/addgene:103005; RRID: Addgene 103005) (Chan et al., 2017 (link)). The AAV particles were precipitated with polyethylene glycol (PEG 8000, Sigma-Aldrich, St. Louis, MO; Cat# P2139-500G) and purified on an iodixanol gradient (OptiPrep, Sigma-Aldrich, Cat# D1556-250ML) with ultracentrifugation for 2.5 hr at 350,000 × g at 18°C. We filtered and concentrated the virus in PBS using Amicon Ultra-15 centrifugation filters (Millipore, Burlington, MA; #UFC905024). The viral titer was measured with the AAVpro(R) Titration Kit (Takara, #6233) and AAV was stored single-use aliquots at –80°C until injection.