2.2 Production and Preparation of rAAV Samples
The rAAV samples were of the AAV8 serotype and produced by Ultragenyx using triple transfection of HEK293 cells following procedures similar to those described in the literature.[32,33] Briefly, three plasmids, an AAV helper plasmid containing the Rep and Cap genes, an adenovirus helper plasmid containing the adenovirus helper genes, and a transgene plasmid containing the sequence to be packaged, were transfected into HEK293 cells. Prior to harvest, a nuclease treatment is performed. After 5 days, the cells were harvested and clarified through a series of depth filters. The clarified harvest was subsequently concentrated and diafiltered via tangential flow filtration (TFF, 300 kDa MWCO membrane) and further purified over affinity chromatography. The AEX load material has an empty-, intermediate-, and full-AAV capsid ratio of 88:6:6.