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.