High Titer Recombinant AAV Vector Production in Adherent and Suspension Cells

摘要

Disclosed are an in vitro method of producing a recombinant AAV virion in a mammalian host cell that comprises a functional adenoviral E1A gene (e.g., a HEK 293 cell) and rAAV virions made by the method.

主权项

1. An in vitro method of producing a recombinant AAV virion in a mammalian host cell, comprising incubating the cell in a transfection medium comprising:
(i) an accessory construct comprising a plasmid (pHelper) comprising adenoviral E2, E4Orf6, and VAI RNA genes operably linked to an origin of replication element and one or more other regulatory sequences; (ii) an AAV helper construct comprising a plasmid (pTrans) comprising AAV rep and AAV cap coding regions operably linked to one or more regulatory sequences; and (iii) a AAV vector comprising a plasmid (pCis), comprising AAV inverted terminal repeats flanking a heterologous gene of interest operably linked to one or more regulatory sequences, wherein the ratio of pHelper:pTrans:pCis is 1:1 to 5:0.009 to 0.36 (weight:weight:weight).