High titer recombinant AAV vector production in adherent and suspension cells
AssigneeAmgen Inc.
InventorsHuiren Zhao, Grace Ki Jeong Lee, Thomas Wolfe, Cherylene Plewa, Jackie Z. Sheng
Life of this patent
Estimating the term…
Abstract
The present invention is directed to an in vitro method of producing a recombinant AAV virion in a mammalian host cell that comprises a functional adenoviral E1A gene and rAAV virions made by the method. Host cells are transfected with varying ratios of plasmids comprising the E1A gene and virions.
Claims14
An in vitro method of producing a recombinant AAV virion in a mammalian host cell, wherein the mammalian host cell is a HEK 293 cell, and wherein the cell is suspended in a transfection media, comprising contacting the cell with polyethylenimine (“PEI”) and:
- (i) an accessory construct, wherein said accessory construct is a plasmid (pHelper) consisting essentially of 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, wherein said AAV helper construct is a plasmid (pTrans) consisting essentially of AAV2 rep and AAV1, AAV2, AAV5, AAV8, or AAV9 cap coding regions operably linked to one or more regulatory sequences;
- and
- (iii) an AAV vector construct, wherein said AAV vector construct is a plasmid (pCis) consisting essentially of AAV2 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 1:5:(0.009-0.36) (weight:weight:weight), the PEI is Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000), and the ratio of PEI:DNA is about 3:1.
An in vitro method of producing a recombinant AAV virion in a mammalian host cell, wherein the mammalian host cell is a HEK 293 cell, and wherein the cell adheres to a solid substrate in a transfection media, comprising contacting the cell with calcium phosphate and:
- an accessory construct, wherein said accessory construct is a plasmid (pHelper) consisting essentially of 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, wherein said AAV helper construct is a plasmid (pTrans) consisting essentially of AAV2 rep and AAV1, AAV2, AAV5, AAV8, or AAV9 cap coding regions operably linked to one or more regulatory sequences;
- and
- (iii) an AAV vector construct, wherein said AAV vector construct is a plasmid (pCis) consisting essentially of AAV2 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 1:5:(0.009-0.36) (weight:weight:weight).
Dependencies are inferred from the claim text.