CPC C12N 7/00 (2013.01) [A61K 9/007 (2013.01); A61K 9/0019 (2013.01); A61K 35/76 (2013.01); C12N 15/86 (2013.01); C12N 15/90 (2013.01); C12N 2740/15043 (2013.01); C12N 2740/15051 (2013.01); C12N 2750/14143 (2013.01); C12N 2750/14151 (2013.01)] | 26 Claims |
1. A method for automated production of an adeno-associated virus (AAV) viral vector, comprising:
(a) introducing an engineered mammalian AAV viral producer cell into a fully enclosed cell engineering system, the engineering mammalian AAV viral producer cell including integrated into its genome:
i. an adenovirus helper gene comprising E2A and E4Orf6 genes under control of a first derepressible promoter;
ii. an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter;
iii. a viral-associated, non-coding RNA under control of a third derepressible promoter; and
iv. a repressor element of the first, second and third derepressible promoters;
(b) transducing the mammalian AAV viral producer cell with a vector encoding a gene of interest to produce a transduced viral producer cell;
(c) treating the mammalian AAV viral producer cell with a binding partner of the repressor element;
(d) activating the first, second and third derepressible promoters;
(e) expanding the transduced viral producer cell and producing the AAV viral vector within the transduced viral producer cell; and
(f) isolating the viral vector,
wherein (a) through (f) are performed in a closed and automated process, and
wherein the transducing comprises viral infection, electroporation, liposome transfection, or membrane disruption.
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