US 11,718,833 B2
Automated production of viral vectors
Matthew Hewitt, Walkersville, MD (US); Young Shin, Walkersville, MD (US); Bingnan Gu, Manvel, TX (US); Caitlin M. Guenther, Houston, TX (US); and Anandita Seth, Sugar Land, TX (US)
Assigned to LONZA WALKERSVILLE, INC., Walkersville, MD (US)
Filed by LONZA WALKERSVILLE, INC., Walkersville, MD (US)
Filed on Dec. 19, 2019, as Appl. No. 16/720,227.
Claims priority of provisional application 62/949,848, filed on Dec. 18, 2019.
Claims priority of provisional application 62/890,904, filed on Aug. 23, 2019.
Claims priority of provisional application 62/866,092, filed on Jun. 25, 2019.
Claims priority of provisional application 62/783,589, filed on Dec. 21, 2018.
Prior Publication US 2020/0208121 A1, Jul. 2, 2020
Int. Cl. A61K 48/00 (2006.01); C12N 15/86 (2006.01); C12N 7/00 (2006.01); A61K 38/00 (2006.01); A61K 39/00 (2006.01); C12N 15/90 (2006.01); A61K 35/76 (2015.01); A61K 9/00 (2006.01)
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.