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US 12,180,511 B2Granted
Automated production of viral vectors
AssigneeLonza Walkersville, Inc.
InventorsMatthew Hewitt, Young Shin, Bingnan Gu, Caitlin M. Guenther, Anandita Seth
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Abstract
The present disclosure provides an automated method of producing viral vectors, utilizing engineered viral vector-producing cell lines within a fully-enclosed cell engineering system. Exemplary viral vectors that can be produced include lentivirus vectors, adeno-associated virus vectors, baculovirus vectors and retrovirus vectors.
Claims17
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 engineered 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 an AAV viral 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 AAV viral vector,
- wherein (a) through (f) are performed in a closed and automated process, and
- wherein the closed and automated process further comprises:
- (g) monitoring with one or more of a temperature sensor, a pH sensor, a glucose sensor, a lactose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor, and
- (h) automatically adjusting media circulation based on the monitoring.
13 more elements
A method for automated production of an adeno-associated virus (AAV) viral vector, comprising:
- (a) transducing a mammalian cell stably expressing one or more nucleic acids encoding TetR and/or TetR-KRAB with:
- i. a first nucleic acid encoding an adenovirus helper gene comprising an E2A gene, an E4Orf gene and a viral-associated non-coding RNA, under control of a first derepressible promoter;
- ii. a second nucleic acid encoding an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter;
- and
- iii. optionally, a third nucleic acid encoding a gene of interest under control of a third derepressible promoter;
- (b) treating the mammalian cell with a binding partner of the TetR;
- (c) activating the first, second and third derepressible promoters;
- (d) expanding the transduced cell and producing the AAV viral vector within the transduced cell;
- and
- (e) isolating the AAV viral vector,
- wherein (a) through (e) are performed in a closed and automated process, and
- wherein the gene of interest is a gene of therapeutic interest.
9 more elements
A method for automated production of a lentiviral vector, comprising:
- (a) introducing a mammalian cell into a fully enclosed cell engineering system;
- (b) transducing the mammalian cell with:
- i. a first nucleic acid encoding a lentiviral regulator of expression of virion proteins (REV) gene under control of a first promoter and an envelope glycoprotein gene under control of a second promoter;
- ii. a second nucleic acid encoding a gene of interest under control of a third promoter;
- and
- iii. a third nucleic acid encoding a lentiviral group specific antigen (GAG) gene and a lentiviral polymerase (POL) gene both under control of a fourth promoter,
- (c) expanding the transduced cell and producing the lentiviral vector within the transduced cell;
- and
- (d) isolating the lentiviral vector,
- wherein (a) through (d) are performed in a closed and automated process, and
- wherein the closed and automated process further comprises:
- (a) monitoring with one or more of a temperature sensor, a pH sensor, a glucose sensor, a lactose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor, and
- (b) automatically adjusting media circulation based on the monitoring.
10 more elements
Dependencies are inferred from the claim text.