Automated production of viral vectors
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.
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.
2. The method of claim 1 , wherein the engineered mammalian AAV viral producer cell is a mammalian cell culture.
3. The method of claim 2 , wherein the mammalian cell culture is a suspension culture.
4. The method of claim 1 , wherein the engineered mammalian AAV viral producer cell is a Chinese hamster ovary (CHO) cell.
5. The method of claim 1 , wherein the engineered mammalian AAV viral producer cell is a human cell.
6. The method of claim 1 , wherein each of the derepressible promoters comprise a functional promoter and two tetracycline operator sequences (TetO 2 ), and wherein the repressor element is a tetracycline repressor protein.
7. The method of claim 1 , wherein the gene of interest is a gene of therapeutic interest.
8. The method of claim 1 , wherein an amount of AAV viral vectors produced is at least about 10 10 viral vectors.
9. 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 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 one or more of a temperature, a pH level, a glucose level, a lactose level, an oxygen level, a carbon dioxide level, and an optical density.
10. The method of claim 9 , wherein the transducing comprises viral infection, electroporation, liposome transfection, or membrane disruption.
11. 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, a 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 viral vector,
wherein (a) through (e) are performed in a closed and automated process, and
wherein the transducing comprises viral infection, electroporation, liposome transfection, or membrane disruption.
12. The method of claim 11 , wherein the mammalian cell is a mammalian cell culture.
13. The method of claim 11 , wherein the treating comprises treating with doxycycline.
14. The method of claim 11 , wherein the gene of interest is a gene of therapeutic interest.
15. The method of claim 11 , wherein an amount of AAV viral vectors produced is at least about 10 10 viral vectors.
16. The method of claim 11 , 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 one or more of a temperature, a pH level, a glucose level, a lactose level, an oxygen level, a carbon dioxide level, and an optical density.
17. The method of claim 11 , wherein the transducing comprises viral infection.
18. A method for automated production of a lentiviral vector, comprising:
(a) introducing an engineered mammalian lentiviral vector producer cell into a fully enclosed cell engineering system, the engineering mammalian lentiviral vector producer cell including integrated into its genome a nucleic acid seqquence comprising:
i. a lentiviral regulator of expression of virion proteins (REV) gene under control of a first promoter;
ii. a lentiviral envelope gene under control of a second promoter; and
iii. a lentiviral group specific antigen (GAG) gene and a lentiviral polymerase (POL) gene both under control of a third promoter,
wherein the nucleic acid sequence is flanked on both the 5′ and 3′ ends by sequences resulting from the recombination of transposon-specific inverted terminal repeats (ITRs);
(b) transducing the mammalian lentiviral vector producer cell with a vector encoding a gene of interest to produce a transduced viral producer cell;
(c) activating the first, second and third promoters;
(d) expanding the transduced viral producer cell and producing the lentiviral vector within the transduced viral producer cell; and
(e) isolating the viral vector,
wherein (a) through (e) 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 one or more of a temperature, a pH level, a glucose level, a lactose level, an oxygen level, a carbon dioxide level, and an optical density.
19. A method for automated production of a lentiviral vector, comprising:
(a) introducing a mammalian cell into a fully enclosed cell engineering system;
(b) transducing a 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 viral 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 one or more of a temperature sensor, a pH level, a glucose level, a lactose level, an oxygen level, a carbon dioxide level, and an optical density.
20. An AAV viral vector produced by the method of claim 1 .
21. The method of claim 18 , wherein the transducing comprises viral infection, electroporation, liposome transfection, or membrane disruption.
22. The method of claim 19 , wherein the transducing comprises viral infection, electroporation, liposome transfection, or membrane disruption.
23. An AAV viral vector produced by the method of claim 9 .
24. An AAV viral vector produced by the method of claim 11 .
25. A lentiviral vector produced by the method of claim 18 .
26. A lentiviral vector produced by the method of claim 19 .