Strictly controlled inducible expression systems for biopharmaceutical production using stable cell lines

Abstract

The present disclosure relates to tightly controlled inducible expression systems useful for the inducible production of one or more RNAs or proteins of interest, including the production of biologics such as recombinant proteins, vaccines or viral vectors. Also provided are cell lines and kits useful for the production of said RNAs or proteins, as well as methods for making said cell lines and for inducing the production of said RNAs or proteins.

Claims

1. a) A first expression cassette comprising a nucleic acid molecule encoding a ket repressor protein operably linked to a constitutive promoter and a polyadenylation signal; b) A second expression cassette comprising a nucleic acid molecule encoding a cummermycin chimeric transactivator protein operably linked to a cummermycin-inducible promoter and a polyadenylation signal; and c) i) A nucleic acid molecule comprising a cummermycin-inducible promoter, a cloning site, and a polyadenylation signal, wherein the cloning site is for inserting a nucleic acid molecule encoding a first target RNA or protein operably linked to the cummermycin-inducible promoter and the polyadenylation signal, or ii) A nucleic acid molecule encoding a first target RNA or protein, operably linked to a cummermycin-inducible promoter and a polyadenylation signal. Third expression cassette including An inducible expression system that includes [the specified element].

2. The expression system according to claim 1, wherein the constitutive promoter is selected from the group consisting of the human ubiquitin C (UBC) promoter, the human elongation factor 1α (EF1A) promoter, the human phosphoglycerate kinase 1 (PGK) promoter, the Simian virus 40 early promoter (SV40), the β-actin promoter, the cytomegalovirus immediate early promoter (CMV), the hybrid CMV enhancer / β-actin promoter (CAG), and variants thereof.

3. The expression system according to claim 1 or 2, wherein the cumermycin-inducible promoter further comprises a three-part leader (TPL) and / or a major late promoter (MLP) enhancer.

4. The expression system according to claim 3, wherein the cumermycin-inducible promoter comprises the nucleotide sequence described in SEQ ID NO: 11 or a functional variant thereof.

5. The expression system according to any one of claims 1 to 4, wherein the third expression cassette comprises a nucleic acid molecule encoding a first target RNA or protein, operably linked to a cummermycin-inducible promoter and a polyadenylation signal.

6. The expression system according to claim 5, which encodes one or more components of a viral vector.

7. The expression system according to any one of claims 1 to 6, further comprising a fourth expression cassette containing a nucleic acid molecule encoding a second target RNA or protein, which is operably linked to a promoter and a polyadenylation signal.

8. The expression system according to claim 7, further comprising a fifth expression cassette containing a nucleic acid molecule encoding a third target RNA or protein, operably linked to a promoter and a polyadenylation signal.

9. The expression system according to claim 7 or 8, wherein the promoter of the fourth and / or fifth expression cassette is a cumermycin-inducible promoter.

10. The third expression cassette encodes lentiviral REV protein, the promoter of the fourth expression cassette is a cummermycin-inducible promoter, the fourth expression cassette encodes viral envelope protein, and the fifth expression cassette encodes lentiviral Gag / pol, or The expression system according to claim 8 or 9, wherein the third expression cassette encodes a viral envelope protein, the promoter of the fourth expression cassette is a cummermycin-inducible promoter, the fourth expression cassette encodes lentiviral Gag / pol, and the fifth expression cassette encodes lentiviral REV protein.

11. The expression system according to claim 10, wherein the viral envelope protein is VSVg.

12. The expression system according to claim 11, wherein the VSVg is VSVg-Q96H-I57L.

13. The expression system according to any one of claims 7 to 9, wherein the third expression cassette encodes Rep40 or Rep52, the fourth expression cassette encodes Rep68 or Rep78, and the fourth expression cassette is under the control of a cummermycin-inducible promoter.

14. A cell comprising the expression system described in any one of claims 1 to 13.

15. The cell according to claim 14, which is a human cell.

16. The cell according to claim 14, which is a human embryonic kidney (HEK)-293 cell or derivative thereof, Chinese hamster ovary (CHO) cell or derivative thereof, VERO cell or derivative thereof, HeLa cell or derivative thereof, A549 cell or derivative thereof, stem cell or derivative thereof, or neuron or derivative thereof.

17. A method for producing a target RNA or protein, comprising culturing the cells described in any one of claims 14 to 16 in the presence of a kumeto effector molecule and a kumermycin effector molecule, wherein the third expression cassette encodes the target RNA or protein and the target RNA or protein is produced.

18. The method according to claim 17, wherein the kmet effector molecule is kmet, and kmet is present at a concentration of 1 to 200 μg / ml, and the cumermycin effector molecule is cumermycin, and cumermycin is present at a concentration of 1 to 30 nM.

19. The method according to claim 17 or 18, wherein the cells are grown in a suspension and / or in the absence of serum.

20. A virus-packaging cell comprising the expression system described in any one of claims 10 to 13.

21. The viral packaging cell according to claim 20, further comprising a viral construct having the target gene.

22. A method for producing a viral vector, a) Introducing a viral construct containing the target gene into the viral packaging cell described in claim 20; and b) Producing a viral vector by culturing the cells in the presence of a cummermycin effector molecule and a cummermycin effector molecule. Methods that include...

23. A method for producing a viral vector, a) To obtain the virus-packaging cells described in claim 21, and b) Producing a viral vector by culturing the cells in the presence of a cummermycin effector molecule and a cummermycin effector molecule. A method that includes this.

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