Nucleic acids encoding vitamin K expoxide reductase subunit 1 and vitamin K dependent protein expression and methods of using same

Patent metadata

Field	Value
Publication number	US-9617523-B2
Application number	US-34229906-A
Country	US
Kind code	B2
Filing date	Jan 27, 2006
Priority date	Feb 28, 2005
Publication date	Apr 11, 2017
Grant date	Apr 11, 2017

Abstract

The present invention relates to a host organism containing recombinant nucleic acids coding for the vitamin K reductase complex subunit 1 (VKORC1) and recombinant nucleic acids coding for a vitamin K dependent (VKD) protein, wherein both the recombinant VKORC1 and the recombinant VKD protein are expressed in said host organism. Further, the present invention relates to a cell culture system comprising cells which contain said recombinant nucleic acids and to methods for improving the productivity of recombinant VKD protein expression in a host organism being cultured in suitable systems.

First claim

What is claimed is:

1. A recombinant mammalian cell comprising an exogenous recombinant nucleic acid encoding human vitamin K reductase complex subunit 1 (VKORC1) inserted into a plasmid and an exogenous recombinant nucleic add coding for a vitamin K dependent (VKD) procoagulant blood factor protein, wherein both the VKORC1 and the VKD protein are expressed in the mammalian cell, and wherein the mammalian cell does not overexpress γ-carboxylase;
wherein the mammalian cell is selected from the group consisting of CHO cells, NSO cells, sp20 cells, Perc6 cells, SkHep cells, BHK cells, Hela cells, Vero cells, COS cells, and HEK293 cells, and
wherein the procoagulant blood factor selected from the group consisting of Factor II, Factor VII, Factor IX and Factor X.

2. The recombinant mammalian cell of claim 1 , wherein the VKD procoagulant blood factor protein is FVII or FIX.

3. The mammalian cell of claim 1 , wherein the procoagulant blood factor is Factor IX, which is human Factor IX.

4. The mammalian cell of claim 1 , wherein either the recombinant nucleic acid coding for the VKORC1 protein or the recombinant nucleic acid coding for the VKD protein or both the recombinant nucleic acids coding for the VKORC1 and the VKD proteins are expressed via an expression mode selected from the group consisting of induced, transient, and permanent expression.

5. A cell culture system comprising mammalian cells which have inserted therein an exogenous recombinant nucleic acid encoding a human vitamin K reductase complex subunit 1 (VKORC1) inserted into a plasmid and an exogenous recombinant nucleic acid coding for a vitamin K dependent (VKD) protein, wherein both the VKORC1 and the VKD protein encoded by the exogenous recombinant nucleic acids are expressed in said cells; wherein the cells do not overexpress γ-carboxylase; wherein the mammalian cells are selected from the group consisting of CHO cells, NSO cells, sp20 cells, Perc6 cells, SkHep cells, BHK cells, Hela cells, Vero cells, COS cells, and HEK293 cells; and
wherein the VKD protein is a procoagulant blood factor selected from the group consisting of Factor II, Factor VII, Factor IX and Factor X.

6. The cell culture system of claim 5 , wherein the procoagulant blood Factor protein is Factor IX, which is human Factor IX.

7. The cell culture system of claim 5 , wherein the VKD procoagulant blood factor protein is FVII or FIX.

8. A method for improving the productivity of recombinant vitamin K dependent (VKD) protein expression in a mammalian cell comprising the steps of:
(a) providing a mammalian cell in culture;
(b) inserting an exogenous recombinant nucleic acid coding for a VKD procoagulant blood factor protein into the mammalian cell of step (a);
(c) inserting an exogenous recombinant nucleic acid comprising the nucleotide sequence encoding a human vitamin K reductase complex subunit 1 (VKORC1) inserted into a plasmid into the mammalian cell of step (a); and
(d) expressing the recombinant nucleic acids of steps (b) and (c); wherein the mammalian cell does not overexpress γ-carboxylase;
wherein the mammalian cell is selected from the group consisting of CHO cells, NSO cells, sp20 cells, Perc6 cells, SkHep cells, BHK cells, Hela cells, Vero cells, COS cells, and HEK293 cells, and
wherein the procoagulant blood factor selected from the group consisting of Factor II, Factor VII, Factor IX and Factor X.

9. The method of claim 8 , wherein the VKD procoagulant blood factor protein is FVII or FIX.

10. A method for improving the productivity of recombinant vitamin K dependent (VKD) protein expression in a mammalian cell comprising the steps of:
(a) providing a mammalian cell comprising an exogenous recombinant nucleic acid coding for a VKD procoagulant blood factor protein integrated into its genome;
(b) inserting an exogenous recombinant nucleic acid comprising the nucleotide sequence of a recombinant nucleic acid encoding a human vitamin K reductase complex subunit 1 (VKORC1) inserted into a plasmid into the mammalian cell of step (a); and
(c) expressing the recombinant nucleic acids of steps (a) and (b);
wherein the mammalian cell does not overexpress γ-carboxylase;
wherein the mammalian cell is selected from the group consisting of CHO cells, NSO cells, sp20 cells, Perc6 cells, SkHep cells, BHK cells, Hela cells, Vero cells, COS cells, and HEK293 cells; and
wherein the procoagulant blood factor selected from the group consisting of Factor II, Factor VII, Factor IX and Factor X.

11. The method of claim 10 , wherein the recombinant nucleic acid coding for the VKD procoagulant blood factor protein is stably expressed.

12. The method of claim 10 , wherein the VKD procoagulant blood factor protein is FVII or FIX.

13. A method for improving the productivity of recombinant vitamin K dependent (VKD) procoagulant blood factor protein expression in a mammalian cell in culture, the method comprising the steps of:
(a) providing a mammalian cell comprising an exogenous recombinant nucleic acid comprising the nucleotide sequence encoding human vitamin K reductase complex subunit 1 (VKORC1) inserted into a plasmid integrated into its genome;
(b) inserting an exogenous recombinant nucleic acid coding for a VKD procoagulant blood factor protein into the mammalian cell of step (a); and
(c) expressing the recombinant nucleic acids of steps (a) and (b)
wherein the mammalian does not overexpress γ-carboxylase;
wherein the mammalian cell is selected from the group consisting of CHO cells, NSO cells, sp20 cells, Perc6 cells, SkHep cells, BHK cells, Hela cells, Vero cells, COS cells, and HEK293 cells, and
wherein the procoagulant blood factor selected from the group consisting of Factor II, Factor VII, Factor IX and Factor X.

14. The method of claim 13 , wherein the recombinant nucleic acid coding for VKORC1 is stably expressed.

15. The method of claim 13 , wherein the VKD procoagulant blood factor protein is FVII or FIX.

Assignees

• Scheiflinger Friedrich
• Boehm Ernst
• Baxalta GmbH
• Baxalta Inc

Inventors

• Scheiflinger Friedrich
• Boehm Ernst

Classifications

• A61P7/04

  Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents · CPC title

• C12N9/0093 Primary

  acting on CH or CH2 groups (1.17) · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title

• C12Y117/00

  Oxidoreductases acting on CH or CH2 groups (1.17) · CPC title

• C12Y304/21022

  Coagulation factor IXa (3.4.21.22) · CPC title