Mammalian expression vector

Abstract

The invention provides vector nucleic acid for expressing at least one polypeptide of interest in a mammalian cell, comprising (a) at least one expression cassette (POI) for expressing a polypeptide of interest; (b) an expression cassette (MSM) comprising a mammalian selectable marker gene; (c) an expression cassette (MASM) comprising a mammalian amplifiable, selectable marker gene; wherein the expression cassette (POI) is flanked 5′ by the expression cassette (MASM), the expression cassette (MSM) is located 3′ from the expression cassette (POI) and wherein the expression cassettes (MASM), (POI) and (MSM) are arranged in the same 5′ to 3′ orientation. Also provided are host cells, comprising said vector and methods for producing a polypeptide using respective host cells.

The invention claimed is: 
     
       1. A vector nucleic acid suitable for expressing at least one polypeptide of interest in a mammalian cell, comprising
 (a) at least one expression cassette (POI) suitable for expressing a polypeptide of interest;   (b) an expression cassette (MSM) comprising a mammalian selectable marker gene wherein said mammalian selectable marker gene is an antibiotic resistance gene; and   (c) an expression cassette (MASM) comprising a mammalian amplifiable, selectable marker gene;   wherein each said expression cassette (POI), (MSM), and (MASM) comprises at least one promoter or promoter/enhancer element, the expression cassette (POI) is flanked 5′ by the expression cassette (MASM), the expression cassette (MSM) is located 3′ from the expression cassette (POI) and the expression cassettes (MASM), (POI), and (MSM) are arranged in the same 5′ to 3′ orientation   and wherein   the vector is circular and the expression cassette (MASM) is arranged 3′ of the expression cassette (MSM) and wherein said circular vector comprises a unique linearization restriction site for linearizing the vector which is located between the expression cassettes (MSM) and (MASM);   or   wherein the vector is linearized via a unique linearization restriction site located between the expression cassettes (MSM) and (MASM); and   wherein the vector is selected from the group consisting of   (a) a circular or linear vector nucleic acid comprising the following genetic elements in the indicated arrangement, wherein the 5′ to 3′ direction is indicated by the →:
 I) Promoter of the (MASM) expression cassette (→) 
 II) Gene encoding the mammalian amplifiable selectable marker of the (MASM) expression cassette (→) 
 III) Intron of the (MASM) expression cassette (→) 
 IV) PolyA site of the (MASM) expression cassette (→) 
 V) Promoter of the (POI) expression cassette (→) 
 VI) Intron of the (POI) expression cassette (→) 
 VII) Polynucleotide encoding a polypeptide of interest, which is inserted in the (POI) expression cassette (→) 
 VIII) PolyA site of the (POI) expression cassette (→) 
 IX) Promoter of the (POI′) expression cassette (→) 
 X) Intron of the (POI′) expression cassette (→) 
 XI) Polynucleotide encoding an additional polypeptide of interest, which is inserted in the (POI′) expression cassette (→) 
 XII) PolyA site of the (POI′) expression cassette (→) 
 XIII) Promoter of the (MSM) expression cassette (→) 
 XIV) Gene encoding the mammalian selectable marker of the (MSM) expression cassette (→) 
 XV) PolyA site of the (MSM) expression cassette (→) 
 XVI) PSM expression cassette (→)or (←) 
 XVII) Linearization restriction site if the vector nucleic acid is circular; and 
   (b) a vector nucleic acid as shown as Seq. ID No. 1 or Seq. ID No. 16.    
     
     
       2. A vector nucleic acid suitable for expressing at least one polypeptide of interest in a mammalian cell, comprising
 (a) at least one expression cassette (POI) suitable for expressing a polypeptide of interest;   (b) an expression cassette (MSM) comprising a mammalian selectable marker gene wherein said mammalian selectable marker gene is an antibiotic resistance gene; and   (c) an expression cassette (MASM) comprising a mammalian amplifiable, selectable marker gene;   wherein each said expression cassette (POI), (MSM), and (MASM) comprises at least one promoter or promoter/enhancer element, the expression cassette (POI) is flanked 5′ by the expression cassette (MASM), the expression cassette (MSM) is located 3′ from the expression cassette (POI) and the expression cassettes (MASM), (POI), and (MSM) are arranged in the same 5′ to 3′ orientation   and wherein   the vector is circular and the expression cassette (MASM) is arranged 3′ of the expression cassette (MSM) and wherein said circular vector comprises a unique linearization restriction site for linearizing the vector which is located between the expression cassettes (MSM) and (MASM);   or   wherein the vector is linearized via a unique linearization restriction site located between the expression cassettes (MSM) and (MASM); and   wherein the vector is selected from the group consisting of   (a) a circular or linear vector nucleic acid comprising the following genetic elements in the following order and indicated arrangement, wherein the 5′ to 3′ direction is indicated by the →:
 I) Promoter of the (MASM) expression cassette (→) 
 II) Gene encoding the mammalian amplifiable selectable marker of the (MASM) expression cassette (→) 
 III) Intron of the (MASM) expression cassette (→) 
 IV) PolyA site of the (MASM) expression cassette (→) 
 V) Promoter of the (POI) expression cassette (→) 
 VI) Intron of the (POI) expression cassette (→) 
 VII) Polynucleotide encoding a polypeptide of interest, which is inserted in the (POI) expression cassette (→) 
 VIII) PolyA site of the (POI) expression cassette (→) 
 IX) Promoter of the (POI′) expression cassette (→) 
 X) Intron of the (POI′) expression cassette (→) 
 XI) Polynucleotide encoding an additional polypeptide of interest, which is inserted in the (POI′) expression cassette (→) 
 XII) PolyA site of the (POI′) expression cassette (→) 
 XIII) Promoter of the (MSM) expression cassette (→) 
 XIV) Gene encoding the mammalian selectable marker of the (MSM) expression cassette (→) 
 XV) PolyA site of the (MSM) expression cassette (→) 
 XVI) Prokaryotic selectable marker gene (PSM) expression cassette (→)or (←) 
 XVII) Linearization restriction site if the vector nucleic acid is circular, 
   wherein the expression cassette POI′ as indicated by elements IX to XII is optional; and   (b) a vector nucleic acid as shown as Seq. ID No. 1 or Seq. ID No. 16.   
     
     
       3. The vector nucleic acid of claim 2, wherein the expression cassette POI′ as indicated by elements IX to XII is present. 
     
     
       4. The vector nucleic acid according to claim 2, wherein said expression cassette (MSM) comprises a gene encoding an enzymatically functional neomycin phosphotransferase and wherein said expression cassette (MASM) comprises a gene encoding an enzymatically functional dihydrofolate reductase (DHFR). 
     
     
       5. The vector nucleic acid according to claim 3 for expressing an immunoglobulin molecule comprising in each expression cassette (POI) and (POI′) a polynucleotide encoding either a light or a heavy chain of an immunoglobulin molecule or functional fragments thereof, wherein each expression cassette (POI) and (POI′) comprises one of said polynucleotides. 
     
     
       6. The vector nucleic acid according to claim 2, wherein said expression cassettes comprise an enhancer and/or an intron. 
     
     
       7. The vector nucleic acid according to claim 2, wherein the expression cassette (POI) and/or the expression cassette (POI′) if present comprises a CMV promoter/enhancer and/or wherein the expression cassettes (MSM) and (MASM) comprise a SV40 promoter/enhancer or a SV40 promoter. 
     
     
       8. The vector nucleic acid according to claim 2, wherein the prokaryotic selectable marker gene (PSM) provides an antibiotic resistance, wherein said antibiotic is selected from the group consisting of ampicillin, kanamycin, tetracycline and chloramphenicol. 
     
     
       9. The vector nucleic acid according to claim 2, wherein the expression cassette (MASM) comprises a DHFR gene selected from the group consisting of wildtype DHFR, a DHFR having a reduced MTX sensitivity compared to wildtype DHFR and a DHFR having an enhanced MTX sensitivity compared to wildtype DHFR. 
     
     
       10. A mammalian host cell comprising a vector nucleic acid according to claim 2, wherein the vector is linearized via a unique linearization site located between the expression cassettes (MSM) and (MASM) and is stably integrated into the genome of the mammalian host cell. 
     
     
       11. The mammalian host cell according to claim 10, wherein said host cell is selected from the group consisting of COP, L, C127, Sp2/0, NS-0, NS-1, NIH3T3, PC12, PC12h, BHK and CHO, COS1, COS3, COS7, CV1, Vero, HeLa, HEK-293, retina-derived PER-C6, cells derived from diploid fibroblasts, myeloma cells and HepG2. 
     
     
       12. The mammalian host cell of claim 10, wherein the expression cassette POI′ as indicated by elements IX to XII is present in the vector nucleic acid. 
     
     
       13. The mammalian host cell according to claim 10, wherein said vector nucleic acid comprises the expression cassette (MSM) comprising a gene encoding an enzymatically functional neomycin phosphotransferase and wherein said expression cassette (MASM) comprises a gene encoding an enzymatically functional dihydrofolate reductase (DHFR). 
     
     
       14. The mammalian host cell according to claim 12, for expressing an immunoglobulin molecule, the vector nucleic acid comprising in each expression cassette (POI) and (POI′) a polynucleotide encoding either a light or a heavy chain of an immunoglobulin molecule or functional fragments thereof, wherein each expression cassette (POI) and (POI′) comprises one of said polynucleotides. 
     
     
       15. The mammalian host cell according to claim 10, wherein said vector nucleic acid comprises expression cassettes comprising an enhancer and/or an intron. 
     
     
       16. The mammalian host cell according to claim 10, wherein expression cassette (POI) and/or expression cassette (POI′) if present comprises a CMV promoter/enhancer and/or wherein the expression cassettes (MSM) and (MASM) comprise a SV40 promoter/enhancer or a SV40 promoter. 
     
     
       17. The mammalian host cell according to claim 15, wherein at least expression cassette (POI) comprises an intron which is arranged between the promoter and the start codon of the polynucleotide for expressing the polypeptide of interest. 
     
     
       18. The mammalian host cell according to claim 15, wherein the expression cassette (MASM) comprises an intron which is located 3′ of the mammalian amplifiable selectable marker gene. 
     
     
       19. The mammalian host cell according to claim 10, wherein a prokaryotic selectable marker gene (PSM) of the vector nucleic acid provides an antibiotic resistance, wherein said antibiotic is selected from the group consisting of ampicillin, kanamycin, tetracycline and chloramphenicol. 
     
     
       20. The mammalian host cell according to claim 10, wherein the expression cassette (MASM) of the vector nucleic acid comprises a DHFR gene selected from the group consisting of wildtype DHFR, a DHFR having a reduced MTX sensitivity compared to wildtype DHFR and a DHFR having an enhanced MTX sensitivity compared to wildtype DHFR. 
     
     
       21. A linearized vector nucleic acid suitable for expressing at least one polypeptide of interest in a mammalian cell, comprising
 (a) at least one expression cassette (POI) suitable for expressing a polypeptide of interest;   (b) an expression cassette (MSM) comprising a mammalian selectable marker gene wherein said mammalian selectable marker gene is an antibiotic resistance gene;   (c) an expression cassette (MASM) comprising a mammalian amplifiable, selectable marker gene; and   (d) an expression cassette (PSM) comprising a prokaryotic selectable marker gene;   wherein each said expression cassette (POI), (MSM), and (MASM) comprises at least one promoter or promoter/enhancer element, and the expression cassettes (MASM), (POI), and (MSM) are arranged in the same 5′ to 3′ orientation and wherein the expression cassettes (a) to (d) are arranged in the following order:
 I) Gene encoding the mammalian amplifiable selectable marker of the (MASM) expression cassette 
 II) Polynucleotide encoding a polypeptide of interest, which is inserted in the (POI) expression cassette 
 III) Gene encoding the mammalian selectable marker of the (MSM) expression cassette 
 IV) Prokaryotic selectable marker gene (PSM) expression cassette. 
   
     
     
       22. The linearized vector nucleic acid according to claim 21, having one or more of the following characteristics:
 i) the expression cassette (MASM) comprises a gene encoding an enzymatically functional dihydrofolate reductase (DHFR);   ii) the expression cassette (MSM) comprises a gene encoding an enzymatically functional neomycin phosphotransferase and wherein said expression cassette (MASM) comprises a gene encoding an enzymatically functional dihydrofolate reductase (DHFR);   iii) the expression cassettes (POI), (MSM), and (MASM) comprise a promoter and/or transcription termination site;   iv) the expression cassette for expressing the polypeptide of interest (POI) comprises a stronger promoter and/or enhancer than the expression cassettes for expressing the mammalian selectable markers;   v) the expression cassettes (POI), (MSM), and (MASM) comprise an enhancer and/or an intron; and   vi) the expression cassette (MASM) comprises an intron which is located 3′ of the mammalian amplifiable selectable marker gene.   
     
     
       23. The linearized vector nucleic acid of claim 21, comprising at least one additional expression cassette (POI′) for expressing an additional polypeptide of interest, wherein the additional expression cassette (POI′) is located between the expression cassette (POI) and the expression cassette (MSM), and wherein the expression cassette (POI′) is arranged in the same 5′ to 3′ orientation as the expression cassettes (POI) and (MSM). 
     
     
       24. The linearized vector nucleic acid of claim 23, comprising in each expression cassette (POI) and (POI′) a polynucleotide encoding either a light or a heavy chain of an immunoglobulin molecule or functional fragments thereof, wherein each expression cassette (POI) and (POI′) comprises one of said polynucleotides. 
     
     
       25. The linearized vector nucleic acid of claim 23, wherein the expression cassette (POI) and/or the expression cassette (POI′) comprises a CMV promoter/enhancer and/or wherein the expression cassettes (MSM) and (MASM) comprise a SV40 promoter/enhancer or a SV40 promoter. 
     
     
       26. The linearized vector nucleic acid according to claim 21, having one or more of the following characteristics:
 i) the prokaryotic selectable marker gene provides an antibiotic resistance, wherein said antibiotic is selected from the group consisting of ampicillin, kanamycin, tetracycline, and chloramphenicol; and   ii) the expression cassette (PSM) is arranged in the same 5′ to 3′ orientation as the expression cassettes (POI), (MSM), and (MASM).   
     
     
       27. A mammalian host cell comprising the linearized vector nucleic acid of claim 21. 
     
     
       28. The mammalian host cell of claim 27, wherein the linearized vector nucleic acid is stably integrated into the genome of the mammalian host cell.