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.

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;   (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.   
     
     
         2 . The vector nucleic acid according to  claim 1 , wherein the expression cassette (POI) comprises the polynucleotide encoding the polypeptide of interest. 
     
     
         3 . The vector nucleic acid according to  claim 1 , which is circular and wherein the expression cassette (MSM) is arranged 3′ of the expression cassette (POI) and wherein the expression cassette (MASM) is arranged 3′ of the expression cassette (MSM). 
     
     
         4 . The vector nucleic acid according to  claim 3 , wherein the vector comprises a unique linearization restriction site for linearizing the vector, wherein said linearization restriction site is located between the expression cassettes (MSM) and (MASM). 
     
     
         5 . The vector nucleic acid according to  claim 1 , 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 (DH FR). 
     
     
         6 . The vector nucleic acid according to  claim 1 , wherein the vector comprises at least one additional expression cassette (POI′) for expressing an additional polypeptide of interest and 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). 
     
     
         7 . The vector nucleic acid according to  claim 6  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. 
     
     
         8 . The vector nucleic acid according to  claim 1 , wherein said expression cassettes comprise a promoter and/or transcription termination site. 
     
     
         9 . The vector nucleic acid according to  claim 1 , wherein said expression cassettes comprise an enhancer and/or an intron. 
     
     
         10 . The vector nucleic acid according to  claim 8 , 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. 
     
     
         11 . The vector nucleic acid according to  claim 9 , wherein at least the 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. 
     
     
         12 . The vector nucleic acid according to  claim 9 , wherein the expression cassette (MASM) comprises an intron which is located 3′ of the mammalian amplifiable selectable marker gene. 
     
     
         13 . The vector nucleic acid according to  claim 1 , comprising at least one additional expression cassette (PSM) comprising a prokaryotic selectable marker gene, wherein the expression cassette (PSM) is located between the expression cassettes (MSM) and (MASM). 
     
     
         14 . The vector nucleic acid according to  claim 13 , wherein the prokaryotic selectable marker gene provides an antibiotic resistance, wherein said antibiotic is selected from the group consisting of ampicillin, kanamycin, tetracycline and chloramphenicol. 
     
     
         15 . The vector nucleic acid according to  claim 5 , wherein the DHFR gene is selected from the group consisting of wildtype DHFR, a DHFR variant having a reduced MTX sensitivity compared to wildtype DHFR and a DHFR variant having an enhanced MTX sensitivity compared to wildtype DHFR. 
     
     
         16 . The vector nucleic acid according to  claim 1 , 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; 
   (b) a vector nucleic acid as shown as Seq. ID No. 1 or Seq. ID No. 16 or a derivative thereof, comprising the same arrangement of genetic elements.   
     
     
         17 . A method for producing a vector nucleic acid according to  claim 1 , wherein said method comprises arranging at least the following genetic elements
 (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;
 such that 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. 
   
     
     
         18 . The method according to  claim 17  for producing a circular vector nucleic acid, comprising arranging the genetic elements such that the expression cassette (MSM) is arranged 3′ of the expression cassette (POI) and wherein the expression cassette (MASM) is arranged 3′ of the expression cassette (MSM). 
     
     
         19 . A mammalian host cell comprising a vector nucleic acid according to  claim 1 . 
     
     
         20 . The mammalian host cell according to  claim 19 , 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. 
     
     
         21 . A method for producing a host cell according to  claim 19 , wherein the host cell is transfected with the vector nucleic acid according to  claim 1 . 
     
     
         22 . A method for producing a polypeptide of interest, said method comprising, culturing at least one host cell according to  claim 19  in a cell culture medium under conditions allowing expression of said polypeptide of interest. 
     
     
         23 . The method according to  claim 22 , wherein said polypeptide of interest is secreted into the cell culture medium and isolated from the cell culture medium. 
     
     
         24 . The method according to  claim 22 , wherein the polypeptide of interest is an immunoglobulin molecule or a functional fragment thereof. 
     
     
         25 . A polypeptide obtained by the method according to  claim 22 . 
     
     
         26 . The polypeptide according to  claim 25 , wherein said polypeptide is an immunoglobulin molecule or a functional fragment thereof.