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US RE49,347 E1Granted
Mammalian expression vector
AssigneeNovartis AG
InventorsHans-Peter Knopf, Burkhard Wilms
Life of this patent
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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.
Claims28
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.
28 more elements
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.
29 more elements
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.
7 more elements
Dependencies are inferred from the claim text.