METHOD OF PRODUCING FACTOR VIII PROTEINS BY RECOMBINANT METHODS
Provided herein are methods and compositions for producing Factor VIII proteins. Such methods include introducing into a cell a nucleic acid molecule encoding a Factor VIII protein operably linked to a promoter, wherein the promoter is characterized by the ability to produce commercially viable Factor VIII protein; and incubating the cell under conditions for producing commercially viable Factor VIII protein. Also provided are nucleic acid molecules which encode a Factor VIII protein operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter, which may be used in the methods provided herein.
1 . A nucleic acid molecule encoding a Factor VIII protein, wherein the Factor VIII protein comprises a substitution of a cysteine residue, wherein the cysteine residue is involved in a disulfide bond in the Factor VIII protein.
2 . The nucleic acid molecule of claim 1 , wherein the nucleic acid is operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter.
3 . The nucleic acid molecule of claim 1 , which encodes a heterologous protein.
4 . The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule comprises a cDNA which encodes the Factor VIII protein.
5 . The nucleic acid molecule of claim 1 , wherein the Factor VIII protein comprises wild-type Factor VIII.
6 . The nucleic acid molecule of claim 1 , wherein the Factor VIII protein comprises a deletion of the B-domain, followed by an addition of an amino acid spacer containing at least one N-linked glycosylation site, wherein the amino acid spacer containing the at least one N-linked glycosylation site facilitates the secretion or expression of the B-domain-deletion Factor VIII protein.
7 . The nucleic acid molecule of claim 6 , wherein the Factor VIII protein comprises an amino acid sequence inserted at position 750 when the protein is aligned with the wild-type Factor VIII, the inserted amino acid sequence consisting of a 226 amino acid spacer containing 6 N-linked glycosylation sites, thereby partially replacing the B domain of the modified Factor VIII protein.
8 . The nucleic acid molecule of claim 1 , wherein the Factor VIII protein comprises a substitution of phenylalanine at position 309 to serine when the protein is aligned with the wild-type Factor VI II.
9 . The nucleic acid molecule of claim 7 , wherein the Factor VIII protein comprises a substitution of phenylalanine at position 309 to serine when the protein is aligned with the wild-type Factor VI II.
10 . The nucleic acid molecule of claim 7 , wherein the Factor VIII protein comprises a substitution of cysteine at position 1899 to glycine when the protein is aligned with the wild-type Factor VIII.
11 . The nucleic acid molecule of claim 7 , wherein the Factor VIII protein comprises a substitution of cysteine at position 1903 to glycine when the protein is aligned with the wild-type Factor VIII.
12 . The nucleic acid molecule of claim 10 , wherein the Factor VIII protein further comprises a substitution of cysteine at position 1903 to glycine when the protein is aligned with the wild-type Factor VIII.
13 . The nucleic acid molecule of claim 9 , wherein the Factor VIII protein comprises a substitution of cysteine at position 1899 to glycine when the protein is aligned with the wild-type Factor VIII.
14 . The nucleic acid molecule of claim 9 , wherein the Factor VIII protein comprises a substitution of cysteine at position 1903 to glycine when the protein is aligned with the wild-type Factor VIII.
15 . The nucleic acid molecule of claim 13 , wherein the Factor VIII protein further comprises a substitution of cysteine at position 1903 to glycine when the protein is aligned with the wild-type Factor VIII.
16 . A method for producing a recombinant Factor VIII protein, comprising the steps of:
introducing into a cell a nucleic acid molecule encoding a Factor VIII protein operably linked to a promoter and incubating the cell under conditions for producing Factor VIII protein;
wherein the promoter is characterized by the ability to produce commercially viable Factor VIII protein, and wherein the Factor VIII protein comprises a substitution of a cysteine residue and the cysteine residue is involved in a disulfide bond in the Factor VIII protein.
17 . The method of claim 16 , wherein the cell is a mammalian cell.
18 . The method of claim 17 , wherein the mammalian cell is selected from the group consisting of a COS-1, CHO, and HEK 293 cell.
19 . The method of claim 16 , wherein the nucleic acid molecule comprises a cDNA which encodes the Factor VIII protein.
20 . The method of claim 16 , wherein the nucleic acid molecule is operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter.
21 . The method of claim 16 , wherein the Factor VIII protein comprises a heterologous sequence.
22 . The method of claim 1 , wherein the Factor VIII protein comprises a deletion of the B-domain, followed by an addition of an amino acid spacer containing at least one N-linked glycosylation site, wherein the amino acid spacer containing the at least one N-linked glycosylation site facilitates the secretion or expression of the B-domain-deletion Factor VIII protein.
23 . The method of claim 22 , wherein the Factor VIII protein comprises an amino acid sequence inserted at position 750 when the protein is aligned with the wild-type Factor VIII, the inserted amino acid sequence consisting of a 226 amino acid spacer containing 6 N-linked glycosylation sites, thereby partially replacing the B domain of the modified Factor VIII protein.
24 . The method of claim 16 , wherein the Factor VIII protein comprises a substitution of phenylalanine at position 309 to serine when the protein is aligned with the wild-type Factor VI II.
25 . The method of claim 23 , wherein the Factor VIII protein comprises a substitution of phenylalanine at position 309 to serine when the protein is aligned with the wild-type Factor VI II.
26 . The method of claim 23 , wherein the Factor VIII protein comprises a substitution of cysteine at position 1899 to glycine when the protein is aligned with the wild-type Factor VIII.
27 . The method of claim 23 , wherein the Factor VIII protein comprises a substitution of cysteine at position 1903 to glycine when the protein is aligned with the wild-type Factor VIII.
28 . The method of claim 26 , wherein the Factor VIII protein further comprises a substitution of cysteine at position 1903 to glycine when the protein is aligned with the wild-type Factor VIII.
29 . The method of claim 25 , wherein the Factor VIII protein comprises a substitution of cysteine at position 1899 to glycine when the protein is aligned with the wild-type Factor VIII.
30 . The method of claim 25 , wherein the Factor VIII protein comprises a substitution of cysteine at position 1903 to glycine when the protein is aligned with the wild-type Factor VIII.
31 . The method of claim 29 , wherein the Factor VIII protein further comprises a substitution of cysteine at position 1903 to glycine when the protein is aligned with the wild-type Factor VIII.