Recombinant factor viii having increased stability

Abstract

The present invention relates to a recombinant factor VIII that includes one or more mutations that result in enhanced stability of both factor VIII and factor VIIIa. Methods of making and using the recombinant factor VIII, and pharmaceutical compositions containing the same are also disclosed. The present invention further relates to an isolated nucleic acid molecule that encodes the recombinant factor VIII, as well as DNA expression systems and host cells containing the isolated nucleic acid molecule.

What is claimed: 
     
         1 . An isolated nucleic acid molecule encoding a recombinant factor VIII, wherein said recombinant factor VIII comprises one or more mutations that result in enhanced stability of both factor VIII and factor VIIIa, wherein the one or more mutations comprise substitution of one or more charged amino acid residues with a hydrophobic amino acid residue at either or both of the A1 A2 or A2A3 domain interfaces. 
     
     
         2 . The isolated nucleic acid molecule according to  claim 1 , wherein the one or more mutations comprise a substitution of a Glu287 residue of wildtype factor VIII, a substitution of an Asp302 residue of wildtype factor VIII, a substitution of an Asp519 residue of wildtype factor VIII, a substitution of a Glu665 residue of wildtype factor VIII, a substitution of a Glu1984 residue of wildtype factor VIII, or combinations thereof. 
     
     
         3 . The isolated nucleic acid molecule according to  claim 2 , wherein the substitution of the Asp302 residue is D302A. 
     
     
         4 . The isolated nucleic acid molecule according to  claim 2 , wherein the substitution of the Glu287 residue is E287A. 
     
     
         5 . The isolated nucleic acid molecule according to  claim 2 , wherein the substitution of the Glu665 residue is E665A or E665V. 
     
     
         6 . The isolated nucleic acid molecule according to  claim 2 , wherein the substitution of the Asp519 residue is D519A or D519V. 
     
     
         7 . The isolated nucleic acid molecule according to  claim 2 , wherein the substitution of the Glu1984 residue is E1984A or E1984V. 
     
     
         8 . The isolated nucleic acid molecule according to  claim 1 , wherein the one or more mutations comprise two or more substitutions selected from the Glu665 residue, the Asp519 residue, and the Glu1984 residue. 
     
     
         9 . The isolated nucleic acid molecule according to  claim 8 , wherein the two or more substitutions include D519V/E665V, D519A/E665V, D519V/E1984A, E665V/E1984A, E665A/E1984V, D519A/E665V/E1984A, D519V/E665V/E1984A, or D519V/E665V/E1984V. 
     
     
         10 . The isolated nucleic acid molecule according to  claim 1 , wherein the recombinant factor VIII further comprises one or more of (i) factor IXa and/or factor X binding domains modified to enhance the affinity of the recombinant factor VIII for one or both of factor IXa and factor X; (ii) modified sites that enhance secretion in culture; (iii) modified serum protein binding sites that enhance the circulating half-life thereof; (iv) at least one glycosylation recognition sequence that is effective in decreasing antigenicity and/or immunogenicity thereof; and (v) a modified calcium-binding site that improves activity of the recombinant factor VIIIa. 
     
     
         11 . The isolated nucleic acid molecule according to  claim 1 , wherein the nucleic acid is RNA. 
     
     
         12 . The isolated nucleic acid molecule according to  claim 1 , wherein nucleic acid is DNA. 
     
     
         13 . The isolated nucleic acid molecule according to  claim 1 , wherein the one or more charged amino acid residues is not interdomain hydrogen bonded. 
     
     
         14 . A recombinant expression system comprising a DNA molecule of  claim 12 . 
     
     
         15 . The recombinant expression system according to  claim 14 , wherein the recombinant expression system is a viral vector. 
     
     
         16 . The recombinant expression system according to  claim 15 , wherein the viral vector is an adeno-associated viral vector. 
     
     
         17 . A recombinant host cell comprising the isolated nucleic acid molecule according to  claim 1 . 
     
     
         18 . The recombinant host cell according to  claim 17 , wherein the recombinant host cell is a dermal fibroblast. 
     
     
         19 . An implantable device comprising a plurality of the recombinant host cells according to  claim 17  contained within the device. 
     
     
         20 . A recombinant host cell comprising a DNA molecule of  claim 12 . 
     
     
         21 . A method of treating an animal for hemophilia A, said method comprising:
 administering to an animal exhibiting hemophilia A an effective amount of the nucleic acid molecule of  claim 1 , whereby the animal expresses the recombinant factor VIII and exhibits effective blood clotting following vascular injury.   
     
     
         22 . The method according to  claim 21 , wherein said animal is a mammal. 
     
     
         23 . The method according to  claim 22 , wherein said mammal is selected from the group consisting of human, rat, mouse, guinea pig, dog, cat, monkey, chimpanzee, orangutan, cow, horse, sheep, pig, goat, rabbit, and chicken. 
     
     
         24 . The method according to  claim 21 , wherein said nucleic acid molecule comprises a viral vector. 
     
     
         25 . The method according to  claim 24 , wherein said viral vector is an adeno-associated viral vector. 
     
     
         26 . A method of treating an animal for hemophilia A, said method comprising:
 administering to an animal exhibiting hemophilia A a recombinant host cell of  claim 17 , whereby the animal expresses the recombinant factor VIII and exhibits effective blood clotting following vascular injury.   
     
     
         27 . The method according to  claim 26 , wherein said administering comprises:
 implanting into the animal a device containing a plurality of said recombinant host cells.   
     
     
         28 . The method according to  claim 26 , wherein the recombinant host cell is a dermal fibroblast. 
     
     
         29 . The method according to  claim 26 , wherein said animal is a mammal. 
     
     
         30 . The method according to  claim 29 , wherein said mammal is selected from the group consisting of human, rat, mouse, guinea pig, dog, cat, monkey, chimpanzee, orangutan, cow, horse, sheep, pig, goat, rabbit, and chicken. 
     
     
         31 . An isolated nucleic acid encoding a recombinant factor VIII comprising one or more mutations that result in enhanced stability of both factor VIII and factor VIIIa, wherein the one or more mutations are selected from the group consisting of substitution of a Glu residue with a hydrophobic amino acid residue at a position corresponding to amino acid 287, 665, and/or 1984 of SEQ ID NO: 2, substitution of an Asp residue with a hydrophobic amino acid residue at a position corresponding to amino acid 302 and/or 519 of SEQ ID NO: 2, and combinations of two or more of said substitutions, and wherein the position of the substitution or substitutions of said recombinant factor VIII aligns with amino acids 287, 302, 519, 665, and/or 1984 of SEQ ID NO: 2 upon alignment of the amino acid sequence of the recombinant factor VIII with the amino acid sequence of SEQ ID NO: 2. 
     
     
         32 . The isolated nucleic acid molecule according to  claim 31 , wherein nucleic acid is DNA. 
     
     
         33 . A recombinant expression system comprising a DNA molecule of  claim 32 . 
     
     
         34 . A recombinant host cell comprising the isolated nucleic acid molecule according to  claim 31 . 
     
     
         35 . A method of treating an animal for hemophilia A, said method comprising:
 administering to an animal exhibiting hemophilia A an effective amount of the nucleic acid molecule of  claim 31 , whereby the animal expresses the recombinant factor VIII and exhibits effective blood clotting following vascular injury.   
     
     
         36 . A method of treating an animal for hemophilia A, said method comprising:
 administering to an animal exhibiting hemophilia A a recombinant host cell of  claim 34 , whereby the animal expresses the recombinant factor VIII and exhibits effective blood clotting following vascular injury.