Inactivation Resistant Factor VIII

Abstract

The present invention provides novel purified and isolated nucleic acid sequences encoding procoagulant-active FVIII proteins. The nucleic acid sequences of the present invention encode amino acid sequences corresponding to known human FVIII sequences, wherein residue Phe309 is mutated. The nucleic acid sequences of the present invention also encode amino acid sequences corresponding to known human FVIII sequences, wherein the APC cleavage sites, Arg336 and Ile562, are mutated. The nucleic acid sequences of the present invention further encode amino acid sequences corresponding to known human FVIII sequences, wherein the B-domain is deleted, the von Willebrand factor binding site is deleted, a thrombin cleavage site is mutated, an amino acid sequence spacer is inserted between the A2- and A3-domains. Methods of producing the FVIII proteins of the invention, nucleotide sequences encoding such proteins, pharmaceutical compositions containing the nucleotide sequences or proteins, as well as methods of treating patients suffering from hemophilia, are also provided.

1 - 65 . (canceled) 
     
     
         66 . An isolated nucleic acid comprising a nucleotide sequence encoding a modified human FVIII polypeptide, wherein said modification comprises a truncated B domain comprising at least 29 amino acids from the amino-terminal end of the B domain containing at least one N-linked glycosylation site and at least one mutation in the A1 domain, wherein the nucleic acid sequence corresponds to known human FVIII nucleic acid sequences and wherein said modified human FVIII polypeptide retains FVIII-type procoagulant activity. 
     
     
         67 . The nucleic acid sequence of  claim 66 , wherein said mutation in the A1 domain comprises a substitution of the Phenylalanine residue at 309 with a Serine residue. 
     
     
         68 . The nucleic acid sequence of  claim 67 , wherein said truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites. 
     
     
         69 . The isolated nucleic acid of  claim 66  operably linked to a promoter. 
     
     
         70 . A vector comprising the nucleic acid of  claim 69 . 
     
     
         71 . A host cell comprising the vector of  claim 70 . 
     
     
         72 . A composition comprising the nucleic acid of  claim 66 . 
     
     
         73 . The composition of  claim 72 , further comprising a parenterally acceptable vehicle or excipient. 
     
     
         74 . An isolated modified human FVIII polypeptide, wherein said modification comprises a truncated B domain comprising at least 29 amino acids from the amino-terminal end of the B domain containing at least one N-linked glycosylation site and at least one mutation in the A1 domain, wherein the modified polypeptide corresponds to an amino acid sequence corresponding to known human FVIII sequences, and wherein said modified human FVIII polypeptide retains FVIII-type procoagulant activity. 
     
     
         75 . The modified human FVIII polypeptide of  claim 74 , wherein said mutation in the A1 domain comprises a substitution of the Phenylalanine residue at 309 with a Serine residue. 
     
     
         76 . The modified human FVIII polypeptide of  claim 75 , wherein said truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites. 
     
     
         77 . A composition comprising the polypeptide of  claim 74 . 
     
     
         78 . The composition of  claim 77 , further comprising a parenterally acceptable vehicle or excipient. 
     
     
         79 . A method for treating a patient for hemophilia comprising the step of administering to the patient a therapeutically effective amount of a nucleic acid comprising the nucleotide sequence encoding a modified human FVIII polypeptide, wherein said modification comprises a truncated B domain comprising at least 29 amino acids from the amino-terminal end of the B domain containing at least one N-linked glycosylation site and at least one mutation in the A1 domain, wherein the nucleic acid sequence corresponds to known human FVIII nucleic acid sequences, and wherein said modified human FVIII polypeptide retains FVIII-type procoagulant activity. 
     
     
         80 . The method of  claim 79 , wherein said mutation in the A1 domain comprises a substitution of the Phenylalanine residue at 309 with a Serine residue. 
     
     
         81 . The method of  claim 80 , wherein said truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites. 
     
     
         82 . A method for treating a patient for hemophilia comprising the step of administering to the patient a therapeutically effective amount of a protein comprising a modified human FVIII polypeptide, wherein said modification comprises a truncated B domain comprising at least 29 amino acids from the amino-terminal end of the B domain containing at least one N-linked glycosylation site and at least one mutation in the A1 domain, wherein the modified polypeptide corresponds to an amino acid sequence corresponding to known human FVIII sequences, and wherein said modified human FVIII polypeptide retains FVIII-type procoagulant activity. 
     
     
         83 . The method of  claim 82 , wherein said mutation in the A1 domain comprises a substitution of the Phenylalanine residue at 309 with a Serine residue. 
     
     
         84 . The method of  claim 83 , wherein said truncated B domain comprises 226 amino acids from the amino terminal end of the B domain containing 6 N-linked glycosylation sites. 
     
     
         85 . An isolated nucleic acid comprising a nucleotide sequence encoding a modified human FVIII polypeptide, wherein said modification comprises a truncated B domain comprising 226 amino acids from the amino-terminal end of the B domain containing 6 N-linked glycosylation sites and a mutation at Phe309 comprising a substitution of the Phenyalanine residue at 309 with a Serine residue, wherein the nucleic acid sequence corresponds to known human FVIII nucleic acid sequences and wherein said modified human FVIII polypeptide retains FVIII-type procoagulant activity. 
     
     
         86 . The isolated nucleic acid of  claim 85  operably linked to a promoter. 
     
     
         87 . A vector comprising the nucleic acid of  claim 86 . 
     
     
         88 . A host cell comprising the vector of  claim 87 . 
     
     
         89 . A composition comprising the nucleic acid of  claim 85 . 
     
     
         90 . The composition of  claim 89 , further comprising a parenterally acceptable vehicle or excipient. 
     
     
         91 . An isolated modified human FVIII polypeptide, wherein said modification comprises a truncated B domain comprising 226 amino acids from the amino-terminal end of the B domain containing 6 N-linked glycosylation sites and a mutation at Phe309 comprising a substitution of the Phenylalanine residue at 309 with a Serine residue, wherein the modified polypeptide corresponds to an amino acid sequence corresponding to known human FVIII sequences, and wherein said modified human FVIII polypeptide retains FVIII-type procoagulant activity. 
     
     
         92 . A composition comprising the polypeptide of  claim 91 . 
     
     
         93 . The composition of  claim 92 , further comprising a parenterally acceptable vehicle or excipient. 
     
     
         94 . A method for treating a patient for hemophilia comprising the step of administering to the patient a therapeutically effective amount of a nucleic acid comprising the nucleotide sequence encoding a modified human FVIII polypeptide, wherein said modification comprises a truncated B domain comprising 226 amino acids from the amino-terminal end of the B domain containing 6 N-linked glycosylation and a mutation at Phe309 comprising a substitution of the Phenylalanine residue at 309 with a Serine residue, wherein the nucleic acid sequence corresponds to known human FVIII nucleic acid sequences, and wherein said modified human FVIII polypeptide retains FVIII-type procoagulant activity. 
     
     
         95 . A method for treating a patient for hemophilia comprising the step of administering to the patient a therapeutically effective amount of a protein comprising a modified human FVIII polypeptide, wherein said modification comprises a truncated B domain comprising 226 amino acids from the amino-terminal end of the B domain containing 6 N-linked glycosylation sites and a mutation at Phe309 comprising a substitution of the Phenylalanine residue at 309 with a Serine residue, wherein the modified polypeptide corresponds to an amino acid sequence corresponding to known human FVIII sequences, and wherein said modified human FVIII polypeptide retains FVIII-type procoagulant activity.