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 may encode amino acid sequences corresponding to known human FVIII sequences, wherein residue Phe309 is mutated. The nucleic acid sequences also may 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. Provided herein are 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.

1 - 65 . (canceled) 
     
     
         66 . 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. 
     
     
         67 . The modified human FVIII polypeptide 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 modified human FVIII polypeptide 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 . A composition comprising the polypeptide of  claim 66 . 
     
     
         70 . The composition of  claim 69 , further comprising a parenterally acceptable vehicle or excipient. 
     
     
         71 . 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. 
     
     
         72 . A composition comprising the polypeptide of  claim 71 . 
     
     
         73 . The composition of  claim 72 , further comprising a parenterally acceptable vehicle or excipient.