Factor viii compositions and methods

Abstract

The present invention provides methods of increasing the half-life and/or specific activity of factor VIII. More specifically, the invention provides methods of increasing the half-life and/or specific activity of factor VIII by substituting one or more amino acids in the A2 domain. It further provides methods for producing such factor VIII mutants. The invention also provides polynucleotides encoding the mutant factor VIII, and methods of treating hemophilia using the polypeptides and polynucleotides of the invention.

1 - 4 . (canceled) 
     
     
         5 . An isolated polynucleotide encoding a mutant factor VIII comprising the amino acid sequence of SEQ ID NO:5, further comprising an amino acid substitution at position Lys(466) and/or position Arg(471) and optionally at one or more positions selected from the group consisting of Lys(380), Arg(484), Tyr(487), Ser(488), Arg(489), Arg(490), His(497), His(499), Lys(523), and Lys(556). 
     
     
         6 - 10 . (canceled) 
     
     
         11 . An isolated polynucleotide encoding a mutant factor VIII comprising the amino acid sequence of SEQ ID NO:5, further comprising an amino acid substitution at position Lys(466) and/or position Arg(471) and optionally at one or more positions selected from the group consisting of Lys(380), Arg(484), Tyr(487), Ser(488), Arg(489), Arg(490), His(497), His(499), Lys(523), and Lys(556), further comprising an amino acid substitution at one or more additional positions in the A2 domain. 
     
     
         12 - 16 . (canceled) 
     
     
         17 . A polynucleotide encoding a mutant factor VIII comprising the amino acid sequence of SEQ ID NO:5, further comprising a substitution at one or more positions selected from the group consisting of Lys(377), His(378), and Lys(466), and optionally at one or more positions selected from the group consisting of Lys (380), Ser(488), Arg(489), Arg(490), Leu(491), Lys(493), Lys(496), His(497), His(499), Lys(512), Lys(523), and Lys(556). 
     
     
         18 - 22 . (canceled) 
     
     
         23 . A polynucleotide encoding a mutant factor VIII comprising the amino acid sequence of SEQ ID NO:5, further comprising a substitution at one or more positions selected from the group consisting of Lys(377), His(378), and Lys(466), and optionally at one or more positions selected from the group consisting of Lys(380), Ser(488), Arg(489), Arg(490), Leu(491), Lys(493), Lys(496), His(497), His(499), Lys(512), Lys(523), and Lys(556), further comprising the amino acid substitution at one or more additional positions in the A2 domain. 
     
     
         24 - 32 . (canceled) 
     
     
         33 . The polynucleotide of any one of  claims 5 ,  11 ,  17  or  23  wherein said polynucleotide is in a vector. 
     
     
         34 . The polynucleotide of  claim 33 , wherein said vector is an expression vector capable of expressing said mutant factor VIII. 
     
     
         35 . The polynucleotide of any one of  claims 5 ,  11 ,  17  or  23  wherein said polynucleotide is a genetic construct operatively linked to a promoter sequence and to a polyadenylation signal sequence. 
     
     
         36 . The polynucleotide of any one of  claims 5 ,  11 ,  17  or  23  wherein said polynucleotide is in a host cell.