Mutant factor viii compositions and methods

Abstract

In one aspect, present invention provides a recombinant mutant human factor VIII having increased expression and/or secretion as compared to wild-type factor VIII. In certain embodiments, the recombinant factor VIII includes one or more amino acid substitution(s) selected from the group consisting of I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and L152. In other aspects, the present invention provides FVIII encoding nucleic acids, FVIII-expression vectors, as well as methods of using the modified FVIII genes in the treatment of FVIII deficiencies, such as hemophilia A.

What is claimed is: 
     
         1 . An isolated human factor VIII polypeptide mutant comprising one or more amino acid substitution(s) selected from the group consisting of I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and L152. 
     
     
         2 . The mutant of  claim 1 , wherein the one or more amino acid substitution(s) are selected from the group consisting I86V, Y105F, A108S, D115E, Q117H, F129L, G132K, H134Q, M147T and L152P. 
     
     
         3 . The mutant of  claim 1 , comprising amino acid substitutions in each of amino acids I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and L152. 
     
     
         4 . The mutant of  claim 3 , comprising each of the amino acid substitutions I86V, Y105F, A108S, D115E, Q117H, F129L, G132K, H134Q, M147T and L152P 
     
     
         5 . The mutant of  claim 1 , wherein the one or more amino acid substitution(s) are selected from the group consisting of I86, A108, G132, M147 and L152P. 
     
     
         6 . The mutant of  claim 5 , wherein the one or more amino acid substitution(s) are selected from the group consisting of I86V, A108S, G132K, M147T and L152P. 
     
     
         7 . The mutant of  claim 5 , comprising amino acid substitutions in each of amino acids I86, A108, G132, M147 and L152. 
     
     
         8 . The mutant of  claim 7 , comprising amino acid substitutions in each of amino acids I86V, A108S, G132K, M147T and L152P. 
     
     
         9 . The mutant of any one of  claims 1  to  8 , further comprising a deletion in the B domain of human factor VIII. 
     
     
         10 . The mutant of  claim 9 , further comprising the a2 and/or a3 domain(s) of human factor VIII. 
     
     
         11 . An isolated polynucleotide encoding the mutant of any one of  claims 1  to  10 . 
     
     
         12 . An expression vector comprising the polynucleotide of  claim 11 . 
     
     
         13 . A host cell comprising the polynucleotide of  claim 11 . 
     
     
         14 . A host cell comprising the expression vector of  claim 12 . 
     
     
         15 . A pharmaceutical composition comprising the expression vector of  claim 14 . 
     
     
         16 . A method for treating a patient with a factor VIII deficiency comprising:
 administering to the patient in need thereof the pharmaceutical composition of  claim 15  in an amount effective for treating the factor VIII deficiency.   
     
     
         17 . A method for expressing a human factor VIII polypeptide mutant comprising:
 (a) transforming a host cell with an expression vector comprising the nucleic acid of  claim 12 ;   (b) growing the host cell under conditions suitable for expressing the human factor VIII polypeptide mutant; and   (c) purifying the human factor VIII polypeptide mutant from host cells expressing said mutant.