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 186, 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.

1 - 63 . (canceled) 
     
     
         64 . An isolated polynucleotide encoding a polypeptide, wherein the polypeptide comprises wildtype human factor VIII with one or more amino acid substitution(s) at positions I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152. 
     
     
         65 . The isolated polynucleotide of  claim 64 , wherein the amino acid substitutions are selected from the group consisting of I86V, I86L, I86M, Y105F, Y105W, A108S, A108G, A108T, A108P, D115E, D115N, D115H, D115Q, D115R, D115K, Q117H, Q117N, Q117E, Q117D, Q117R, Q117K, F129L, F129V, F1291, F129M, F129P, F129T, F129K, G132K, G132E, G132D, G132R, G132T, G132M, G132N, G132S, G132W, H134Q, H134G, H134Y, H134N, H134E, H134D, H134R, H134K, M147T, M147A, M147G, M147S, M147P, L152P, L152S, L152G and L152T. 
     
     
         66 . The isolated polynucleotide of  claim 64 , wherein the polypeptide comprises amino acid substitutions A108S, M147T, and L152P. 
     
     
         67 . The isolated polynucleotide of  claim 65 , wherein the amino acid substitutions are selected from the group consisting of I86V, Y105F, A108S, D115E, Q117H, F129L, G132K, H134Q, M147T and L152P. 
     
     
         68 . The isolated polynucleotide of  claim 66 , wherein the polypeptide comprises the amino acid substitution I86V. 
     
     
         69 . The isolated polynucleotide of  claim 65 , wherein the amino acid substitutions are selected from the group consisting of I86V, A108S, G132K, M147T and L152P. 
     
     
         70 . The isolated polynucleotide of  claim 65 , wherein the amino acid substitutions are selected from the group consisting of A108S, M147T, and L152P. 
     
     
         71 . The isolated polynucleotide of  claim 70 , wherein the polypeptide further comprises amino acid substitutions I86V and G132K. 
     
     
         72 . The isolated polynucleotide of  claim 64 , wherein the polypeptide comprises a deletion in the B domain. 
     
     
         73 . The isolated polynucleotide of  claim 64 , wherein the polypeptide comprises the a2 and/or a3 domain(s) of human factor VIII. 
     
     
         74 . An isolated polynucleotide comprising SEQ ID NO: 1, wherein the polynucleotide encodes a human factor VIII polypeptide comprising one or more amino acid substitution(s) at positions I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152. 
     
     
         75 . The isolated polynucleotide of  claim 74 , wherein the amino acid substitutions are selected from the group consisting of I86V, I86L, I86M, Y105F, Y105W, A108S, A108G, A108T, A108P, D115E, D115N, D115H, D115Q, D115R, D115K, Q117H, Q117N, Q117E, Q117D, Q117R, Q117K, F129L, F129V, F1291, F129M, F129P, F129T, F129K, G132K, G132E, G132D, G132R, G132T, G132M, G132N, G132S, G132W, H134Q, H134G, H134Y, H134N, H134E, H134D, H134R, H134K, M147T, M147A, M147G, M147S, M147P, L152P, L152S, L152G and L152T. 
     
     
         76 . The isolated polynucleotide of  claim 75 , wherein the amino acid substitutions are selected from the group consisting of I86V, Y105F, A108S, D115E, Q117H, F129L, G132K, H134Q, M147T and L152P. 
     
     
         77 . The isolated polynucleotide of  claim 74 , wherein the polypeptide comprises a deletion in the B domain. 
     
     
         78 . An expression vector comprising the polynucleotide of  claim 64 . 
     
     
         79 . A host cell comprising the polynucleotide of  claim 64 . 
     
     
         80 . A host cell comprising the expression vector of  claim 79 . 
     
     
         81 . A pharmaceutical composition comprising the expression vector of  claim 80 . 
     
     
         82 . A method for treating a patient with a factor VIII deficiency comprising: administering to the patient in need thereof the pharmaceutical composition of  claim 81  in an amount effective for treating the factor VIII deficiency. 
     
     
         83 . A method for expressing a human factor VIII polypeptide mutant comprising: (a) transforming a host cell with an expression vector comprising the polynucleotide of  claim 64 ; (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.