Viral vectors encoding recombinant fix with increased expression for gene therapy of hemophilia b

Abstract

Codon-altered polynucleotides encoding Factor IX variants for expression in mammalian cells are provided, including sequences with high nucleotide sequence identity to the CS02, CS03, CSO4, CS05, and CS06 sequences. Mammalian gene therapy vectors, such as adeno-associated viral vectors, and methods for treating hemophilia B using those vectors are also provided.

1 - 35 . (canceled) 
     
     
         36 . A method for treating hemophilia B comprising administering to a patient in need thereof a nucleic acid composition comprising a Factor IX polynucleotide encoding a Factor IX protein, said Factor IX polynucleotide comprising a nucleic acid sequence that is least 97% identical to the nucleic acid sequence of SEQ ID NO: 17. 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . A method for producing an adeno-associated virus (AAV) particle comprising introducing a nucleic acid composition comprising a Factor IX polynucleotide encoding a Factor IX protein, said Factor IX polynucleotide comprising a nucleic acid sequence that is least 97% identical to the nucleic acid sequence of SEQ ID NO: 17, into a mammalian host cell, wherein the nucleic acid composition is competent for replication in the mammalian host cell. 
     
     
         40 . The method of  claim 36 , wherein the Factor IX polynucleotide comprises a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 17. 
     
     
         41 . The method of  claim 36 , wherein the Factor IX polynucleotide has no more than 10 CpG dinucleotides. 
     
     
         42 . The method of  claim 36 , wherein the Factor IX polynucleotide has no more than 3 CpG dinucleotides. 
     
     
         43 . The method of  claim 36 , wherein the Factor IX polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 17. 
     
     
         44 . The method of  claim 36 , wherein the Factor IX polynucleotide encodes for leucine at the amino acid position corresponding to residue 384 of the full-length wild type Factor IX polypeptide (SEQ ID NO: 2), which is an arginine in the wild type Factor IX polypeptide. 
     
     
         45 . The method of  claim 36 , wherein the Factor IX protein encoded by the Factor IX polynucleotide has from 1 to 10 amino acid substitutions as compared to SEQ ID NO: 12. 
     
     
         46 . The method of  claim 36 , wherein the Factor IX protein encoded by the Factor IX polynucleotide has the amino acid sequence of SEQ ID NO: 12. 
     
     
         47 . The method of  claim 36 , wherein the Factor IX polynucleotide further comprises a pre-pro-leader polynucleotide encoding a pre-pro-leader peptide, said pre-pro-leader peptide comprising the amino acid sequence of SEQ ID NO: 36. 
     
     
         48 . The method of  claim 47 , wherein the pre-pro-leader polynucleotide has the nucleic acid sequence of SEQ ID NO: 23. 
     
     
         49 . The method of  claim 36 , wherein the Factor IX polynucleotide has a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 9. 
     
     
         50 . The method of  claim 36 , wherein the Factor IX polynucleotide has the nucleic acid sequence of SEQ ID NO: 9. 
     
     
         51 . The method of  claim 36 , wherein the nucleic acid composition further comprises a liver-specific promoter element operably linked to the Factor IX polynucleotide. 
     
     
         52 . The method of  claim 51 , wherein the liver-specific promoter element comprises one copy of a promoter polynucleotide, said promoter polynucleotide comprising a nucleic acid sequence that is least 95% identical to SEQ ID NO: 39. 
     
     
         53 . The method of  claim 51 , wherein the liver-specific promoter element comprises three copies of a promoter polynucleotide, said promoter polynucleotide comprising a nucleic acid sequence that is least 95% identical to SEQ ID NO: 39. 
     
     
         54 . The method of  claim 53 , wherein said promoter polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 39. 
     
     
         55 . The method of  claim 54 , wherein the nucleic acid composition further comprises an intron operatively linked to the Factor IX polynucleotide. 
     
     
         56 . The method of  claim 55 , wherein the intron comprises an MVM intron polynucleotide comprising a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 53. 
     
     
         57 . The method of  claim 56 , wherein said MVM intron polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 53. 
     
     
         58 . The method of  claim 57 , wherein said intron is positioned between a promoter element and the translation initiation site of the nucleotide sequence encoding a Factor IX polypeptide. 
     
     
         59 . The method of  claim 36 , wherein the nucleic acid composition comprises a polynucleotide having the nucleic acid sequence of SEQ ID NO: 40. 
     
     
         60 . The method of  claim 36 , wherein the nucleic acid composition comprises a mammalian gene therapy vector. 
     
     
         61 . The method of  claim 60 , wherein the mammalian gene therapy vector is an adeno-associated virus (AAV) vector. 
     
     
         62 . The method of  claim 61 , wherein said adeno-associated virus vector is a serotype 8 adeno-associated virus (AAV-8) vector. 
     
     
         63 . The method of  claim 61 , wherein the mammalian gene therapy vector comprises a single-stranded polynucleotide encoding the Factor IX protein.