NZ784737B2 - Optimized factor VIII genes - Google Patents

Abstract

The present disclosure provides codon optimized Factor VIII sequences, vectors, and host cells comprising codon optimized Factor VIII sequences, polypeptides encoded by codon optimized Factor VIII sequences, and methods of producing such polypeptides. The present disclosure also provides methods of treating bleeding disorders such as hemophilia comprising administering to the subject a codon optimized Factor VIII nucleic acid sequence or the polypeptide encoded thereby.

Claims (17)

WHAT IS CLAIMED IS:

1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a ptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence comprising nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 71.

2. The isolated nucleic acid molecule of claim 1, n the nucleotide sequence further comprises a c acid sequence encoding a signal e, wherein the nucleic acid sequence encoding a signal peptide comprises at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to: (i) nucleotides 1 to 57 of SEQ ID NO: 1; (ii) nucleotides 1 to 57 of SEQ ID NO: 2; (iii) nucleotides 1 to 57 of SEQ ID NO: 3; (iv) nucleotides 1 to 57 of SEQ ID NO: 4; (v) nucleotides 1 to 57 of SEQ ID NO: 5; (vi) nucleotides 1 to 57 of SEQ ID NO: 6; (vii) tides 1 to 57 of SEQ ID NO: 70; (viii) nucleotides 1 to 57 of SEQ ID NO: 71; or (ix) nucleotides 1 to 57 of SEQ ID NO: 68.

3. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises one or more property selected from the group consisting of: (a) the human codon adaptation index of the c acid molecule or a portion thereof is increased relative to SEQ ID NO: 16; (b) the frequency of optimal codons of the nucleotide sequence or a portion thereof is sed relative to SEQ ID NO:16; (c) the nucleotide sequence or a portion thereof contains a higher tage of G/C nucleotides compared to the percentage of G/C nucleotides in SEQ ID NO: 16; (d) the relative synonymous codon usage of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO: 16; (e) the effective number of codons of the nucleotide ce or a portion thereof is reduced relative SEQ ID NO: 16; (f) the tide sequence contains fewer r matrix attachment regions/autonomously replicating sequences (MARS/ARS) sequences relative to SEQ ID NO: 16; (g) the nucleotide sequence contains fewer destabilizing ts ve to SEQ ID NO: 16; and (h) any combination thereof.

4. The isolated nucleic acid molecule of claim 1, further sing a heterologous nucleotide sequence encoding a heterologous amino acid sequence, optionally wherein the heterologous amino acid sequence: comprises an immunoglobulin constant region or a portion thereof, XTEN, transferrin, albumin, or a PAS sequence; and/or is linked to the N-terminus or the inus of the amino acid sequence encoded by the nucleotide sequence or ed between two amino acids in the amino acid sequence encoded by the nucleotide sequence at one or more insertion sites corresponding to the amino acid position of mature human FVIII (SEQ ID NO: 15).

5. The isolated nucleic acid molecule of claim 1, wherein the FVIII polypeptide is a full length FVIII or a B domain deleted FVIII.

6. The isolated nucleic acid molecule of claim 5, wherein the logous amino acid sequence is an immunoglobulin constant region or a portion thereof, XTEN, transferrin, albumin, or a PAS sequence.

7. The isolated nucleic acid molecule of claim 5, wherein the insertion site corresponds to amino acid residue 3, 18, 22, 26, 40, 60, 65, 81, 116, 119, 130, 188, 211, 216, 220, 224, 230, 333, 336, 339, 375, 378, 399, 403, 409, 416, 442, 487, 490, 494, 500, 518, 599, 603, 713, 745, 1656, 1711, 1720, 1725 of SEQ ID NO: 15.

8. Use of the nucleic acid molecule of any one of of claims 1 to 7 in the cture of a medicament for treating a bleeding disorder.

9. A vector comprising the nucleic acid molecule of any one of claims 2 to 7.

10. Use of the vector of claim 9 in the manufacture of a medicament for treating a bleeding disorder.

11. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence comprising SEQ ID NO: 72.

12. The isolated nucleic acid molecule of claim 11, n the FVIII polypeptide is a full length FVIII or a B domain deleted FVIII.

13. A vector comprising the nucleic acid molecule of either one of claims 11 or 12.

14. The vector of claim 13, wherein the vector is a lentiviral vector.

15. Use of the c acid le of either one of claims 11 or 12 in the manufacture of a medicament for treating a bleeding disorder.

16. Use of the vector of either one of claims 13 or 14 in the manufacture of a medicament for treating a bleeding er.

17. Use of a vector comprising the nucleic acid molecule of claim 1 in the manufacture of a medicament for treating a bleeding disorder. : coFVIII-3 — SE ID NO: 1 ATGCAGATCGAACTGAGCACCTGCTTCTTTCTGTGCCTGCTGAGGTTTTGCTTTAGCGCCACCAGGAGATACTATCTGGGCGC CGTGGAACTGAGCTGGGACTATATGCAGTCTGATCTGGGCGAACTGCCAGTGGATGCCAGGTTTCCCCCCAGAGTGCCCAAAA GCTTTCCCTTTAATACCAGCGTGGTGTATAAGAAAACCCTGTTTGTGGAATTCACTGATCATCTGTTTAATATCGCCAAGCCC CCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCCGAAGTGTATGATACCGTGGTCATCACCCTGAAAAACATGGC CAGCCATCCAGTGAGCCTGCATGCTGTGGGCGTCTCCTATTGGAAAGCCTCTGAAGGCGCCGAGTATGATGATCAGACCAGCC AGAGGGAAAAAGAAGATGATAAAGTCTTTCCTGGGGGCAGCCATACCTATGTCTGGCAGGTCCTGAAAGAAAATGGCCCCATG GATCCCCTGTGCCTGACCTATAGCTATCTGAGCCATGTGGACCTGGTGAAGGATCTGAACAGCGGCCTGATTGGGGC CCTGCTGGTGTGCAGGGAAGGCAGCCTGGCCAAAGAAAAAACCCAGACCCTGCATAAGTTTATCCTGCTGTTTGCCGTGTTTG ATGAAGGCAAAAGCTGGCATTCTGAAACCAAAAACAGCCTGATGCAGGACAGGGATGCCGCCTCTGCCAGGGCCTGGCCCAAA ATGCATACCGTGAATGGCTATGTGAATAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAAAGCGTGTATTGGCATGTGAT GGGCACCACCCCCGAAGTGCATAGCATCTTTCTGGAAGGCCATACCTTCCTGGTCAGGAACCACAGGCAGGCCAGCC TGGAAATCAGCCCCATCACCTTCCTGACCGCCCAGACCCTGCTGATGGATCTGGGCCAGTTTCTGCTGTTTTGCCACATCTCC AGCCATCAGCATGATGGCATGGAAGCCTATGTGAAAGTCGATAGCTGCCCCGAAGAACCCCAGCTGAGGATGAAAAACAATGA AGAAGCCGAAGACTATGATGATGATCTGACTGATTCTGAAATGGATGTGGTCAGGTTTGATGATGATAATAGCCCCAGCTTTA TCCAGATCAGGAGCGTGGCCAAAAAACATCCCAAGACCTGGGTGCATTATATCGCTGCTGAGGAAGAAGATTGGGACTATGCC GTGCTGGCCCCTGATGATAGGAGCTATAAAAGCCAGTATCTGAACAATGGCCCCCAGAGG TTGGCAGGAAGTATAA AAAAGTCAGGTTTATGGCCTACACTGAT AAACCTTCAAGACCAGGGAAGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCC ATGGCGAAGTGGGGGACACCCTGCTGATCATCTTTAAAAATCAGGCCAGCAGGCCCTAT TATCTATCCCCATGGC ATCACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAAGGCGTGAAACATCTGAAAGATTTTCCCATCCTGCCTGGCGA AATCTTTAAGTATAAATGGACTGTGACTGTGGAAGATGGCCCCACCAAAAGCG TCCCAGGTGCCTGACCAGGTATTATTCCA GCTTTGTGAATATGGAACGCGATCTGGCCTCTGGCCTGATTGGCCCCCTGCTG TCTGCTATAAAGAGTCTGTGGACCAGAGG GGCAATCAGATCATGAGCGATAAAAGGAATGTCATCCTGTTCTCTGTCTTTGATGAGAATAGGAGCTGGTACCTGACCGAAAA CATCCAGAGGTTTCTGCCCAATCCCGCCGGCGTGCAGCTGGAAGATCCCGAGTTTCAGGCCAGCAATATCATGCATAGCATCA ATGGCTATGTCTTTGATAGCCTGCAGCTGAGCGTGTGCCTGCATGAGGTGGCCTATTGGTATATCCTGAGCATCGGCGCCCAG ACCGATTTTCTGAGCGTGTTTTTCTCTGGCTATACCTTTAAACATAAAATGGTGTATGAGGACACCCTGACCCTGTTTCCCTT CTCTGGCGAAACCGTGTTTATGAGCATGGAAAATCCCGGCCTGTGGATCCTGGGCTGCCACAACAGCGATTTCAGGAACAGGG GCATGACTGCCCTGCTGAAAGTCTCCAGCTGCGATAAAAACACTGGGGACTATTATGAGGACAGCTATGAGGACATCAGCGCC TATCTGCTGAGCAAGAACAATGCCATCGAACCCAGGAGCTTTAGCCAGAATCCCCCAGTGCTGAAAAGGCATCAGAGGGAAAT CACCAGGACCACCCTGCAGTCTGATCAGGAAGAAATCGACTATGATGATACCATCAGCGTGGAAATGAAGAAAGAAGATTTTG ATATCTATGATGAAGATGAAAATCAGAGCCCCAGGAGCTTTCAGAAGAAAACCAGGCATTACTTCATCGCTGCTGTGGAAAGG CTGTGGGACTATGGCATGTCCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAGTCTGGCAGCGTGCCCCAGTTTAAAAAAGT CGTGTTTCAGGAGTTTACCGATGGCAGCTTTACCCAGCCCCTGTATAGGGGCGAACTGAATGAACATCTGGGCCTGCTGGGCC CCTACATCAGGGCCGAAGTGGAAGATAATATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTTTATTCCAGC CTGATCAGCTATGAGGAAGATCAGAGGCAGGGGGCTGAGCCCAGGAAAAACTTTGTGAAACCCAATGAAACCAAGACCTACTT TTGGAAAGTCCAGCATCATATGGCCCCCACCAAGGATGAATTTGATTGCAAAGCCTGGGCCTACTTCTCTGATGTGGACCTGG AAAAAGATGTGCATAGCGGCCTGATTGGCCCCCTGCTGGTGTGCCACACCAATACCCTGAACCCTGCCCATGGCAGGCAGGTG ACTGTGCAGGAGTTTGCCCTGTTCTTTACCATCTTTGATGAAACCAAAAGCTGGTACTTCACCGAAAACATGGAAAGGAACTG CAGGGCCCCCTGCAACATCCAGATGGAAGATCCCACCTTTAAAGAAAATTATAGGTTCCATGCCATCAATGGCTATATCATGG ATACCCTGCCTGGCCTGGTCATGGCCCAGGACCAGAGGATCAGGTGGTATCTGCTGAGCATGGGCAGCAATGAAAACATCCAT AGCATCCATTTCTCTGGCCATGTCTTTACCGTCAGGAAAAAAGAAGAGTATAAAATGGCCCTGTATAATCTGTACCCTGGGGT GTTTGAAACCGTGGAAATGCTGCCCAGCAAAGCCGGCATCTGGAGGGTGGAATGCCTGATTGGCGAACATCTGCATGCTGGCA TGAGCACCCTGTTTCTGGTGTATAGCAATAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCATATCAGGGATTTTCAGATC ACTGCCTCTGGCCAGTATGGCCAGTGGGCCCCCAAACTGGCCAGGCTGCATTATTCCGGAAGCATCAATGCCTGGAGCACCAA AGAACCCTTTAGCTGGATCAAAGTCGATCTGCTGGCCCCCATGATCATCCATGGCATCAAGACCCAGGGGGCCAGGCAGAAAT TTTCCAGCCTGTACATCAGCCAGTTTATCATCATGTATAGCCTGGATGGCAAAAAATGGCAGACCTATAGGGGCAATAGCACC CTGATGGTGTTCTTTGGCAATGTGGACAGCAGCGGCATCAAACATAATATCTTTAATCCCCCCATCATCGCCAGGTA TATCAGGCTGCATCCCACCCATTATAGCATCAGGAGCACCCTGAGGATGGAACTGATGGGCTGCGATCTGAACAGCTGCAGCA TGCCCCTGGGCATGGAAAGCAAAGCCATCAGCGATGCCCAGATCACTGCCTCCAGCTACTTCACTAATATGTTTGCCACCTGG AGCCCCAGCAAAGCCAGGCTGCATCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCAGGTCAACAATCCCAAAGAATGGCTGCA GGTCGATTTTCAGAAAACCATGAAAGTCACTGGGGTGACCACCCAGGGGGTGAAAAGCCTGCTGACCAGCATGTATGTGAAAG AGTTTCT ATCTCCAGCAGCCAGGATGGCCATCAGTGGACCCTGTTCTTTCAGAATGGCAAAGTCAAAGTCTTTCAGGGCAAT CAGGACAGCTTTACCCCTGTGGTGAATAGCCTGGATCCCCCCCTGCTGACCAGGTATCTGAGGATCCATCCCCAGAGCTGGGT GCATCAGATCGCCCTGAGGATGGAAGTGCTGGGCTGCGAAGCCCAGGACCTGTACTGA : coFVIII-4 - SE ID NO: 2 ATGCAGATCGAGCTGAGCACGTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCAGCGCCACCAGGAGGTACTACCTGGGCGC CGTGGAGCTGAGCTGGGACTACATGCAGAGCGACCTGGGCGAGCTGCCCGTGGACGCCAGGTTCCCCCCCAGGGTGCCCAAGA GCTTCCCCTTCAACACGAGCGTGGTGTACAAGAAGACCCTGTTCGTGGAGTTCACCGACCATCTGTTCAATATCGCCAAGCCC AGGCCCCCCTGGATGGGGCTGCTGGGGCCCACGATCCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGC CAGCCACCCCGTGAGCCTGCACGCCGTGGGCGTGAGCTACTGGAAGGCCAGCGAGGGCGCCGAGTACGACGACCAGACCAGCC AGAGGGAGAAGGAGGACGACAAGGTGTTCCCCGGCGGCAGCCACACCTACGTGTGGCAGGTGCTGAAGGAGAATGGGCCCATG GACCCCCTGTGCCTGACCTACTCTTACCTGAGCCACGTGGATCTGGTGAAGGACCTGAACAGCGGCCTGATCGGCGC CCTGCTGGTGTGCAGGGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCG ACGAGGGCAAGAGCTGGCACAGCGAGACCAAGAACAGCCTGATGCAGGATAGGGACGCCGCCAGCGCCAGGGCCTGGCCCAAG ATGCACACCGTGAACGGCTACGTGAACAGGTCTCTGCCCGGCCTGATCGGCTGCCACAGGAAGAGCGTGTACTGGCACGTGAT CGGCATGGGGACCACCCCCGAGGTGCACAGCATCTTCCTGGAGGGCCACACGTTCCTGGTGAGGAATCACAGGCAGGCCAGCC TGGAGATCAGCCCGATCACCTTCCTGACCGCCCAGACCCTGCTGATGGACCTGGGGCAGTTCCTGCTGTTCTGCCATATCAGC TCTCACCAGCACGACGGCATGGAGGCCTACGTGAAGGTGGATAGCTGCCCCGAGGAGCCCCAGCTGAGGATGAAGAACAACGA GGAGGCCGAGGACTACGACGACGACCTGACCGACAGCGAGATGGACGTGGTGAGGTTCGACGACGACAATAGCCCGAGCTTCA TCCAGATCAGGAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCATTACATCGCCGCCGAGGAGGAGGATTGGGACTACGCC CCCCTGGTGCTGGCCCCCGACGACAGGTCTTACAAGAGCCAGTACCTGAACAACGGGCCCCAGAGGATCGGCAGGAAGTACAA GAAGGTGAGGTTCATGGCCTACACCGACGAGACCTTCAAGACCAGGGAGGCGATCCAGCACGAGAGCGGGATCCTGGGGCCCC r1GC"G"ACGGCGAGGTGGGCGACACGCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCGTACAATATCTACCCCCACGGG A"CACCGACG"GAGGCCCCTGTACTCTAGGAGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCCATCC"GCCCGGCGA G r1CT"CAAG"ACAAGTGGACCGTGACCGTGGAGGACGGGCCCACGAAGAGCGACCCCAGGTGCCTGACCAGG"AC"ACAGCT C"""CGTGAACA"GGAGAGGGACCTGGCCAGCGGCCTGATCGGGCCCCTGCTGATCTGCTACAAGGAGAGCG"GGA"CAGAGG CAGA"CATGAGCGACAAGAGGAACGTGATCCTGTTCAGCGTGTTCGACGAGAATAGG"C""GGTACC"GACCGAGAA TA"CCAGAGG""CC"GCCCAACCCCGCCGGCGTGCAGCTGGAGGATCCCGAGTTCCAGGCCAGCAACATCA"GCACAGCATCA ACGGCTACGTG""CGACAGCC"GCAGCTGAGCGTGTGCCTGCACGAGGTGGCCTACTGGTACA"CC"GAGCA"CGGCGCCCAG ACCGACT"CC"GAGCG"GTTC"TCAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACCC"G"TCCCCTr1 CAGCGGCGAGACCG"G"TCATGAGCATGGAGAACCCCGGCCTGTGGATCCTGGGCTGCCATAACTCCGACT"CAGGAA"AGGG GCA"GACCGCCC"GCTGAAGG"GAGCTCTTGCGACAAGAACACCGGCGACTACTACGAGGA"AGC"ACGAGGA"A"CAGCGCC TACCTGC"GAGCAAGAACAACGCCATCGAGCCCAGGTCTTTCAGCCAGAACCCCCCCGTGC"GAAGAGGCACCAGAGGGAGAr1 CACCAGGACGACCC"GCAGAGCGACCAGGAGGAGATCGACTACGACGACACGATCAGCGTGGAGA"GAAGAAGGAGGA"TTCG ACA"C"ACGACGAGGACGAGAATCAGAGCCCCAGGTCTTTCCAGAAGAAGACCAGGCATTACTTCATCGCCGCCG"GGAGAGG C"G"GGGAC"ACGGCATGAGCAGCTCTCCCCACGTGCTGAGGAATAGGGCCCAGAGCGGCAGCGTGCCCCAG"TCAAGAAGGr1 CCAGGAGT"CACCGACGGCAGCTTCACCCAGCCCCTGTACAGGGGCGAGCTGAACGAGCACCTGGGCC"GCTGGGGC CC"ACATCAGGGCCGAGGTGGAGGATAACATCATGGTGACCTTCAGGAATCAGGCCAGCAGGCCCTATAGCT"C"ATAGC"Cr1 C"GA"CAGC"ACGAGGAGGATCAGAGGCAGGGCGCCGAGCCCAGGAAGAACTTCGTGAAGCCCAACGAGACCAAGACC"ACr1r1 C"GGAAGGTGCAGCACCACATGGCCCCCACGAAGGACGAGTTCGACTGCAAGGCCTGGGCCTACTTCAGCGACG”GGA"C"GG AGAAGGACGHGCACAGCGGCCTGATCGGGCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCCGCCCACGGCAGGCAGG"G ACCG”GCAGGAGTTCGCCCTGTTCTTCACCATCTTCGACGAGACCAAGAGCTGGTACTTCACCGAGAATATGGAGAGGAAHHG CAGGGCCCCCTGCAATATCCAGATGGAGGACCCGACCTTCAAGGAGAATTACAGGTTCCACGCCATCAACGGCTACATCAHGG TGCCCGGCCTGGTCATGGCCCAGGATCAGAGGATCAGGTGGTATCTGCTGAGCATGGGGAGCAACGAGAATATCCAC AGCATCCACTTCAGCGGCCACGTGTTCACCGTGAGGAAGAAGGAGGAGTACAAGATGGCCCTGTACAATCTGTACCCCGGCGT GTTCGAGACCGTGGAGATGCTGCCCAGCAAGGCCGGGATCTGGAGGGTGGAGTGCCTGATCGGCGAGCACCTGCACGCCGGCA TGAGCACGCTGTTCCTGGTGTACTCTAACAAGTGCCAGACCCCCCTGGGGATGGCCAGCGGCCACATCAGGGACTTCCAGATC ACCGCCAGCGGCCAGTACGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTATTCCGGAAGCATCAACGCCTGGAGCACGAA GGAGCCCTTCAGCTGGATCAAGGTGGATCTGCTGGCCCCCATGATCATCCACGGGATCAAGACCCAGGGCGCCAGGCAGAAGT TCAGCTCTCTGTATATCAGCCAGTTCATCATCATGTACTCTCTGGACGGCAAGAAGTGGCAGACCTACAGGGGCAACAGCACC CTGATGGTGTTCTTCGGCAACGTGGACTCTAGCGGGATCAAGCACAATATCTTCAACCCCCCCATCATCGCCAGGTA CATCAGGCTGCACCCCACCCATTACTCTATCAGGTCTACCCTGAGGATGGAGCTGATGGGCTGCGACCTGAACAGCTGCAGCA TGCCCCTGGGGATGGAGAGCAAGGCCATCAGCGACGCCCAGATCACCGCCAGCTCTTACTTCACCAACATGTTCGCCACCTGG AGCCCGAGCAAGGCCAGGCTGCACCTGCAGGGCAGGTCTAACGCCTGGAGGCCCCAGGTGAACAACCCCAAGGAGTGGCTGCA GGTGGATTTCCAGAAGACC TGAAGGTGACCGGCGTGACCACGCAGGGCGTGAAGAGCCTGCTGACCAGCATGTACGTGAAGG AG"TCCT ATCAGCTCTAGCCAGGACGGCCACCAGTGGACCCTGTTCTTCCAGAACGGCAAGGTGAAGGTGTTCCAGGGCAAC CAGGATAGCTTCACCCCCGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGGTATCTGAGGATCCACCCCCAGAGCTGGGT GCACCAGA"CGCCCTGAGGATGGAGGTGCTGGGCTGCGAGGCCCAGGATCTGTATTGA : coFVIII-5 — SE ID NO: 70 ATGCAAATCGAACTGAGCACCTGTTTCTTCCTCTGCCTGCTGAGATTCTGTTTCTCCGCGACCCGCCGATACTACCTGGGAGCAGTGG AGCTCTCCTGGGATTACATGCAGAGCGACCTTGGGGAGCTGCCCGTGGATGCCAGGTTCCCTCCCCGGGTGCCAAAGTCGTTTCCGTT CAACACCTCCGTGGTGTACAAGAAAACTCTGTTCGTGGAGTTCACCGACCACCTGTTCAATATCGCCAAGCCCAGACCTCCCTGGATG GGGCTGTTGGGACCTACCATCCAAGCGGAGGTGTACGACACTGTGGTCATCACTCTGAAGAACATGGCCTCGCATCCCGTGTCCCTGC ACGCCGTGGGAGTGTCTTACTGGAAAGCGTCCGAGGGGGCCGAATACGACGACCAGACCTCGCAGAGAGAAAAGGAAGATGACAAGGT GTTCCCAGGAGGATCGCACACCTACGTGTGGCAAGTGTTGAAGGAGAACGGCCCAATGGCCTCCGACCCGCTGTGCCTGACCTACTCG TACCTGTCCCACGTGGACCTCGTGAAGGACCTCAACTCGGGACTGATTGGAGCCCTGCTGGTCTGCAGGGAAGGCTCACTGGCGAAAG CTCAGACCTTGCACAAGTTCATTCTGCTGTTCGCTGTGTTCGACGAGGGGAAGTCGTGGCACAGCGAGACTAAGAACTCCCT GATGCAAGATAGAGATGCCGCCTCCGCCCGGGCCTGGCCTAAGATGCACACCGTGAACGGTTACGTGAACCGCTCCCTCCCTGGCCTG ATTGGATGCCACCGGAAGTCCGTGTACTGGCACGTGATCGGGATGGGGACCACCCCCGAGGTGCACAGCATCTTCCTGGAAGGTCACA CATTTCTCGTGCGCAACCACCGGCAGGCCTCCCTGGAAATCAGCCCCATTACCTTCCTCACTGCCCAGACTCTGCTGATGGACCTGGG ACAGTTCCTGCTGTTCTGCCATATCTCCTCCCACCAACATGACGGAATGGAGGCATACGTGAAGGTCGATTCCTGCCCTGAGGAACCC CAGCTCCGCATGAAGAACAATGAGGAAGCCGAGGACTACGACGACGACCTGACGGATAGCGAGATGGATGTGGTCCGGTTCGATGACG ATAACAGCCCTTCCTTCATCCAAATTCGCTCGGTGGCAAAGAAGCACCCCAAGACCTGGGTGCATTACATTGCGGCGGAAGAAGAGGA CTGGGATTATGCCCCGCTTGTCCTCGCTCCTGACGACCGGAGCTACAAGAGCCAGTACCTGAACAACGGTCCACAGAGGATCGGTAGA AAGTACAAGAAGGTCCGCTTCATGGCCTATACCGACGAAACCTTCAAAACTAGAGAGGCCATCCAACACGAATCCGGCATCCTGGGCC CGC"CTTGTACGGAGAAGTCGGCGACACCCTTCTCATTATCTTCAAGAACCAGGCTTCCCGGCCGTACAACATCTATCCGCATGGGAT CAC"GACGTGCGCCCACTGTACTCGCGGCGCCTGCCCAAGGGTGTCAAACACCTGAAGGATTTTCCGATCCTTCCGGGAGAAATCT"C AAGTGGACCGTGACCGTGGAAGATGGCCCAACTAAGTCTGACCCTAGATGCCTCACCCGCTACTACTCATCC""CG"CAACA r1GGAGCGCGACC"GGCCAGCGGACTGATCGGCCCGCTGCTGATTTGCTACAAGGAATCAGTGGACCAACGGGGAAACCAGATCATG"C GGA"AAGAGGAACGTCATCCTCTTCTCCGTGTTTGACGAAAACCGGTCGTGGTACCTGACTGAAAACATCCAGCGG""CC"CCCCAAC CCCGCGGGCG"GCAGC"GGAAG TCCTGAGTTTCAGGCATCAAACATCATGCACTCCATTAACGGCTACGTG"TCG ""CGC"GCAGC r1GAGCG"G"G"C"GCACGAAGTGGCCTACTGGTACATCCTGTCCATTGGTGCCCAGACTGACTTCCTG"CCG"GT""""C"CCGGC"A CACG"TCAAGCACAAGA"GG"G"ACGAGGACACCCTGACCCTCTTCCCTTTTTCCGGCGAAACTGTG"""A"GAGCA"GGAGAATCCC GGCC"G"GGA"C""GGGCTGCCACAACAGCGACTTCCGTAACAGAGGAATGACTGCGCTGCTCAAGG"G"CCAGC"GCGACAAGAACA CCGGAGAC"A""A"GAGGAC"CATACGAGGACATCTCCGCCTACCTCCTGTCCAAGAATAACGCCA""GAACCTCGGAGC""CAGCCA GAACCCACCCG"GCTTAAGAGACATCAACGGGAGATCACTAGGACCACCCTGCAGTCAGACCAGGAGGAAA"CGACTACGA"GACACC A"CTCGG"CGAGA"GAAGAAGGAGGACTTTGACATCTACGACGAAGATGAAAACCAGAGCCCGAGG"CG"TCCAAAAGAAAACCCGCC AC"AC"T"AT"GC"GC"G"CGAGCGGCTGTGGGACTACGGAATGTCGTCCTCGCCGCACGTGCTCCGCAACCGAGCCCAGAGCGGCTC GG"GCCGCAA""CAAGAAGG"CGTGTTCCAGGAGTTCACTGACGGGAGCTTCACTCAGCCTTTGTACCGGGGAGAACTCAA"GAACAr1 C"CGGCC"CC"CGGACC""ACATCAGAGCAGAAGTGGAAGATAACATCATGGTCACTTTCCGTAACCAAGCCAGCCGCCCG"ACTCGr1 "CC"CCCTCA"""CT"ACGAAGAGGACCAGCGGCAGGGCGCAGAACCGCGCAAGAACTTCGTGAAGCCCAACGAAACCAAGAC C"ACT"C"GGAAAGTGCAGCATCATATGGCCCCGACTAAGGACGAGTTTGACTGCAAAGCCTGGGCCTACT"CTCCGATG"GGACTTG GAGAAGGACG"CCAC"CCGGCCTCATCGGTCCCCTGCTCGTGTGCCATACCAATACCCTGAACCCCGCACACGGTCGCCAGGTCACCG r1GCAGGAG'TCGCTC”GHTCHTCACTATCTTCGACGAAACTAAGTCCTGGTACTTCACCGAGAACATGGAGAGGAACTGCAGAGCCCC C"GTAACA"CCAGATGGAGGACCCGACGTTCAAGGAAAACTACCGGTTCCACGCCATTAACGGATACATCAHGGATACGC"GCCGGGT C"TGTGATGGCCCAGGA”CAACGGATCAGATGGTACTTATTGTCGATGGGCAGCAACGAGAACATCCACTCHATTCACTTCTCCGGTC A"GTGTTCACTGTGCGGAAGAAGGAAGAGTACAAGATGGCCCTGTACAACCTTTATCCCGGAGTGTTCGAAACTGTGGAAATGCTGCC GGCCGGCATTTGGCGCGTGGAGTGTTTGATTGGAGAACATCTCCATGCGGGGATGTCAACCCTGTHCCTGGTGTATAGCAAC CAGACTCCGCT”GGGATGGCGTCAGGACACATTAGGGATTTCCAGATCACTGCGTCCGGCCAGTACGGCCAATGGGCCCCTA AGCTGGCCCGCCTGCATHACTCCGGATCCATTAACGCCTGGTCAACCAAGGAGCCATTCTCCTGGATCAAGGTGGACCTTCTGGCCCC CATGATTATCCACGGAATTAAGACCCAGGGGGCCCGGCAGAAGTTCTCCTCACTGTACATCAGCCAGTTCATAATCATGTACTCCCTG GACGGAAAGAAGTGGCAAACCTACAGGGGGAACAGCACCGGCACACTGATGGTCTTTTTCGGAAATGTGGACTCCTCCGGGATTAAGC ATAACATCTTCAACCCTCCGATTATCGCTCGGTACATTAGACTTCACCCTACCCACTACAGCATTCGCTCCACCCTGCGGATGGAACT GATGGGCTGCGATCTGAACTCGTGCAGCATGCCGTTGGGAATGGAGTCCAAAGCAATTTCCGACGCGCAGATCACCGCCTCGTCCTAC TTTACCAACATGTTCGCCACGTGGTCACCGTCCAAGGCCCGGCTGCACCTCCAGGGAAGATCCAACGCATGGCGGCCACAGGTCAACA ACCCTAAGGAGTGGCTCCAGGTGGACTTCCAGAAAACCATGAAGGTCACCGGAGTCACAACCCAGGGAGTGAAGTCGCTGCTGACTTC TATGTACGTCAAGGAGTTCCTGATCTCCAGCAGCCAGGACGGGCACCAGTGGACCCTGTTCTTCCAAAATGGAAAGGTCAAGGTGTTT CAGGGCAATCAGGATTCATTCACCCCGGTGGTGAACTCCCTTGATCCACCCCTCCTGACCCGCTACCTTCGCATCCACCCACAGTCCT GGGTGCACCAGATCGCGCTGAGGATGGAGGTCCTGGGATGCGAAGCCCAGGACCTGTACTGA : coFVIII-6 — SE ID NO: 71 ATTGAGCTGTCCACTTGTTTCTTCCTGTGCCTCCTGCGCTTCTGTTTCTCCGCCACTCGCCGGTACTACCTTGGAGCCGTGG AGCTTTCATGGGACTACATGCAGAGCGACCTGGGCGAACTCCCCGTGGATGCCAGATTCCCCCCCCGCGTGCCAAAGTCCTTCCCCTT TAACACCTCCGTGGTGTACAAGAAAACCCTCTTTGTCGAGTTCACTGACCACCTGTTCAACATCGCCAAGCCGCGCCCACCTTGGATG GGCCTCCTGGGACCGACCATTCAAGCTGAAGTGTACGACACCGTGGTGATCACCCTGAAGAACATGGCGTCCCACCCCGTGTCCCTGC ATGCGGTCGGAGTGTCCTACTGGAAGGCCTCCGAAGGAGCTGAGTACGACGACCAGACTAGCCAGCGGGAAAAGGAGGACGATAAAGT GTTCCCGGGCGGCTCGCATACTTACGTGTGGCAAGTCCTGAAGGAAAACGGACCTATGGCATCCGATCCTCTGTGCCTGACTTACTCC TACCTTTCCCATGTGGACCTCGTGAAGGACCTGAACAGCGGGCTGATTGGTGCACTTCTCGTGTGCCGCGAAGGTTCGCTCGCTAAGG AAAAGACCCAGACCCTCCATAAGTTCATCCTTTTGTTCGCTGTGTTCGATGAAGGAAAGTCATGGCATTCCGAAACTAAGAACTCGCT GGACCGGGATGCCGCCTCAGCCCGCGCCTGGCCTAAAATGCATACAGTCAACGGATACGTGAATCGGTCACTGCCCGGGCTC ATCGGTTGTCACAGAAAGTCCGTGTACTGGCACGTCATCGGCATGGGCACTACGCCTGAAGTGCACTCCATCTTCCTGGAAGGGCACA CCTTCCTCGTGCGCAACCACCGCCAGGCCTCTCTGGAAATCTCCCCGATTACCTTTCTGACCGCCCAGACTCTGCTCATGGACCTGGG GCAGTTCCTTCTCTTCTGCCACATCTCCAGCCATCAGCACGACGGAATGGAGGCCTACGTGAAGGTGGACTCATGCCCGGAAGAACCT CAGTTGCGGATGAAGAACAACGAGGAGGCCGAGGACTATGACGACGATTTGACTGACTCCGAGATGGACGTCGTGCGGTTCGATGACG ACAACAGCCCCAGCTTCATCCAGATTCGCAGCGTGGCCAAGAAGCACCCCAAAACCTGGGTGCACTACATCGCGGCCGAGGAAGAAGA TTGGGACTACGCCCCGTTGGTGCTGGCACCCGATGACCGGTCGTACAAGTCCCAGTATCTGAACAATGGTCCGCAGCGG TTGGCAGA AAGTACAAGAAAGTGCGGTTCATGGCGTACACTGACGAAACGTTTAAGACCCGGGAGGCCATTCAACATGAGAGCGGCATTCTGGGAC CACTGCTGTACGGAGAGGTCGGCGATACCCTGCTCATCATCTTCAAAAACCAGGCCTCCCGGCCTTACAACATCTACCCTCACGGAAT CACCGACGTGCGGCCACTCTACTCGCGGCGCCTGCCGAAGGGCGTCAAGCACCTGAAAGACTTCCCTATCCTGCCGGGCGAAATCT"C AAG"ATAAGTGGACCGTCACCGTGGAGGACGGGCCCACCAAGAGCGATCCTAGGTGTCTGACTCGGTACTACTCCAGC"”CG"GAACA r1GGAACGGGACC"GGCATCGGGACTCATTGGACCGCTGCTGATCTGCTACAAAGAGTCGGTGGATCAACGCGGCAACCAGATCATG"C CGACAAGCGCAACGTGATCCTGTTCTCCGTGTTTGATGAAAACAGATCCTGGTACCTCACTGAAAACATCCAGAGGT"CC"CCCAAAC CCCGCAGGAG"GCAAC"GGAGGACCCTGAGTTTCAGGCCTCGAATATCATGCACTCGATTAACGGTTACGTG""CGAC"CGC"GCAGC r1GAGCG"G"GCC""CCA"GAAGTCGCTTACTGGTACATTCTGTCCATCGGCGCCCAGACTGACTTCCTGAGCG"GT"C""T"CCGGT"A CACC"T"AAGCACAAGA"GG"G"ACGAAGATACCCTGACCCTGTTCCCTTTCTCCGGCGAAACGG"G""CA"G"CGA"GGAGAACCCG GGTC"G"GGA""C"GGGATGCCACAACAGCGACTTTCGGAACCGCGGAATGACTGCCCTGCTGAAGG"G"CC"CA"GCGACAAGAACA CCGGAGAC"AC"ACGAGGAC"CCTACGAGGATATCTCAGCCTACCTCCTGTCCAAGAACAACGCGA"CGAGCCGCGCAGC"TCAGCCA GCC"G"GCTGAAGAGGCACCAGCGAGAAATTACCCGGACCACCCTCCAATCGGATCAGGAGGAAA"CGACTACGACGACACC A"CTCGG"GGAAA"GAAGAAGGAAGATTTCGATATCTACGACGAGGACGAAAATCAGTCCCCTCGC"CA"TCCAAAAGAAAACTAGAC AC"AC"""A"CGCCGCGG"GGAAAGACTGTGGGACTATGGAATGTCATCCAGCCCTCACGTCCT"CGGAACCGGGCCCAGAGCGGATC GG"GCC"CAG""CAAGAAAG"GGTGTTCCAGGAGTTCACCGACGGCAGCTTCACCCAGCCGCTG"ACCGGGGAGAACTGAACGAACAC C"GGGCC"GC"CGG"CCC"ACATCCGCGCGGAAGTGGAGGATAACATCATGGTGACCTTCCGTAACCAAGCATCCAGACC"TACTCCr1 r1C"A""CC"CCCTG r1CTCA"ACGAGGAGGACCAGCGCCAAGGCGCCGAGCCCCGCAAGAACTTCGTCAAGCCCAACGAGAC"AAGAC C"AC""C"GGAAGG"CCAACACCATATGGCCCCGACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACT"CTCCGACG"GGACCTr1 GAGAAGGA"G"CCA""CCGGCCTGATCGGGCCGCTGCTCGTGTGTCACACCAACACCCTGAACCCAGCGCAHGGACGCCAGG"CACCG r1CCAGGAG'T"GCTC”GTTCHTCACCATTTTTGACGAAACTAAGTCCTGGTACTTCACCGAGAATATGGAGCGAAACTGTAGAGCGCC C"GCAATA"CCAGA”GGAAGATCCGACTTTCAAGGAGAACTATAGATTCCACGCCATCAACGGGTACATCAHGGATACTC"GCCGGGG C"GGTCATGGCCCAGGATCAGAGGATTCGGTGGTACTTGCTGTCAATGGGATCGAACGAAAACATTCACTCCATTCACTTCTCCGGTC ACGTGTTCAC”GTGCGCAAGAAGGAGGAGTACAAGATGGCGCTGTACAATCTGTACCCCGGGGTGTTCGAAACTGTGGAGATGCTGCC GGCCGGCAHCTGGAGAGTGGAGTGCCTGATCGGAGAGCACCTCCACGCGGGGATGTCCACCCTCTHCCTGGTGTACTCGAAT AAGTGCCAGACCCCGCTGGGCATGGCCTCGGGCCACATCAGAGACTTCCAGATCACAGCAAGCGGACAATACGGCCAATGGGCGCCGA AGCTGGCCCGCTTGCACTACTCCGGATCGATCAACGCATGGTCCACCAAGGAACCGTTCTCGTGGATTAAGGTGGACCTCCTGGCCCC TATGATTATCCACGGAATTAAGACCCAGGGCGCCAGGCAGAAGTTCTCCTCCCTGTACATCTCGCAATTCAHCATCATGTACAGCCTG GACGGGAAGAAGTGGCAGACTTACAGGGGAAACTCCACCGGCACCCTGATGGTCTTTTTCGGCAACGTGGATTCCTCCGGCATTAAGC ACAACATCTTCAACCCACCGATCATAGCCAGATATATTAGGCTCCACCCCACTCACTACTCAATCCGCTCAACTCTTCGGATGGAACT CATGGGGTGCGACCTGAACTCCTGCTCCATGCCGTTGGGGATGGAATCAAAGGCTATTAGCGACGCCCAGATCACCGCGAGCTCCTAC AACATGTTCGCCACCTGGAGCCCCTCCAAGGCCAGGCTGCACTTGCAGGGACGGTCAAATGCCTGGCGGCCGCAAGTGAACA ATCCGAAGGAATGGCTTCAAGTGGATTTCCAAAAGACCATGAAAGTGACCGGAGTCACCACCCAGGGAGTGAAGTCCCTTCTGACCTC GATGTATGTGAAGGAGTTCCTGATTAGCAGCAGCCAGGACGGGCACCAGTGGACCCTGTTCTTCCAAAACGGAAAGGTCAAGGTGTTC CAGGGGAACCAGGACTCGTTCACACCCGTGGTGAACTCCCTGGACCCCCCACTGCTGACGCGGTACTTGAGGATTCATCCTCAGTCCT GGGTCCATCAGATTGCATTGCGAATGGAAGTCCTGGGCTGCGAGGCCCAGGACCTGTACTGA : l-52 - SE ID NO: 3 ATGCAAATCGAAC"GAGCACCTGTTTCTTCCTCTGCCTGCTGAGATTCTGTTTCTCCGCGACCCGCCGATAC"ACCTGGGAGC AG"GGAGC"CTCC"GGGATTACATGCAGAGCGACCTTGGGGAGCTGCCCGTGGATGCCAGGTTCCCTCCCCGGGTGCCAAAGT CG"TTCCG""CAACACCTCCGTGGTGTACAAGAAAACTCTGTTCGTGGAGTTCACCGACCACCTGTTCAA"A"CGCCAAGCCC AGACCTCCC"GGAHGGGGCTGTTGGGACCTACCATCCAAGCGGAGGTGTACGACACTGTGGTCATCACTC"GAAGAACA"GGC CTCGCATCCCGTG”CCCTGCACGCCGTGGGAGTGTCTTACTGGAAAGCGTCCGAGGGGGCCGAATACGACGACCAGACC"CGC AGAGAGAAAAGGAAGATGACAAGGTGTTCCCAGGAGGATCGCACACCTACGTGTGGCAAGTGTTGAAGGAGAACGGCCCAATG GCCTCCGACCCGC”GTGCCTGACCTACTCGTACCTGTCCCACGTGGACCTCGTGAAGGACCTCAACTCGGGACTGATTGGAGC CCTGCTGGTCTGCAGGGAAGGCTCACTGGCGAAAGAAAAGACTCAGACCTTGCACAAGTTCATTCTGCTGTTCGCTGTGHTCG ACGAGGGGAAGTCGTGGCACAGCGAGACTAAGAACTCCCTGATGCAAGATAGAGATGCCGCCTCCGCCCGGGCCTGGCCHAAG ATGCACACCGTGAACGGTTACGTGAACCGCTCCCTCCCTGGCCTGATTGGATGCCACCGGAAGTCCGTGTACTGGCACGTGAT CGGGATGGGGACCACCCCCGAGGTGCACAGCATCTTCCTGGAAGGTCACACATTTCTCGTGCGCAACCACCGGCAGGCCTCCC TGGAAATCAGCCCCATTACCTTCCTCACTGCCCAGACTCTGCTGATGGACCTGGGACAGTTCCTGCTGTTCTGCCATATCTCC TCCCACCAACATGACGGAATGGAGGCATACGTGAAGGTCGATTCCTGCCCTGAGGAACCCCAGCTCCGCATGAAGAACAATGA GGAAGCCGAGGACTACGACGACGACCTGACGGATAGCGAGATGGATGTGGTCCGGTTCGATGACGATAACAGCCCTTCCTTCA TCCAAATTCGCTCGGTGGCAAAGAAGCACCCCAAGACCTGGGTGCATTACATTGCGGCGGAAGAAGAGGACTGGGATTATGCC CCGCTTGTCCTCGCTCCTGACGACCGGAGCTACAAGAGCCAGTACCTGAACAACGGTCCACAGAGGATCGGTAGAAAGTACAA GAAGGTCCGCTTCATGGCCTATACCGACGAAACCTTCAAAACTAGAGAGGCCATCCAACACGAATCCGGCATCCTGGGCCCGC r1C""G"ACGGAGAAGTCGGCGACACCCTTCTCATTATCTTCAAGAACCAGGCTTCCCGGCCGTACAACATCTATCCGCA"GGG ATCAC"GACG"GCGCCCACTGTACTCGCGGCGCCTGCCCAAGGGTGTCAAACACCTGAAGGATTTTCCGATCC""CCGGGAGA AA"CT"CAAG"ACAAGTGGACCGTGACCGTGGAAGATGGCCCAACTAAGTCTGACCCTAGATGCCTCACCCGC"ACTAC"CAT CC""CGTCAACATGGAGCGCGACCTGGCCAGCGGACTGATCGGCCCGCTGCTGATTTGCTACAAGGAATCAGTGGACCAACGG GGAAACCAGA"CATGTCGGATAAGAGGAACGTCATCCTCTTCTCCGTGTTTGACGAAAACCGGTCGTGGTACCTGACCGAGAA CATCCAGAGGTTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCCGAGTTCCAGGCCAGCAACATCATGCACAGCATCA ATGGCTACGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAGGTGGCCTACTGGTACATCCTGAGCATCGGCGCCCAG ACCGACTTCCTGAGCGTGTTCTTCTCTGGCTACACCTTCAAGCACAAGATGGTGTATGAGGACACCCTGACCCTGTTCCCCTT CAGCGGGGAGACTGTCTTCATGAGCATGGAGAACCCTGGCCTGTGGATCCTGGGCTGCCACAACAGCGACTTCAGGAACAGGG GCATGACTGCCCTGCTGAAAGTCTCCAGCTGTGACAAGAACACCGGGGACTACTACGAGGACAGCTACGAGGACATCAGCGCC TACCTGCTGAGCAAGAACAATGCCATCGAGCCCAGGAGCTTCTCTCAGAACCCCCCAGTGCTGAAGAGGCACCAGAGGGAGAT CACCAGGACCACCCTGCAGTCTGACCAGGAGGAGATCGACTATGATGACACCATCAGCGTGGAGATGAAGAAGGAGGACTTCG ACATCTACGACGAGGACGAGAACCAGAGCCCCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTGCTGCTGTGGAGAGG CTGTGGGACTATGGCATGTCCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAGTCTGGCAGCGTGCCCCAGTTCAAGAAAGT CGTGTTCCAGGAGTTCACCGACGGCAGCTTCACCCAGCCCCTGTACAGAGGGGAGCTGAACGAGCACCTGGGCCTGCTGGGCC CCTACATCAGGGCCGAGGTGGAGGACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGC CTGATCAGCTACGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAGAACTTTGTGAAGCCCAATGAAACCAAGACCTACTT CTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGACGAGTTCGACTGCAAGGCCTGGGCCTACTTCTCTGACGTGGACCTGG AGAAGGACGTGCACTCTGGCCTGATTGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTG ACTGTGCAGGAGTTCGCCCTGTTCTTCACCATCTTCGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTG CAGGGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGG ACACCCTGCCTGGCCTGGTCATGGCCCAGGACCAGAGGATCAGGTGGTATCTGCTGAGCATGGGCAGCAACGAGAACATCCAC CACTTCTCTGGCCACGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCTGGGGT AACCGTGGAGATGCTGCCCAGCAAGGCCGGCATCTGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCACGCCGGCA TGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATC ACTGCCTCTGGCCAGTACGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCCGGAAGCATCAATGCCTGGAGCACCAA GGAGCCCTTCAGCTGGATCAAAGTGGACCTGCTGGCCCCCATGATCATCCACGGCATCAAGACCCAGGGGGCCAGGCAGAAGT TCTCCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACAGGGGCAACAGCACC GGCACCCTGATGGTGTTCTTCGGCAACGTGGACAGCAGCGGCATCAAGCACAACATCTTCAACCCCCCCATCATCGCCAGATA CATCAGGCTGCACCCCACCCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCA TGCCCCTGGGCATGGAGAGCAAGGCCATCTCTGACGCCCAGATCACTGCCTCCAGCTACTTCACCAACATGTTTGCCACCTGG AGCCCCAGCAAGGCCAGGCTGCACCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCAGGTCAACAACCCCAAGGAGTGGCTGCA CTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTACGTGAAGG AGTTCCTGATCTCCAGCAGCCAGGACGGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAAC CAGGACAGCTTCACCCCTGTGGTCAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATCCACCCCCAGAGCTGGGT GCACCAGATCGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGA Solid underline = coFVIII—52—NT58 (SEQ ID NO: 7) Qgghgguqngg?ing: coFVIII—52—CT (SEQ ID NO: 8) : l-62 - SE ID NO: 4 AT"GAGC"GTCCACTTGTTTCTTCCTGTGCCTCCTGCGCTTCTGTTTCTCCGCCACTCGCCGGTAC"ACCTTGGAGC CG"GGAGC"""CA"GGGACTACATGCAGAGCGACCTGGGCGAACTCCCCGTGGATGCCAGATTCCCCCCCCGCGTGCCAAAGT CC"TCCCC"""AACACCTCCGTGGTGTACAAGAAAACCCTCTTTGTCGAGTTCACTGACCACCTGTTCAACA"CGCCAAGCCG CGCCCACC""GGA”GGGCCTCCTGGGACCGACCATTCAAGCTGAAGTGTACGACACCGTGGTGATCACCCTGAAGAACA"GGC GTCCCACCCCGTG”CCCTGCATGCGGTCGGAGTGTCCTACTGGAAGGCCTCCGAAGGAGCTGAGTACGACGACCAGACTAGCC AGCGGGAAAAGGAGGACGATAAAGTGTTCCCGGGCGGCTCGCATACTTACGTGTGGCAAGTCCTGAAGGAAAACGGACC"ATG GCATCCGA”CCTC”GTGCCTGACTTACTCCTACCTTTCCCATGTGGACCTCGTGAAGGACCTGAACAGCGGGCTGATTGGTGC ACTTCTCGHGTGCCGCGAAGGTTCGCTCGCTAAGGAAAAGACCCAGACCCTCCATAAGTTCATCCTTTTGTTCGCTGTGHTCG ATGAAGGAAAGTCATGGCATTCCGAAACTAAGAACTCGCTGATGCAGGACCGGGATGCCGCCTCAGCCCGCGCCTGGCCHAAA ATGCATACAGTCAACGGATACGTGAATCGGTCACTGCCCGGGCTCATCGGTTGTCACAGAAAGTCCGTGTACTGGCACGTCAT CGGCATGGGCACTACGCCTGAAGTGCACTCCATCTTCCTGGAAGGGCACACCTTCCTCGTGCGCAACCACCGCCAGGCCTCTC TGGAAATCTCCCCGATTACCTTTCTGACCGCCCAGACTCTGCTCATGGACCTGGGGCAGTTCCTTCTCTTCTGCCACATCTCC AGCCATCAGCACGACGGAATGGAGGCCTACGTGAAGGTGGACTCATGCCCGGAAGAACCTCAGTTGCGGATGAAGAACAACGA GGAGGCCGAGGACTATGACGACGATTTGACTGACTCCGAGATGGACGTCGTGCGGTTCGATGACGACAACAGCCCCAGCTTCA TCCAGATTCGCAGCGTGGCCAAGAAGCACCCCAAAACCTGGGTGCACTACATCGCGGCCGAGGAAGAAGATTGGGACTACGCC GTGCTGGCACCCGATGACCGGTCGTACAAGTCCCAGTATCTGAACAATGGTCCGCAGCGGATTGGCAGAAAGTACAA GAAAGTGCGGTTCATGGCGTACACTGACGAAACGTTTAAGACCCGGGAGGCCATTCAACATGAGAGCGGCATTCTGGGACCAC r1GC"G"ACGGAGAGGTCGGCGATACCCTGCTCATCATCTTCAAAAACCAGGCCTCCCGGCCTTACAACATCTACCC"CACGGA ATCACCGACG"GCGGCCACTCTACTCGCGGCGCCTGCCGAAGGGCGTCAAGCACCTGAAAGACTTCCCTATCC"GCCGGGCGA AA"CT"CAAG"A"AAGTGGACCGTCACCGTGGAGGACGGGCCCACCAAGAGCGATCCTAGGTGTCTGAC"CGG"AC"ACTCCA GC""CGTGAACA"GGAACGGGACCTGGCATCGGGACTCATTGGACCGCTGCTGATCTGCTACAAAGAG"CGGTGGA"CAACGC GGCAACCAGA"CATGTCCGACAAGCGCAACGTGATCCTGTTCTCCGTGTTTGATGAAAACAGATCCTGGTACCTGACCGAGAA CATCCAGAGGTTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCCGAGTTCCAGGCCAGCAACATCATGCACAGCATCA ATGGCTACGTGTTCGACAGCCTGCAGCTGAGCGTGTGCCTGCACGAGGTGGCCTACTGGTACATCCTGAGCATCGGCGCCCAG ACCGACTTCCTGAGCGTGTTCTTCTCTGGCTACACCTTCAAGCACAAGATGGTGTATGAGGACACCCTGACCCTGTTCCCCTT CAGCGGGGAGACTGTCTTCATGAGCATGGAGAACCCTGGCCTGTGGATCCTGGGCTGCCACAACAGCGACTTCAGGAACAGGG GCATGACTGCCCTGCTGAAAGTCTCCAGCTGTGACAAGAACACCGGGGACTACTACGAGGACAGCTACGAGGACATCAGCGCC TACCTGCTGAGCAAGAACAATGCCATCGAGCCCAGGAGCTTCTCTCAGAACCCCCCAGTGCTGAAGAGGCACCAGAGGGAGAT CACCAGGACCACCCTGCAGTCTGACCAGGAGGAGATCGACTATGATGACACCATCAGCGTGGAGATGAAGAAGGAGGACTTCG ACATCTACGACGAGGACGAGAACCAGAGCCCCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTGCTGCTGTGGAGAGG CTGTGGGACTATGGCATGTCCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAGTCTGGCAGCGTGCCCCAGTTCAAGAAAGT CGTGTTCCAGGAGTTCACCGACGGCAGCTTCACCCAGCCCCTGTACAGAGGGGAGCTGAACGAGCACCTGGGCCTGCTGGGCC CCTACATCAGGGCCGAGGTGGAGGACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGC CTGATCAGCTACGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAGAACTTTGTGAAGCCCAATGAAACCAAGACCTACTT CTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGACGAGTTCGACTGCAAGGCCTGGGCCTACTTCTCTGACGTGGACCTGG AGAAGGACGTGCACTCTGGCCTGATTGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTG ACTGTGCAGGAGTTCGCCCTGTTCTTCACCATCTTCGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTG CAGGGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGG ACACCCTGCCTGGCCTGGTCATGGCCCAGGACCAGAGGATCAGGTGGTATCTGCTGAGCATGGGCAGCAACGAGAACATCCAC AGCATCCACTTCTCTGGCCACGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCTGGGGT GTTCGAAACCGTGGAGATGCTGCCCAGCAAGGCCGGCATCTGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCACGCCGGCA TGAGCACCCTGTTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATC ACTGCCTCTGGCCAGTACGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCCGGAAGCATCAATGCCTGGAGCACCAA GGAGCCCTTCAGCTGGATCAAAGTGGACCTGCTGGCCCCCATGATCATCCACGGCATCAAGACCCAGGGGGCCAGGCAGAAGT TCTCCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACAGGGGCAACAGCACC GGCACCCTGATGGTGTTCTTCGGCAACGTGGACAGCAGCGGCATCAAGCACAACATCTTCAACCCCCCCATCATCGCCAGATA CATCAGGCTGCACCCCACCCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCA TGCCCCTGGGCATGGAGAGCAAGGCCATCTCTGACGCCCAGATCACTGCCTCCAGCTACTTCACCAACATGTTTGCCACCTGG AGCCCCAGCAAGGCCAGGCTGCACCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCAGGTCAACAACCCCAAGGAGTGGCTGCA GGTGGACTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTACGTGAAGG AGTTCCTGATCTCCAGCAGCCAGGACGGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAAC CAGGACAGCTTCACCCCTGTGGTCAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATCCACCCCCAGAGCTGGGT GCACCAGATCGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGA Solid underline = I—62—NT58 (SEQ ID NO: 9) Qgghgguqngg?ing: coFVIII—62—CT (SEQ ID NO: 10) : coFVlll-25 - SE ID NO: 5 AT"GAGC"GAGCACCTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAGATAC"ACCTGGGCGC CG"GGAGC"GAGC"GGGACTACATGCAGTCTGACCTGGGCGAGCTGCCAGTGGACGCCAGGTTCCCCCCCAGAGTGCCCAAGA GC"TCCCC""CAACACCAGCGTGGTGTACAAGAAGACCCTGTTCGTGGAGTTCACTGACCACCTGTTCAACA"CGCCAAGCCC AGGCCCCCC"GGA”GGGCCTGCTGGGCCCCACCATCCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGC CAGCCACCCCGTC”CCCTGCACGCCGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGCGCCGAGTACGACGACCAGACCAGCC AGAGGGAGAAGGAGGACGACAAGGTGTTCCCTGGGGGCAGCCACACCTACGTGTGGCAGGTCCTGAAGGAGAACGGCCCCATG GCCTCTGACCCCC”GTGCCTGACCTACAGCTACCTGAGCCACGTGGACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGC CCTGCTGGTGTGCAGGGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCG ACGAGGGCAAGAGCTGGCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGACGCCGCCTCTGCCAGGGCCTGGCCCAAG ATGCACACCGTCAACGGCTACGTCAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGAGCGTGTACTGGCATGTGAT CGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTCAGGAACCACAGGCAGGCCAGCC TGGAGATCAGCCCCATCACCTTCCTGACCGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCTCC AGCCACCAGCACGACGGCATGGAGGCCTACGTGAAAGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAACGA GGAGGCCGAGGACTATGATGACGACCTGACCGACAGCGAGATGGACGTGGTCAGGTTCGACGACGACAACAGCCCCAGCTTCA TCCAGATCAGGAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATCGCTGCTGAGGAGGAGGACTGGGACTATGCC CCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCTGAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAA GAAAGTCAGGTTCATGGCCTACACTGAT AAACCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCC r1GC"G"ACGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTACCCCCA’"GGC ATCACCGACG"GAGGCCCCTGTACAGCAGGAGGCTGCCTAAGGGGGTGAAGCACCTGAAAGACTTCCCCA"CC"GCCTGGGGA GA"CT"CAAG"ACAAGTGGACTGTGACTGTGGAGGACGGCCCCACCAAGAGCGACCCCAGGTGCCTGACCAGA"ACTACAGCA GC""CGTCAACATGGAGAGGGACCTGGCCTCTGGCCTGATTGGCCCCCTGCTGATCTGCTACAAGGAGTC"GTGGACCAGAGG GGCAACCAGA"CATGAGCGACAAGAGGAACGTGATCCTGTTCTCTGTCTTCGACGAGAACAGGAGCTGGTACCTGACTGAAAA CATCCAGCGGTTCCTCCCCAACCCCGCGGGCGTGCAGCTGGAAGATCCTGAGTTTCAGGCATCAAACATCATGCACTCCATTA ACGTGTTCGATTCGCTGCAGCTGAGCGTGTGTCTGCACGAAGTGGCCTACTGGTACATCCTGTCCATTGGTGCCCAG ACTGACTTCCTGTCCGTGTTTTTCTCCGGCTACACGTTCAAGCACAAGATGGTGTACGAGGACACCCTGACCCTCTTCCCTTT TTCCGGCGAAACTGTGTTTATGAGCATGGAGAATCCCGGCCTGTGGATCTTGGGCTGCCACAACAGCGACTTCCGTAACAGAG GAATGACTGCGCTGCTCAAGGTGTCCAGCTGCGACAAGAACACCGGAGACTATTATGAGGACTCATACGAGGACATCTCCGCC TACCTCCTGTCCAAGAATAACGCCATTGAACCTCGGAGCTTCAGCCAGAACCCACCCGTGCTTAAGAGACATCAACGGGAGAT CACTAGGACCACCCTGCAGTCAGACCAGGAGGAAATCGACTACGATGACACCATCTCGGTCGAGATGAAGAAGGAGGACTTTG ACATCTACGACGAAGATGAAAACCAGAGCCCGAGGTCGTTCCAAAAGAAAACCCGCCACTACTTTATTGCTGCTGTCGAGCGG CTGTGGGACTACGGAATGTCGTCCTCGCCGCACGTGCTCCGCAACCGAGCCCAGAGCGGCTCGGTGCCGCAATTCAAGAAGGT CGTGTTCCAGGAGTTCACTGACGGGAGCTTCACTCAGCCTTTGTACCGGGGAGAACTCAATGAACATCTCGGCCTCCTCGGAC CTTACATCAGAGCAGAAGTGGAAGATAACATCATGGTCACTTTCCGTAACCAAGCCAGCCGCCCGTACTCGTTCTACTCCTCC CTCATTTCTTACGAAGAGGACCAGCGGCAGGGCGCAGAACCGCGCAAGAACTTCGTGAAGCCCAACGAAACCAAGACCTACTT CTGGAAAGTGCAGCATCATATGGCCCCGACTAAGGACGAGTTTGACTGCAAAGCCTGGGCCTACTTCTCCGATGTGGACTTGG AGAAGGACGTCCACTCCGGCCTCATCGGTCCCCTGCTCGTGTGCCATACCAATACCCTGAACCCCGCACACGGTCGCCAGGTC CAGGAGTTCGCTCTGTTCTTCACTATCTTCGACGAAACTAAGTCCTGGTACTTCACCGAGAACATGGAGAGGAACTG CAGAGCCCCCTGTAACATCCAGATGGAGGACCCGACGTTCAAGGAAAACTACCGGTTCCACGCCATTAACGGATACATCATGG ATACGCTGCCGGGTCTTGTGATGGCCCAGGATCAACGGATCAGATGGTACTTATTGTCGATGGGCAGCAACGAGAACATCCAC CACTTCTCCGGTCATGTGTTCACTGTGCGGAAGAAGGAAGAGTACAAGATGGCCCTGTACAACCTTTATCCCGGAGT GTTCGAAACTGTGGAAATGCTGCCGTCGAAGGCCGGCATTTGGCGCGTGGAGTGTTTGATTGGAGAACATCTCCATGCGGGGA TGTCAACCCTGTTCCTGGTGTATAGCAACAAGTGCCAGACTCCGCTTGGGATGGCGTCAGGACACATTAGGGATTTCCAGATC ACTGCGTCCGGCCAGTACGGCCAATGGGCCCCTAAGCTGGCCCGCCTGCATTACTCCGGATCCATTAACGCCTGGTCAACCAA ATTCTCCTGGATCAAGGTGGACCTTCTGGCCCCCATGATTATCCACGGAATTAAGACCCAGGGGGCCCGGCAGAAGT TCTCCTCACTGTACATCAGCCAGTTCATAATCATGTACTCCCTGGACGGAAAGAAGTGGCAAACCTACAGGGGGAACAGCACC GGCACACTGATGGTCTTTTTCGGAAATGTGGACTCCTCCGGGATTAAGCATAACATCTTCAACCCTCCGATTATCGCTCGGTA CATTAGACTTCACCCTACCCACTACAGCATTCGCTCCACCCTGCGGATGGAACTGATGGGCTGCGATCTGAACTCGTGCAGCA TGCCGTTGGGAATGGAGTCCAAAGCAATTTCCGACGCGCAGATCACCGCCTCGTCCTACTTTACCAACATGTTCGCCACGTGG TCACCGTCCAAGGCCCGGCTGCACCTCCAGGGAAGATCCAACGCATGGCGGCCACAGGTCAACAACCCTAAGGAGTGGCTCCA GGTGGACTTCCAGAAAACCATGAAGGTCACCGGAGTCACAACCCAGGGAGTGAAGTCGCTGCTGACTTCTATGTACGTCAAGG AGTTCCTGATCTCCAGCAGCCAGGACGGGCACCAGTGGACCCTGTTCTTCCAAAATGGAAAGGTCAAGGTGTTTCAGGGCAAT CAGGATTCATTCACCCCGGTGGTGAACTCCCTTGATCCACCCCTCCTGACCCGCTACCTTCGCATCCACCCACAGTCCTGGGT GCACCAGATCGCGCTGAGGATGGAGGTCCTGGGATGCGAAGCCCAGGACCTGTACTGA Solid underline = coFVIII—25—NT58 (SEQ ID NO: 11) Qgghgguqngg?ing: coFVIII—25—CT (SEQ ID NO: 12) : coFVlll-26 — SE ID NO: 6 ATGCAGAT"GAGC"GAGCACCTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAGATAC"ACCTGGGCGC CG"GGAGC"GAGC"GGGACTACATGCAGTCTGACCTGGGCGAGCTGCCAGTGGACGCCAGGTTCCCCCCCAGAGTGCCCAAGA GC"TCCCC""CAACACCAGCGTGGTGTACAAGAAGACCCTGTTCGTGGAGTTCACTGACCACCTGTTCAACA"CGCCAAGCCC AGGCCCCCC"GGA”GGGCCTGCTGGGCCCCACCATCCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTGAAGAACATGGC CAGCCACCCCGTC”CCCTGCACGCCGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGCGCCGAGTACGACGACCAGACCAGCC AGAGGGAGAAGGAGGACGACAAGGTGTTCCCTGGGGGCAGCCACACCTACGTGTGGCAGGTCCTGAAGGAGAACGGCCCCATG GCCTCTGACCCCC”GTGCCTGACCTACAGCTACCTGAGCCACGTGGACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGC CCTGCTGGTGTGCAGGGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCG ACGAGGGCAAGAGCTGGCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGACGCCGCCTCTGCCAGGGCCTGGCCCAAG ATGCACACCGTCAACGGCTACGTCAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGAGCGTGTACTGGCATGTGAT CGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTCAGGAACCACAGGCAGGCCAGCC TGGAGATCAGCCCCATCACCTTCCTGACCGCCCAGACCCTGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCTCC AGCCACCAGCACGACGGCATGGAGGCCTACGTGAAAGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAACGA GGAGGCCGAGGACTATGATGACGACCTGACCGACAGCGAGATGGACGTGGTCAGGTTCGACGACGACAACAGCCCCAGCTTCA TCCAGATCAGGAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATCGCTGCTGAGGAGGAGGACTGGGACTATGCC CCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCTGAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAA GAAAGTCAGGTTCATGGCCTACACTGAT AAACCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCC r1GC"G"ACGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTACCCCCA’"GGC ATCACCGACG"GAGGCCCCTGTACAGCAGGAGGCTGCCTAAGGGGGTGAAGCACCTGAAAGACTTCCCCA"CC"GCCTGGGGA GA"CT"CAAG"ACAAGTGGACTGTGACTGTGGAGGACGGCCCCACCAAGAGCGACCCCAGGTGCCTGACCAGA"ACTACAGCA GC""CGTCAACATGGAGAGGGACCTGGCCTCTGGCCTGATTGGCCCCCTGCTGATCTGCTACAAGGAGTC"GTGGACCAGAGG GGCAACCAGA"CATGAGCGACAAGAGGAACGTGATCCTGTTCTCTGTCTTCGACGAGAACAGGAGCTGGTACCTCACTGAAAA CATCCAGAGGTTCCTCCCAAACCCCGCAGGAGTGCAACTGGAGGACCCTGAGTTTCAGGCCTCGAATATCATGCACTCGATTA ACGGTTACGTGTTCGACTCGCTGCAGCTGAGCGTGTGCCTCCATGAAGTCGCTTACTGGTACATTCTGTCCATCGGCGCCCAG ACTGACTTCCTGAGCGTGTTCTTTTCCGGTTACACCTTTAAGCACAAGATGGTGTACGAAGATACCCTGACCCTGTTCCCTTT CTCCGGCGAAACGGTGTTCATGTCGATGGAGAACCCGGGTCTGTGGATTCTGGGATGCCACAACAGCGACTTTCGGAACCGCG GAATGACTGCCCTGCTGAAGGTGTCCTCATGCGACAAGAACACCGGAGACTACTACGAGGACTCCTACGAGGATATCTCAGCC CTGTCCAAGAACAACGCGATCGAGCCGCGCAGCTTCAGCCAGAACCCGCCTGTGCTGAAGAGGCACCAGCGAGAAAT TACCCGGACCACCCTCCAATCGGATCAGGAGGAAATCGACTACGACGACACCATCTCGGTGGAAATGAAGAAGGAAGATTTCG ATATCTACGACGAGGACGAAAATCAGTCCCCTCGCTCATTCCAAAAGAAAACTAGACACTACTTTATCGCCGCGGTGGAAAGA CTGTGGGACTATGGAATGTCATCCAGCCCTCACGTCCTTCGGAACCGGGCCCAGAGCGGATCGGTGCCTCAGTTCAAGAAAGT GGTGTTCCAGGAGTTCACCGACGGCAGCTTCACCCAGCCGCTGTACCGGGGAGAACTGAACGAACACCTGGGCCTGCTCGGTC CCTACATCCGCGCGGAAGTGGAGGATAACATCATGGTGACCTTCCGTAACCAAGCATCCAGACCTTACTCCTTCTATTCCTCC CTGATCTCATACGAGGAGGACCAGCGCCAAGGCGCCGAGCCCCGCAAGAACTTCGTCAAGCCCAACGAGACTAAGACCTACTT CTGGAAGGTCCAACACCATATGGCCCCGACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACTTCTCCGACGTGGACCTTG AGAAGGATGTCCATTCCGGCCTGATCGGGCCGCTGCTCGTGTGTCACACCAACACCCTGAACCCAGCGCATGGACGCCAGGTC CAGGAGTTTGCTCTGTTCTTCACCATTTTTGACGAAACTAAGTCCTGGTACTTCACCGAGAATATGGAGCGAAACTG TAGAGCGCCCTGCAATATCCAGATGGAAGATCCGACTTTCAAGGAGAACTATAGATTCCACGCCATCAACGGGTACATCATGG ATACTCTGCCGGGGCTGGTCATGGCCCAGGATCAGAGGATTCGGTGGTACTTGCTGTCAATGGGATCGAACGAAAACATTCAC TCCATTCACTTCTCCGGTCACGTGTTCACTGTGCGCAAGAAGGAGGAGTACAAGATGGCGCTGTACAATCTGTACCCCGGGGT GTTCGAAACTGTGGAGATGCTGCCGTCCAAGGCCGGCATCTGGAGAGTGGAGTGCCTGATCGGAGAGCACCTCCACGCGGGGA TGTCCACCCTCTTCCTGGTGTACTCGAATAAGTGCCAGACCCCGCTGGGCATGGCCTCGGGCCACATCAGAGACTTCCAGATC ACAGCAAGCGGACAATACGGCCAATGGGCGCCGAAGCTGGCCCGCTTGCACTACTCCGGATCGATCAACGCATGGTCCACCAA GGAACCGTTCTCGTGGATTAAGGTGGACCTCCTGGCCCCTATGATTATCCACGGAATTAAGACCCAGGGCGCCAGGCAGAAGT CCCTGTACATCTCGCAATTCATCATCATGTACAGCCTGGACGGGAAGAAGTGGCAGACTTACAGGGGAAACTCCACC GGCACCCTGATGGTCTTTTTCGGCAACGTGGATTCCTCCGGCATTAAGCACAACATCTTCAACCCACCGATCATAGCCAGATA TATTAGGCTCCACCCCACTCACTACTCAATCCGCTCAACTCTTCGGATGGAACTCATGGGGTGCGACCTGAACTCCTGCTCCA TGCCGTTGGGGATGGAATCAAAGGCTATTAGCGACGCCCAGATCACCGCGAGCTCCTACTTCACTAACATGTTCGCCACCTGG AGCCCCTCCAAGGCCAGGCTGCACTTGCAGGGACGGTCAAATGCCTGGCGGCCGCAAGTGAACAATCCGAAGGAATGGCTTCA AGTGGATTTCCAAAAGACCATGAAAGTGACCGGAGTCACCACCCAGGGAGTGAAGTCCCTTCTGACCTCGATGTATGTGAAGG TGATTAGCAGCAGCCAGGACGGGCACCAGTGGACCCTGTTCTTCCAAAACGGAAAGGTCAAGGTGTTCCAGGGGAAC CAGGACTCGTTCACACCCGTGGTGAACTCCCTGGACCCCCCACTGCTGACGCGGTACTTGAGGATTCATCCTCAGTCCTGGGT CCATCAGATTGCATTGCGAATGGAAGTCCTGGGCTGCGAGGCCCAGGACCTGTACTGA Solid underline = I—26—NT58 (SEQ ID NO: 13) Qgghgguqngg?ing: coFVIII—26—CT (SEQ ID NO: 14) :BDD-FVIII non-o timized-" arental" Nucleotide Seguence (SEQ ID NO: 16) ATGCAAATAGAGCTCTCCACCTGCTTCTITCTGTGCCTT'ITGCGATI'CTGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTG GAACTGTCATGGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGATTTCCTCCTAGAGTG CCAAAATC'I'ITI'CC A'I'I'CAACACCTCAGTCGTGTACAAAAAGACTCTGTTTGTAGAATTCACGGATCACCTTITCAACATCGCTAAGCCAAGGCCACCCT GGATGGGTCTGCTAGGTCCTACCATCCAGGCTGAGGTTTATGATACAGTGGTCATTACACTTAAGAACATGGCTTCCCATCCTGTC AGTCTTCATGCTGTTGGTGTATCCTACTGGAAAGCTTCTGAGGGAGCTGAATATGATGATCAGACCAGTCAAAGGGAGAAAGAA GATGATAAAGTCTTCCCTGGTGGAAGCCATACATATGTCTGGCAGGTCCTGAAAGAGAATGGTCCAATGGCCTCTGACCCACTGT GCCTTACCTACTCATATCTITCTCATGTGGACCTGGTAAAAGACTI'GAATTCAGGCCTCATTGGAGCCCTACTAGTATGTAGAGAA GGGAGTCTGGCCAAGGAAAAGACACAGACCTTGCACAAA'ITI'ATACTACTTITTGCTGTATITGATGAAGGGAAAAGTTGGCACT CAGAAACAAAGAACTCCTTGATGCAGGATAGGGATGCTGCATCTGCTCGGGCCTGGCCTAAAATGCACACAGTCAATGG'I'I'ATGT AAACAGGTCTCTGCCAGGTCTGATTGGATGCCACAGGAAATCAGTCTATI'GGCATGTGA'I'I'GGAATGGGCACCACTCCTGAAGTG CACTCAATATI'CCTCGAAGGTCACACATI'TCTI'GTGAGGAACCATCGCCAGGCGTCC'I'I'GGAAATCTCG CCAATAAC'ITI'CCTI'ACT GCTCAAACACTCTI'GATGGACCTI'GGACAGTI'TCTACTGTITI'GTCATATCTC'I'I'CCCACCAACATGATGGCATGGAAGC'ITATGTC AAAGTAGACAGCTGTCCAGAGGAACCCCAACTACGAATGAAAAATAATGAAGAAGCGGAAGACTATGATGATGATC'I'I'ACTGAT TCTGAAATGGATGTGGTCAGGTI'I'GATGATGACAACTCTCCTTCCTTI'ATCCAAATI'CGCTCAG'I'I'GCCAAGAAGCATCCTAAAACT TGGGTACATTACATTGCTGCTGAAGAGGAGGACTGGGACTATGCTCCCTTAGTCCTCGCCCCCGATGACAGAAGTTATAAAAGTC AATATTTGAACAATGGCCCTCAGCGGATTGGTAGGAAGTACAAAAAAGTCCGATI'I'ATGGCATACACAGATGAAACCT'I'I'AAGAC TCGTGAAGCTATTCAGCATGAATCAGGAATCTI'GGGACCTTI'ACT'ITATGGGGAAGTI'GGAGACACACTG'I'I'GA'I'I'ATA'ITI'AAGA ATCAAGCAAGCAGACCATATAACATCTACCCTCACGGAATCACTGATGTCCGTCC'ITI'GTA'I'I'CAAGGAGATTACCAAAAGGTGTA AAACA'I'I'TGAAGGATTTTCCAATTCTGCCAGGAGAAATATTCAAATATAAATGGACAGTGACTGTAGAAGATGGGCCAACTAAAT CAGATCCTCGGTGCCTGACCCGCTATTACTCTAGTTTCGTTAATATGGAGAGAGATCTAGCTTCAGGACTCATTGGCCCTCTCCTCA TCTGCTACAAAGAATCTGTAGATCAAAGAGGAAACCAGATAATGTCAGACAAGAGGAATGTCATCCTG'ITI'I'CTGTATI'I'GATGA GAACCGAAGCTGGTACCTCACAGAGAATATACAACGCTITCTCCCCAATCCAGCTGGAGTGCAGCTTGAGGATCCAGAG'I'I'CCAA GCCTCCAACATCATGCACAGCATCAATGGCTATG'I'I'TITGATAGTITGCAGTTGTCAGTTTGTTTGCATGAGGTGGCATACTGGTA AAGCATTGGAGCACAGACTGACTTCCT'ITCTGTCTTCTTCTCTGGATATACCTTCAAACACAAAATGGTCTATGAAGACAC ACTCACCCTA'I'I'CCCATI'CTCAGGAGAAACTGTC'I'I'CATGTCGATGGAAAACCCAGGTCTATG GA'I'I'CTGGGGTGCCACAACTCAG AC'ITI'CGGAACAGAGGCATGACCGCCTTACTGAAGGTTI'CTAG'ITGTGACAAGAACACTGGTGATTA'I'I'ACGAGGACAGTTATGA AGATAT'I'I'CAGCATACTTGCTGAGTAAAAACAATGCCA'ITGAACCAAGAAGCTTCTCTCAAAACCCACCAGTCTTGAAACGCCATC AACGGGAAATAACTCGTACTACTCTI'CAGTCAGATCAAGAGGAAATI'GACTATGATGATACCATATCAGTTGAAATGAAGAAGGA AGATTTTGACA'ITI'ATGATGAGGATGAAAATCAGAGCCCCCGCAGCTTTCAAAAGAAAACACGACACTATTTTATTGCTGCAGTGG AGAGGCTCTGG GA'I'I'ATG GGATGAGTAGCTCCCCACATGTI'CTAAGAAACAGGGCTCAGAGTGGCAGTGTCCCTCAGTTCAAGA AAG'I'I'G'ITI'TCCAG GAATI'TACTGATGGCTCCTTTACTCAGCCCTI'ATACCGTGGAGAACTAAATGAACA'ITI'GGGACTCCTGGGG CCATATATAAGAGCAGAAG'I'I'GAAGATAATATCATGGTAACTI'TCAGAAATCAGGCCTCTCGTCCCTA'I'I'CCTI'CTATTCTAGCCTT A'ITI'CTI'ATGAGGAAGATCAGAGGCAAGGAGCAGAACCTAGAAAAAACTITGTCAAGCCTAATGAAACCAAAACTTACT'ITI'GGA AAGTGCAACATCATATGGCACCCACTAAAGATGAGTTTGACTGCAAAGCCTGGGCTTATTTCTCTGATGTTGACCTGGAAAAAGAT GTGCACTCAGGCCTGATTGGACCCCTTCTGGTCTGCCACACTAACACACTGAACCCTGCTCATGGGAGACAAGTGACAGTACAGG AA'I'I'TGCTCTGTTTITCACCATCTITGATGAGACCAAAAGCTGGTACITCACTGAAAATATGGAAAGAAACTGCAGGGCTCCCTGC CAGATGGAAGATCCCACTITTAAAGAGAATTATCGCTTCCATGCAATCAATGGCTACATAATGGATACACTACCTGG C'I'I' AGTAATGGCTCAGGATCAAAGGATTCGATGGTATCTGCTCAGCATGGGCAGCAATGAAAACATCCATTCTATTCATITCAGTGGA TTCACTGTACGAAAAAAAGAGGAGTATAAAATGGCACTGTACAATCTCTATCCAGGTGTTITTGAGACAGTGGAAATGT TACCATCCAAAGCTGGAATTTGGCGGGTGGAATGCCTTA'ITGGCGAGCATCTACATGCTGGGATGAGCACACTITTTCTGGTGTA CAGCAATAAGTGTCAGACTCCCCTGGGAATGGCTTCTGGACACATI'AGAGATTTTCAGATTACAGCTTCAGGACAATATGGACAG TGGGCCCCAAAGCTGGCCAGACTTCATTATTCCGGATCAATCAATGCCTGGAGCACCAAGGAGCCC'I'I'TI'CTTGGATCAAGGTGG ATCTGTI'G ATGATTATI'CACGGCATCAAGACCCAGGGTGCCCGTCAGAAG'I'I'CTCCAGCCTCTACATCTCTCAG'ITI'ATCA TCATGTATAGTCTI'GATGGGAAGAAGTGGCAGACTI'ATCGAGGAAATTCCACTGGAACC'I'I'AATGGTCTTC'I'I'TGGCAATGTGGAT TCATCTGGGATAAAACACAATAT'I'I'TTAACCCTCCAATTATTGCTCGATACATCCG'ITI'GCACCCAACTCA'I'I'ATAGCATTCGCAGC ACTCTTCGCATGGAGTI'GATGGGCTGTGATI'I'AAATAGTI'GCAGCATGCCATI'GGGAATGGAGAGTAAAGCAATATCAGATGCAC AGA'I'I'ACTG CTTCATCCTACTTTACCAATATGT'ITGCCACCTGGTCTCCTI'CAAAAGCTCGAC'I'I'CACCTCCAAGGGAGGAGTAATG CCTGGAGACCTCAGGTGAATAATCCAAAAGAGTGGCTGCAAGTGGACTTCCAGAAGACAATGAAAGTCACAGGAGTAACTACTC AGGGAGTAAAATCTCTGCTTACCAGCATGTATGTGAAGGAGTTCCTCATCTCCAGCAGTCAAGATGGCCATCAGTGGACTCTCTI'T TTTCAGAATGGCAAAGTAAAGGTTITI'CAGGGAAATCAAGACTCCTI'CACACCTGTGGTGAACTCTCTAGACCCACCGTTACTGAC TCGCTACCTTCGAATTCACCCCCAGAG'I'I'GGGTGCACCAGATTGCCCTGAGGATGGAGGTTCTGGGCTGCGAGGCACAGGACCTC VIII = B Domain-Deleted FVIII 10/44 :BDD-FV||I non-o timized"' arental" Amino Acid ce (SEQ ID NO:17) ATRRYYLGAVELSWDYMQSDLG ELPVDARFPPRVPKSFPFNTSVVYKKTLFVE FTDH LF N IAKPRPPWMG LLG PTIQAEVYDTVVITLK N MASH PVSLHAVGVSYWKASEGAEYDDQTSQRE KEDDKVFPGGSHTYVWQVLKENG PMASDPLCLTYSYLSHVDLVKDLNSG LIGA LLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKM HTVNGYVNRSLPGLIGCHRKSVYWHVIG MGT TPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLM DLGQFLLFCHISSHQHDGM EAYVKVDSCPEEPQLRM KNNEEAEDYDDDLT DSE M DVVRFDDDNSPSF IQI RSVAKKH PKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLN NG PQRIG RKYKKVRFMAYTDETFKTR EAIQH ESG | LG PLLYG EVG DTLLI | FKNQASRPYNIYPHGITDVRPLYSRRLPKGVKH LKDFPI LPG E| FKYKWTVTVE DG PTKSDPRC LTRYY SSFVNM ERDLASGLIGPLLICYKESVDQRGNQI MSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVF DSLQLSVCLH I LSIGAQTDFLSVFFSGYTF KH KMVYEDTLTLFPFSG ETVFMSM EN PG LWI LGCH NSDFRN RG MTALLKVSSC DKNTG DYYEDSYEDISAYLLSKN NAI EPRSFSQN HQREITR'ITLQSDQEE | DYDDTISVE M KKEDF DIYDE DE NQSPRSFQKKTR HYFIAAVERLWDYG MSSSPHVLRN VPQFKKVVFQEFTDGSFTQPLYRG ELN EH LG LLG PYI RAEVEDN | MVTFRNQASRPYSF YSSLISYEE DQRQGAE PRKN FVKPN ETKTYFWKVQHH MAPTKDEFDCKAWAYFSDVD LE KDVHSG L|G PLLVCHTNTLN PAHG RQVT VQE FALFFTI FDETKSWYFTENM ERNCRAPCNIQM EDPTFKENYRFHAINGYI MDTLPGLVMAQDQRI RWYLLSMGSNENIHSIHFSG HVFTVRKKE EYKMALYN LYPGVFETVEM LPSKAG IWRVECLIGEHLHAGMSTLFLVYSN KCQTP LG MASG H | RDFQITASGQYGQWA PKLARLHYSGSI NAWSTKEPFSWI KVDLLAPM| | HG | QKFSSLYISQFIIMYSLDG KKWQTYRG NSTGTLMVFFG NVDSSG | KH NI FNPPI IARYI RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQG PQVNNP DFQKTM KVTGVTI'QGVKSLLTSMYVKE FLISSSQDG HQWTLFFQNG KVKVFQG NQDSFTPVVNSLDPPLLTRYLRI H PQSW VHQIALRM EVLGCEAQDLY *BDD-FVIII = B Domain-Deleted FVIII 11?44 FIG 2A— Non 0 timized BDD FVIII Preferred t