CpG REDUCED FACTOR VIII VARIANTS, COMPOSITIONS AND METHODS AND USES FOR TREATMENT OF HEMOSTASIS DISORDERS

Abstract

CpG reduced nucleic acid variants encoding FVIII protein and methods of use thereof are disclosed. In particular embodiments, CpG reduced nucleic acid variants encoding FVIII are expressed more efficiently by cells, are secreted at increased levels by cells over wild-type Factor VIII proteins, exhibit enhanced expression and/or activity over wild-type Factor VIII proteins or are packaged more efficiently into viral vectors.

Claims

1. A nucleic acid variant encoding Factor VIII (FVIII) having a B domain deletion, wherein the nucleic acid variant has 92% or greater identity to SEQ ID NO:7.

2. The nucleic acid variant of claim 1, wherein the nucleic acid variant has 93% or greater sequence identity to SEQ ID NO:7.

3. The nucleic acid variant of claim 1, wherein the nucleic acid variant has 94% or greater sequence identity to SEQ ID NO:7.

4. The nucleic acid variant of claim 1, wherein the nucleic acid variant has 95% or greater sequence identity to SEQ ID NO:7.

5. The nucleic acid variant of claim 1, wherein the nucleic acid variant has 95%-100% sequence identity to SEQ ID NO:7.

6. The nucleic acid variant of claim 1, wherein the nucleic acid variant has 20 or fewer, 15 or fewer, or 10 or fewer cytosine-guanine dinucleotides (CpGs).

7. The nucleic acid variant of claim 1, wherein the nucleic acid variant has no more than 5 cytosine-guanine dinucleotides (CpGs).

9. The nucleic acid variant of claim 1, wherein the nucleic acid variant has 4, 3, 2, 1 or 0 cytosine-guanine dinucleotides (CpGs).

10. The nucleic acid variant of claim 1, wherein the nucleic acid variant encodes SEQ ID NO:25 having a deletion of one or more amino acids of the sequence SFSQNPPVLKRHQR (SEQ ID NO:29), or a deletion of the entire sequence SFSQNPPVLKRHQR.

11. The nucleic acid variant of claim 1, wherein the nucleic acid variant encodes SEQ ID NO:25.

12. A nucleic acid variant encoding Factor VIII (FVIII) having a B domain deletion (FVIII-BDD), wherein the nucleic acid variant has fewer cytosine-guanine dinucleotides (CpG) than SEQ ID NO: 19.

13. The nucleic acid variant of claim 12, wherein said FVIII-BDD is mammalian.

14. A nucleic acid variant encoding human Factor VIII having a B domain deletion (hFVIII-BDD), wherein the nucleic acid variant has no more than 2 cytosine-guanine dinucleotides (CpGs).

15. The nucleic acid variant of claim 14, wherein the nucleic acid variant has 1 cytosine-guanine dinucleotide (CpG).

16. The nucleic acid variant of claim 14, wherein the nucleic acid variant has no cytosine-guanine dinucleotides (CpGs).

17. The nucleic acid variant of any of claims 1-16, wherein the encoded FVIII-BDD or hFVIII-BDD is identical to hFVIII-BDD encoded by SEQ ID NO: 19.

18. The nucleic acid variant of any of claims 1-17, wherein the nucleic acid variant is distinct from FVIII-V3 (SEQ ID NO:20) and CO3 (SEQ ID NO:21).

19. The nucleic acid variant of any of claims 1-18, wherein the nucleic acid variant encodes SEQ ID NO:25 having a deletion of one or more amino acids of the sequence SFSQNPPVLKRHQR (SEQ ID NO:29), or a deletion of the entire sequence SFSQNPPVLKRHQR.

20. The nucleic acid variant of any of claims 1-18, wherein the nucleic acid variant encodes SEQ ID NO:25.

21. A vector comprising the nucleic acid variant of any of claims 1-20.

22. An expression vector comprising the nucleic acid variant of any of claims 1-20.

23. The expression vector of claim 22, selected from the group consisting of an adenovirus-associated virus (AAV) vector, a retroviral vector, an adenoviral vector, a plasmid, or a lentiviral vector.

24. The expression vector of claim 23, wherein said AAV vector comprises an AAV serotype or an AAV pseudotype, wherein said AAV pseudotype comprise an AAV capsid serotype different from an ITR serotype.

25. The expression vector of 23 or 24, further comprising an intron, an expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and/or a filler polynucleotide sequence.

26. The expression vector of claim 25, wherein the intron is within or flanks the nucleic acid variant.

27. The expression vector of claim 25, wherein the expression control element is operably linked to the nucleic acid variant.

28. The expression vector of claim 25, wherein the AAV ITR(s) flanks the 5 or 3 terminus of the nucleic acid variant.

29. The expression vector of claim 25, wherein the filler polynucleotide sequence flanks the 5 or 3terminus of the nucleic acid variant.

30. The expression vector of any of claims claim 25-29, wherein the intron, expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and/or a filler polynucleotide sequence has been modified to have reduced cytosine-guanine dinucleotides (CpGs).

31. The expression vector of any of claims claim 25-29, wherein the intron, expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and/or a filler polynucleotide sequence has been modified to have 20 or fewer, 15 or fewer, 10 or fewer, 5 or fewer or 0 cytosine-guanine dinucleotides (CpGs).

32. The expression vector of any of claims 25, 27, 30 and 31, wherein the expression control element comprises a constitutive or regulatable control element, or a tissue-specific expression control element or promoter.

33. The expression vector of any of claims 25, 27, 30 and 31, wherein the expression control element comprises an element that confers expression in liver.

34. The expression vector of any of claims 25, 27, 30 and 31, wherein the expression control element comprises a TTR promoter or mutant TTR promoter.

35. The expression vector of claim 34, wherein the mutant TTR promoter comprises SEQ ID NO:22.

36. The expression vector of any of claims claim 25-35, wherein the ITR comprises one or more ITRs of any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes, or a combination thereof.

37. The expression vector of any of claims claim 24-30, wherein the vector comprises an ITR, a promoter, a polyA signal and/or intron sequence set forth in SEQ ID NO:23.

38. An AAV vector comprising the nucleic acid variant of any of claims 1-20 or the expression vector of any of claims 25-37.

39. The AAV vector of claim 38, wherein the AAV vector comprises a modified or variant AAV VP1, VP2 and/or VP3 capsid sequence, or wild-type AAV VP1, VP2 and/or VP3 capsid sequence.

40. The AAV vector of claim 38, wherein the AAV vector comprises a modified or variant AAV VP1, VP2 and/or VP3 capsid sequence having 90% or more identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 VP1, VP2 and/or VP3 sequences.

41. The AAV vector of claim 38, wherein the AAV vector comprises a VP1, VP2 or VP3 capsid sequence selected from any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes.

42. The AAV vector of claim 38, wherein the AAV vector comprises a capsid having 90% or more sequence identity to LK03 capsid (SEQ ID NO:27).

43. The AAV vector of claim 38, wherein the AAV vector comprises a capsid having 90% or more sequence identity to SPK capsid (SEQ ID NO:28).

44. The AAV vector of claim 38, wherein the AAV vector comprises LK03 capsid (SEQ ID NO:27).

45. The AAV vector of claim 38, wherein the AAV vector comprises SPK capsid (SEQ ID NO:28).

46. The AAV vector of claim 38, wherein the AAV vector comprises the nucleic acid variant SEQ ID NO:7 and LK03 capsid sequence (SEQ ID NO:27).

47. The AAV vector of claim 38, wherein the AAV vector comprises the nucleic acid variant SEQ ID NO:7 and SPK capsid (SEQ ID NO:28).

48. The AAV vector of claim 38, wherein the AAV vector comprises the nucleic acid variant and one or more of a mutated TTR promoter (TTRmut), synthetic intron, poly A and ITR in SEQ ID NO:23.

49. The AAV vector of claim 38, wherein the AAV vector comprises the nucleic acid variant and one or more of a mutated TTR promoter (TTRmut), synthetic intron, poly A and ITR in SEQ ID NO:23 and LK03 capsid sequence (SEQ ID NO:27) or SPK capsid (SEQ ID NO:28).

50. A host cell comprising the nucleic acid variant of any of claims 1-20, or the vector or expression vector of any of claims 21-37.

51. The host cell of claim 50, said host cells expressing the FVIII encoded by said nucleic acid variant.

52. A host cell comprising the AAV vector of any of claims 38-49.

53. The host cell of claim 52, said host cells producing the AAV vector of any of claims 38-49.

54. A pharmaceutical composition comprising the nucleic acid variant of any of claims 1-20, the vector or expression vector of any of claims 21-37, or the AAV vector of any of claims 38-49 in a biologically compatible carrier or excipient.

55. The nucleic acid variant of any claims 1-20, the vector or expression vector of any of claims 21-37, or the AAV vector of any of claims 38-49 encapsulated in a liposome or mixed with phospholipids or micelles.

56. The pharmaceutical composition of claim 54 or 55, further comprising empty capsid AAV.

57. The pharmaceutical composition of claim 54 or 55, further comprising empty capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and/or AAV-Rh74 serotype.

58. The pharmaceutical composition of claim 54 or 55, further comprising empty capsid AAV of the same serotype as the AAV vector administered.

59. The pharmaceutical composition of claim 54 or 55, wherein the empty capsid is LK03 capsid (SEQ ID NO:27) or SPK capsid (SEQ ID NO:28).

60. The pharmaceutical composition of any of claims 56-59, wherein the ratio of said empty capsids to said AAV vector is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

61. A method for delivering or transferring a nucleic acid sequence into a mammal or a mammalian cell, comprising administering or contacting the nucleic acid variant of any of claims 1-20, vector or expression vector of any of claims 21-37, or the AAV vector of any of claims 38-49 to said mammal or mammalian cell, thereby delivering or transferring the nucleic acid sequence into the mammal or mammalian cell.

62. A method of treating a mammal in need of Factor VIII, comprising: (a) providing the nucleic acid variant of any of claims 1-20, vector or expression vector of any of claims 21-37, or the AAV vector of any of claims 38-49; and (b) administering an amount of the nucleic acid variant of any of claims 1-20, vector or expression vector of any of claims 21-37, or the AAV vector of any of claims 38-49 to the mammal wherein said Factor VIII is expressed in the mammal.

63. The method of claim 61 or 62, wherein said Factor VIII encoded by the nucleic acid variant is expressed in a cell, tissue or organ of said mammal.

64. The method of claim 63, wherein, the cell comprises a secretory cell.

65. The method of claim 63, wherein the cell comprises an endocrine cell or an endothelial cell.

66. The method of claim 63, wherein the cell comprises a hepatocyte, a sinusoidal endothelial cell, a megakaryocyte, a platelet or hematopoetic stem cell.

67. The method of claim 63, wherein the tissue or organ of said mammal comprises liver.

68. The method of any of claims 61-67, wherein the mammal produces an insufficient amount of Factor VIII protein, or a defective or aberrant Factor VIII protein.

69. The method of any of claims 61-67, wherein the mammal has hemophilia A.

70. The method of any of claims 61-67, wherein the Factor VIII encoded by the nucleic acid variant is expressed at levels having a beneficial or therapeutic effect on the mammal.

71. A method for treatment of a hemostasis related disorder in a patient in need thereof comprising administration of a therapeutically effective amount of the nucleic acid variant of any of claims 1-20, vector or expression vector of any of claims 21-37, or the AAV vector of any of claims 38-49 in a biologically acceptable carrier to the patient.

72. The method of claims 61, 62 or 71, wherein said mammal or said patient has a disorder selected from the group consisting of hemophilia A, von Willebrand diseases and bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC) and over-anticoagulation treatment disorders.

73. The method of any of claims 61-72, wherein the nucleic acid variant of any of claims 1-20, vector or expression vector of any of claims 21-37, or the AAV vector of any of claims 38-49 is delivered to said mammal or said patient intravenously, intraarterially, intramuscularly, subcutaneously, intra-cavity, or by intubation, or via catheter.

74. The method of any of claims 61-72, wherein FVIII is expressed at levels without substantially increasing risk of thrombosis.

75. The method of claim 74, wherein said thrombosis risk is determined by measuring fibrin degradation products.

76. The method of any of claims 61-72, wherein FVIII is expressed at levels greater than 1% of the levels of FVIII found in a subject that does not have hemophilia A.

77. The method of any of claims 61-72, wherein FVIII is expressed at levels greater than 3% of the levels of FVIII found in a subject that does not have hemophilia A.

78. The method of any of claims 61-72, wherein activity of FVIII is detectable for at least 1, 2, 3 or 4 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months, or at least 1 year.

79. The method of any of claims 61-72, wherein FVIII is expressed at levels greater than 1% or 3% of the levels of FVIII found in a subject that does not have hemophilia A for at least 1, 2, 3 or 4 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months, or at least 1 year.

80. The method of any of claims 61-72, wherein FVIII is expressed at levels having a therapeutic effect for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, weeks or months.

81. The method of any of claims 61-72, wherein said FVIII is present in the mammal or patient at levels of about 20% FVIII activity or greater than 20% activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months.

82. The method of any of claims 61-72, wherein said FVIII is expressed at levels at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36.sup.%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of normal FVIII levels.

83. The method of any of claims 61-72, wherein the AAV vector is administered at a dose of less than 1?10.sup.12 vector genomes per kilogram (vg/kg) of the mammal or patient, and said FVIII is produced in the mammal or patient at levels of about 20% activity or greater than 20% activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months

84. The method of any of claims 61-72, wherein the AAV vector is administered at a dose of about 5?10.sup.11 vector genomes per kilogram (vg/kg) of the mammal or patient, and said FVIII is produced in the mammal or patient at levels of about 20% activity or greater than 20% activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months.

85. The method of any of claims 61-84, wherein said mammal or said patient is human.

86. The method of any of claims 61-85, wherein said mammal, said patient or said human is sero-positive or sero-negative for AAV.

87. The method of any of claims 61-86, further comprising administering AAV empty capsid to said mammal or said patient.

88. The method of any of claims 61-86, further comprising administering empty capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and/or AAV-Rh74 serotype.

89. The method of any of claims 61-86, further comprising administering empty capsid AAV of the same serotype as the AAV vector administered.

90. The method of claim 89, wherein the empty capsid is LK03 capsid (SEQ ID NO:27) or SPK capsid (SEQ ID NO:28).

91. The method of any of claims 87-90, wherein the ratio of said empty capsids to said AAV vector is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

92. The method of any of claims 61-91, further comprising administering an immunosuppressive agent.

93. The method of any of claims 61-91, further comprising administering an immunosuppressive agent after the AAV vector is administered.

94. The method of any of claims 61-91, further comprising administering an immunosuppressive agent from a time period within 1 hour to up to 45 days after the AAV vector is administered.

95. The method of any of claims 92-94, wherein the immunosuppressive agent comprises a steroid, cyclosporine (e.g., cyclosporine A), mycophenolate, Rituximab or a derivative thereof.

96. The method of any of claims 61-95, wherein the AAV vector is administered in a range from about 1?10.sup.8 to about 1?10.sup.14 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.

97. The method of any of claims 61-95, wherein the AAV vector is administered in a range from about 1?10.sup.9 to about 1?10.sup.13 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.

98. The method of any of claims 61-95, wherein the AAV vector is administered in a range from about 1?10.sup.10 to about 1?10.sup.12 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.

99. The method of any of claims 61-95, wherein the AAV vector is administered in a range from about 1?10.sup.11 to about 1?10.sup.12 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.

100. The method of any of claims 61-95, wherein the AAV vector is administered in a range from about 1?10.sup.12 to about 1?10.sup.13 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.

101. The method of any of claims 61-95, wherein the AAV vector is administered in a range from about 1?10.sup.13 to about 1?10.sup.14 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.

102. The method of any of claims 61-95, wherein the AAV vector is administered in a range from about 5?10.sup.11 to about 1?10.sup.12 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.

103. The method of any of claims 61-95, wherein the AAV vector is administered at a dose of about 5?10.sup.11 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.

104. The method of any of claims 61-103, further comprising analyzing or monitoring the mammal for the presence or amount of AAV antibodies, an immune response against AAV, FVIII antibodies, an immune response against FVIII, FVIII amounts, FVIII activity level, amounts or levels of one or more liver enzymes or frequency, and/or severity or duration of bleeding episodes.

105. A method of producing FVIII protein comprising expressing in a cell the nucleic acid variant as claimed in any of claims 1-20, or the vector or expression vector of any of claims 21-37, and recovering said FVIII protein produced by the cells.

106. The method of claim 105, further comprising purifying or isolating said FVIII protein produced by the cells.

Description

DESCRIPTION OF DRAWINGS

[0032] FIG. 1 shows human FVIII (hFVIII) levels 24 hour following hydrodynamic tail vein (HTV) injection of 50 ?g of plasmid, for 18 different clones (X01-X18 corresponding to SEQ ID Nos: 1-18, respectively) and FVIII-CO3 (SEQ ID NO:21).

[0033] FIGS. 2A-2C show FVIII levels in hemophilia A/CD4.sup.?/? mice after AAV vector administration of FVIII (A) CO3 (SEQ ID NO:21), X09 (SEQ ID NO:9), X12 (SEQ ID NO: 12) and X16 (SEQ ID NO: 16); (B) CO3 (SEQ ID NO:21), X01 (SEQ ID NO: 1) and X11 (SEQ ID NO: 11); or (C) CO3 (SEQ ID NO:21), X07 (SEQ ID NO:7) and X10 (SEQ ID NO: 10).

[0034] FIGS. 3A-3B show levels of hFVIII antigen in ng/ml (B) or % total antigen (C) in plasma of NOD/SCID mice following intravenous administration of either vehicle (circle), 4?10.sup.10 (square), 8?10.sup.10 (triangle), or 1.6?10.sup.11 vg/mouse (inverted triangle) of AAV-SPK-8005-hFVIII over the course of 87 days. Lines represent hFVIII averages?SD in each cohort. Human FVIII plasma levels were assayed by ELISA and ng/ml FVIII was converted to % normal FVIII levels by assuming 150 ng/ml is equivalent to 100% activity.

[0035] FIG. 3C shows levels of D-dimers in plasma of NOD/SCID mice following intravenous administration of either vehicle, 4?10.sup.10, 8?10.sup.10 or 1.6?10.sup.11 vg/mouse of AAV-SPK-8005-hFVIII as illustrated, left to right at each timepoint, x-axis. Bars represent averages?SD of mice in each cohort. D-dimer levels were assayed by ELISA.

[0036] FIG. 4 shows NHP Study design.

[0037] FIGS. 5A-5D show hFVIII antigen levels in NHPs following intravenous administration of either 2?10.sup.12 (A), 5?10.sup.12 (B) or 1?10.sup.13 vg/kg (C) of AAV-SPK-8005. Lines represent individual animals. Human FVIII plasma levels were assayed by ELISA and represent repeated measurements, obtained by serial bleeding, on the same group of animals during the course of the study (n=2-3 animals per cohort). Human FVIII levels measured in vehicle-treated animals are shown in open squares in all three graphs. ?=Development of inhibitors against FVIII.

[0038] FIGS. 6A-6C show ALT levels in NHPs, at 2?10.sup.12 (A), 5?10.sup.12 (B) or 1?10.sup.13 vg/kg (C) of AAV-SPK-8005.

[0039] FIGS. 7A-7C show D-Dimer levels in NHPs. D-dimer antigen concentration in plasma of NHPs following intravenous administration of either 2?10.sup.12 (A), 5?10.sup.12 (B) or 1?10.sup.13 vg/kg (C) of AAV-SPK-8005. The dotted line indicates 500 ng/ml, the upper limit of normal for D-dimers in humans.

[0040] FIG. 8 shows a data summary of FVIII levels in the three doses of AAV-SPK-8005.

[0041] FIGS. 9A-9D show levels of hFVIII in plasma of cynomolgus macaques following intravenous administration of either 2?10.sup.12 (A), 6?10.sup.12 (B) or 2?10.sup.13 (vg/kg) (C) of AAV-SPK-8011 (LK03 capsid)-hFVIII. Lines represent individual animals. hFVIII plasma levels were assayed by ELISA and represent repeated measurements, obtained by serial bleeding, on the same group of animals during the course of the study (n=3 animals per cohort). Human FVIII levels measured in vehicle-treated animals are shown in open squares (n=2). ?=Time when development of inhibitors against FVIII was detected in each individual animal.

[0042] FIG. 10 shows a comparison of FVIII levels achieved with AAV-SPK-8011 (LK03 capsid)-hFVIII to the reported levels of FVIII delivered by way of AAV vectors with AAV5 and AAV8 capsids. AAV5: http://www.biomarin.com/pdf/BioMarin_R&D_Day_4_20_2016.pdf, slide 16. AAV8: McIntosh J et al. Blood 2013; 121(17):3335-44.

[0043] FIG. 11 shows AAV-SPK (SEQ ID NO:28) and AAV-LK03 (SEQ ID NO:27) tissue biodistribution in non-human primates, predominanyl in kidney, spleen and liver (3.sup.rd bar for each tissue).

[0044] FIG. 12 shows hepatic and splenic FVIII expression after systemic administration of AAV-SPK-8005 into mice.

[0045] FIG. 13 shows transduction efficiency of the AAV-LK03 capsid analyzed in vitro. X-axis, cynomolgus (left vertical bar), human (right vertical bar).

[0046] FIGS. 14A-14B show plasma concentration of hFIX in rabbits after AAV administration. Rabbits received intravenous injection of hFIX vectors AAV-SPK or AAV-LK03 at doses of (A) 1?10.sup.12 vg/kg (low dose, n=4) or (B) 1?10.sup.13 vg/kg (high dose, n=3-5). Human FIX levels between groups were compared using a 2-tailed Mann-Whitney test. No significant differences were observed. Animals 5 and 15 in the low dose cohorts were excluded from the analysis due to misinjection. Animals 9 and 10 were also excluded from the graph as they developed neutralizing antibodies against human FIX.

[0047] FIGS. 15A-15B show a time course of antibody formation to human FIX (anti-FIX). Rabbits received intravenous injection of of hFIX vectors AAV-SPK or AAV-LK03 at doses of (A) 1?10.sup.12 vg/kg (low dose, n=4) or (B) 1?10.sup.13 vg/kg (high dose, n=3-5). The data are shown for each individual animal.

DETAILED DESCRIPTION

[0048] Disclosed herein are CpG reduced nucleic acid variants encoding FVIII, distinct from wild-type nucleic acid that encode FVIII. Such CpG reduced nucleic acid variants encoding FVIII can be expressed at increased levels in cells and/or animals, which in turn can provide increased FVIII protein levels in vivo. Also disclosed are CpG reduced nucleic acid variant encoding FVIII that can provide for greater biological activity in vitro and/or in vivo. Exemplary CpG reduced nucleic acid variant encoding FVIII can exhibit one or more of the following: 1) increased expression in cells and/or animals; 2) increased activity; and 3) a therapeutic effect at lower AAV doses than wild-type hFVIII.

[0049] The terms polynucleotide and nucleic acid are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Polynucleotides include genomic DNA, cDNA and antisense DNA, and spliced or unspliced mRNA, rRNA tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Polynucleotides include naturally occurring, synthetic, and intentionally modified or altered polynucleotides (e.g., variant nucleic acid). Polynucleotides can be single, double, or triplex, linear or circular, and can be of any length. In discussing polynucleotides, a sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5 to 3 direction.

[0050] As used herein, the terms modify or variant and grammatical variations thereof, mean that a nucleic acid, polypeptide or subsequence thereof deviates from a reference sequence. Modified and variant sequences may therefore have substantially the same, greater or less expression, activity or function than a reference sequence, but at least retain partial activity or function of the reference sequence. A particular example of a modification or variant is a CpG reduced nucleic acid variant encoding FVIII.

[0051] A nucleic acid or polynucleotide variant refers to a modified sequence which has been genetically altered compared to wild-type. The sequence may be genetically modified without altering the encoded protein sequence. Alternatively, the sequence may be genetically modified to encode a variant protein. A nucleic acid or polynucleotide variant can also refer to a combination sequence which has been codon modified to encode a protein that still retains at least partial sequence identity to a reference sequence, such as wild-type protein sequence, and also has been codon-modified to encode a variant protein. For example, some codons of such a nucleic acid variant will be changed without altering the amino acids of the protein (FVIII) encoded thereby, and some codons of the nucleic acid variant will be changed which in turn changes the amino acids of the protein (FVIII) encoded thereby.

[0052] The term variant Factor VIII (FVIII) refers to a modified FVIII which has been genetically altered as compared to unmodified wild-type FVIII (e.g., SEQ ID NO: 19) or FVIII-BDD. Such a variant can be referred to as a nucleic acid variant encoding Factor VIII (FVIII). A particular example of a variant is a CpG reduced nucleic acid encoding FVIII or FVIII-BDD protein. The term variant need not appear in each instance of a reference made to CpG reduced nucleic acid encoding FVIII. Likewise, the term CpG reduced nucleic acid or the like may omit the term variant but it is intended that reference to CpG reduced nucleic acid includes variants at the genetic level.

[0053] FVIII constructs having reduced CpG content can exhibit improvements compared to wild-type FVIII or FVIII-BDD in which CpG content has not been reduced, and do so without modifications to the nucleic acid that result in amino acid changes to the encoded FVIII or FVIII-BDD protein. When comparing expression, if the CpG reduced nucleic acid encodes a FVIII protein that retains the B-domain, it is appropriate to compare it to wild-type FVIII expression; and if the CpG reduced nucleic acid encodes a FVIII protein without a B-domain, it is compared to expression of wild-type FVIII that also has a B-domain deletion.

[0054] A variant Factor VIII (FVIII) can also mean a modified FVIII protein such that the modified protein has an amino acid alteration compared to wild-type FVIII. Again, when comparing activity and/or stability, if the encoded variant FVIII protein retains the B-domain, it is appropriate to compare it to wild-type FVIII; and if the encoded variant FVIII protein has a B-domain deletion, it is compared to wild-type FVIII that also has a B-domain deletion.

[0055] A variant FVIII can include a portion of the B-domain. Thus, FVIII-BDD includes a portion of the B-domain. Typically, in FVIII-BDD most of the B-domain is deleted.

[0056] A variant FVIII can include an SQ sequence set forth as SFSQNPPVLKRHQR (SEQ ID NO:29). Typically, such a variant FVIII with an SQ (FVIII/SQ) has a BDD, e.g., at least all or a part of BD is deleted. Variant FVIII, such as FVIII-BDD can have all or a part of the SQ sequence, i.e. all or a part of SEQ ID NO:29. Thus, for example, a variant FVIII-BDD with an SQ sequence (SFSQNPPVLKRHQR, SEQ ID NO:29) can have all or just a portion of the amino acid sequence SFSQNPPVLKRHQR. For example, FVIII-BDD can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acid residues of SFSQNPPVLKRHQR included. Thus, SFSQNPPVLKRHQR with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 internal deletions as well as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino- or carboxy terminal deletions are included in the variant FVIII proteins set forth herein.

[0057] The polypeptides, proteins and peptides encoded by the nucleic acid or polynucleotide sequences, include full-length native (FVIII) sequences, as with naturally occurring wild-type proteins, as well as functional subsequences, modified forms or sequence variants so long as the subsequence, modified form or variant retain some degree of functionality of the native full-length protein. For example, a CpG reduced nucleic acid encoding FVIII protein can have a B-domain deletion as set forth herein and retain clotting function. In methods and uses of the invention, such polypeptides, proteins and peptides encoded by the nucleic acid sequences can be but are not required to be identical to the endogenous protein that is defective, or whose expression is insufficient, or deficient in the treated mammal.

[0058] Non-limiting examples of modifications include one or more nucleotide or amino acid substitutions (e.g., 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100, 100-150, 150-200, 200-250, 250-500, 500-750, 750-850 or more nucleotides or residues). An example of a nucleic acid modification is CpG reduction. In certain embodiments, a CpG reduced nucleic acid encoding FVIII, such as human FVIII protein, has 10 or fewer CpGs compared to wild-type sequence encoding human Factor FVIII; or has 5 or fewer CpGs compared to wild-type sequence encoding human Factor FVIII; or has no more than 5 CpGs in the CpG reduced nucleic acid encoding FVIII.

[0059] An example of an amino acid modification is a conservative amino acid substitution or a deletion (e.g., subsequences or fragments) of a reference sequence, e.g. FVIII, such as FVIII with a B-domain deletion. In particular embodiments, a modified or variant sequence retains at least part of a function or activity of unmodified sequence.

[0060] All mammalian and non-mammalian forms of nucleic acid encoding proteins, including other mammalian forms of the CpG reduced nucleic acid encoding FVIII and FVIII proteins disclosed herein are expressly included, either known or unknown. Thus, the invention includes genes and proteins from non-mammals, mammals other than humans, and humans, which genes and proteins function in a substantially similar manner to the FVIII (e.g., human) genes and proteins described herein.

[0061] The term vector refers to small carrier nucleic acid molecule, a plasmid, virus (e.g., AAV vector), or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid. Such vectors can be used for genetic manipulation (i.e., cloning vectors), to introduce/transfer polynucleotides into cells, and to transcribe or translate the inserted polynucleotide in cells. An expression vector is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell. A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), intron, ITR(s), selectable marker (e.g., antibiotic resistance), polyadenylation signal.

[0062] A viral vector is derived from or based upon one or more nucleic acid elements that comprise a viral genome. Particular viral vectors include lentivirus, pseudo-typed lentivirus and parvo-virus vectors, such as adeno-associated virus (AAV) vectors. Also provided are vectors comprising a CpG reduced nucleic acid encoding FVIII.

[0063] The term recombinant, as a modifier of vector, such as recombinant viral, e.g., lenti- or parvo-virus (e.g., AAV) vectors, as well as a modifier of sequences such as recombinant polynucleotides and polypeptides, means that the compositions have been manipulated (i.e., engineered) in a fashion that generally does not occur in nature. A particular example of a recombinant vector, such as an AAV vector would be where a polynucleotide that is not normally present in the wild-type viral (e.g., AAV) genome is inserted within the viral genome. An example of a recombinant polynucleotide would be where a CpG reduced nucleic acid encoding a FVIII protein is cloned into a vector, with or without 5, 3 and/or intron regions that the gene is normally associated within the viral (e.g., AAV) genome. Although the term recombinant is not always used herein in reference to vectors, such as viral and AAV vectors, as well as sequences such as polynucleotides, recombinant forms including polynucleotides, are expressly included in spite of any such omission.

[0064] A recombinant viral vector or AAV vector is derived from the wild type genome of a virus, such as AAV by using molecular methods to remove the wild type genome from the virus (e.g., AAV), and replacing with a non-native nucleic acid, such as a CpG reduced nucleic acid encoding FVIII. Typically, for AAV one or both inverted terminal repeat (ITR) sequences of AAV genome are retained in the AAV vector. A recombinant viral vector (e.g., AAV) is distinguished from a viral (e.g., AAV) genome, since all or a part of the viral genome has been replaced with a non-native sequence with respect to the viral (e.g., AAV) genomic nucleic acid such as a CpG reduced nucleic acid encoding FVIII. Incorporation of a non-native sequence therefore defines the viral vector (e.g., AAV) as a recombinant vector, which in the case of AAV can be referred to as a rAAV vector.

[0065] A recombinant vector (e.g., lenti-, parvo-, AAV) sequence can be packaged-referred to herein as a particle for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo. Where a recombinant vector sequence is encapsidated or packaged into an AAV particle, the particle can also be referred to as a rAAV. Such particles include proteins that encapsidate or package the vector genome. Particular examples include viral envelope proteins, and in the case of AAV, capsid proteins.

[0066] A vector genome refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form a viral (e.g., AAV) particle. In cases where recombinant plasmids are used to construct or manufacture recombinant vectors, the vector genome does not include the portion of the plasmid that does not correspond to the vector genome sequence of the recombinant plasmid. This non vector genome portion of the recombinant plasmid is referred to as the plasmid backbone, which is important for cloning and amplification of the plasmid, a process that is needed for propagation and recombinant virus production, but is not itself packaged or encapsidated into virus (e.g., AAV) particles. Thus, a vector genome refers to the nucleic acid that is packaged or encapsidated by virus (e.g., AAV).

[0067] A transgene is used herein to conveniently refer to a nucleic acid that is intended or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a gene that encodes a polypeptide or protein (e.g., a CpG reduced nucleic acid encoding Factor VIII).

[0068] In a cell having a transgene, the transgene has been introduced/transferred by way of vector, such as AAV, transduction or transfection of the cell. The terms transduce and transfect refer to introduction of a molecule such as a nucleic acid into a cell or host organism. The transgene may or may not be integrated into genomic nucleic acid of the recipient cell. If an introduced nucleic acid becomes integrated into the nucleic acid (genomic DNA) of the recipient cell or organism it can be stably maintained in that cell or organism and further passed on to or inherited by progeny cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid may exist in the recipient cell or host organism extrachromosomally, or only transiently.

[0069] A transduced cell is a cell into which the transgene has been introduced. Accordingly, a transduced cell (e.g., in a mammal, such as a cell or tissue or organ cell), means a genetic change in a cell following incorporation of an exogenous molecule, for example, a nucleic acid (e.g., a transgene) into the cell. Thus, a transduced cell is a cell into which, or a progeny thereof in which an exogenous nucleic acid has been introduced. The cell(s) can be propagated and the introduced protein expressed, or nucleic acid transcribed. For gene therapy uses and methods, a transduced cell can be in a subject.

[0070] An expression control element refers to nucleic acid sequence(s) that influence expression of an operably linked nucleic acid. Control elements, including expression control elements as set forth herein such as promoters and enhancers, Vector sequences including AAV vectors can include one or more expression control elements. Typically, such elements are included to facilitate proper heterologous polynucleotide transcription and if appropriate translation (e.g., a promoter, enhancer, splicing signal for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons etc.). Such elements typically act in cis, referred to as a cis acting element, but may also act in trans.

[0071] Expression control can be at the level of transcription, translation, splicing, message stability, etc. Typically, an expression control element that modulates transcription is juxtaposed near the 5 end (i.e., upstream) of a transcribed nucleic acid. Expression control elements can also be located at the 3 end (i.e., downstream) of the transcribed sequence or within the transcript (e.g., in an intron). Expression control elements can be located adjacent to or at a distance away from the transcribed sequence (e.g., 1-10, 10-25, 25-50, 50-100, 100 to 500, or more nucleotides from the polynucleotide), even at considerable distances. Nevertheless, owing to the length limitations of certain vectors, such as AAV vectors, expression control elements will typically be within 1 to 1000 nucleotides from the transcribed nucleic acid.

[0072] Functionally, expression of operably linked nucleic acid is at least in part controllable by the element (e.g., promoter) such that the element modulates transcription of the nucleic acid and, as appropriate, translation of the transcript. A specific example of an expression control element is a promoter, which is usually located 5 of the transcribed sequence e.g., a CpG reduced nucleic acid encoding FVIII. A promoter typically increases an amount expressed from operably linked nucleic acid as compared to an amount expressed when no promoter exists.

[0073] An enhancer as used herein can refer to a sequence that is located adjacent to the heterologous polynucleotide. Enhancer elements are typically located upstream of a promoter element but also function and can be located downstream of or within a sequence (e.g., a CpG reduced nucleic acid encoding FVIII). Hence, an enhancer element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream of a CpG reduced nucleic acid encoding FVIII. Enhancer elements typically increase expressed of an operably linked nucleic acid above expression afforded by a promoter element.

[0074] An expression construct may comprise regulatory elements which serve to drive expression in a particular cell or tissue type. Expression control elements (e.g., promoters) include those active in a particular tissue or cell type, referred to herein as a tissue-specific expression control elements/promoters. Tissue-specific expression control elements are typically active in specific cell or tissue (e.g., liver). Expression control elements are typically active in particular cells, tissues or organs because they are recognized by transcriptional activator proteins, or other regulators of transcription, that are unique to a specific cell, tissue or organ type. Such regulatory elements are known to those of skill in the art (see, e.g., Sambrook et al. (1989) and Ausubel et al. (1992)).

[0075] The incorporation of tissue specific regulatory elements in the expression constructs of the invention provides for at least partial tissue tropism for the expression of a CpG reduced nucleic acid encoding FVIII. Examples of promoters that are active in liver are the TTR promoter, human alpha 1-antitrypsin (hAAT) promoter; albumin, Miyatake, et al. J. Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig, et al., Gene Ther. 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot, et al., Hum. Gene. Ther., 7:1503-14 (1996)], among others. An example of an enhancer active in liver is apolipoprotein E (apoE) HCR-1 and HCR-2 (Allan et al., J. Biol. Chem., 272:29113-19 (1997)).

[0076] Expression control elements also include ubiquitous or promiscuous promoters/enhancers which are capable of driving expression of a polynucleotide in many different cell types. Such elements include, but are not limited to the cytomegalovirus (CMV) immediate early promoter/enhancer sequences, the Rous sarcoma virus (RSV) promoter/enhancer sequences and the other viral promoters/enhancers active in a variety of mammalian cell types, or synthetic elements that are not present in nature (see, e.g., Boshart et al, Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic 0-actin promoter and the phosphoglycerol kinase (PGK) promoter.

[0077] Expression control elements also can confer expression in a manner that is regulatable, that is, a signal or stimuli increases or decreases expression of the operably linked heterologous polynucleotide. A regulatable element that increases expression of the operably linked polynucleotide in response to a signal or stimuli is also referred to as an inducible element (i.e., is induced by a signal). Particular examples include, but are not limited to, a hormone (e.g., steroid) inducible promoter. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal or stimuli present; the greater the amount of signal or stimuli, the greater the increase or decrease in expression. Particular non-limiting examples include zinc-inducible sheep metallothionine (MT) promoter; the steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (WO 98/10088); the tetracycline-repressible system (Gossen, et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline-inducible system (Gossen, et al., Science. 268:1766-1769 (1995); see also Harvey, et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)); the RU486-inducible system (Wang, et al., Nat. Biotech. 15:239-243 (1997) and Wang, et al., Gene Ther. 4:432-441 (1997)]; and the rapamycin-inducible system (Magari, et al., J. Clin. Invest. 100:2865-2872 (1997); Rivera, et al., Nat. Medicine. 2:1028-1032 (1996)). Other regulatable control elements which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, development.

[0078] Expression control elements also include the native elements(s) for the heterologous polynucleotide. A native control element (e.g., promoter) may be used when it is desired that expression of the heterologous polynucleotide should mimic the native expression. The native element may be used when expression of the heterologous polynucleotide is to be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. Other native expression control elements, such as introns, polyadenylation sites or Kozak consensus sequences may also be used.

[0079] The term operably linked means that the regulatory sequences necessary for expression of a coding sequence are placed in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector. This definition is also sometimes applied to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule wherein a hybrid nucleic acid molecule is generated.

[0080] In the example of an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element modulates expression of the nucleic acid. More specifically, for example, two DNA sequences operably linked means that the two DNAs are arranged (cis or trans) in such a relationship that at least one of the DNA sequences is able to exert a physiological effect upon the other sequence.

[0081] Accordingly, additional elements for vectors include, without limitation, an expression control (e.g., promoter/enhancer) element, a transcription termination signal or stop codon, 5 or 3 untranslated regions (e.g., polyadenylation (polyA) sequences) which flank a sequence, such as one or more copies of an AAV ITR sequence, or an intron.

[0082] Further elements include, for example, filler or stuffer polynucleotide sequences, for example to improve packaging and reduce the presence of contaminating nucleic acid. AAV vectors typically accept inserts of DNA having a size range which is generally about 4 kb to about 5.2 kb, or slightly more. Thus, for shorter sequences, inclusion of a stuffer or filler in order to adjust the length to near or at the normal size of the virus genomic sequence acceptable for AAV vector packaging into virus particle. In various embodiments, a filler/stuffer nucleic acid sequence is an untranslated (non-protein encoding) segment of nucleic acid. For a nucleic acid sequence less than 4.7 Kb, the filler or stuffer polynucleotide sequence has a length that when combined (e.g., inserted into a vector) with the sequence has a total length between about 3.0-5.5 Kb, or between about 4.0-5.0 Kb, or between about 4.3-4.8 Kb.

[0083] An intron can also function as a filler or stuffer polynucleotide sequence in order to achieve a length for AAV vector packaging into a virus particle. Introns and intron fragments that function as a filler or stuffer polynucleotide sequence also can enhance expression.

[0084] The phrase hemostasis related disorder refers to bleeding disorders such as hemophilia A, hemophilia A patients with inhibitory antibodies, deficiencies in coagulation Factors, VII, VIII, IX and X, XI, V, XII, II, von Willebrand factor, combined FV/FVIII deficiency, vitamin K epoxide reductase C1 deficiency, gamma-carboxylase deficiency; bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over-anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e. FXa inhibitors); and platelet disorders such as, Bernard Soulier syndrome, Glanzman thromblastemia, and storage pool deficiency.

[0085] The term isolated, when used as a modifier of a composition, means that the compositions are made by the hand of man or are separated, completely or at least in part, from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane.

[0086] With reference to nucleic acids of the invention, the term isolated refers to a nucleic acid molecule that is separated from one or more sequences with which it is immediately contiguous (in the 5 and 3 directions) in the naturally occurring genome (genomic DNA) of the organism from which it originates. For example, the isolated nucleic acid may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the DNA of a prokaryote or eukaryote.

[0087] With respect to RNA molecules of the invention, the term isolated primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a substantially pure form (the term substantially pure is defined below).

[0088] With respect to protein, the term isolated protein or isolated and purified protein is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in substantially pure form.

[0089] The term isolated does not exclude combinations produced by the hand of man, for example, a recombinant vector (e.g., rAAV) sequence, or virus particle that packages or encapsidates a vector genome and a pharmaceutical formulation. The term isolated also does not exclude alternative physical forms of the composition, such as hybrids/chimeras, multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or derivatized forms, or forms expressed in host cells produced by the hand of man.

[0090] The term substantially pure refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). The preparation can comprise at least 75% by weight, or about 90-99% by weight, of the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

[0091] The phrase consisting essentially of when referring to a particular nucleotide sequence or amino acid sequence means a sequence having the properties of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.

[0092] The term oligonucleotide, as used herein refers to primers and probes, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, such as more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application for which the oligonucleotide is used.

[0093] The term probe as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

[0094] The probes herein are selected to be substantially complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to specifically hybridize or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5 or 3 end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.

[0095] The term specifically hybridize refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed substantially complementary). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.

[0096] The term primer as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to act functionally as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH, the primer may be extended at its 3 terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product.

[0097] The primer may vary in length depending on the particular conditions and requirements of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able to anneal with the desired template strand in a manner sufficient to provide the 3 hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5 end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.

[0098] The term identity, homology and grammatical variations thereof, mean that two or more referenced entities are the same, when they are aligned sequences. Thus, by way of example, when two polypeptide sequences are identical, they have the same amino acid sequence, at least within the referenced region or portion. Where two polynucleotide sequences are identical, they have the same polynucleotide sequence, at least within the referenced region or portion. The identity can be over a defined area (region or domain) of the sequence. An area or region of identity refers to a portion of two or more referenced entities that are the same. Thus, where two protein or nucleic acid sequences are identical over one or more sequence areas or regions they share identity within that region. An aligned sequence refers to multiple polynucleotide or protein (amino acid) sequences, often containing corrections for missing or additional bases or amino acids (gaps) as compared to a reference sequence.

[0099] The identity can extend over the entire length or a portion of the sequence. In certain embodiments, the length of the sequence sharing the percent identity is 2, 3, 4, 5 or more contiguous nucleic acids or amino acids, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. contiguous nucleic acids or amino acids. In additional embodiments, the length of the sequence sharing identity is 21 or more contiguous nucleic acids or amino acids, e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, etc. contiguous nucleic acids or amino acids. In further embodiments, the length of the sequence sharing identity is 41 or more contiguous nucleic acids or amino acids, e.g. 42, 43, 44, 45, 45, 47, 48, 49, 50, etc., contiguous nucleic acids or amino acids. In yet further embodiments, the length of the sequence sharing identity is 50 or more contiguous nucleic acids or amino acids, e.g., 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-150, 150-200, 200-250, 250-300, 300-500, 500-1,000, etc. contiguous nucleic acids or amino acids.

[0100] As set forth herein, CpG reduced nucleic acid variants encoding FVIII will be distinct from wild-type but may exhibit sequence identity with wild-type FVIII protein with, or without B-domain. In CpG reduced nucleic acid variants encoding FVIII, at the nucleotide sequence level, a CpG reduced nucleic acid encoding FVIII will typically be at least about 70% identical, more typically about 75% identical, even more typically about 80%-85% identical to wild-type FVIII encoding nucleic acid. Thus, for example, a CpG reduced nucleic acid encoding FVIII may have 75%-85% identity to wild-type FVIII encoding gene, or to each other, i.e., X01 vs. X02, X03 vs. X04, etc. as set forth herein.

[0101] At the amino acid sequence level, a variant such as a variant FVIII protein will be at least about 70% identical, more typically about 75% identical, or 80% identical, even more typically about 85 identity, or 90% or more identity. In other embodiments, a variant such as a variant FVIII protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence, e.g. wild-type FVIII protein with or without B-domain.

[0102] To determine identity, if the FVIII (CpG reduced nucleic acid encoding FVIII) retains the B-domain, it is appropriate to compare identity to wild-type FVIII. If the FVIII (CpG reduced nucleic acid encoding FVIII) has a B-domain deletion, it is appropriate to compare identity to wild-type FVIII that also has a B-domain deletion.

[0103] The terms homologous or homology mean that two or more referenced entities share at least partial identity over a given region or portion. Areas, regions or domains of homology or identity mean that a portion of two or more referenced entities share homology or are the same. Thus, where two sequences are identical over one or more sequence regions they share identity in these regions. Substantial homology means that a molecule is structurally or functionally conserved such that it has or is predicted to have at least partial structure or function of one or more of the structures or functions (e.g., a biological function or activity) of the reference molecule, or relevant/corresponding region or portion of the reference molecule to which it shares homology.

[0104] The extent of identity (homology) or percent identity between two sequences can be ascertained using a computer program and/or mathematical algorithm. For purposes of this invention comparisons of nucleic acid sequences are performed using the GCG Wisconsin Package version 9.1, available from the Genetics Computer Group in Madison, Wis. For convenience, the default parameters (gap creation penalty=12, gap extension penalty=4) specified by that program are intended for use herein to compare sequence identity. Alternately, the Blastn 2.0 program provided by the National Center for Biotechnology Information (found on the world wide web at ncbi.nlm.nih.gov/blast/; Altschul et al., 1990, J Mol Biol 215:403-410) using a gapped alignment with default parameters, may be used to determine the level of identity and similarity between nucleic acid sequences and amino acid sequences. For polypeptide sequence comparisons, a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAM100, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantitate extent of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol. Biol. 147:195 (1981)). Programs for quantitating protein structural similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

[0105] Nucleic acid molecules, expression vectors (e.g., vector genomes), plasmids, including CpG reduced nucleic acid variants encoding FVIII of the invention may be prepared by using recombinant DNA technology methods. The availability of nucleotide sequence information enables preparation of isolated nucleic acid molecules of the invention by a variety of means. For example, CpG reduced nucleic acid variants encoding FVIII can be made using various standard cloning, recombinant DNA technology, via cell expression or in vitro translation and chemical synthesis techniques. Purity of polynucleotides can be determined through sequencing, gel electrophoresis and the like. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides having shared structural features, for example, using an expression library; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to a nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of a subtracted nucleic acid library.

[0106] Nucleic acids of the invention may be maintained as DNA in any convenient cloning vector. In a one embodiment, clones are maintained in a plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, Calif.), which is propagated in a suitable E. coli host cell. Alternatively, nucleic acids may be maintained in vector suitable for expression in mammalian cells. In cases where post-translational modification affects coagulation function, nucleic acid molecule can be expressed in mammalian cells.

[0107] CpG reduced nucleic acid variants encoding FVIII of the invention include cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double-stranded. Thus, this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid of the invention. Such oligonucleotides are useful as probes for detecting FVIII expression.

[0108] A B-domain deleted, CpG reduced nucleic acid variant encoding FVIII of the invention, optionally having amino acid substitutions, deletions or additions, may be prepared in a variety of ways, according to known methods. The protein may be purified from appropriate sources, e.g., transformed bacterial or animal cultured cells or tissues which express engineered FVIII by immune-affinity purification.

[0109] The availability of CpG reduced nucleic acid variants encoding FVIII enables production of FVIII using in vitro expression methods known in the art. For example, a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such as pSP64 or pSP65 for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocyte lysates. In vitro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wis. or BRL, Rockville, Md.

[0110] Alternatively, larger quantities of FVIII may be produced by expression in a suitable prokaryotic or eukaryotic expression system. For example, a CpG reduced nucleic acid variant encoding FVIII, for example, may be inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli or a mammalian cell line such as baby hamster kidney (BHK), CHO or Hela cells. Alternatively, tagged fusion proteins comprising FVIII can be generated. Such FVIII-tagged fusion proteins are encoded by part or all of a DNA molecule, ligated in the correct codon reading frame to a nucleotide sequence encoding a portion or all of a desired polypeptide tag which is inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli or a eukaryotic cell, such as, but not limited to, yeast and mammalian cells.

[0111] Vectors such as those described herein optionally comprise regulatory elements necessary for expression of the DNA in the host cell positioned in such a manner as to permit expression of the encoded protein in the host cell. Such regulatory elements required for expression include, but are not limited to, promoter sequences, enhancer sequences and transcription initiation sequences as set forth herein and known to the skilled artisan.

[0112] A FVIII encoded by a CpG reduced nucleic acid variant, produced by gene expression in a recombinant prokaryotic or eukaryotic system, may be purified according to methods known in the art. In an embodiment, a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium. If expression/secretion vectors are not used, an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein or nickel columns for isolation of recombinant proteins tagged with 6-8 histidine residues at their N-terminus or C-terminus. Alternative tags may comprise the FLAG epitope, GST or the hemagglutinin epitope. Such methods are commonly used by skilled practitioners.

[0113] FVIII proteins, prepared by the aforementioned methods, may be analyzed according to standard procedures. For example, such proteins may be assessed for altered coagulation properties according to known methods.

[0114] Accordingly, the invention also provides methods of making a polypeptide (as disclosed), the method including expression from nucleic acid encoding the polypeptide (generally nucleic acid). This may conveniently be achieved by culturing a host cell, containing such a vector, under appropriate conditions which cause or allow production of the polypeptide. Polypeptides may also be produced in in vitro systems.

[0115] Methods and uses of the invention of the invention include delivering (transducing) nucleic acid (transgene) into host cells, including dividing and/or non-dividing cells. The nucleic acids, recombinant vector (e.g., rAAV), methods, uses and pharmaceutical formulations of the invention are additionally useful in a method of delivering, administering or providing a protein to a subject in need thereof, as a method of treatment. In this manner, the nucleic acid is transcribed and the protein may be produced in vivo in a subject. The subject may benefit from or be in need of the protein because the subject has a deficiency of the protein, or because production of the protein in the subject may impart some therapeutic effect, as a method of treatment or otherwise.

[0116] Vectors including lenti- or parvo-virus vector (e.g., AAV) sequences, recombinant virus particles, methods and uses may be used to deliver a CpG reduced nucleic acid variant encoding FVIII with a biological effect to treat or ameliorate one or more symptoms associated with a FVIII deficiency or abnormality. Recombinant lenti- or parvo-virus vector (e.g., AAV) sequences, plasmids, recombinant virus particles, methods and uses may be used to provide therapy for various disease states involving or due to a FVIII deficiency or abnormality.

[0117] Invention nucleic acids, vectors, expression vectors (e.g., rAAV), and recombinant virus particles, methods and uses permit the treatment of genetic diseases, e.g., a FVIII deficiency. For deficiency state diseases, gene transfer can be used to bring a normal gene into affected tissues for replacement therapy, as well as to create animal models for the disease using antisense mutations. For unbalanced disease states, gene transfer could be used to create a disease state in a model system, which could then be used in efforts to counteract the disease state. The use of site-specific integration of nucleic acid sequences to correct defects is also possible.

[0118] In particular embodiments, CpG reduced nucleic acid variants encoding FVIII may be used, for example, as therapeutic and/or prophylactic agents (protein or nucleic acid) which modulate the blood coagulation cascade or as a transgene in gene. For example, CpG reduced nucleic acid variants encoding FVIII may have similar coagulation activity as wild-type FVIII, or altered coagulation activity compared to wild-type FVII. Cell-based strategies allow continuous expression of CpG reduced nucleic acid variants encoding FVIII in hemophilia A patients. As disclosed herein, certain modifications of FVIII molecules (nucleic acid and protein) result in increased expression at the nucleic acid level, increased coagulation activity thereby effectively improving hemostasis.

[0119] CpG reduced nucleic acid variants encoding FVIII may be used for a variety of purposes in accordance with the invention. In one embodiment, a nucleic acid delivery vehicle (i.e., an expression vector) for modulating blood coagulation is provided wherein the expression vector comprises a CpG reduced nucleic acid variants encoding FVIII as described herein. Administration of FVIII-encoding expression vectors to a patient results in the expression of FVIII protein which serves to alter the coagulation cascade. In accordance with the invention, expression of CpG reduced nucleic acid variants encoding FVIII protein as described herein, or a functional fragment, increases hemostasis.

[0120] In additional embodiments of the invention, compositions and methods are provided for administration of a viral vector comprising a CpG reduced nucleic acid variant encoding FVIII. In one embodiment, the expression vector comprising CpG reduced nucleic acid variant encoding FVIII is a viral vector.

[0121] Expression vectors comprising CpG reduced nucleic acid variants encoding FVIII may be administered alone, or in combination with other molecules useful for modulating hemostasis. According to the invention, vectors, expression vectors or combination of therapeutic agents may be administered to the patient alone or in a pharmaceutically acceptable or biologically compatible compositions.

[0122] Viral vectors such as lenti- and parvo-virus vectors, including AAV serotypes and variants thereof provide a means for delivery of nucleic acid into cells ex vivo, in vitro and in vivo, which encode proteins such that the cells express the encoded protein. AAV are viruses useful as gene therapy vectors as they can penetrate cells and introduce nucleic acid/genetic material so that the nucleic acid/genetic material may be stably maintained in cells. In addition, these viruses can introduce nucleic acid/genetic material into specific sites, for example. Because AAV are not associated with pathogenic disease in humans, AAV vectors are able to deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV pathogenesis or disease.

[0123] Viral vectors which may be used in the invention include, but are not limited to, adeno-associated virus (AAV) vectors of multiple serotypes (e.g., AAV-1 to AAV-12, and others) and hybrid/chimeric AAV vectors, lentivirus vectors and pseudo-typed lentivirus vectors (e.g., Ebola virus, vesicular stomatitis virus (VSV), and feline immunodeficiency virus (FIV)), herpes simplex virus vectors, adenoviral vectors (with or without tissue specific promoters/enhancers), vaccinia virus vectors, retroviral vectors, lentiviral vectors, non-viral vectors and others.

[0124] AAV and lentiviral particles may be used to advantage as vehicles for effective gene delivery. Such virions possess a number of desirable features for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with these vectors also demonstrated no sustained toxicity and immune responses were minimal or undetectable. AAV are known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis or by transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints and hematopoietic stem cells. Non-viral vectors, for example, based on plasmid DNA or minicircles, are also suitable gene transfer vectors for a large gene as that encoding FVIII.

[0125] It may be desirable to introduce a vector that can provide, for example, multiple copies of a desired gene and hence greater amounts of the product of that gene. Improved AAV and lentiviral vectors and methods for producing these vectors have been described in detail in a number of references, patents, and patent applications, including: Wright J. F. (Hum Gene Ther 20:698-706, 2009) a technology used for the production of clinical grade vector at Children's Hospital of Philadelphia. Lentiviral vector can also be produced at CHOP and the other vectors are available through the Lentivirus vector production core laboratory by NHLBI Gene Therapy Resource Program (GTRP)Lentivirus Vector Production Core Laboratory.

[0126] Accordingly, in various embodiments of the invention a vector includes a lenti- or parvo-viral vector, such as an adeno-viral vector. In particular embodiments, a recombinant vector is a parvovirus vector. Parvoviruses are small viruses with a single-stranded DNA genome. Adeno-associated viruses (AAV) are in the parvovirus family.

[0127] Accordingly, the invention provides viral vectors that include CpG reduced nucleic acid variants encoding FVIII. For example, a recombinant AAV vector can include CpG reduced nucleic acid variants encoding FVIII, where the encoded FVIII protein optionally has B-domain deletion. Vector delivery or administration to a subject (e.g., mammal) therefore provides FVIII to a subject such as a mammal (e.g., human).

[0128] Direct delivery of vectors or ex-vivo transduction of human cells followed by infusion into the body will result in FVIII expression thereby exerting a beneficial therapeutic effect on hemostasis. In the context of invention Factor VIII described herein, such administration enhances pro-coagulation activity.

[0129] AAV vectors and lentiviral vectors do not typically include viral genes associated with pathogenesis. Such vectors typically have one or more of the wild type AAV genes deleted in whole or in part, for example, rep and/or cap genes, but retain at least one functional flanking ITR sequence, as necessary for the rescue, replication, and packaging of the recombinant vector into an AAV vector particle. For example, only the essential parts of vector e.g., the ITR and LTR elements, respectively are included. An AAV vector genome would therefore include sequences required in cis for replication and packaging (e.g., functional ITR sequences)

[0130] Recombinant AAV vector, as well as methods and uses thereof, include any viral strain or serotype. As a non-limiting example, a recombinant AAV vector can be based upon any AAV genome, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -rh74, -rh10 or AAV-2i8, for example. Such vectors can be based on the same strain or serotype (or subgroup or variant), or be different from each other. As a non-limiting example, a recombinant AAV vector based upon one serotype genome can be identical to one or more of the capsid proteins that package the vector. In addition, a recombinant AAV vector genome can be based upon an AAV (e.g., AAV2) serotype genome distinct from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector genome can be based upon AAV2, whereas at least one of the three capsid proteins could be a AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 or variant thereof, for example.

[0131] In particular embodiments, adeno-associated virus (AAV) vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 and AAV-2i8, as well as variants (e.g., capsid variants, such as amino acid insertions, additions, substitutions and deletions) thereof, for example, as set forth in WO 2013/158879 (International Application PCT/US2013/037170), WO 2015/013313 (International Application PCT/US2014/047670) and US 2013/0059732 (U.S. Pat. No. 9,169,299, discloses LK01, LK02, LK03, etc.).

[0132] AAV variants include variants and chimeras of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 and AAV-2i8 capsid. Accordingly, AAV vectors and AAV variants (e.g., capsid variants) that include (encapsidate or package) CpG reduced nucleic acid variants encoding FVIII, are provided.

[0133] AAV and AAV variants (e.g., capsid variants) serotypes (e.g., VP1, VP2, and/or VP3 sequences) may or may not be distinct from other AAV serotypes, including, for example, AAV1-AAV12, Rh74 or Rh10 (e.g., distinct from VP1, VP2, and/or VP3 sequences of any of AAV1-AAV12, Rh74 or Rh100 serotypes).

[0134] As used herein, the term serotype is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes). Despite the possibility that AAV variants including capsid variants may not be serologically distinct from a reference AAV or other AAV serotype, they differ by at least one nucleotide or amino acid residue compared to the reference or other AAV serotype.

[0135] Under the traditional definition, a serotype means that the virus of interest has been tested against serum specific for all existing and characterized serotypes for neutralizing activity and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates of are discovered and/or capsid mutants generated, there may or may not be serological differences with any of the currently existing serotypes. Thus, in cases where the new virus (e.g., AAV) has no serological difference, this new virus (e.g., AAV) would be a subgroup or variant of the corresponding serotype. In many cases, serology testing for neutralizing activity has yet to be performed on mutant viruses with capsid sequence modifications to determine if they are of another serotype according to the traditional definition of serotype. Accordingly, for the sake of convenience and to avoid repetition, the term serotype broadly refers to both serologically distinct viruses (e.g., AAV) as well as viruses (e.g., AAV) that are not serologically distinct that may be within a subgroup or a variant of a given serotype.

[0136] AAV vectors therefore include gene/protein sequences identical to gene/protein sequences characteristic for a particular serotype. As used herein, an AAV vector related to AAV1 refers to one or more AAV proteins (e.g., VP1, VP2, and/or VP3 sequences) that has substantial sequence identity to one or more polynucleotides or polypeptide sequences that comprise AAV1. Analogously, an AAV vector related to AAV8 refers to one or more AAV proteins (e.g., VP1, VP2, and/or VP3 sequences) that has substantial sequence identity to one or more polynucleotides or polypeptide sequences that comprise AAV8. An AAV vector related to AAV-Rh74 refers to one or more AAV proteins (e.g., VP1, VP2, and/or VP3 sequences) that has substantial sequence identity to one or more polynucleotides or polypeptide sequences that comprise AAV-Rh74. Such AAV vectors related to another serotype, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8, can therefore have one or more distinct sequences from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 and AAV-2i8, but can exhibit substantial sequence identity to one or more genes and/or proteins, and/or have one or more functional characteristics of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 (e.g., such as cell/tissue tropism). Exemplary non-limiting AAV variants include capsid variants of any of VP1, VP2, and/or VP3.

[0137] In various exemplary embodiments, an AAV vector related to a reference serotype has a polynucleotide, polypeptide or subsequence thereof that includes or consists of a sequence at least 80% or more (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 (e.g., such as an ITR, or a VP1, VP2, and/or VP3 sequences).

[0138] Compositions, methods and uses of the invention include AAV sequences (polypeptides and nucleotides), and subsequences thereof that exhibit less than 100% sequence identity to a reference AAV serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, or AAV-2i8, but are distinct from and not identical to known AAV genes or proteins, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8, genes or proteins, etc. In one embodiment, an AAV polypeptide or subsequence thereof includes or consists of a sequence at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to any reference AAV sequence or subsequence thereof, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 (e.g., VP1, VP2 and/or VP3 capsid or ITR). In certain embodiments, an AAV variant has 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions.

[0139] Recombinant AAV vectors, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 and variant, related, hybrid and chimeric sequences, can be constructed using recombinant techniques that are known to the skilled artisan, to include one or more nucleic acid sequences (transgenes) flanked with one or more functional AAV ITR sequences.

[0140] In one embodiment of the invention, CpG reduced nucleic acid variants encoding FVIII, vector or expression vector, may be administered to a patient via infusion in a biologically compatible carrier, for example, via intravenous injection. The CpG reduced nucleic acid variants encoding FVIII, vectors and expression vectors of the invention may optionally be encapsulated into liposomes or mixed with other phospholipids or micelles to increase stability of the molecule. CpG reduced nucleic acid variants encoding FVIII, vectors and expression vectors of the invention, may be administered alone or in combination with other agents known to modulate hemostasis (e.g., Factor V, Factor Va or derivatives thereof).

[0141] An appropriate composition in which to deliver FVIII may be determined by a medical practitioner upon consideration of a variety of physiological variables, including, but not limited to, the patient's condition and hemodynamic state. A variety of compositions well suited for different applications and routes of administration are well known in the art and are described hereinbelow.

[0142] A preparation containing purified FVIII protein, produced by expression of CpG reduced nucleic acid variants encoding FVIII, vectors and expression vectors of the invention, contains a physiologically acceptable matrix and may be formulated as a pharmaceutical preparation. The preparation can be formulated using substantially known prior art methods, it can be mixed with a buffer containing salts, such as NaCl, CaCl.sub.2, and amino acids, such as glycine and/or lysine, and in a pH range from 6 to 8. Until needed, the purified preparation containing FVIII can be stored in the form of a finished solution or in lyophilized or deep-frozen form.

[0143] A preparation can be stored in lyophilized form and is dissolved into a visually clear solution using an appropriate reconstitution solution. Alternatively, the preparation according to the invention can also be made available as a liquid preparation or as a liquid that is deep-frozen. The preparation according to the invention may optionally be especially stable, i.e., it can be allowed to stand in dissolved form for a prolonged time prior to administration or delivery.

[0144] The preparation according to the invention can be made available as a pharmaceutical preparation with FVIII activity in the form of a one-component preparation or in combination with other factors in the form of a multi-component preparation. Prior to processing the purified protein into a pharmaceutical preparation, the purified protein is subjected to the conventional quality controls and fashioned into a therapeutic form of presentation. In particular, during the recombinant manufacture, the purified preparation is tested for the absence of cellular nucleic acids as well as nucleic acids that are derived from the expression vector, such as is described in EP 0 714 987.

[0145] The pharmaceutical protein preparation may be used at dosages of between 30-100 IU/kg (One I.U is 100 ng/ml) at as single daily injection or up to 3 times/day for several days. Patients may be treated immediately upon presentation at the clinic with a bleed. Alternatively, patients may receive a bolus infusion every eight to twelve hours, or if sufficient improvement is observed, a once daily infusion of the FVIII.

[0146] Accordingly, invention nucleic acids, vectors, recombinant vectors (e.g., rAAV), and recombinant virus particles and other compositions, agents, drugs, biologics (proteins) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are useful for, among other things, administration and delivery to a subject in vivo or ex vivo.

[0147] In particular embodiments, pharmaceutical compositions also contain a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and which may be administered without undue toxicity.

[0148] As used herein the term pharmaceutically acceptable and physiologically acceptable mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact. A pharmaceutically acceptable or physiologically acceptable composition is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing substantial undesirable biological effects. Thus, such a pharmaceutical composition may be used, for example in administering a nucleic acid, vector, viral particle or protein to a subject.

[0149] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugars and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

[0150] The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding, free base forms. In other cases, a preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0151] Pharmaceutical compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions.

[0152] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0153] Compositions suitable for parenteral administration comprise aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non-limiting illustrative examples include water, buffered saline, Hanks' solution, Ringer's solution, dextrose, fructose, ethanol, animal, vegetable or synthetic oils. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[0154] Additionally, suspensions of the active compounds may be prepared as appropriate oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0155] Cosolvents and adjuvants may be added to the formulation. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Adjuvants include, for example, surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

[0156] After pharmaceutical compositions have been prepared, they may be placed in an appropriate container and labeled for treatment. For administration of FVIII-containing vectors or polypeptides, such labeling would include amount, frequency, and method of administration.

[0157] Pharmaceutical compositions and delivery systems appropriate for the compositions, methods and uses of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20.sup.th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18.sup.th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12.sup.th ed., Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11.sup.th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).

[0158] The invention also provides methods for introducing CpG reduced nucleic acid variants encoding FVIII into a cell or an animal. In a particular embodiment, the invention provides methods for modulating hemostasis. In one embodiment, a method includes contact or administration of an individual (patient or subject such as a mammal) with a nucleic acid delivery vehicle (e.g., an AAV vector) comprising CpG reduced nucleic acid variant encoding FVIII under conditions wherein the FVIII polypeptide is expressed in the individual. In another embodiment, a method includes providing cells of an individual (patient or subject such as a mammal) with a nucleic acid delivery vehicle (e.g., an AAV vector) comprising a CpG reduced nucleic acid variant encoding FVIII under conditions wherein the FVIII polypeptide is expressed in the individual.

[0159] From the foregoing, it can be seen that CpG reduced nucleic acid variants encoding FVIII may be used in the treatment of disorders associated with deficient, insufficient or aberrant blood coagulation.

[0160] Compositions of CpG reduced nucleic acid variants encoding FVIII, including vectors, recombinant vectors (e.g., rAAV), and recombinant virus particles can be administered, and methods and uses of the invention can be provided, in a sufficient or effective amount to a subject in need thereof. An effective amount or sufficient amount refers to an amount that provides, in single or multiple doses, alone or in combination, with one or more other compositions (therapeutic or immunosupprosive agents such as a drug), treatments, protocols, or therapeutic regimens agents, a detectable response of any duration of time (long or short term), an expected or desired outcome in or a benefit to a subject of any measurable or detectable degree or for any duration of time (e.g., for minutes, hours, days, months, years, or cured).

[0161] Doses can vary and depend upon the type, onset, progression, severity, frequency, duration, or probability of the disease to which treatment is directed, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan. The dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy and the status of the subject. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.

[0162] The dose to achieve a therapeutic effect, e.g., the dose in vector genomes/per kilogram of body weight (vg/kg), will vary based on several factors including, but not limited to: route of administration, the level of heterologous polynucleotide expression required to achieve a therapeutic effect, the specific disease treated, any host immune response to the viral vector, a host immune response to the heterologous polynucleotide or expression product (protein), and the stability of the protein expressed. One skilled in the art can determine a rAAV/vector genome dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors. Generally, doses will range from at least 1?10.sup.8, or more, for example, 1?10.sup.9, 1?10.sup.10, 1?10.sup.11, 1?10.sup.12, 1?10.sup.13 or 1?10.sup.14, or more, vector genomes per kilogram (vg/kg) of the weight of the subject, to achieve a therapeutic effect. AAV dose in the range of 1?10.sup.10-1?10.sup.11 in mice, and 1?10.sup.12-1?10.sup.13 in dogs have been effective.

[0163] Using hemophilia B as an example, generally speaking, it is believed that, in order to achieve a therapeutic effect, a blood coagulation factor concentration that is greater than 1% of factor concentration found in a normal individual is needed to change a severe disease phenotype to a moderate one. A severe phenotype is characterized by joint damage and life-threatening bleeds. To convert a moderate disease phenotype into a mild one, it is believed that a blood coagulation factor concentration greater than 5% of normal is needed. FVIII levels in normal humans are about 150-200 ng/ml plasma, but may be less (e.g., range of about 100-150 ng/ml) or greater (e.g., range of about 200-300 ng/ml) and still considered normal due to functioning clotting as determined, for example, by an activated partial thromboplastin time (aPTT) one-stage clotting assay. Thus, a therapeutic effect can be achieved by expression of FVIII such that the total amount of FVIII in the subject/human is greater than 1% of the FVIII present in normal subjects/humans, e.g., 1% of 100-300 ng/ml.

[0164] With respect to treating such a hemophilic subject, a typical dose is at least 1?10.sup.10 vector genomes (vg) per kilogram (vg/kg) of the weight of the subject, or between about 1?10.sup.10 to 1?10.sup.11 vg/kg of the weight of the subject, or between about 1?10.sup.11 to 1?10.sup.12 vg/kg of the weight of the subject, or between about 1?10.sup.12 to 1?10.sup.13 vg/kg of the weight of the subject, to achieve a desired therapeutic effect. AAV vector doses can be at a level, typically at the lower end of the dose spectrum, such that there is not a substantial immune response against the FVIII or AAV vector.

[0165] The doses of an effective amount or sufficient amount for treatment (e.g., to ameliorate or to provide a therapeutic benefit or improvement) typically are effective to provide a response to one, multiple or all adverse symptoms, consequences or complications of the disease, one or more adverse symptoms, disorders, illnesses, pathologies, or complications, for example, caused by or associated with the disease, to a measurable extent, although decreasing, reducing, inhibiting, suppressing, limiting or controlling progression or worsening of the disease is a satisfactory outcome.

[0166] An effective amount or a sufficient amount can but need not be provided in a single administration, may require multiple administrations, and, can but need not be, administered alone or in combination with another composition (e.g., agent), treatment, protocol or therapeutic regimen. For example, the amount may be proportionally increased as indicated by the need of the subject, type, status and severity of the disease treated or side effects (if any) of treatment. In addition, an effective amount or a sufficient amount need not be effective or sufficient if given in single or multiple doses without a second composition (e.g., another drug or agent), treatment, protocol or therapeutic regimen, since additional doses, amounts or duration above and beyond such doses, or additional compositions (e.g., drugs or agents), treatments, protocols or therapeutic regimens may be included in order to be considered effective or sufficient in a given subject. Amounts considered effective also include amounts that result in a reduction of the use of another treatment, therapeutic regimen or protocol, such as administration of recombinant clotting factor protein (e.g., FVIII) for treatment of a clotting disorder (e.g., hemophilia A).

[0167] Accordingly, methods and uses of the invention also include, among other things, methods and uses that result in a reduced need or use of another compound, agent, drug, therapeutic regimen, treatment protocol, process, or remedy. For example, for a blood clotting disease, a method or use of the invention has a therapeutic benefit if in a given subject a less frequent or reduced dose or elimination of administration of a recombinant clotting factor protein to supplement for the deficient or defective (abnormal or mutant) endogenous clotting factor in the subject. Thus, in accordance with the invention, methods and uses of reducing need or use of another treatment or therapy are provided.

[0168] An effective amount or a sufficient amount need not be effective in each and every subject treated, nor a majority of treated subjects in a given group or population. An effective amount or a sufficient amount means effectiveness or sufficiency in a particular subject, not a group or the general population. As is typical for such methods, some subjects will exhibit a greater response, or less or no response to a given treatment method or use.

[0169] The term ameliorate means a detectable or measurable improvement in a subject's disease or symptom thereof, or an underlying cellular response. A detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression, or duration of the disease, or complication caused by or associated with the disease, or an improvement in a symptom or an underlying cause or a consequence of the disease, or a reversal of the disease. For HemA, an effective amount would be an amount that reduces frequency or severity of acute bleeding episodes in a subject, for example, or an amount that reduces clotting time as measured by a clotting assay, for example.

[0170] Accordingly, pharmaceutical compositions of the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended therapeutic purpose. Determining a therapeutically effective dose is well within the capability of a skilled medical practitioner using the techniques and guidance provided in the invention.

[0171] Therapeutic doses will depend on, among other factors, the age and general condition of the subject, the severity of the aberrant blood coagulation phenotype, and the strength of the control sequences regulating the expression levels of CpG reduced nucleic acid variants encoding FVIII. Thus, a therapeutically effective amount in humans will fall in a relatively broad range that may be determined by a medical practitioner based on the response of an individual patient to vector-based FVIII treatment. Such doses may be alone or in combination with an immunosuppressive agent or drug.

[0172] Compositions such as pharmaceutical compositions may be delivered to a subject, so as to allow production of a biologically active protein (e.g., Factor VIII (FVIII) encoded by CpG reduced nucleic acid variant) or by inducing continuous expression of the FVIII transgene in vivo by gene- and or cell-based therapies or by ex-vivo modification of the patient's or donor's cells. In a particular embodiment, pharmaceutical compositions comprising sufficient genetic material to enable a recipient to produce a therapeutically effective amount of a FVIII polypeptide can influence hemostasis in the subject.

[0173] The compositions may be administered alone. In certain embodiments, CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle provides a therapeutic effect without an immunosuppressive agent. The therapeutic effect of FVIII optionally is sustained for a period of time, e.g., 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, or 30-50 days or more, for example, 50-75, 75-100, 100-150, 150-200 days or more without administering an immunosuppressive agent. Accordingly, in certain embodiments CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle provide a therapeutic effect without administering an immunosuppressive agent for a period of time.

[0174] The compositions may be administered in combination with at least one other agent. In certain embodiments, CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle are administered in conjunction with one or more immunosuppressive agents prior to, substantially at the same time or after administering a CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle. In certain embodiments, CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle are administered in conjunction with one or more immunosuppressive agents after a period of time following administering a CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle, e.g., 1-12, 12-24 or 24-48 hours, or 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30, 30-50, or more than 50 days following administering a CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle. Such administration of immunosuppressive agents after a period of time following administering a CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle if there is a decrease in FVIII after the initial expression levels for a period of time, e.g., 20-25, 25-30, 30-50, 50-75, 75-100, 100-150, 150-200 or more than 200 days following administering a CpG reduced nucleic acid variant encoding FVIII, vector, expression vector/recombinant vector (e.g., rAAV), or recombinant virus particle.

[0175] In certain embodiments, an immunosuppressive agent is an anti-inflammatory agent. In certain embodiments, an immunosuppressive agent is a steroid. In certain embodiments, an immunosuppressive agent is cyclosporine (e.g., cyclosporine A), mycophenolate, Rituximab or a derivative thereof. Additional particular agents include a stabilizing compound.

[0176] Compositions may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents (e.g., co-factors) which influence hemostasis.

[0177] Factor VIII, alone or in combination with other agents may be administered or contacted or directly infused into a patient in an appropriate biological carrier as described herein. Vectors and expression vectors of the invention comprising a CpG reduced nucleic acid variant encoding FVIII, may be administered to a patient by a variety of means to achieve and optionally maintain for a period of time a prophylactically and/or therapeutically effective level of FVIII polypeptide. One of skill in the art could readily determine specific protocols for using the FVIII encoding expression vectors of the invention for the therapeutic treatment of a particular patient.

[0178] Protocols for the generation of adenoviral vectors and administration to patients have been described in U.S. Pat. Nos. 5,998,205; 6,228,646; 6,093,699; 6,100,242; and International Patent Application Nos. WO 94/17810 and WO 94/23744, which are incorporated herein by reference in their entirety. In particular, for example, AAV vectors are employed to deliver Factor VIII (FVIII) encoded by CpG reduced nucleic acid variants to a patient in need thereof.

[0179] Factor VIII (FVIII) encoded by CpG reduced nucleic acid variants delivered by way of AAVvectors of the invention may be administered to a patient by any means known.

[0180] Methods and uses of the invention include delivery and administration systemically, regionally or locally, or by any route, for example, by injection or infusion. Delivery of the pharmaceutical compositions in vivo may generally be accomplished via injection using a conventional syringe, although other delivery methods such as convection-enhanced delivery are envisioned (See e.g., U.S. Pat. No. 5,720,720). For example, compositions may be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intravenously, intra-pleurally, intraarterially, orally, intrahepatically, via the portal vein, or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications. A clinician specializing in the treatment of patients with blood coagulation disorders may determine the optimal route for administration of the adenoviral-associated vectors comprising CpG reduced nucleic acid variants encoding FVIII based on a number of criteria, including, but not limited to: the condition of the patient and the purpose of the treatment (e.g., enhanced or reduced blood coagulation).

[0181] Invention methods and uses can be combined with any compound, agent, drug, treatment or other therapeutic regimen or protocol having a desired therapeutic, beneficial, additive, synergistic or complementary activity or effect. Exemplary combination compositions and treatments include second actives, such as, biologics (proteins), agents (e.g., immunosuppressive agents) and drugs. Such biologics (proteins), agents, drugs, treatments and therapies can be administered or performed prior to, substantially contemporaneously with or following any other method or use of the invention, for example, a therapeutic method of treating a subject for a blood clotting disease such as HemA.

[0182] The compound, agent, drug, treatment or other therapeutic regimen or protocol can be administered as a combination composition, or administered separately, such as concurrently or in series or sequentially (prior to or following) delivery or administration of a nucleic acid, vector, recombinant vector (e.g., rAAV), or recombinant virus particle. The invention therefore provides combinations in which a method or use of the invention is in a combination with any compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition, set forth herein or known to one of skill in the art. The compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition can be administered or performed prior to, substantially contemporaneously with or following administration of a nucleic acid, vector, recombinant vector (e.g., rAAV), or recombinant virus particle of the invention, to a subject.

[0183] The invention is useful in animals including human and veterinary medical applications. Suitable subjects therefore include mammals, such as humans, as well as non-human mammals. The term subject refers to an animal, typically a mammal, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and experimental animals (mouse, rat, rabbit, guinea pig). Human subjects include fetal, neonatal, infant, juvenile and adult subjects. Subjects include animal disease models, for example, mouse and other animal models of blood clotting diseases such as HemA and others known to those of skill in the art.

[0184] Subjects appropriate for treatment in accordance with the invention include those having or at risk of producing an insufficient amount or having a deficiency in a functional gene product (e.g., FVIII protein), or produce an aberrant, partially functional or non-functional gene product (e.g., FVIII protein), which can lead to disease. Subjects appropriate for treatment in accordance with the invention also include those having or at risk of producing an aberrant, or defective (mutant) gene product (protein) that leads to a disease such that reducing amounts, expression or function of the aberrant, or defective (mutant) gene product (protein) would lead to treatment of the disease, or reduce one or more symptoms or ameliorate the disease. Target subjects therefore include subjects having aberrant, insufficient or absent blood clotting factor production, such as hemophiliacs (e.g., hemophilia A).

[0185] Subjects can be tested for an immune response, e.g., antibodies against AAV. Candidate hemophilia subjects can therefore be screened prior to treatment according to a method of the invention. Subjects also can be tested for antibodies against AAV after treatment, and optionally monitored for a period of time after treatment. Subjects developing antibodies can be treated with an immunosuppressive agent, or can be administered one or more additional amounts of AAV vector.

[0186] Subjects appropriate for treatment in accordance with the invention also include those having or at risk of producing antibodies against AAV. AAV vectors can be administered or delivered to such subjects using several techniques. For example, empty capsid AAV (i.e., AAV lacking a FVIII nucleic acid) can be delivered to bind to the AAV antibodies in the subject thereby allowing the AAV vector bearing CpG reduced nucleic acid variant encoding FVIII to transform cells of the subject. Amounts of empty capsid AAV to administer can be calibrated based upon the amount of AAV antibodies produced in a particular subject. Empty capsid can be of any AAV serotype, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8.

[0187] Alternatively or in addition to, AAV vector can be delivered by direct intramuscular injection (e.g., one or more slow-twitch fibers of a muscle). In another alternative, a catheter introduced into the femoral artery can be used to delivery AAV vectors to liver via the hepatic artery. Non-surgical means can also be employed, such as endoscopic retrograde cholangiopancreatography (ERCP), to deliver AAV vectors directly to the liver, thereby bypassing the bloodstream and AAV antibodies. Other ductal systems, such as the ducts of the submandibular gland, can also be used as portals for delivering AAV vectors into a subject that develops or has preexisting anti-AAV antibodies.

[0188] Administration or in vivo delivery to a subject can be performed prior to development of an adverse symptom, condition, complication, etc. caused by or associated with the disease. For example, a screen (e.g., genetic) can be used to identify such subjects as candidates for invention compositions, methods and uses. Such subjects therefore include those screened positive for an insufficient amount or a deficiency in a functional gene product (e.g., FVIII protein), or that produce an aberrant, partially functional or non-functional gene product (e.g., FVIII protein).

[0189] Administration or in vivo delivery to a subject in accordance with the methods and uses of the invention as disclosed herein can be practiced within 1-2, 2-4, 4-12, 12-24 or 24-72 hours after a subject has been identified as having the disease targeted for treatment, has one or more symptoms of the disease, or has been screened and is identified as positive as set forth herein even though the subject does not have one or more symptoms of the disease. Of course, methods and uses of the invention can be practiced 1-7, 7-14, 14-21, 21-48 or more days, months or years after a subject has been identified as having the disease targeted for treatment, has one or more symptoms of the disease, or has been screened and is identified as positive as set forth herein.

[0190] A unit dosage form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect). Unit dosage forms may be within, for example, ampules and vials, which may include a liquid composition, or a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo. Individual unit dosage forms can be included in multi-dose kits or containers. Recombinant vector (e.g., rAAV) sequences, recombinant virus particles, and pharmaceutical compositions thereof can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.

[0191] Subjects can be tested for FVIII amounts or FVIII activity to determine if such subjects are appropriate for treatment according to a method of the invention. Candidate hemophilia subjects can be tested for FVIII amounts or activity prior to treatment according to a method of the invention. Subjects also can be tested for amounts of FVIII or FVIII activity after treatment according to a method of the invention. Such treated subjects can be monitored after treatment for FVIII amounts or FVIII activity, periodically, e.g., every 1-4 weeks or 1-6 months.

[0192] Subjects can be tested for one or more liver enzymes for an adverse response or to determine if such subjects are appropriate for treatment according to a method of the invention. Candidate hemophilia subjects can therefore be screened for amounts of one or more liver enzymes prior to treatment according to a method of the invention. Subjects also can be tested for amounts of one or more liver enzymes after treatment according to a method of the invention. Such treated subjects can be monitored after treatment for elevated liver enzymes, periodically, e.g., every 1-4 weeks or 1-6 months.

[0193] Exemplary liver enzymes include alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), but other enzymes indicative of liver damage can also be monitored. A normal level of these enzymes in the circulation is typically defined as a range that has an upper level, above which the enzyme level is considered elevated, and therefore indicative of liver damage. A normal range depends in part on the standards used by the clinical laboratory conducting the assay.

[0194] Subjects can be monitored for bleeding episodes to determine if such subjects are eligible for or responding to treatment, and/or the amount or duration of responsiveness. Subjects can be monitored for bleeding episodes to determine if such subjects are in need of an additional treatment, e.g., a subsequent AAV vector administration or administration of an immunosuppressive agent, or more frequent monitoring. Hemophilia subjects can therefore be monitored for bleeding episodes prior to and after treatment according to a method of the invention. Subjects also can be tested for frequency and severity of bleeding episodes during or after treatment according to a method of the invention.

[0195] The invention provides kits with packaging material and one or more components therein. A kit typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein. A kit can contain a collection of such components, e.g., a nucleic acid, recombinant vector, virus (e.g., AAV) vector, or virus particle and optionally a second active, such as another compound, agent, drug or composition.

[0196] A kit refers to a physical structure housing one or more components of the kit. Packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).

[0197] Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels or inserts can include information identifying manufacturer, lot numbers, manufacture location and date, expiration dates. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location and date. Labels or inserts can include information on a disease for which a kit component may be used. Labels or inserts can include instructions for the clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimes described herein.

[0198] Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, complications or reactions, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects or complications could also occur when the subject has, will be or is currently taking one or more other medications that may be incompatible with the composition, or the subject has, will be or is currently undergoing another treatment protocol or therapeutic regimen which would be incompatible with the composition and, therefore, instructions could include information regarding such incompatibilities.

[0199] Labels or inserts include printed matter, e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component. Labels or inserts can additionally include a computer readable medium, such as a bar-coded printed label, a disk, optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media or memory type cards.

[0200] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0201] All patents, patent applications, publications, and other references, GenBank citations and ATCC citations cited herein are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will control.

[0202] Various terms relating to the biological molecules of the invention are used hereinabove and also throughout the specification and claims.

[0203] All of the features disclosed herein may be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature serving a same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, disclosed features (e.g., CpG reduced nucleic acid variants encoding FVIII, vector, plasmid, expression/recombinant vector (e.g., rAAV) sequence, or recombinant virus particle) are an example of a genus of equivalent or similar features.

[0204] As used herein, the singular forms a, and, and the include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a nucleic acid includes a plurality of such nucleic acids, reference to a vector includes a plurality of such vectors, and reference to a virus or particle includes a plurality of such viruses/particles.

[0205] As used herein, all numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to 80% or more identity, includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc., and so forth.

[0206] Reference to an integer with more (greater) or less than includes any number greater or less than the reference number, respectively. Thus, for example, a reference to less than 100, includes 99, 98, 97, etc. all the way down to the number one (1); and less than 10, includes 9, 8, 7, etc. all the way down to the number one (1).

[0207] As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to a numerical range, such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so forth. Reference to a range of 1-50 therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so forth.

[0208] Reference to a series of ranges includes ranges which combine the values of the boundaries of different ranges within the series. Thus, to illustrate reference to a series of ranges, for example, of 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-850, includes ranges of 1-20, 1-30, 1-40, 1-50, 1-60, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 50-75, 50-100, 50-150, 50-200, 50-250, 100-200, 100-250, 100-300, 100-350, 100-400, 100-500, 150-250, 150-300, 150-350, 150-400, 150-450, 150-500, etc.

[0209] The invention is generally disclosed herein using affirmative language to describe the numerous embodiments and aspects. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures. For example, in certain embodiments or aspects of the invention, materials and/or method steps are excluded. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include aspects that are not expressly excluded in the invention are nevertheless disclosed herein.

[0210] A number of embodiments of the invention have been described. Nevertheless, one skilled in the art, without departing from the spirit and scope of the invention, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, the following examples are intended to illustrate but not limit the scope of the invention claimed in any way.

Example 1

[0211] Disclosed herein are gene constructs for use in gene therapy methods to treat hemophilia. In addition, these factor VIII (FVIII) encoding gene constructs may be useful in vitro in the setting of protein expression systems, to produce recombinant FVIII protein for administration. Each gene construct can optionally include one or more of an expression control (e.g., promoter) element, factor VIII gene and other regulatory features required for expression of the gene, such as introns, ITRs, stop codons, poly A signals, etc.

Example 2

CpG Reduced Factor VIII DNA Sequences and Certain Vector Constructs, Plasmid Constructs and AAV Vector Producing Cell Lines.

[0212] 18 different CpG reduced nucleic acid variants encoding FVIII (SEQ ID NOs: 1-18) were produced and assessed in expression assays. CpG reduced human FVIII cDNA constructs were generated with a mutant transthyretin (TTRmut) promoter (SEQ ID NO:22).

[0213] AAV-SPK-8011 expression cassette has the CpG reduced FVIII-X07 nucleic acid sequence and the LK03 capsid for packaging. LK03 capsid has substantial homology to AAV3, a non-pathogenic, naturally replication deficient single-stranded DNA virus.

[0214] Packaging plasmid pLK03 is a 7,484 bp plasmid construct that carries the AAV2 Rep and AAV-LK03 Cap genes under the control of AAV2 p5 promoter, bacterial origin of replication and gene conferring resistance to Kanamycin in bacterial cells. In this construct, the p5 rep promoter has been moved 3 of the cap gene to reduce the potential for formation of wild-type or pseudo wild type AAV species, and to increase yield of the vector.

[0215] The cloned DNA for gene transfer is a gene expression cassette, packaged into the AAV-LK03 capsid as a single-stranded genome, encoding human coagulation factor VIII (hFVIII) under control of a liver-specific promoter. The expression plasmid is referred to as pAAV-TTRmut-hFVIII-X07. It was modified by the introduction of 4 point mutations in the TTR promoter, and the coding region optimized to increase expression of human FVIII. The AAV expression cassette contains the following elements: [0216] AAV2 ITR [0217] Transthyretin (TTR) promoter: A liver-specific transthyretin (TTR) promoter with 4 point mutations that increase gene expression compared with the wild type promoter (Costa et al. 1991) [0218] Synthetic intron: Derived from human elongation factor EF-1 alpha gene [0219] FVIII coding sequence: B-domain deleted, codon-optimized human FVIII coding sequence. [0220] Rabbit beta globin poly A signal sequence (Levitt et al. 1989). [0221] AAV2 ITR

[0222] Three DNA plasmid constructs are used to transfect human embryo kidney 293 cells to produce the SPK-8011 vector by a helper virus-free process (Matsushita et al. 1998): [0223] The gene cassette (hFVIII coding sequence and associated regulatory elements) is cloned into a plasmid to give the vector plasmid, pAAV-TTRmut-hFVIII-X07. [0224] The AAV viral genome (rep and cap) lacking the viral ITRs is cloned into a plasmid to give the AAV packaging plasmid, pLK03, providing the required AAV2 rep and AAV-LK03 cap genes in trans for AAV vector packaging. The viral promoter (p5) for the rep gene was relocated in the plasmid in order to prevent formation of replication competent AAV by non-homologous recombination. [0225] Three genes from adenovirus-2 are cloned into a third plasmid (pCCVC-AD2HP) providing the necessary helper virus genes for vector production. Plasmid pCCVC-AD2HPv2 is an 11,832 bp plasmid construct that carries three adenovirus genes, E2A, E4 and the VA RNAs to provide helper functions necessary for replication and encapsidation of AAV vector. Plasmid pCCVC-AD2HPv2 is a derivative of pCCVC-AD2HP in which the DrdI-DrdI 1882 bp restriction fragment containing the Amp.sup.R gene and part of the pUC ori sequence has been removed and replaced with the DrdI-DrdI fragment from plasmid pAAV2-hRPE65v2 containing the entire Kan.sup.R gene and part of the pUC ori sequence.

[0226] The cell substrate used for AAV vector production is a derivative of primary human embryonic kidney cells (HEK) 293. The HEK293 cell line is a permanent line transformed by sheared human adenovirus type 5 (Ad5) DNA (Graham et al. 1977). The Working Cell Bank is derived from a characterized HEK293 Master Cell Bank from the Center for Cellular and Molecular Therapeutics (CCMT) at The Children's Hospital of Philadelphia (CHOP).

Example 3

Evaluation of AAV-hFVIII Vectors in Mice.

[0227] FVIII transgene constructs (hFVIII) were packaged into adeno-associated viral (AAV) vectors and delivered to mice. In brief, groups of 4 hemophilia A/CD4.sup.?/? mice were injected at 8-10 weeks of age with 4?10.sup.12 vg/kg of AAV-hFVIII vectors. Immunodeficient mice were used to enable quantification of FVIII plasma levels, as the inhibitory antibodies to FVIII that are generated in normal mice prevent long-term analysis of FVIII expression.

[0228] Levels of FVIII expression were determined and in several instances were higher than expression provided by the CO3 sequence (SEQ ID NO:21) encoding hFVIII. As shown in FIG. 2, vectors including AAV-SPK-8005 expressed higher hFVIII levels compared to reference AAV-CO3vector. The data surprisingly reveal that several of the DNA sequences expressed higher levels of FVIII than a codon-optimized sequence (CO3, SEQ ID NO:21) encoding FVIII.

[0229] AAV-Spark8005 (also designated SPK-8005), rather than AAV-LK03-hFVIII (also designated AAV-LK03-hFVIII and SPK-8011), was used in this study to ensure efficient transduction (i.e.; hFVIII transgene expression) of mouse hepatocytes. Thus, this study was designed to evaluate the safety of sustained hFVIII expression, and not the safety of the AAV-LK03 capsid.

[0230] The three doses of AAV-SPK-8005-hFVIII used (4?10.sup.10, 8?10.sup.10, 1.6?10.sup.11 vg/mouse; approximately 1.6?10.sup.12, 3.2?10.sup.12, 6.4?10.sup.12 vg/kg, based on mouse weight of 25 g) were chosen to generate approximately 5-25, 25-75, and 50-150% hFVIII antigen levels, respectively. The study involved 350 male NOD/SCID mice (Table 1) and was divided into two sub-studies: Main study (n=270) and Bioanalysis study (n=80). In the Main study, 60 mice were treated with either vehicle or one of the three doses of vector (4?10.sup.10, 8?10.sup.10, 1.6?10.sup.11 vg/mouse). Ten mice were used for day 29/30 assessments of clinical chemistries, 10 were used for hematology, and coagulation assessments were made on the remaining 10 animals. These 30 mice were sacrificed on day 29 or 30. The other group of 30 mice that were treated with either vehicle or one of the three vector doses was handled similarly at the day 87 timepoint, and they were sacrificed on day 87. Upon termination, gross pathology observations were performed on all animals in the Main study and comprehensive histopathology was performed on 10 animals/cohort per timepoint (hematology subset). Another cohort of 30 na?ve mice was used for background control clinical pathology measurements.

[0231] In the Bioanalysis study, 20 mice were injected with vehicle or one of the three vector doses. These animals were bled prior to test article injection and serially on days 15, 30, 60, and 87. The intended volume of plasma collected for each sample should have been sufficient for determination of both hFVIII antigen and D-dimer levels. However, due to insufficient plasma volume collections, only a single assay was performed on individual mouse plasma at all timepoints, with the exception of the terminal timepoint. Thus, some mice were evaluated for circulating levels of hFVIII antigen and others for D-dimer levels. Since more plasma is required to perform the hFVIII ELISA (minimum of 50 uL) than the D-dimer ELISA (minimum of 20 uL), the choice of assay was dictated by the volume of plasma collected.

TABLE-US-00001 TABLE 1 Mouse study design No. of Mice Dose Dose Dose Main Study Group Level Volume Concentration Day 29/30 Day 87 Bioanalysis No. Test Material (vg/mouse) (?L/mouse) (vg/mL) Subset Subset Study.sup.a Na?ve.sup.b None Na na na na na na 1 Control Article 0 200 0 30 30 20 2 AAV-SPK-8005.sup.c 4 ? 10.sup.10 200 2 ? 10.sup.11 30 30 20 3 AAV-SPK-8005.sup.c 8 ? 10.sup.10 200 4 ? 10.sup.11 30 30 20 4 AAV-SPK-8005.sup.c 1.6 ? 10.sup.11 200 8 ? 10.sup.11 30 30 20 .sup.aBlood was collected from all mice at predose and on Days 15, 30, 60, and 87 of study. .sup.bBlood was collected from 30 total mice (10 na?ve mice per clinical pathology evaluation) Clinical Pathology-Main Study for background control levels. .sup.cAAV-SPK-8005-hFVIII is also designated SPK-8005 na = Not applicable

[0232] Plasma FVIII Antigen Levels:

[0233] As shown in FIGS. 3A-3B, a dose-response was observed in the circulating levels of hFVIII antigen over the course of 87 days. At the low dose of vector (4?10.sup.10 vg/mouse), average hFVIII levels of 64+/?49 ng/ml were seen at day 60 post-injection, and 115+/?60 ng/ml and 273+/58 ng/ml were seen at the mid and high doses, respectively. These antigen levels represent 43, 77, and 182% of normal hFVIII antigen (150 ng/mL is equivalent to 100%). Therefore, in hemostatically normal NOD/SCID mice, total (mouse+human) FVIII levels of 143%, 177% and 282% would be expected at the three dose levels, respectively. Thus, using AAV-SPK-8005-hFVIII, sustained and supraphysiological levels of hFVIII were observed in the plasma of immunodeficient mice, making this study appropriate for assessing safety of long-term expression of hFVIII.

[0234] D-Dimer Levels:

[0235] In order to assess the potential for thrombogenesis due to sustained expression of hFVIII in hemostatically normal, but immunodeficient mice, D-dimer antigen levels were measured. The average predose level of D-dimers among 50 na?ve mice was 8.8+/?2.9 ng/ml. The data in FIG. 3C represent average D-dimer levels in the four dose cohorts. There was no statistical difference in D-dimer levels between cohorts at all five timepoints (1 way ANOVA p=0.46). It was concluded that sustained expression of hFVIII at levels has high as 194% of normal (day 30), and for at least 87 days, is not associated with an elevated level of D-dimers in this strain of mice.

[0236] Clinical and Anatomical Pathology:

[0237] There were nine animals (6 Main study and 3 Bioanalysis study) either euthanized early or found dead during the course of this study.

[0238] The six Main study animals were evaluated histopathologically, and malignant lymphomas were observed in four of these six mice, including one vehicle control-injected mouse. (Group 1 animal 7729, Group 3 animal 7871, Group 3 animal 7880, and Group 3 animal 7874). The biological significance of the neoplastic findings was considered to be equivocal. Statistical significance of individual group comparisons to the control group was considered unlikely. A high spontaneous frequency of thymic lymphomas, as well as neoplastic enlargements of spleens and lymph nodes are known to occur in this strain (Prochazka, Gaskins, Shultz, & Leiter, 1992).

[0239] Non-neoplastic findings related to the test article were not present in these six mice. The microscopic findings observed were considered incidental and of the nature commonly observed in this strain and age of mice, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of AAV-SPK-8005-hFVIII.

[0240] The remaining 234 mice included in the Main study survived to the scheduled timepoints. No adverse or AAV-SPK-8005-hFVIII-related clinical observations occurred in the mice throughout the study. All clinical observations of scab formation, fur loss or thin cover and bent tail were considered unrelated to administration of AAV-SPK-8005-hFVIII, because these observations are common in this mouse species and/or occurred across groups. Body weights and body weight gains were comparable among dose groups and unaffected by administration of AAV-SPK-8005-hFVIII. An apparent significant (p<0.05 or p<0.01) reduction in Group 4 mean body weights from Day 32 to study completion was attributed to redistribution of the group weights (some heavier animals euthanized in Group 4 as compared to Group 1) after the Day 29/30 euthanasia, and was not related to AAV-SPK-8005-hFVIII administration. Group 4 mice gained weight in a comparable manner to the other groups throughout the study. All other significant (p<0.05 or p<0.01) differences in mean body weights or body weight gains were not considered related to AAV-SPK-8005-hFVIII, because the increases and decreases were sporadic with no dose-dependence and were considered related to normal fluctuations in mouse body weights.

[0241] Clinical pathology was performed on the Main study animals. Clinical chemistry parameters were analyzed on 10 mice/cohort per time point (day 29/30 and day 87). Coagulation assessments were performed on another group of 10 mice/cohort, and hematology measurements were made on the other group of 10 mice/cohort. Gross pathology was performed on all animals and histopathology was performed on the group of 10 mice utilized for hematology assessments. There were no AAV-SPK-8005-hFVIII-related changes in hematology or clinical chemistry parameters in mice from either the Day 29/30 and Day 87 euthanasia timepoints. In general, where significant (p<0.05 or p<0.01) differences in hematology and clinical chemistry parameters as compared to the control values existed, the differences were not related to AAV-SPK-8005-hFVIII, because corresponding parameters were unaffected and the observations were not dose-dependent. All changes in clinical chemistry and hematology parameters were sporadic, attributed to a single animal, of a magnitude of change commonly observed in laboratory animals and/or within the clinical pathology parameters assessed for the na?ve animals.

[0242] Changes in coagulation parameters were observed in mice administered AAV-SPK-8005-hFVIII. A dose-dependent reduction in mean aPTT was observed at the Day 29/30 timepoint, with Group 3 and 4 values significantly (p<0.05 or p<0.01) different from control values. A significant (p<0.01) reduction in mean aPTT values was also observed in all AAV-SPK-8005-hFVIII groups as compared to the control group at the Day 87 timepoint. Reduced mean prothrombin time was also observed in the AAV-SPK-8005-hFVIII groups as compared to the control group at Days 29/30 and 87, however the reduction was only statistically significant (p<0.05 or p<0.01) for Groups 2 and 3 on day 29/30 and Group 4 on Day 87. Mean fibrinogen values were comparable among dose groups throughout the study. These effects are considered related to the pharmacologic effect of AAV-SPK-8005-hFVIII, and not considered adverse. As shown in FIGS. 3A-3C and discussed above, all mice injected with AAV-SPK-8005-hFVIII expressed hFVIII antigen and thus, supraphysiological levels of total FVIII are predicted to circulate in the plasma of these hemostatically normal mice. These levels would be expected to have an effect on coagulation parameters, such as reduced aPTT and prothrombin times.

[0243] A group of 120 Main study mice (30/cohort) were sacrificed on day 29 or 30 of the study. No gross pathology observations related to AAV-SPK-8005-hFVIII were made on these mice. Analysis of organ weights revealed that the absolute weights of heart and kidney differed between the 10 control and vector-injected animals sacrificed on day 29; however, this was not observed between the 10 control and vector-injected animals sacrificed on day 30, so the significance of this finding is unclear. There was no microscopic correlate to the statistically significant increase in heart and kidney absolute weights (and these weights as a percent of brain weight) observed on day 29. Furthermore, heart and kidney weight as a percent of body weight were not significantly different from controls. There was a significant increase in mean absolute lung weight in Group 2 animals, but this was considered incidental and unrelated AAV-SPK-8005-hFVIII because there was no dose dependence. No other organ weight changes were noted at Day 29/30.

[0244] Upon histopathological analyses on Day 29/30, there were five animals with neoplastic findings. A bronchioloalveolar adenoma was observed in one Group 2 animal (7824). Malignant lymphoma was observed in one Group 2 animal (7838), one Group 3 animal (7885), and one group 4 animal (7941). Adenoma was observed in stomach in one Group 4 animal (7942). No neoplastic findings were observed in Group 1. The biological significance of the neoplastic findings is considered to be equivocal. Statistical significance of individual group comparisons to the control group is unlikely. However, it is noteworthy that neoplastic findings were only observed in treated animals at Day 29/30. In the absence of historical control data for NOD SCID mice at a comparable age, these neoplastic findings are inconclusive.

[0245] No test article-related non-neoplastic microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of mice, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of AAV-SPK-8005-hFVIII.

[0246] Another group of 120 Main study mice (30/cohort) were sacrificed 87 days post-injection and analyzed in a similar manner. Although no gross pathology observations considered related to AAV-SPK-8005-hFVIII were seen, lesions were observed in four mice (one enlarged thymus not analyzed histologically, one enlarged thymus correlated to malignant lymphoma, one enlarged spleen not analyzed histologically, one discolored testis). In contrast to what was observed at day 29/30, decreased heart weights, not increased weights were observed. In addition, decreases in liver weights were seen. The statistically significant changes in heart weight were small and not clearly related to dose. The statistical significant change in absolute liver weight was small and the liver weights to body and brain weight were comparable among groups. Therefore the slight changes were interpreted as incidental and unrelated to administration of AAV-SPK-8005-hFVIII. No other organ weight changes were noted at Day 87.

[0247] Histopathology performed on mice on day 87 post-injection identified four animals with neoplastic findings. Malignant lymphoma was observed in one Group 2 animal (7808) and three Group 3 animals (7868, 7869 and 7870). No neoplastic findings were observed in Group 1. The biological significance of the neoplastic findings is considered to be equivocal. Statistical significance of individual group comparisons to the control group is unlikely. However, it is noteworthy that neoplastic findings were only observed in treated animals at Day 87. In the absence of historical control data for NOD SCID mice at a comparable age these neoplastic findings are inconclusive.

[0248] With regards to non-neoplastic changes, no test article-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of mice, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of AAV-SPK-8005-hFVIII.

[0249] Conclusions:

[0250] A single administration of AAV-SPK-8005-hFVIII at doses of 4?10.sup.10, 8?10.sup.10, or 1.6?10.sup.11 vg/mouse, or control article, by intravenous injection to male NOD/SCID mice was well tolerated. AAV-SPK-8005-hFVIII did not result in any test article-related mortality, adverse clinical observations or changes in body weight. There were no toxicologically important differences in organ weights, hematology or coagulation parameters and no treatment-related gross pathology or histopathology findings in the male mice at Days 29/30 or Day 87. The reductions in mean aPTT and prothrombin time that were observed at both euthanasia timepoints were considered related to the supraphysiologic levels of FVIII that were expressed in these hemostatically normal mice, and were not adverse. Within the Main study (terminal evaluations), malignancies were observed in nine out of 60 vector-injected mice, or 15% of the animals. Seven of these nine mice had lymphomas, which were most commonly seen in lymph nodes. This immunodeficient mouse strain is known to have a high spontaneous frequency of lymphomas (Prochazka et al., 1992), and a life span of just 8.5 months. Thus, the frequency of tumors seen in this study is unlikely related to AAV-SPK-8005-hFVIII administration. The purpose of this study was to evaluate the safety of sustained expression of hFVIII over the course of approximately three months. It was not designed to evaluate the AAV-SPK capsid. AAV-SPK and an immunodeficient mouse strain were used to ensure high level expression of hFVIII. Administration of AAV-SPK-8005-hFVIII to NOD/SCID mice resulted in sustained and high levels of hFVIII. Thus, this study was appropriate for assessing the safety of long-term expression of hFVIII.

Example 4

Evaluation of AAV-SPK-8005 and AAV-SPK-8011 (LK03 Capsid, FVIII-X07 (SEQ ID NO: 7)) Vectors in Non-Human Primates (NHPs).

[0251] Based on the results in mice, FVIII transgene constructs packaged into adeno-associated viral (AAV) vectors were delivered to non-human primates (NHPs).

[0252] In brief, a dose-ranging study in male cynomolgus macaques administered a single intravenous infusion of AAV-SPK-8005 or AAV-SPK-8011 (LK03 capsid). Expression of hFVIII was evaluated over 8 weeks. The animal groups and dose levels of each are shown in FIG. 4.

[0253] NHPs received an intravenous infusion via the saphenous vein using a calibrated infusion pump over approximately 30 minutes. Macaques were prescreened for neutralizing antibodies against the AAV capsid. All treated animals were initially determined to have a <1:3 titer before vector administration. This was done to ensure successful hepatic transduction, as even low titers inhibit vector uptake by liver cells after systemic delivery (Jiang et al. 2006). All animals were also negative for the presence of neutralizing antibodies against FVIII before gene transfer.

[0254] Plasma levels of hFVIII were measured by a human-specific ELISA that does not detect the cynomolgus endogenous FVIII. All the animals in the study, with the exception of one macaque in the mid dose cohort, express hFVIII following vector delivery. Human factor VIII antigen levels peaked at around 1-2 weeks following vector administration. At one week after gene transfer, NHPs transduced with 2?10.sup.12 vg/kg of AAV-SPK-8005 expressed hFVIII antigen levels of 13.2?3% (average?standard error of the mean). At one week after gene transfer, average hFVIII levels in two of the three animals in the next treatment cohort (5?10.sup.12 vg/kg) were 27?0.2%. Human FVIII could not be detected in the third macaque in that cohort at any time point. Upon re-testing of baseline plasma samples it was determined that this animal was in fact positive for the presence of anti-AAV antibodies and that the initially determined titer of <1:3 was incorrect. Finally, at the highest tested dose of 1?10.sup.13 vg/kg, peak hFVIII antigen levels of 54.1?15.6% were observed after AAV infusion.

[0255] As anticipated by studies in NHPs expressing human FIX, human FVIII expression declined in approximately one third of the animals around week 4, concomitant with the appearance of inhibitor antibodies to hFVIII in these 3 macaques (labeled with a c symbol in FIG. 5). Development of species-specific antibodies to hFVIII has been previously documented in non-human primates, and is likely due to differences in several amino acid residues between the human transgene product and the endogenous cynomolgus FVIII (McIntosh, J. et al., Blood 121:3335-44 (2013)).

[0256] To assess potential thrombogenesis due to continuous expression of human FVIII, D-dimer antigen levels were measured in this study. It should be noted that reports on the clinical relevance or even the normal values of D-dimer antigen levels in cynomolgus macaques are scarce; as a reference, the normal range for D-dimers in humans is below 500 ng/ml. Since the animals express endogenous cynomolgus FVIII, production of hFVIII as a result of hepatic gene transfer will result in supraphysiological levels of FVIII activity.

[0257] The animal that was dosed at 5?10.sup.12 vg/kg but did not express human FVIII had a peak of 863 ng/ml two weeks after AAV infusion. The rest of the animals did not show any significant increase in D-dimer antigen levels compared to baseline values. Taken together, these results suggest that expression of human FVIII, at the levels targeted in this study, is not associated with an increased risk of thrombosis.

[0258] Four weeks after vector administration, no vector-related changes were apparent. Liver function tests showed normal values, with minor fluctuations that appeared to be unrelated to vector dose, as they were present prior to dosing in most cases (FIG. 6).

[0259] D-dimer levels up to week 5 are shown in FIG. 7. One animal in the high dose cohort had a slight (577 ng/ml), transient elevation in D-dimer levels one week after vector administration, when circulating human FVIII peaked at around 100%; the D-dimer levels rapidly returned to normal after this single elevate measurement. Notably, there was no correlation between D-dimer levels and hFVIII antigen levels (FIG. 7, bottom panels).

[0260] For AAV-SPK-8011 (LK03 capsid) vector, three cohorts of cynomolgus macaques (n=3) were treated with increasing doses of AAV-SPK-8011 (LK03 capsid) (2?10.sup.12, 6?10.sup.12 and 2?10.sup.13 (vg/kg); FIG. 4). Animals were monitored for clinical observations, body weights clinical pathology (clinical chemistry, hematology, coagulation, urinalysis). In addition, hFVIII antigen levels, FVIII inhibitory antibodies and D-dimer levels were assessed throughout the study.

[0261] The hFVIII antigen data is shown in FIG. 9. Average hFVIII antigen levels peaked around week 2-3 with 22.3?6.2% hFVIII seen in the low dose cohort and 61.6?15.7% and 153?58.1% observed in the mid and high dose cohorts, respectively, using 150 ng/ml as the 100% normal hFVIII antigen level (FIGS. 9A-9D). Thus, the LK03 AAV capsid serotype efficiently transduces NHP hepatocytes in vivo unlike mouse liver.

[0262] FVIII expression levels attained with AAV-SPK-8011 (LK03 capsid) were compared to reported levels of FVIII attained with AAV5 and AAV8 capsid based AAV vectors for delivery of FVIII. A comparison revealed levels of FVIII achieved with AAV-SPK-8011 (LK03 capsid) were greater than the reported levels of FVIII delivered by way of AAV vectors with AAV5 and AAV8 capsids (FIG. 10).

[0263] Humoral response to hFVIII in plasma of cynomolgus macaques was measured following administration of either 2?10.sup.12, 6?10.sup.12 or 2?10.sup.13 vg/kg of AAV-SPK-8011 (LK03 capsid). The animals were assessed for anti-hFVIII IgG antibodies by ELISA at baseline and at the indicated time points.

[0264] Despite the therapeutic hFVIII levels observed soon after gene transfer, in most animals the levels began to decline around week 4. This was consistent with previous studies using another AAV-hFVIII vector, and correlated with an increase in anti-hFVIII antibodies. Generation of anti-FVIII antibodies has also been observed by others following hepatic AAV-hFVIII gene transfer in NHPs (McIntosh, J. et al., Blood 121:3335-44 (2013)).

Example 5

Biodistribution of AAV-LK03 Capsid in Non-Human Primates (NHPs).

[0265] Biodistribution of the AAV-LK03 capsid in non-human primates was evaluated in a non-GLP study. Intravenous administration of an AAV-LK03-encapsidated vector encoding human coagulation factor IX (AAV-LK03-hFIX) showed that the two main target tissues are the liver and the spleen (FIG. 11). The splenic tropism is not a unique characteristic of AAV-LK03. For example, the AAV5 capsid, which has been used in several liver-directed gene therapy trials (e.g. NCT02396342, NCT02082860, NCT02576795) with a strong safety record, targets the spleen with the same if not higher efficacy than it targets the liver of non-human primates (Paneda et al. 2013). The SPK-8011 expression cassette uses the mouse transthyretin or TTR promoter, which is considered liver-specific (Costa, 1991). To further support the liver-specific nature of the promoter, a PCR-based expression analysis measured vector-derived FVIII expression in the livers and spleens of mice after administration of a different AAV vector packaging the same expression cassette as SPK-8011 (i.e. AAV-SPK-8005). As shown in FIG. 12, human FVIII expression in the spleen is several orders of magnitude lower compared with that derived from hepatocytes.

[0266] This is the first clinical study to use AAV-LK03, although studies have been conducted using other AAV vectors including several for hemophilia B (NCT02396342, NCT01620801 NCT00076557, NCT02484092, NCT02618915, NCT00979238, NCT01687608) and one for hemophilia A (NCT02576795). A study conducted by St. Jude Children's Research Hospital in collaboration with University College London utilized an AAV8 vector carrying a self-complementary genome encoding a codon-optimized human factor IX cDNA, scAAV2/8-LP1-hFIXco. Ten subjects who received the vector have had stable factor IX levels of 1-6% through a median of 3.2 years and all participants have either discontinued or reduced the use of prophylactic factor replacement (Nathwani et al. 2014). A clinical study for hemophilia A used an AAV5 encapsidated vector encoding human FVIII (NCT02576795). Preliminary data presented in 2016 demonstrate increases in FVIII activity after gene transfer in several subjects ranging from from 2-60% with follow-up of up to 16 weeks (BioMarin, April 2016).

Example 6

Transduction Efficiency of AAV-LK03 Capsid Analyzed in an In Vitro Setting.

[0267] Primary hepatocytes from cynomolgus macaque and human origin were transduced with an AAV-LK03 vector expressing luciferase at four different multiplicities of infection (MOI) ranging from 500 to 62,500 vector genomes per cell. Seventy-two hours after transduction, luciferase expression was analyzed.

[0268] The AAV-LK03 capsid uniquely demonstrated significantly higher efficiency in transducing human hepatocytes in culture. In the representative example shown in FIG. 13, LK03 demonstrated approximately 5-fold higher efficiency in transducing human hepatocytes as compared to non-human primate hepatocytes in vitro. Importantly, these results are consistent across multiple MOIs and replicate studies.

Example 7

Assessment of Germline Transmission of Vector-Encoded Sequences.

[0269] Assessment of the potential for germline transmission of vector-encoded sequences is critical for clinical translation of gene transfer strategies. This study was designed with the following goals: (1) to evaluate dissemination of AAV-SPK and AAV-LK03 to semen and to determine the kinetics of vector clearance; and (2) to ensure that AAV administration to rabbits was successful, which was confirmed by analysis of human factor IX antigen and anti-FIX antibodies in plasma.

[0270] In this study, a rabbit model was used to analyze vector dissemination to the semen of two vector capsids, namely AAV-SPK and AAV-LK03 (Table 2). Dissemination of AAV-SPK to semen showed both dose-dependent and time-dependent kinetics, with the higher dose showing elevated levels of vector sequences in semen for a longer time. The kinetics were very similar to what has been seen previously with AAV8 vectors (Favaro P, et al., Molecular Therapy 17:1022-1030 (2009)). In contrast, limited dissemination to semen occurred with the AAV-LK03 vector. This is unlikely due to lower over-all vector exposure in AAV-LK03 injected mice, since the levels of hFIX expressed from AAV-LK03 were similar or higher than those seen with the AAV-SPK vector, and the ability to mediate liver-derived hFIX expression can be used as a surrogate for gene transfer.

TABLE-US-00002 TABLE 2 Study design Group Dose Level No. of No. Test Material (vg/kg) Animals 1 AAV-SPK-hFIX.C16 1 ? 10.sup.12 5 2A AAV-SPK-hFIX.C16 1 ? 10.sup.13 3 2B* AAV-SPK-hFIX.C19-PD 1 ? 10.sup.13 2 3 AAV-LK03-hFIX.C16 1 ? 10.sup.12 5 4 AAV-LK03-hFIX.C16 1 ? 10.sup.13 5 5 Vehicle N/A 2 *Two different hFIX coding sequences were used in the AAV-SPK cohorts, i.e. three animals received AAV-SPK-hFIX.C16 and two animals were treated with AAV-SPK-hFIX.C19-Padua (PD). Since the main goal of this study was to assess germline transmission of the two novel AAV capsids, this was considered acceptable.
The main differences between the hFIX.C16 and hFIX.C19-Padua transgenes are that the latter is codon-optimized and encodes a high specific activity hFIX variant.

Methods

[0271] Animals and Vectors:

[0272] New Zealand white rabbits were obtained from Covance Research Products (Denver, Pa.) and treated at 6 months of age with AAV vectors produced at the Children's Hospital of Philadelphia Vector Core. The test and control articles were administered via the marginal ear vein.

[0273] Semen Collection:

[0274] An artificial vagina (AV), developed by researchers at Argus Research Lab, Inc. (Horsham, Pa.) was used for semen collection. The AV is lined with a condom from which the tip is removed and a collection tube is added, and the AV is filled with warm water (55? C.). Semen samples were obtained from a practiced buck stimulated by a teaser doe. Samples were collected prior to injection and at 1, 2, 4, 6, 8, and 10 weeks and 3-8 months post-injection. Semen samples were shipped to Charles River Laboratories (Reno, Nev.) for analysis of vector copy number using a validated real-time quantitative PCR assay.

[0275] Blood Sample Collection:

[0276] Blood was collected by medial auricular artery or marginal ear vein puncture prior to AAV administration and at multiple time points (pre, 1 week and 1-6 months post-injection). Each sample was placed on ice following collection, processed to plasma and. stored in an ?80? C. freezer until shipment to the Sponsor, where it was also kept in an ?80? C. freezer until the assay was performed.

[0277] Human Factor IX Levels:

[0278] Levels of human FIX (hFIX) protein in rabbit plasma were quantified using a sandwich-style FIX ELISA kit (Affinity Biologicals, FIX:EIA) as follows: first, the wells of a microtiter plate were coated with a capture antibody that recognizes hFIX and that does not cross-react with endogenous rabbit FIX (1:1000 dilution). Reference plasma with a known human hFIX concentration was diluted to generate a standard curve (the highest standard [500 ng/ml] was serially diluted down to 7.8 ng/ml). Sample plasmas were diluted depending on the expected concentration so that the absorbance values fell within the range of the standard curve. After addition of the samples to the wells, the plate was incubated at room temperature for 90 minutes and then washed three times. A horseradish peroxidase (HRP)-conjugated secondary antibody to hFIX was added to the plate to bind to the captured FIX (1:100 dilution). After washing the plate to remove unbound conjugated antibody, the peroxidase activity was measured following incubation with 1-Step Ultra TMB Substrate (Thermo Scientific, catalog number 34028). The reaction was stopped with 1M sulfuric acid and read on a SpectraMax M2e microplate reader at an absorbance setting of 450 nm. The absorbance value obtained is proportional to the concentration of hFIX present in the sample.

[0279] Anti-hFIX Antibody Levels:

[0280] The anti-hFIX assay is conceptually and methodologically similar to the hFIX ELISA described above. In short, plates were coated with 1 ?g/ml of recombinant hFIX (Benefix, Wyeth). After incubation of plasma samples, a goat anti-rabbit IgG HRP-conjugated antibody (SIGMA, A4914) is used for detection. Samples with an IgG level two-fold higher than baseline readings were considered positive.

Results

Vector Dissemination to Semen

[0281] New Zealand rabbits were injected with AAV-SPK or AAV-LK03 (n=5 per group) vectors expressing hFIX under the control of the ApoE/hAAT liver-specific promoter at two doses: 1?10.sup.12 vg/kg (low dose) or 1?10.sup.13 vg/kg (high dose). Semen samples from all rabbits were obtained prior to injection and at 1, 2, 4, 6, 8, and 10 weeks and 3-8 months post-injection. Genomic DNA was purified from semen samples and analyzed for the presence of hFIX sequences using a quantitative polymerase chain reaction (Q-PCR) assay. The validated assay was developed by Charles River Laboratories (Reno, Nev.). Semen samples were considered to be positive if they had detectable hFIX levels above the lower limit of quantitation (LLOQ) (10 copies/reaction or 50 copies/?g at approximately 200 ng/reaction). Semen samples from rabbits that were negative for hFIX vector sequences on at least three consecutive timepoints were not analyzed further.

[0282] Pretreatment semen DNA from all vector and vehicle-injected animals was negative for hFIX sequences. The semen from rabbits injected with the low dose of AAV-SPK-hFIX (1?10.sup.12 vg/kg) was in general negative for hFIX sequences, except for three animals that had low levels at weeks 1-4 (maximum 3151 copies/?g DNA or ?1?10.sup.?2 copies/haploid genome). None of the samples collected beyond week 4 were positive for vector sequences (Table 3). At the high dose of AAV-SPK-hFIX (1?10.sup.13 vg/kg), higher levels of vector were present (maximum 178,352 copies/?g DNA or 0.59 copies/haploid genome), and it took longer to clear, up to 5 month between the five animals (Table 3). With the exception of one animal (week 1), rabbits treated with the low dose of AAV-LK03-hFIX showed no dissemination of hFIX sequences to semen (Table 3). In addition, very little vector dissemination to semen was observed at a ten-fold higher dose, with three animals lacking any hFIX sequences at all timepoints and two animals showing low levels at week 2 (maximum: 392 copies/ug DNA or 1.3?10.sup.?3 copies/haploid genome), but not at later timepoints (Table 3). Among the two vehicle-injected animals, one had a spurious finding at week 1 (56 copies/ug DNA) and at month 5 (96 copies/?g DNA). These values are near the LLOQ, and most likely represent contamination at the semen collection or DNA preparation step.

TABLE-US-00003 TABLE 3 Detection of hFIX DNA sequences in rabbit semen following AAV-SPK and AAV-LK03 administration as a function of time. Vector Dose Pre W 1 W 2 W 4 W 6 W 8 W 10 M 3 M 4 M 5 M 6 M 7 M 8 SPK low 0/5 3/5 3/5 1/5 0/5 0/5 0/5 0/5 0/5 0/5 Ndt Ndt Ndt SPK high 0/5 5/5 4/5 4/5 3/5 1/5 2/5 0/5 1/5 0/5 0/5 0/5 0/5 LK03 low 0/5 1/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Ndt Ndt Ndt LK03 high 0/5 2/5 2/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Ndt Ndt Ndt Number of animals out of 5 with positive semen samples. W = week; M = month; Ndt = not determined

Plasma Human FIX Antigen Levels

[0283] Circulating hFIX levels were measured in plasma samples from the animals described above at the indicated timepoints (FIGS. 14A-14B and Table 4).

TABLE-US-00004 TABLE 4 Human FIX expression levels (ng/ml) following vector administration Day after injection Animal Capsid Transgene vg/kg 0 7 28 56 94 112 147 175 1 SPK FIX.C16 1E+12 ND ND 49.0 39.7 70.6 126.1 147.9 102.4 2 SPK FIX.C16 1E+12 ND ND 50.0 104.9 139.5 147.0 171.8 198.4 3 SPK FIX.C16 1E+12 ND ND 36.4 76.5 95.0 114.8 114.7 78.9 4 SPK FIX.C16 1E+12 ND ND 148.2 254.4 214.5 291.5 236.7 274.9 5 SPK FIX.C16 1E+12 ND ND ND ND 31.3 10.5 13.4 ND 6 SPK FIX.C16 1E+13 ND 347.9 2341.0 1224.5 1102.2 1031.6 959.0 830.7 7 SPK FIX.C16 1E+13 ND 1564.1 14174.2 5311.2 3281.9 3300.9 2405.9 2640.8 8 SPK FIX.C16 1E+13 ND 2344.8 756.5 1515.0 8305.2 10907.0 5838.9 4352.8 9 SPK FIX.C19-PD 1E+13 ND 103.3 234.4 ND 40.4 83.4 316.6 381.9 10 SPK FIX.C19-PD 1E+13 ND 642.2 2873.5 ND 31.6 14.1 14.1 ND 11 LK03 FIX.C16 1E+12 ND 333.1 604.9 1659.2 2151.8 1914.7 1358.4 1120.9 12 LK03 FIX.C16 1E+12 ND 2138.4 532.4 3199.8 1306.6 985.3 732.0 593.4 13 LK03 FIX.C16 1E+12 ND 2465.9 45.3 84.9 134.4 168.6 127.0 84.9 14 LK03 FIX.C16 1E+12 ND 886.1 289.0 636.4 551.7 547.4 582.3 410.4 15 LK03 FIX.C16 1E+12 ND ND ND 35.0 109.0 30.2 24.8 ND 16 LK03 FIX.C16 1E+13 ND 90.7 404.6 2228.9 1265.4 899.5 715.6 693.2 17 LK03 FIX.C16 1E+13 ND 424.7 546.6 490.2 695.9 437.0 964.4 821.4 18 LK03 FIX.C16 1E+13 ND 1255.6 1787.2 6079.2 4628.5 1874.0 1576.0 2226.4 19 LK03 FIX.C16 1E+13 ND 518.4 8917.2 2772.7 2905.7 1195.0 1877.0 1899.3 20 LK03 FIX.C16 1E+13 ND 2615.0 10782.8 8075.5 6908.0 6630.8 6226.0 5489.2 21 Vehicle FIX.C16 N/A ND ND ND 28.9 31.1 16.1 ND ND 22 Vehicle FIX.C16 N/A ND ND ND 29.2 30.6 11.9 ND ND ND = Not detected

[0284] In the low dose cohorts, the AAV-LK03 vector appeared to be a more potent vector compared with AAV-SPK, as measured by circulating hFIX levels. Six months after treatment with AAV-LK03 or AAV-SPK, average hFIX levels were 552?217 ng/ml vs. 164?45 ng/ml, respectively (FIG. 14A). However, this difference did not reach statistical significance, likely due to the limited number of animals. Interestingly, no hFIX expression was detected seven days after administration of the AAV-SPK vector, whereas robust expression derived from the AAV-LK03 was observed at the same time point. The low hFIX levels in two of the animals (rabbits #5 and #15), barely detectable above background, might be attributed to failed injections. Eliminating these animals from the analysis did not change the lack of statistical significance.

[0285] The two capsids appeared to be equally potent when tested at the high dose. Specifically, six months after treatment with 1?10.sup.13 vg/kg of AAV-LK03 or AAV-SPK, average hFIX levels were 2226?868 ng/ml vs. 2052?909 ng/ml, respectively (FIG. 14B). Of note, two different hFIX coding sequences were used in the AAV-SPK group, i.e. three animals received AAV-SPK-hFIX.C16 and two animals were treated with AAV-SPK-hFIX.C19-Padua (PD). The main differences between the hFIX.C16 and hFIX.C19-PD transgenes are that the latter is codon-optimized and encodes a high specific activity hFIX variant, which affects the biological activity of the protein, but not antigen levels, as measured by ELISA.

Anti-FIX Antibodies

[0286] Based on a report by others, it was anticipated that approximately 20-40% of the animals would develop antibodies against human FIX vectors (Favaro P, et al., Molecular Therapy 17:1022-1030 (2009)). FIGS. 15A-15B and Tables 5A and 5B summarize anti-AAV IgG levels in this study. Interestingly, three out of five animals treated with the low dose of AAV-LK03 were positive for human FIX antibodies one month after vector administration, but the IgG levels declined with time and only one animal was barely twice the baseline levels at the end of the study (Table 5B). The kinetics of anti-FIX IgG appearance and ulterior clearance in this group of rabbits correlates well with the sharp decrease in hFIX levels observed at day 28, which was followed by a rebound in circulating hFIX (FIG. 14A). Also, the high antibody titers against hFIX in the two animals treated with AAV-SPK-hFIX.C19-Padua may explain the low expression levels in these two rabbits.

TABLE-US-00005 TABLE 5A Summary of antibody formation (IgG, ng/ml) to human FIX in individual AAV-injected rabbits Day after injection Animal Capsid Transgene vg/kg 0 7 28 56 94 112 147 175 1 SPK FIX.C16 1.00E+12 1390 1134 2864 8627 1631 1261 1210 1088 2 SPK FIX.C16 1.00E+12 1706 1143 4670 7132 2834 2733 3294 3180 3 SPK FIX.C16 1.00E+12 1904 1128 2919 2394 1964 1792 1753 1688 4 SPK FIX.C16 1.00E+12 1256 1084 789 1692 1463 1034 1367 1457 5 SPK FIX.C16 1.00E+12 1086 1004 701 664 834 956 774 785 6 SPK FIX.C16 1.00E+13 565 836 940 814 1246 721 1326 1592 7 SPK FIX.C16 1.00E+13 792 721 666 709 960 829 909 1084 8 SPK FIX.C16 1.00E+13 1016 863 1729 1705 2539 1619 1406 2143 9 SPK FIX.C19-PD 1.00E+13 768 783 1330 1076 11241 893 37141 12634 10 SPK FIX.C19-PD 1.00E+13 566 541 4556 1398 9356 1270 20050 9167 11 LK03 FIX.C16 1.00E+12 1606 1821 2150 2283 1973 1788 1561 1580 12 LK03 FIX.C16 1.00E+12 813 1391 7993 1603 1087 1505 1702 13 LK03 FIX.C16 1.00E+12 699 N/A 8153 610 680 903 871 1040 14 LK03 FIX.C16 1.00E+12 776 756 534 760 699 709 636 769 15 LK03 FIX.C16 1.00E+12 890 891 2320 693 561 843 972 1102 16 LK03 FIX.C16 1.00E+13 1479 2050 2579 1501 1487 1622 1526 1768 17 LK03 FIX.C16 1.00E+13 1979 1801 1506 1087 1196 837 1025 876 18 LK03 FIX.C16 1.00E+13 2074 1968 1368 1236 1284 1247 1107 1067 19 LK03 FIX.C16 1.00E+13 1131 1270 792 1237 2415 2463 1529 1597 20 LK03 FIX.C16 1.00E+13 967 2065 1250 2537 1927 1459 1343 1603 21 Vehicle FIX.C16 N/A 899 1074 1124 844 853 916 1017 961 22 Vehicle FIX.C16 N/A 477 702 891 471 460 541 536 597 N/A, not available

TABLE-US-00006 TABLE 5B Number of rabbits per group positive for anti-hFIX antibodies over time 1 ? 10.sup.12 vg/kg 1 ? 10.sup.13 vg/kg AAV-SPK AAV-LK03 AAV-SPK AAV-LK03 Day 28 2/5 3/5 1/5 0/5 Day 175 0/5 1/5 4/5 0/5

Conclusion

[0287] Dissemination of AAV-SPK and AAV-LK03 vectors to semen was quantified using a validated assay over the course of up to eight months. AAV-SPK vector sequences were detected in semen of all five rabbits one week after administration of the high vector dose. The majority of the animals cleared the sequences by week 10 and the last detected positive sample occurred at month 5. This is similar to the time course of an AAV8 vector administered to rabbits at the same dose vectors (Favaro P, et al., Molecular Therapy 17:1022-1030 (2009)). In contrast, very limited distribution of AAV-LK03 was observed following a high dose of this vector, with three of five animals showing no vector sequences in semen at any timepoint. The lower dissemination of vector to semen was unlikely due to a lower overall exposure of AAV-LK03 in rabbits. Confirmation that rabbits were successfully injected with each AAV vector was demonstrated by measuring hFIX plasma levels, a surrogate for gene transfer. At the high dose in this study (1?10.sup.13 vg/kg), similar circulating levels of hFIX were observed in animals injected with AAV-LK03 and AAV-SPK, demonstrating that the vectors are equally potent in mediating liver gene transfer.

[0288] Consistent with studies evaluating germline transmission of AAV2 and AAV8 vectors expressing a hFIX transgene, some of the animals develop anti-hFIX antibodies, likely due to the amino acid differences between rabbit and human factor IX.

[0289] These results add to the current body of data on the potential for germline transmission of AAV vectors. AAV-SPK has a similar pattern as the previously investigated serotypes, AAV2 and AAV8 vectors (Favaro P, et al., Molecular Therapy 17:1022-1030 (2009)). That is, there is a dose-dependent dissemination of AAV vector sequences to semen, with complete clearance over time. AAV-LK03, however, differs from AAV2, AAV8, and AAV-SPK, in that very little vector distributes to the semen, potentially making this vector capsid safer than the others in terms of genotoxicity.

Example 8

[0290] A clinical study will be conducted to determine safety and kinetics of a single IV infusion of AAV-FVIII. The AAV capsid that will be used for the AAV vector will have shown in preclinical studies to have had good safety and efficacy, the ability to achieve clinically relevant FVIII activity levels at dose of about 1?10.sup.12 vg/kg or greater, optionally after 1-3 months of vector infusion; and cross reacting neutralizing antibodies (Ab) to the AAV capsid approximately 10% less prevalent than AAV8. The design of a representative clinical study can be as shown in Table 6.

TABLE-US-00007 TABLE 6 AAV-FVIII Clinical Study Design Safety and Tolerability of AAV-FVIII Clinically significant in vital signs, lab values and clinical assessments (including number of bleeds and QoL) from baseline Kinetics of AAV-FVIII Transgene FVIII activity levels and antigen levels at peak and steady-state Dosing Starting, Middle and Highest Dose Cohorts will each include 2-5 subjects Design Open-label, non-randomized, dose escalation Participating countries USA and potentially Europe, Japan and Canada Sample size Up to 15 subjects Eligibility Ages Eligible for Study: 18 Years and older Genders Eligible for Study: Male Accepts Healthy Volunteers: No Inclusion Criteria Able to provide informed consent and comply with requirements of the study Males ?18 y.o. with confirmed diagnosis of hemophilia A (?2 IU/dL or ?2% endogenous factor VIII) Received ?50 exposure days to factor VIII products A minimum of an average of 4 bleeding events per year requiring episodic treatment of factor VIII infusions or prophylactic factor VIII infusions No measurable factor VIII inhibitor as assessed by the central laboratory and have no prior history of inhibitors to factor VIII protein Agree to use reliable barrier contraception until 3 consecutive samples are negative for vector sequences Exclusion Criteria Evidence of active hepatitis B or C Currently on antiviral therapy for hepatitis B or C Have significant underlying liver disease Have serological evidence* of HIV-1 or HIV-2 with CD4 counts ?200/mm3 (* subjects who are HIV+ and stable with CD4 count >200/mm3 and undetectable viral load are eligible to enroll) Have detectable antibodies reactive with variant AAV capsid Participated in a gene transfer trial within the last 52 weeks or an investigational drug within the last 12 weeks Unable or unwilling to comply with study assessments Screening Visit Eligibility evaluation AAV NAb titer is the major screen failure Day 0 Visit FVIII product incremental recovery then vector infusion Follow-up Visits (~17 visits) Safety and kinetic evaluations End-of Study Visit (at about week 52) Final safety evaluation

Example 9

TTR Promoter

[0291] The characterization of the transthyretin (TTR) promoter was originally described in Costa and Grayson 1991, Nucleic Acids Research 19(15):4139-4145. The TTR promoter sequence was a modified sequence, from TATTTGTGTAG to TATTGACTTAG.

TABLE-US-00008 TTRpromoterwith4nucleotidemutation(TTRmut), SEQIDNO:22 GTCTGTCTGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCT AGGCAAGGTTCATATTGACTTAGGTTACTTATTCTCCTTTTGTTGACTAA GTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCA GCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCG TCACACAGATCCACAAGCTCCT

Example 10

CpG Reduced FVIII Encoding Transgene Constructs and Exemplary AAV Capsids

[0292]

TABLE-US-00009 FVIIIencodingCpGreducednucleicacidvariantX01 (SEQIDNO:1) atgcagattgagctgtctacctgcttcttcctgtgcctgctgaggttctgcttctctgct accaggaggtactacctgggggctgtggagctgagctgggattacatgcagtctgacctg ggggagctgcctgtggatgccaggtttccccccagggtgcccaagagcttccccttcaat acctctgtggtgtataagaagaccctgtttgtggagttcactgatcatctgttcaacatt gctaaacccaggcccccctggatggggctgctgggccctaccatccaggctgaggtgtat gacactgtggtgatcactctgaagaacatggctagccatcctgtgtctctgcatgctgtg ggggtgagctactggaaggcttctgagggggctgagtatgatgatcagactagccagagg gagaaggaggatgacaaggtgttccctgggggctctcacacctatgtctggcaggtgctg aaggagaatggccccatggcctctgatcctctgtgtctgacctatagctacctgagccat gtggacctggtgaaggacctgaactctggcctgattggggccctgctggtgtgtagggag gggagcctggccaaggagaagacccagaccctgcacaagttcattctgctgtttgctgtg tttgatgagggcaagagctggcattctgaaaccaagaacagcctgatgcaggacagggat gctgcctctgctagggcctggcccaagatgcacactgtgaatgggtatgtcaataggtct ctgcctggcctgattggctgccacaggaagtctgtgtactggcatgtgattgggatgggc accacccctgaggtgcacagcatctttctggagggccacaccttcctggtgaggaatcac agacaggccagcctggagatcagccccatcaccttcctgactgcccagaccctgctgatg gacctgggccagtttctgctgttctgccacatctctagccaccagcatgatggcatggag gcctatgtgaaggtggactcctgccctgaggagccccagctgaggatgaagaataatgag gaggctgaggactatgatgatgacctgactgactctgagatggatgtggtgagatttgat gatgacaattctcccagcttcattcagatcaggtctgtggccaagaagcatcccaagacc tgggtgcactacattgctgctgaggaggaggactgggactatgcccccctggtgctggcc cctgatgacaggagctataagagccagtacctgaataatggcccccagaggattgggagg aagtataagaaggtgaggttcatggcctatactgatgaaaccttcaagaccagagaggcc atccagcatgagtctgggatcctggggcccctgctgtatggggaggtgggggacaccctg ctgatcatcttcaagaaccaggccagcaggccctacaacatctaccctcatggcatcact gatgtgaggcctctgtacagcagaaggctgcccaagggggtgaagcatctgaaggacttc cccattctgcctggggagattttcaagtacaagtggactgtgactgtggaggatggccca accaagtctgaccctaggtgcctgactaggtactacagcagctttgtgaatatggagagg gacctggcctctggcctgattggccccctgctgatctgctacaaggagtctgtggatcag aggggcaaccagatcatgtctgacaagaggaatgtgatcctgttctctgtgtttgatgag aacaggagctggtacctgactgagaacattcagaggtttctgcccaaccctgctggggtg cagctggaggaccctgaattccaggcctctaacatcatgcacagcattaatggctatgtg tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacattctgagc attggggcccagactgacttcctgtctgtgttcttctctggctacacctttaagcacaag atggtgtatgaggataccctgaccctgtttcctttctctggggagactgtgttcatgagc atggagaaccctggcctgtggatcctgggctgccacaactctgacttcaggaacaggggg atgactgctctgctgaaggtgagcagctgtgataagaacactggggactactatgaggac agctatgaggacatctctgcctatctgctgagcaagaataatgctattgagcccaggagc ttctctcagaacccccctgtgctgaagaggcaccagagggagatcaccagaactactctg cagtctgaccaggaggagattgactatgatgacaccatctctgtggagatgaagaaggag gattttgatatttatgatgaggatgaaaaccagagccccaggagctttcagaagaagact aggcactatttcattgctgctgtggagaggctgtgggactatggcatgtcttctagcccc catgtgctgaggaacagggcccagtctggctctgtgccccagttcaagaaggtggtgttc caggagttcactgatggcagcttcactcagcccctgtacaggggggagctgaatgagcac ctggggctgctgggcccttatatcagggctgaggtggaggataacatcatggtgaccttc aggaaccaggccagcaggccctacagcttctactctagcctgatcagctatgaggaggac cagaggcagggggctgagcccaggaagaactttgtgaagcccaatgagaccaagacttat ttctggaaggtgcagcaccatatggcccccaccaaggatgagtttgattgcaaagcctgg gcctacttctctgatgtggacctggagaaggatgtgcactctgggctgattggccccctg ctggtgtgccacaccaacactctgaaccctgcccatggcaggcaggtgactgtgcaggag tttgccctgttcttcaccatctttgatgagactaagagctggtacttcactgagaacatg gagaggaactgcagggccccctgcaatatccagatggaggaccccacctttaaggaaaat tataggtttcatgccattaatggctacatcatggacaccctgcctggcctggtgatggcc caggaccagaggatcaggtggtacctgctgagcatgggcagcaatgagaacattcacagc atccacttctctggccatgtgttcactgtgaggaagaaggaggagtacaagatggccctg tataatctgtaccctggggtgtttgagactgtggagatgctgcccagcaaggctggcatc tggagggtggagtgcctgattggggagcacctgcatgctggcatgagcaccctgttcctg gtgtattctaacaagtgtcagacccccctgggcatggcctctggccatatcagggacttc cagatcactgcctctggccagtatgggcagtgggcccccaagctggccaggctgcattac tctggcagcatcaatgcctggagcaccaaggagccattcagctggattaaggtggacctg ctggctccaatgattatccatggcatcaagacccagggggccaggcagaagtttagcagc ctgtacatctctcagtttatcatcatgtactctctggatggcaaaaagtggcagacctac aggggcaattctactggcactctgatggtgttctttggcaatgtggacagctctgggatc aagcacaacatctttaacccccctatcattgccaggtacattaggctgcaccccacccat tacagcatcaggagcaccctgaggatggagctgatgggctgtgatctgaacagctgcagc atgcccctgggcatggagagcaaggctatctctgatgcccagattactgccagcagctac ttcaccaatatgtttgccacctggagccccagcaaggccaggctgcacctgcagggcagg tctaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggacttccag aagaccatgaaggtgactggggtgaccacccagggggtgaagagcctgctgactagcatg tatgtgaaggagttcctgatcagcagcagccaggatggccatcagtggaccctgttcttc cagaatggcaaggtgaaggtgttccagggcaatcaggacagcttcacccctgtggtgaac agcctggacccccccctgctgaccagatacctgaggatccacccccagagctgggtgcat cagattgccctgaggatggaggtgctggggtgtgaggcccaggacctgtactga FVIIIencodingCpGreducednucleicacidvariantX02 (SEQIDNO:2) atgcagattgagctgtctacctgctttttcctgtgtctgctgaggttctgcttctctgcc actaggaggtactacctgggggctgtggagctgtcttgggattacatgcagtctgatctg ggggagctgcctgtggatgccaggtttcctcccagggtgcccaagtctttccccttcaat acctctgtggtgtataagaagaccctgtttgtggagtttactgatcacctgttcaacatt gccaagcccaggcccccttggatgggcctgctggggcccaccatccaggctgaggtgtat gacactgtggtgatcaccctgaagaacatggcctctcaccctgtgagcctgcatgctgtg ggggtgagctactggaaggcctctgagggggctgagtatgatgaccagaccagccagagg gagaaggaggatgataaggtgttccctggggggagccacacttatgtgtggcaggtgctg aaggagaatggcccaatggcctctgatcccctgtgcctgacctattcttacctgagccat gtggacctggtgaaggacctgaactctggcctgattggggccctgctggtgtgcagggag ggctctctggctaaggagaagacccagaccctgcacaagttcatcctgctgtttgctgtg tttgatgaggggaagagctggcactctgagaccaagaacagcctgatgcaggacagggat gctgcctctgccagggcctggcccaaaatgcacactgtgaatggctatgtgaataggagc ctgcctggcctgattggctgccacaggaagtctgtgtattggcatgtgattggcatgggc accacccctgaggtgcactctatcttcctggagggccatactttcctggtgaggaatcat aggcaggccagcctggagattagccccattacctttctgactgcccagaccctgctgatg gacctgggccagttcctgctgttttgccacatcagctctcaccagcatgatggcatggag gcctatgtgaaggtggatagctgccctgaggagccccagctgaggatgaagaacaatgag gaggctgaggattatgatgatgatctgactgattctgaaatggatgtggtgaggtttgat gatgacaatagcccctctttcatccagatcaggtctgtggccaagaagcatcctaagacc tgggtgcactacattgctgctgaggaggaggactgggactatgctcccctggtgctggcc cctgatgacaggtcttacaagagccagtacctgaacaatggcccccagagaattgggagg aagtataagaaggtgagattcatggcttacactgatgagaccttcaagactagggaggcc atccagcatgagtctggcattctgggccccctgctgtatggggaggtgggggacaccctg ctgatcatcttcaagaaccaggcctctaggccctacaatatttacccccatgggatcact gatgtgaggcccctgtacagcaggaggctgcctaagggggtgaagcatctgaaggacttc cccatcctgcctggggagatcttcaagtataagtggactgtgactgtggaagatggcccc accaagtctgaccctaggtgcctgaccaggtactactcttcttttgtgaacatggagagg gacctggcctctggcctgattggccccctgctgatctgctacaaggagtctgtggaccag agggggaaccagattatgtctgacaagaggaatgtgattctgttctctgtgtttgatgag aacaggagctggtatctgactgagaacatccagaggttcctgcccaatcctgctggggtg cagctggaggaccctgagttccaggccagcaacatcatgcacagcatcaatgggtatgtg tttgattctctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagc attggggctcagactgatttcctgtctgtgttcttttctggctacacctttaagcataag atggtgtatgaggacactctgaccctgtttcccttctctggggagactgtgtttatgagc atggagaaccctggcctgtggatcctgggctgccacaactctgatttcaggaacaggggc atgactgctctgctgaaggtgtcttcttgtgacaagaacactggggactattatgaggac agctatgaggacatctctgcctacctgctgagcaagaacaatgctattgagcccagatct ttcagccagaacccccctgtgctgaagaggcaccagagggagatcactaggaccaccctg cagtctgaccaggaggagattgactatgatgacactatctctgtggagatgaagaaggag gactttgatatctatgatgaggatgagaaccagtctcccaggagcttccagaaaaagacc aggcactacttcattgctgctgtggagaggctgtgggactatggcatgtcttctagcccc catgtgctgaggaacagggcccagtctgggtctgtgccccagttcaagaaggtggtgttc caggagttcactgatgggagcttcacccagcctctgtacaggggggagctgaatgagcac ctggggctgctgggcccttatattagggctgaggtggaggacaacatcatggtgactttc aggaatcaggcctctaggccctatagcttctacagctctctgatcagctatgaggaggat cagaggcagggggctgagcccaggaagaactttgtgaagcccaatgagaccaagacctac ttctggaaggtgcagcaccacatggctcctaccaaggatgagtttgactgcaaggcctgg gcctacttttctgatgtggacctggagaaggatgtgcactctggcctgattggccccctg ctggtgtgtcataccaacaccctgaaccctgcccatggcaggcaggtgactgtgcaggag tttgccctgttcttcaccatctttgatgagaccaagagctggtactttactgagaacatg gagaggaattgcagagccccttgcaacatccagatggaggacccaaccttcaaagagaac tacaggttccatgccatcaatgggtacatcatggacaccctgcctggcctggtgatggct caggaccagaggatcaggtggtatctgctgagcatgggcagcaatgagaatatccatagc attcacttctctggccatgtgttcactgtgaggaagaaggaggagtacaagatggccctg tataacctgtaccctggggtgtttgagactgtggagatgctgccaagcaaggctgggatt tggagggtggagtgcctgattggggagcacctgcatgctggcatgtctaccctgttcctg gtgtactccaataagtgccagacccccctgggcatggcctctggccacatcagggacttc cagatcactgcctctggccagtatgggcagtgggccccaaagctggccaggctgcactat tctgggagcatcaatgcttggagcaccaaggagcctttcagctggattaaggtggatctg ctggcccccatgatcattcatggcatcaaaacccagggggctagacagaagttttctagc ctgtacatcagccagttcatcatcatgtacagcctggatggcaagaagtggcagacttac aggggcaatagcactggcaccctgatggtgttttttggcaatgtggacagctctggcatc aagcacaacatctttaacccccccattattgccaggtatatcaggctgcatcccacccac tattctattaggtctactctgagaatggagctgatgggctgtgacctgaacagctgtagc atgcccctggggatggagagcaaggctatctctgatgcccagatcactgccagctcttat ttcaccaatatgtttgccacctggtctccctctaaggccaggctgcacctgcagggcagg agcaatgcttggaggccccaggtgaataaccccaaggagtggctgcaggtggacttccag aagaccatgaaggtgactggggtgactacccagggggtgaagtctctgctgactagcatg tatgtgaaggagttcctgatcagcagcagccaggatgggcatcagtggactctgttcttc cagaatggcaaggtgaaggtcttccaggggaaccaggatagcttcactcctgtggtgaac tctctggacccccccctgctgactaggtatctgaggatccacccccagagctgggtgcac cagattgccctgaggatggaggtgctgggctgtgaggcccaggacctgtattga FVIIIencodingCpGreducednucleicacidvariantX03 (SEQIDNO:3) atgcagattgaactgtctacttgtttcttcctgtgcctgctgaggttttgcttctctgct actaggaggtactatctgggggctgtggagctgtcttgggactatatgcagtctgacctg ggggagctgcctgtggatgctaggtttccccccagggtgcccaagagcttcccctttaac acctctgtggtgtataagaagactctgtttgtggagttcactgaccatctgttcaacatt gccaagccaaggcccccctggatgggcctgctgggccccaccatccaggctgaggtgtat gacactgtggtgattactctgaagaacatggccagccatcctgtgagcctgcatgctgtg ggggtgtcttactggaaggcctctgagggggctgagtatgatgaccagacctctcagagg gagaaggaggatgacaaggtgttccctgggggctctcatacctatgtgtggcaggtcctg aaggagaatgggcccatggcctctgaccccctgtgcctgacctactcttatctgtctcat gtggacctggtgaaggacctgaactctggcctgattggggccctgctggtgtgcagggag ggcagcctggctaaggagaagacccagactctgcacaagttcatcctgctgtttgctgtg tttgatgagggcaagagctggcactctgagaccaagaacagcctgatgcaggacagggat gctgcctctgctagggcctggcccaagatgcacactgtgaatgggtatgtgaacaggagc ctgccaggcctgattggctgccataggaagtctgtgtattggcatgtgattgggatgggg actacccctgaggtccacagcattttcctggaggggcatacctttctggtgaggaaccac aggcaggcctctctggagatctctcccattactttcctgactgcccagaccctgctgatg gacctgggccagttcctgctgttctgccacatcagcagccaccagcatgatggcatggag gcctatgtgaaggtggatagctgccctgaggagccccagctgaggatgaaaaacaatgag gaggctgaggattatgatgatgacctgactgattctgagatggatgtggtgaggtttgat gatgataacagccccagcttcatccagattaggtctgtggccaagaagcatcccaagacc tgggtgcactacattgctgctgaggaggaggattgggactatgctcctctggtgctggcc cctgatgacaggagctacaagagccagtacctgaataatggcccccagaggattggcagg aagtataagaaggtgaggttcatggcctacactgatgagacctttaagaccagggaggcc atccagcatgaatctgggatcctgggccccctgctgtatggggaggtgggggacaccctg ctgattatctttaagaaccaggctagcaggccctacaacatttacccccatggcattact gatgtgaggcccctgtacagcaggaggctgcccaagggggtgaagcacctgaaggatttc cccattctgcctggggagatctttaagtacaaatggactgtgactgtggaggatggccct actaagtctgatcccaggtgtctgaccagatactacagcagctttgtgaatatggagagg gacctggcttctggcctgattggccccctgctgatctgctacaaggagtctgtggaccag aggggcaatcagattatgtctgacaagaggaatgtgatcctgttctctgtgtttgatgag aacagaagctggtacctgactgagaacatccagaggttcctgcccaaccctgctggggtg cagctggaggaccctgagttccaggctagcaatatcatgcacagcattaatggctatgtg tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctattggtacattctgagc attggggcccagactgatttcctgtctgtgttcttttctggctacaccttcaagcacaag atggtgtatgaggatactctgaccctgtttcccttctctggggagactgtgttcatgagc atggagaaccctggcctgtggatcctgggctgtcacaactctgacttcaggaacaggggc atgactgccctgctgaaggtgagctcttgtgataagaacactggggactactatgaggac tcttatgaggacatctctgcctacctgctgagcaagaacaatgctattgagcccaggagc ttctctcagaatccccctgtgctgaagaggcatcagagggagatcactaggactaccctg cagtctgaccaggaagagattgactatgatgacaccatctctgtggaaatgaagaaggag gactttgatatctatgatgaggatgaaaaccagagccccaggagcttccagaagaagacc aggcattacttcattgctgctgtggagaggctgtgggactatgggatgagctcttctccc catgtgctgaggaatagggctcagtctggctctgtcccacagttcaagaaggtggtgttt caggagttcactgatggcagcttcactcagcccctgtacaggggggagctgaatgagcat ctgggcctgctggggccctacatcagggctgaggtggaggataacattatggtgactttc aggaaccaggcctctaggccctacagcttctacagcagcctgatcagctatgaggaggac cagaggcagggggctgagcccaggaagaactttgtgaagcccaatgagactaagacctat ttctggaaggtgcagcatcacatggctcccactaaagatgagtttgactgcaaggcctgg gcctacttctctgatgtggatctggagaaggatgtgcattctgggctgattggccctctg ctggtctgccatactaacaccctgaatcctgcccatggcaggcaggtgactgtgcaggag tttgccctgttctttaccatctttgatgagaccaagtcttggtacttcactgagaacatg gagaggaactgcagggccccctgtaacatccagatggaggaccccacctttaaggagaac tacaggttccatgccatcaatggctacatcatggacactctgcctggcctggtgatggcc caggaccagaggatcaggtggtacctgctgtctatgggctctaatgagaacattcattct atccacttctctggccatgtgtttactgtgaggaagaaggaggagtacaagatggccctg tacaatctgtaccctggggtgtttgaaactgtggagatgctgccctctaaggctggcatc tggagggtggagtgcctgattggggaacacctgcatgctggcatgagcaccctgttcctg gtctatagcaataagtgccagacccccctggggatggcctctgggcatatcagagacttc cagatcactgcctctggccagtatggccagtgggcccccaagctggccaggctgcactac tctggcagcattaatgcctggagcaccaaggagcccttctcttggatcaaggtggacctg ctggctcccatgatcatccatgggatcaagacccagggggccaggcagaagttcagcagc ctgtacatctctcagttcatcatcatgtactctctggatggcaagaagtggcagacctac aggggcaatagcactgggaccctgatggtgttctttgggaatgtggacagctctggcatc aagcacaatatcttcaacccccccatcattgccaggtacatcagactgcaccccactcat tacagcatcaggagcactctgaggatggagctgatgggctgtgacctgaatagctgctct atgcccctgggcatggagagcaaggccatttctgatgcccagattactgcctcttcttac ttcactaatatgtttgccacctggagccccagcaaggccaggctgcatctgcaggggagg agcaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggacttccag aagactatgaaggtgactggggtgaccactcagggggtgaagagcctgctgaccagcatg tatgtgaaggagttcctgatctcttctagccaggatgggcaccagtggaccctgtttttc cagaatgggaaggtgaaggtgtttcagggcaatcaggacagctttactcctgtggtgaac agcctggacccccccctgctgactaggtacctgaggattcacccccagagctgggtgcac cagattgccctgaggatggaggtgctgggctgtgaggcccaggatctgtactga FVIIIencodingCpGreducednucleicacidvariantX04 (SEQIDNO:4) atgcagattgagctgtctacctgcttctttctgtgcctgctgaggttctgtttctctgcc actaggaggtattatctgggggctgtggagctgtcctgggactacatgcagtctgatctg ggggagctgcctgtggatgccaggttccctcccagggtgcccaagtctttccctttcaat acctctgtggtgtacaagaagactctgtttgtggagtttactgatcacctgtttaacatt gccaagcccaggcccccctggatggggctgctgggccccaccatccaggctgaggtgtat gacactgtggtgattactctgaagaatatggcttctcaccctgtgagcctgcatgctgtg ggggtgagctactggaaggcctctgagggggctgagtatgatgaccagaccagccagagg gagaaggaggatgacaaggtgttccctgggggcagccacacttatgtgtggcaggtgctg aaggagaatggcccaatggcctctgaccccctgtgcctgacctacagctatctgagccat gtggatctggtgaaggatctgaactctggcctgattggggccctgctggtgtgcagggag ggctctctggccaaggagaagactcagactctgcacaagttcatcctgctgtttgctgtg tttgatgagggcaagagctggcactctgagaccaagaactctctgatgcaggatagggat gctgcttctgccagggcctggcccaagatgcacactgtgaatgggtatgtgaataggagc ctgcctgggctgattgggtgtcacaggaagtctgtgtactggcatgtgattggcatgggc accactcctgaggtgcacagcatctttctggagggccacacttttctggtgaggaatcac aggcaggccagcctggagatcagccccatcaccttcctgactgcccagaccctgctgatg gatctgggccagttcctgctgttttgccatatcagcagccatcagcatgatgggatggag gcttatgtgaaggtggactcttgccctgaggagcctcagctgaggatgaagaataatgaa gaggctgaggactatgatgatgatctgactgactctgagatggatgtggtgaggtttgat gatgacaacagccccagctttatccagattaggtctgtggccaagaagcaccccaagacc tgggtgcattacattgctgctgaggaagaggattgggactatgcccccctggtgctggcc cctgatgacaggagctacaagtctcagtacctgaacaatggccctcagaggattggcagg aagtacaagaaggtgaggttcatggcttacactgatgagaccttcaagaccagggaggcc attcagcatgaatctgggatcctgggccccctgctgtatggggaggtgggggacaccctg ctgattattttcaagaaccaggccagcaggccctacaacatttatcctcatggcattact gatgtgagacccctgtacagcaggaggctgcctaagggggtgaagcacctgaaggacttc cccatcctgcctggggagatcttcaagtacaagtggactgtgactgtggaggatggcccc actaagtctgaccccaggtgcctgactaggtactactccagctttgtgaacatggagagg gacctggcctctggcctgattggccccctgctgatctgctacaaggagtctgtggatcag aggggcaaccagatcatgtctgacaagagaaatgtgatcctgttctctgtgtttgatgag aataggtcttggtacctgactgagaacatccagaggtttctgcctaatcctgctggggtg cagctggaggatcctgagttccaggcctctaacattatgcacagcatcaatgggtatgtg tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagc attggggcccagactgactttctgtctgtgttcttctctggctacacctttaagcataag atggtgtatgaggacaccctgactctgttccccttctctggggagactgtgttcatgagc atggagaacccaggcctgtggatcctgggctgccacaactctgatttcaggaataggggc atgactgccctgctgaaggtgagcagctgtgataagaacactggggactattatgaggat agctatgaggacatctctgcctacctgctgagcaagaacaatgccattgagcccaggagc ttcagccagaatcctcctgtgctgaagaggcaccagagggagatcaccaggaccaccctg cagtctgatcaggaggagattgactatgatgacactatctctgtggagatgaagaaggag gactttgacatctatgatgaggatgagaatcagagccccaggagcttccagaagaagact agacactactttattgctgctgtggagaggctgtgggactatggcatgagctcttctccc catgtgctgagaaacagggcccagtctggctctgtgccccagttcaagaaggtggtcttc caggagttcactgatggctctttcacccagcctctgtatagaggggagctgaatgagcac ctgggcctgctgggcccttacatcagggctgaggtggaggacaatatcatggtgaccttc aggaaccaggctagcaggccctactctttctacagcagcctgatcagctatgaggaggac cagaggcagggggctgagcctaggaagaattttgtgaagcccaatgagaccaagacctac ttctggaaggtgcagcaccacatggctcccactaaggatgagtttgactgcaaggcctgg gcctacttttctgatgtggacctggagaaggatgtgcattctggcctgattggccccctg ctggtctgccacaccaatactctgaaccctgctcatgggagacaggtgactgtgcaggag tttgccctgttcttcaccatctttgatgagaccaagtcctggtactttactgagaacatg gagaggaattgcagggccccttgcaacatccagatggaggaccccaccttcaaggaaaat tataggttccatgccatcaatggctacatcatggacaccctgcctggcctggtgatggcc caggaccagaggatcaggtggtatctgctgtctatgggctctaatgagaacatccacagc atccatttctctggccatgtgttcactgtgaggaagaaggaggagtataagatggctctg tacaacctgtaccctggggtctttgagactgtggagatgctgcccagcaaggctggcatt tggagggtggagtgcctgattggggaacacctgcatgctgggatgagcaccctgttcctg gtgtactctaacaagtgccagaccccactgggcatggcttctggccacatcagggatttc cagattactgcctctggccagtatggccagtgggctcccaagctggctaggctgcactac tctgggagcatcaatgcctggtctactaaggagcctttctcttggatcaaagtggacctg ctggcccctatgatcatccatgggatcaagactcagggggccaggcagaagttcagcagc ctgtacatctctcagttcatcattatgtacagcctggatggcaagaagtggcagacctac aggggcaacagcactggcaccctgatggtgttctttgggaatgtggacagctctgggatt aagcacaacatctttaacccccccatcattgccaggtatatcaggctgcaccctacccac tacagcattaggagcaccctgaggatggagctgatgggctgtgacctgaacagctgcagc atgcccctggggatggagagcaaggccatttctgatgctcagatcactgcttctagctac ttcactaacatgtttgccacctggtctcccagcaaggctagactgcacctgcaggggagg agcaatgcctggaggccccaggtgaataatcccaaggagtggctgcaggtggatttccag aaaaccatgaaggtgactggggtgactacccagggggtgaagtctctgctgaccagcatg tatgtgaaggagttcctgatcagcagcagccaggatgggcatcagtggaccctgttcttt cagaatgggaaggtgaaggtgtttcagggcaatcaggacagcttcacccctgtggtgaac agcctggacccccccctgctgaccaggtacctgaggatccacccccagagctgggtgcat cagattgccctgaggatggaggtgctgggctgtgaggcccaggacctgtactga FVIIIencodingCpGreducednucleicacidvariantX05 (SEQIDNO:5) atgcagattgagctgtctacttgcttcttcctgtgcctgctgaggttctgcttctctgcc actaggaggtattacctgggggctgtggagctgagctgggactatatgcagtctgacctg ggggagctgcctgtggatgccaggtttcctcccagggtgcctaagagcttccccttcaac acctctgtggtgtacaagaagactctgtttgtggagtttactgatcatctgttcaacatt gccaagcccaggcctccttggatggggctgctgggccccaccatccaggctgaggtgtat gacactgtggtgattaccctgaagaatatggccagccatcctgtgagcctgcatgctgtg ggggtgagctattggaaggcctctgagggggctgagtatgatgatcagactagccagagg gagaaggaggatgacaaggtgttccctggggggagccatacctatgtgtggcaggtgctg aaggagaatggccccatggcctctgaccctctgtgcctgacttatagctacctgagccat gtggatctggtgaaggacctgaactctggcctgattggggccctgctggtgtgcagggag ggcagcctggccaaggagaagactcagaccctgcacaagttcatcctgctgtttgctgtg tttgatgaggggaagtcctggcactctgagactaagaacagcctgatgcaggatagggat gctgcttctgccagggcctggcctaagatgcacactgtgaatggctatgtgaataggagc ctgcctggcctgattggctgccataggaagtctgtgtactggcatgtgattgggatgggc accacccctgaggtgcactctattttcctggagggccatactttcctggtgaggaaccat aggcaggccagcctggagatcagccccatcactttcctgactgcccagactctgctgatg gacctgggccagttcctgctgttctgccacatcagcagccatcagcatgatggcatggag gcttatgtgaaggtggacagctgccctgaggagcctcagctgaggatgaagaataatgag gaggctgaggactatgatgatgacctgactgactctgagatggatgtggtgaggtttgat gatgacaactctccctctttcatccagatcaggtctgtggccaagaagcaccctaagacc tgggtgcactacattgctgctgaggaggaggattgggactatgcccccctggtgctggcc ccagatgacaggagctacaagtcccagtacctgaacaatggcccccagaggattggcagg aagtacaagaaggtgaggttcatggcttatactgatgagactttcaagaccagggaggcc atccagcatgagtctggcatcctgggccctctgctgtatggggaggtgggggacaccctg ctgattatcttcaagaaccaggcttctaggccctacaatatctaccctcatggcatcact gatgtgaggcccctgtacagcaggaggctgcccaagggggtgaagcatctgaaggatttc cccatcctgcctggggagatctttaagtataagtggactgtgactgtggaggatggcccc actaagtctgaccccaggtgcctgaccaggtattacagcagctttgtgaacatggagagg gatctggcttctgggctgattggccccctgctgatctgctacaaggagtctgtggaccag aggggcaaccagatcatgtctgacaagaggaatgtgatcctgttctctgtgtttgatgag aataggagctggtacctgactgagaacatccagaggtttctgcccaatcctgctggggtg cagctggaggatcctgagtttcaggcctctaatatcatgcacagcatcaatggctatgtg tttgactctctgcagctgtctgtgtgcctgcatgaggtggcctattggtacatcctgagc attggggcccagactgactttctgtctgtgtttttttctggctacaccttcaagcacaag atggtgtatgaggatactctgactctgttccctttttctggggagactgtgttcatgtct atggagaaccctgggctgtggattctgggctgccacaattctgacttcaggaacagaggc atgactgctctgctgaaggtgagcagctgtgacaagaacactggggactactatgaggac tcttatgaggacatttctgcctacctgctgagcaagaacaatgccattgagcccagaagc ttttctcagaacccccctgtgctgaagaggcaccagagggagatcaccaggaccaccctg cagtctgaccaggaggagattgactatgatgatactatttctgtggagatgaagaaggag gactttgacatctatgatgaggatgagaaccagagccccaggtctttccagaagaagact aggcactactttattgctgctgtggagaggctgtgggactatgggatgtctagctctcct catgtgctgaggaacagggcccagtctggctctgtgccccagtttaaaaaggtggtgttc caggaattcactgatggcagctttacccagcctctgtacaggggggagctgaatgagcac ctggggctgctggggccttacattagggctgaggtggaggacaacatcatggtgaccttc aggaatcaggccagcaggccctactctttctacagcagcctgatctcttatgaggaggac cagaggcagggggctgaacccaggaagaactttgtgaagcccaatgagaccaagacctac ttctggaaggtgcagcaccacatggctcccaccaaggatgagtttgattgcaaggcctgg gcttacttctctgatgtggatctggagaaggatgtgcactctgggctgattggccccctg ctggtgtgccacaccaacactctgaaccctgcccatggcagacaggtgactgtgcaggag tttgccctgttcttcactatctttgatgagactaagagctggtacttcactgagaacatg gagaggaattgcagggccccttgcaacatccagatggaggaccccacctttaaggagaac tacaggtttcatgccattaatggctacatcatggacaccctgcctggcctggtgatggcc caggaccagaggatcaggtggtacctgctgtctatggggagcaatgagaacatccacagc attcacttctctggccatgtgttcactgtgaggaagaaggaggagtacaagatggccctg tacaacctgtaccctggggtgtttgagactgtggagatgctgcccagcaaggctgggatc tggagggtggagtgcctgattggggagcacctgcatgctgggatgagcaccctgttcctg gtgtatagcaacaagtgccagacccccctgggcatggcctctggccacatcagagacttt cagattactgcctctggccagtatgggcagtgggcccccaagctggccaggctgcactat tctggctctattaatgcctggagcactaaggagcccttcagctggattaaggtggacctg ctggctcccatgatcatccatggcatcaagactcagggggccaggcagaagttctcttct ctgtacatcagccagttcattatcatgtactccctggatggcaagaagtggcagacctat aggggcaacagcactggcaccctgatggtgttctttgggaatgtggacagctctggcatc aagcataatatcttcaatccccccatcattgctaggtacatcaggctgcaccccacccac tactctattaggtctaccctgaggatggagctgatgggctgtgacctgaacagctgcagc atgcctctgggcatggagagcaaagccatctctgatgcccagatcactgccagcagctac tttaccaacatgtttgctacttggagccccagcaaggccaggctgcacctgcaggggagg tctaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggacttccag aagactatgaaggtgactggggtgaccacccagggggtgaagagcctgctgacctctatg tatgtgaaggagttcctgattagcagcagccaggatggccaccagtggaccctgtttttc cagaatgggaaggtgaaggtgtttcaggggaaccaggacagcttcactcctgtggtgaac tctctggacccccccctgctgaccaggtatctgaggatccaccctcagagctgggtgcac cagattgccctgaggatggaggtgctgggctgtgaggcccaggacctgtactga FVIIIencodingCpGreducednucleicacidvariantX06 (SEQIDNO:6) atgcagattgagctgagcacctgcttcttcctgtgcctgctgaggttttgcttctctgcc accaggaggtactacctgggggctgtggagctgagctgggattacatgcagtctgacctg ggggagctgcctgtggatgccaggttccctcccagggtgcccaagtctttccccttcaac acttctgtggtgtacaagaagaccctgtttgtggagtttactgaccacctgttcaacatt gccaagcccaggcctccctggatgggcctgctgggccccaccattcaggctgaggtgtat gacactgtggtcatcaccctgaaaaatatggctagccaccctgtgtctctgcatgctgtg ggggtgagctactggaaggcctctgagggggctgagtatgatgaccagactagccagagg gagaaggaggatgacaaggtgttccctgggggcagccacacttatgtgtggcaggtgctg aaagagaatggccccatggcttctgatcccctgtgtctgacctatagctacctgagccat gtggatctggtgaaggacctgaactctggcctgattggggccctgctggtgtgcagggag ggcagcctggctaaggagaagacccagaccctgcataagttcatcctgctgtttgctgtg tttgatgagggcaagagctggcactctgagactaagaacagcctgatgcaggatagggat gctgcttctgccagggcctggcccaagatgcacactgtgaatgggtatgtgaacaggagc ctgcctggcctgattggctgccataggaagtctgtctattggcatgtgattggcatgggc actactcctgaggtgcacagcatctttctggagggccacaccttcctggtgaggaaccac aggcaggccagcctggagatctctcccatcactttcctgactgctcagaccctgctgatg gacctgggccagttcctgctgttctgtcacatctctagccaccagcatgatggcatggag gcctatgtgaaggtggatagctgccctgaggaaccccagctgaggatgaagaacaatgag gaggctgaggattatgatgatgatctgactgattctgagatggatgtggtgaggtttgat gatgacaattctcctagcttcattcagatcagatctgtggccaaaaagcatcctaagact tgggtgcattatattgctgctgaggaggaggattgggattatgcccccctggtgctggct cctgatgataggagctacaagtctcagtacctgaataatgggccccagaggattggcagg aagtacaagaaggtgaggttcatggcctacactgatgagaccttcaagaccagggaggcc attcagcatgagtctgggattctggggcccctgctgtatggggaggtgggggataccctg ctgatcattttcaagaaccaggccagcaggccctacaacatctacccccatgggattact gatgtgaggcccctgtactctaggaggctgcctaagggggtgaagcacctgaaggatttt cctatcctgcctggggaaatcttcaagtacaagtggactgtgactgtggaggatggcccc actaagtctgatcccaggtgtctgaccaggtattatagctcttttgtgaacatggagagg gatctggcctctgggctgattggccctctgctgatctgctacaaggagtctgtggaccag aggggcaaccagatcatgtctgacaagaggaatgtgatcctgttctctgtgtttgatgag aacaggagctggtatctgactgagaacatccagaggtttctgcccaatcctgctggggtg cagctggaggatcctgagttccaggctagcaacatcatgcacagcatcaatgggtatgtg tttgacagcctgcagctgtctgtgtgtctgcatgaggtggcctactggtatatcctgtct attggggcccagactgacttcctgtctgtgtttttttctgggtatacttttaagcacaag atggtgtatgaggacaccctgactctgttccccttctctggggagactgtgtttatgagc atggagaaccctggcctgtggatcctgggctgccacaattctgacttcaggaataggggg atgactgccctgctgaaggtgagcagctgtgataagaatactggggactactatgaggac tcttatgaggacatttctgcctatctgctgtctaagaacaatgccattgaacccaggagc ttctctcagaacccccctgtgctgaagaggcaccagagggaaatcaccagaactactctg cagtctgatcaggaggaaattgactatgatgacactatttctgtggagatgaagaaggag gactttgacatctatgatgaggatgagaaccagagcccaaggagcttccagaagaagact aggcactacttcattgctgctgtggagaggctgtgggactatggcatgagcagcagcccc catgtgctgagaaacagggcccagtctgggtctgtgccccagttcaagaaggtggtgttc caggagttcactgatgggagcttcacccagcccctgtataggggggagctgaatgagcac ctgggcctgctgggcccctatattagggctgaggtggaggacaacatcatggtgaccttc aggaatcaggcctctaggccctacagcttctacagcagcctgattagctatgaggaggat cagaggcagggggctgaacccaggaagaactttgtgaagcccaatgagaccaagacctat ttctggaaggtgcagcatcacatggcccccaccaaggatgagtttgactgcaaggcctgg gcctacttctctgatgtggatctggagaaggatgtgcactctggcctgattggccccctg ctggtgtgccacaccaacaccctgaaccctgctcatggcaggcaggtgactgtgcaggag tttgccctgttcttcaccatctttgatgagactaagtcttggtacttcactgagaatatg gagaggaattgcagggccccctgcaatattcagatggaagaccccaccttcaaggagaat tacaggttccatgccattaatggctacatcatggataccctgcctggcctggtgatggcc caggatcagaggatcaggtggtacctgctgagcatgggcagcaatgagaacatccactct atccacttctctggccatgtgtttactgtgaggaagaaggaggagtataagatggccctg tacaacctgtaccctggggtctttgagactgtggagatgctgccttctaaggctggcatt tggagggtggagtgcctgattggggaacacctgcatgctggcatgtctaccctgttcctg gtgtacagcaataagtgccagacccccctgggcatggcctctgggcatatcagggatttc cagatcactgcctctggccagtatggccagtgggccccaaagctggctaggctgcactac tctgggagcatcaatgcctggagcactaaggagcccttcagctggatcaaggtggacctg ctggcccccatgattatccatgggattaagactcagggggccaggcagaagttcagcagc ctgtacatcagccagttcattatcatgtacagcctggatggcaagaagtggcagacctat aggggcaactctactgggaccctgatggtgttctttgggaatgtggatagctctgggatc aagcacaatatcttcaacccccccatcattgccaggtatatcaggctgcaccccacccac tacagcattaggtctaccctgaggatggagctgatgggctgtgatctgaacagctgtagc atgcctctgggcatggagtctaaggccatttctgatgcccagattactgctagcagctac ttcaccaacatgtttgccacctggtctcccagcaaggccaggctgcatctgcagggcagg tctaatgcttggaggccccaggtgaacaacccaaaggagtggctgcaggtggatttccag aagactatgaaggtgactggggtgaccactcagggggtgaagtctctgctgacctctatg tatgtgaaggagttcctgatctctagcagccaggatggccatcagtggaccctgttcttc cagaatggcaaggtgaaagtgttccagggcaatcaggatagcttcactccagtggtgaac agcctggatccccctctgctgactaggtacctgaggatccacccccagagctgggtgcac cagattgccctgaggatggaggtgctgggctgtgaggcccaggacctgtactga FVIIIencodingCpGreducednucleicacidvariantX07 (SEQIDNO:7) atgcagattgagctgagcacctgcttcttcctgtgtctgctgaggttctgcttctctgcc accaggaggtattacctgggggctgtggagctgagctgggactatatgcagtctgacctg ggggagctgcctgtggatgctaggttcccccccagggtgcccaagagcttcccctttaac acttctgtggtgtacaagaagaccctgtttgtggagttcactgaccacctgttcaacatt gccaagcccaggcccccctggatggggctgctggggcccaccatccaggctgaggtgtat gacactgtggtgatcaccctgaagaacatggccagccaccctgtgagcctgcatgctgtg ggggtgagctactggaaggcttctgagggggctgagtatgatgaccagactagccagagg gagaaggaggatgacaaggtgtttcctgggggcagccatacctatgtgtggcaggtgctg aaggagaatggccccatggcctctgaccccctgtgcctgacctacagctacctgtctcat gtggacctggtgaaggacctgaactctggcctgattggggctctgctggtgtgtagggag ggcagcctggctaaggaaaagacccagaccctgcataagtttatcctgctgtttgctgtg tttgatgagggcaagagctggcactctgagaccaagaacagcctgatgcaggatagggat gctgcctctgccagggcttggcctaagatgcacactgtgaatgggtatgtgaataggagc ctgcctggcctgattggctgccacaggaagtctgtgtactggcatgtgattgggatgggc accacccctgaggtccatagcatcttcctggagggccacactttcctggtgaggaaccac agacaggcctctctggagatctctcccatcaccttcctgactgctcagactctgctgatg gacctgggccagttcctgctgttttgccatattagcagccaccagcatgatgggatggag gcctatgtgaaggtggatagctgccctgaggagcctcagctgaggatgaagaacaatgag gaggctgaagactatgatgatgacctgactgattctgagatggatgtggtgaggtttgat gatgacaatagccccagcttcattcagatcaggtctgtggccaagaaacaccccaagacc tgggtgcactacattgctgctgaggaagaggactgggactatgctcccctggtgctggcc cctgatgataggtcttataagagccagtacctgaacaatgggccccagaggattggcagg aagtacaagaaggtgaggttcatggcctacactgatgaaaccttcaaaaccagggaggcc attcagcatgagtctggcatcctgggccctctgctgtatggggaggtgggggacaccctg ctgatcatcttcaagaaccaggccagcaggccctacaacatctatcctcatggcatcact gatgtgaggcccctgtacagcaggaggctgcccaagggggtgaagcacctgaaagacttc cccatcctgcctggggagatctttaagtataagtggactgtgactgtggaggatggccct accaagtctgaccccaggtgtctgaccaggtactattctagctttgtgaacatggagagg gacctggcctctggcctgattgggcccctgctgatctgctacaaggagtctgtggaccag aggggcaaccagatcatgtctgacaagaggaatgtgatcctgttttctgtgtttgatgag aataggagctggtacctgactgagaacatccagaggtttctgcccaatcctgctggggtg cagctggaggatcctgagttccaggccagcaatatcatgcatagcatcaatggctatgtg tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagc attggggcccagactgactttctgtctgtgttcttttctggctataccttcaagcacaag atggtgtatgaggataccctgaccctgttccccttctctggggagactgtgttcatgagc atggagaatcctgggctgtggatcctggggtgccacaactctgattttaggaacaggggg atgactgccctgctgaaggtgtctagctgtgataagaacactggggactactatgaggac agctatgaggacatttctgcttatctgctgtctaagaataatgccattgagcccagaagc ttcagccagaatccccctgtgctgaagagacatcagagggagatcaccagaactaccctg cagtctgatcaggaggagattgactatgatgacactatctctgtggagatgaagaaggag gactttgacatctatgatgaggatgagaatcagtctcccaggagctttcagaagaagacc agacattacttcattgctgctgtggagaggctgtgggactatggcatgagctctagccct catgtgctgaggaacagggcccagtctggctctgtgccccagttcaagaaggtggtgttc caggaattcactgatggcagcttcacccagcccctgtacaggggggagctgaatgagcac ctgggcctgctggggccttatatcagggctgaggtggaggataatattatggtgactttc aggaaccaggccagcaggccctactctttctatagcagcctgatctcttatgaggaggat cagaggcagggggctgagcctaggaagaactttgtgaagcccaatgagactaagacctac ttctggaaggtccagcaccacatggcccctaccaaggatgagtttgactgcaaggcctgg gcctatttctctgatgtggatctggagaaggatgtccattctgggctgattggccccctg ctggtgtgccacactaacactctgaatcctgcccatggcaggcaggtgactgtccaggag tttgccctgttcttcactatctttgatgagaccaagagctggtactttactgagaacatg gagaggaactgcagagctccttgcaatattcagatggaggaccccaccttcaaggagaat tacaggttccatgccattaatgggtacatcatggacaccctgcctggcctggtgatggct caggaccagaggatcaggtggtacctgctgagcatgggctctaatgagaatatccacagc atccacttctctgggcatgtgttcactgtgaggaagaaggaggagtacaagatggctctg tataatctgtaccctggggtgtttgaaactgtggagatgctgccctctaaggctggcatc tggagggtggagtgcctgattggggagcacctgcatgctggcatgagcaccctgttcctg gtgtacagcaacaagtgccagacccccctgggcatggcctctggccacatcagggacttc cagatcactgcctctggccagtatggccagtgggcccccaagctggccaggctgcactat tctggcagcatcaatgcctggagcaccaaggagcccttcagctggatcaaggtggacctg ctggcccccatgatcattcatggcatcaagacccagggggccaggcagaagttcagctct ctgtacatctctcagttcatcatcatgtactctctggatgggaagaagtggcagacctac aggggcaacagcactggcaccctgatggtgttctttgggaatgtggactcttctggcatc aagcacaacatcttcaatccccccatcattgctaggtatattaggctgcatcccacccac tacagcatcaggtctaccctgaggatggagctgatgggctgtgacctgaactcttgcagc atgcccctgggcatggagtctaaggccatctctgatgcccagattactgccagcagctac ttcaccaacatgtttgccacctggagcccctctaaggccaggctgcatctgcaggggagg agcaatgcctggaggcctcaggtgaacaaccccaaggagtggctgcaggtggatttccag aagaccatgaaggtgactggggtgaccacccagggggtcaagagcctgctgaccagcatg tatgtgaaggagttcctgatcagcagcagccaggatggccaccagtggactctgttcttt cagaatgggaaggtgaaggtgtttcagggcaatcaggactctttcacccctgtggtgaac agcctggacccccccctgctgaccagatacctgaggatccacccccagtcttgggtgcat cagattgccctgaggatggaggtgctgggctgtgaggctcaggatctgtactga FVIIIencodingCpGreducednucleicacidvariantX08 (SEQIDNO:8) atgcagattgagctgagcacttgcttttttctgtgcctgctgaggttttgtttttctgcc accaggaggtactacctgggggctgtggagctgagctgggactatatgcagtctgatctg ggggagctgcctgtggatgccaggttcccccccagggtgcccaagtcttttcccttcaac acctctgtggtgtataagaagaccctgtttgtggagttcactgaccacctgttcaacatt gctaagcctaggcccccctggatgggcctgctgggccctaccattcaggctgaggtgtat gacactgtggtgatcaccctgaagaacatggccagccatcctgtgagcctgcatgctgtg ggggtctcttactggaaggcctctgagggggctgagtatgatgaccagaccagccagaga gagaaggaggatgacaaggtcttccctgggggctctcacacctatgtgtggcaggtgctg aaggaaaatggccccatggcctctgaccccctgtgcctgacctacagctatctgagccat gtggatctggtgaaggacctgaattctggcctgattggggccctgctggtgtgcagggag ggcagcctggccaaggagaagacccagaccctgcacaagtttatcctgctgtttgctgtg tttgatgagggcaagtcttggcactctgagactaagaacagcctgatgcaggacagggat gctgcctctgccagggcctggcccaagatgcacactgtgaatggctatgtgaacaggagc ctgcctgggctgattggctgccacaggaagtctgtgtactggcatgtgattggcatgggc accacccctgaggtgcacagcatcttcctggaaggccacactttcctggtgaggaaccat aggcaggccagcctggagatcagccctatcaccttcctgactgcccagaccctgctgatg gatctggggcagttcctgctgttctgccacatctctagccaccagcatgatgggatggag gcctatgtgaaggtggacagctgcccagaggagcctcagctgaggatgaaaaacaatgaa gaggctgaggattatgatgatgatctgactgactctgagatggatgtggtgagatttgat gatgacaatagccctagctttattcagatcaggtctgtggctaagaagcaccccaagacc tgggtgcattacattgctgctgaggaggaggactgggattatgctcctctggtgctggcc cctgatgataggagctacaagagccagtacctgaataatggccctcagaggattggcagg aagtacaagaaggtgaggttcatggcttacactgatgagaccttcaagactagggaggcc atccagcatgagtctgggatcctggggcccctgctgtatggggaggtgggggacaccctg ctgatcatcttcaagaaccaggctagcaggccttacaacatctatccccatgggatcact gatgtgagacctctgtacagcaggaggctgcccaagggggtcaagcatctgaaagacttc cccatcctgcctggggagatctttaagtataagtggactgtgactgtggaggatgggccc accaagtctgaccccaggtgcctgaccaggtattacagcagctttgtgaacatggagagg gatctggcctctgggctgattggccccctgctgatctgttacaaggaatctgtggatcag aggggcaatcagatcatgtctgacaagaggaatgtgatcctgttctctgtgtttgatgag aataggtcttggtacctgactgaaaacatccagaggttcctgcccaaccctgctggggtc cagctggaggatcctgagttccaggctagcaacatcatgcacagcatcaatgggtatgtg tttgatagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgtct attggggcccagactgacttcctgtctgtgttcttttctggctacaccttcaagcacaag atggtgtatgaggacaccctgaccctgttccccttctctggggagactgtctttatgagc atggagaaccctgggctgtggatcctgggctgccacaactctgatttcaggaataggggc atgactgctctgctgaaggtgagctcttgtgacaagaacactggggattactatgaggac agctatgaggacatttctgcctacctgctgagcaagaacaatgccattgagcctaggagc tttagccagaatcctcctgtcctgaagaggcaccagagggagatcaccaggaccaccctg cagtctgaccaggaggagattgactatgatgataccatctctgtggagatgaagaaggag gactttgacatctatgatgaggatgagaatcagtctcccaggagcttccagaagaagacc aggcactatttcattgctgctgtggagaggctgtgggactatggcatgagcagctctcct catgtgctgaggaatagggctcagtctggctctgtgccccagttcaagaaagtggtgttt caggagttcactgatggctctttcacccagcctctgtataggggggagctgaatgagcac ctggggctgctgggcccctatatcagggctgaggtggaggataacatcatggtgaccttc aggaaccaggcctctaggccctacagcttctatagcagcctgatcagctatgaggaggac cagaggcagggggctgagcccaggaagaactttgtgaagcccaatgagaccaagacttac ttctggaaggtgcagcatcacatggcccccaccaaggatgagtttgactgtaaggcctgg gcctacttctctgatgtggatctggagaaggatgtgcactctggcctgattggccccctg ctggtgtgccataccaatactctgaaccctgctcatggcaggcaggtgactgtgcaggag tttgctctgttcttcactatctttgatgagaccaagtcttggtatttcactgagaatatg gagaggaactgcagggccccctgcaacatccagatggaggaccccacctttaaggagaac tataggtttcatgccatcaatggctacatcatggacaccctgcctggcctggtgatggcc caggatcagaggatcaggtggtacctgctgagcatggggtctaatgagaacatccacagc atccacttctctggccatgtgtttactgtgagaaagaaggaggagtacaagatggctctg tacaatctgtaccctggggtctttgagactgtggagatgctgcctagcaaggctgggatc tggagggtggagtgcctgattggggaacatctgcatgctgggatgtctactctgttcctg gtgtacagcaacaagtgccagacccccctgggcatggcttctggccatatcagggacttt cagattactgcctctgggcagtatggccagtgggcccccaagctggctaggctgcattat tctggcagcatcaatgcctggtctactaaggagcccttcagctggatcaaggtggatctg ctggcccccatgatcatccatggcatcaagacccagggggccaggcagaagtttagctct ctgtacattagccagttcatcatcatgtacagcctggatgggaagaagtggcagacctac aggggcaattctactggcaccctgatggtgttctttggcaatgtggacagctctggcatc aagcacaacatctttaacccccctatcattgctaggtacatcaggctgcatcccacccat tacagcatcaggagcaccctgaggatggagctgatgggctgtgacctgaactcttgcagc atgcccctgggcatggagagcaaggccatttctgatgcccagattactgccagcagctac ttcactaacatgtttgccacctggtctcccagcaaggccaggctgcacctgcagggcagg agcaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggatttccag aagaccatgaaggtgactggggtgaccacccagggggtgaagagcctgctgactagcatg tatgtgaaggagttcctgatcagctctagccaggatggccaccagtggactctgtttttc cagaatggcaaggtgaaggtgttccagggcaaccaggactctttcactcctgtggtgaac agcctggacccccccctgctgaccaggtatctgaggattcacccccagtcttgggtgcat cagattgccctgaggatggaggtgctgggctgtgaggcccaggatctgtactga FVIIIencodingCpGreducednucleicacidvariantX09 (SEQIDNO:9) atgcagattgagctgagcacctgcttcttcctgtgtctgctgagattttgcttttctgcc actaggaggtattacctgggggctgtggagctgtcttgggactacatgcagtctgatctg ggggagctgcctgtggatgccaggttcccacctagggtgcctaagagctttcccttcaat acctctgtggtgtacaagaagaccctgtttgtggagttcactgaccacctgttcaacatt gccaagcctaggcccccctggatgggcctgctgggccctaccatccaggctgaagtgtat gacactgtggtgatcaccctgaagaacatggccagccaccctgtgagcctgcatgctgtg ggggtgtcttactggaaggcctctgagggggctgagtatgatgatcagaccagccagagg gagaaggaagatgacaaggtgttccctgggggcagccacacctatgtctggcaggtgctg aaggagaatggccccatggcctctgatcccctgtgcctgacctactcttacctgagccat gtggacctggtgaaggatctgaattctggcctgattggggccctgctggtgtgcagggag ggcagcctggccaaggagaagacccagaccctgcataagttcatcctgctgtttgctgtg tttgatgaagggaagagctggcactctgagactaagaacagcctgatgcaggacagggat gctgcttctgccagggcctggcccaagatgcacactgtgaatggctatgtgaatagaagc ctgcctggcctgattgggtgccacaggaagtctgtgtactggcatgtgattgggatgggc actacccctgaggtgcatagcatcttcctggaaggccataccttcctggtgaggaatcat aggcaggcttctctggaaatttctcccatcactttcctgactgctcagaccctgctgatg gacctgggccagttcctgctgttctgccacatcagctctcaccagcatgatgggatggag gcctatgtgaaggtggacagctgtcctgaggagccccagctgaggatgaagaacaatgag gaggctgaggactatgatgatgacctgactgactctgagatggatgtggtcaggtttgat gatgacaatagcccctctttcatccagatcaggtctgtggccaagaagcaccccaagact tgggtgcactacattgctgctgaggaggaggattgggattatgcccctctggtgctggcc cctgatgacaggagctataagtctcagtacctgaataatggcccccagaggattgggagg aagtataagaaggtgaggtttatggcctacactgatgagaccttcaagaccagggaggcc atccagcatgagtctggcatcctgggccccctgctgtatggggaggtgggggataccctg ctgatcatcttcaagaaccaggcctctaggccctacaatatctaccctcatggcatcact gatgtgagacccctgtatagcaggaggctgcctaagggggtgaagcacctgaaggacttc cccatcctgcctggggagatcttcaagtataagtggactgtgactgtggaggatggcccc accaagtctgaccccaggtgcctgaccaggtattacagctcttttgtgaacatggagagg gatctggcctctgggctgattggcccactgctgatctgctacaaggagtctgtggatcag aggggcaatcagatcatgtctgacaagaggaatgtgatcctgttttctgtgtttgatgaa aataggtcttggtatctgactgagaacatccagaggtttctgcccaatcctgctggggtg cagctggaggatcctgagtttcaggcctctaatatcatgcattctatcaatggctatgtg tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagc attggggctcagactgacttcctgtctgtgttcttttctggctatactttcaagcacaag atggtgtatgaggacactctgaccctgttccccttctctggggagactgtgttcatgtct atggaaaatcctgggctgtggattctgggctgccacaattctgacttcaggaataggggg atgactgccctgctgaaggtgtctagctgtgataagaacactggggattactatgaggac tcttatgaagatatctctgcctatctgctgagcaagaacaatgccattgagcccaggagc ttcagccagaacccccctgtgctgaagaggcaccagagggagatcaccaggaccactctg cagtctgatcaggaggagattgactatgatgacactatctctgtggagatgaagaaggag gattttgacatttatgatgaggatgagaaccagtctcccaggagcttccagaagaagacc aggcattactttattgctgctgtggagaggctgtgggactatgggatgagcagctctcct catgtgctgaggaacagggcccagtctgggtctgtgccccagttcaagaaggtggtgttc caggagttcactgatgggagcttcacccagcccctgtataggggggagctgaatgagcac ctgggcctgctgggcccctacatcagggctgaggtggaggataatatcatggtgaccttc aggaaccaggctagcaggccttacagcttttacagcagcctgatctcttatgaagaagac cagaggcagggggctgagcccaggaagaattttgtgaagcctaatgagaccaagacttat ttttggaaggtgcagcatcacatggctcctaccaaggatgagtttgactgcaaggcctgg gcctacttttctgatgtggatctggagaaggatgtgcactctggcctgattggccctctg ctggtgtgccatactaacactctgaaccctgcccatgggaggcaggtgactgtgcaggag tttgccctgttcttcactatttttgatgagaccaagtcttggtatttcactgagaacatg gagaggaactgcagggctccctgcaacatccagatggaagaccccaccttcaaggagaac tataggttccatgccatcaatgggtacatcatggataccctgcctggcctggtgatggcc caggatcagaggattaggtggtatctgctgagcatgggctctaatgagaacatccacagc atccatttctctggccatgtgttcactgtgaggaagaaggaggagtacaagatggctctg tacaacctgtatcctggggtgtttgagactgtggagatgctgcccagcaaggctggcatc tggagggtggaatgcctgattggggagcacctgcatgctggcatgagcactctgttcctg gtgtatagcaacaagtgccagacccccctgggcatggcctctggccatatcagggatttc cagatcactgcttctggccagtatggccagtgggcccccaagctggccaggctgcactat tctggcagcatcaatgcctggagcactaaggagcctttttcttggatcaaggtggacctg ctggcccctatgattattcatggcatcaagacccagggggccaggcagaagttctctagc ctgtacatctctcagttcatcattatgtatagcctggatggcaagaagtggcagacctac aggggcaatagcactggcaccctgatggtgttttttgggaatgtggactcttctgggatc aagcacaacatctttaacccccccatcattgccaggtatattaggctgcaccccacccac tacagcatcaggagcaccctgaggatggagctgatgggctgtgatctgaattcttgctct atgcccctgggcatggagagcaaggccatctctgatgcccagatcactgccagctcttac ttcaccaacatgtttgccacctggtctcctagcaaggccaggctgcatctgcagggcagg agcaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggacttccag aagaccatgaaggtgactggggtgaccactcagggggtgaagagcctgctgacctctatg tatgtgaaggagttcctgatcagcagcagccaggatggccaccagtggactctgttcttc cagaatgggaaggtgaaggtgttccagggcaaccaggatagctttacccctgtggtgaac agcctggaccctcctctgctgaccagatacctgaggatccatcctcagagctgggtgcac cagattgccctgaggatggaggtgctgggctgtgaggcccaggatctgtactga FVIIIencodingCpGreducednucleicacidvariantX10 (SEQIDNO:10) atgcagattgagctgagcacttgcttcttcctgtgcctgctgaggttctgcttttctgct actaggaggtactacctgggggctgtggagctgagctgggattacatgcagtctgacctg ggggagctgccagtggatgccaggttcccccccagggtgcccaagtcttttcctttcaac acctctgtggtgtacaagaagaccctgtttgtggagttcactgaccacctgttcaacatt gccaagcccaggcccccctggatggggctgctggggcccaccatccaggctgaggtgtat gacactgtggtgattaccctgaagaacatggctagccaccctgtgagcctgcatgctgtg ggggtgagctattggaaggcctctgagggggctgagtatgatgatcagaccagccagagg gaaaaggaggatgacaaggtgttccctgggggcagccatacttatgtgtggcaggtgctg aaggagaatgggcccatggcctctgaccccctgtgcctgacttacagctatctgagccat gtggacctggtgaaggatctgaactctggcctgattggggctctgctggtgtgcagggag ggcagcctggctaaggagaagactcagactctgcataagttcatcctgctgtttgctgtg tttgatgaaggcaagagctggcactctgagaccaagaactctctgatgcaggatagggat gctgcctctgccagggcttggcccaagatgcacactgtgaatggctatgtgaacaggagc ctgcctggcctgattgggtgccacaggaagtctgtgtactggcatgtgattggcatgggc accacccctgaggtgcacagcattttcctggagggccacaccttcctggtgaggaatcac aggcaggccagcctggagatcagccccatcaccttcctgactgcccagaccctgctgatg gacctggggcagtttctgctgttctgccacatcagcagccatcagcatgatggcatggag gcctatgtgaaggtggactcttgccctgaggagccccagctgaggatgaagaacaatgag gaggctgaggattatgatgatgacctgactgactctgagatggatgtggtgaggtttgat gatgacaatagccccagcttcatccagattaggtctgtggccaagaagcaccctaagacc tgggtgcactacattgctgctgaggaggaggattgggattatgcccccctggtgctggct cctgatgacaggtcttataagagccagtacctgaacaatgggccccagaggattggcagg aagtacaagaaggtgaggttcatggcttacactgatgagaccttcaagactagggaggcc atccagcatgagtctggcatcctgggccccctgctgtatggggaggtgggggataccctg ctgatcatcttcaagaaccaggccagcaggccctacaacatttaccctcatggcatcact gatgtgaggcccctgtacagcaggagactgcccaagggggtgaagcacctgaaggatttt cccattctgcctggggagatcttcaagtacaagtggactgtgactgtggaggatggcccc accaagtctgatcccaggtgcctgactaggtactactcttcttttgtgaatatggagagg gatctggcctctggcctgattggccccctgctgatctgctacaaggagtctgtggaccag aggggcaaccagatcatgtctgacaagaggaatgtgatcctgttctctgtgtttgatgag aataggagctggtacctgactgagaatatccagaggttcctgcctaatcctgctggggtc cagctggaggatcctgagttccaggctagcaacattatgcacagcatcaatggctatgtg tttgattctctgcagctgtctgtgtgcctgcatgaggtggcttactggtacatcctgtct attggggcccagactgatttcctgtctgtgttcttctctggctacactttcaagcataag atggtgtatgaggataccctgaccctgttccccttctctggggagactgtgttcatgtct atggagaaccctggcctgtggatcctgggctgtcataactctgacttcagaaacaggggc atgactgccctgctgaaggtgagcagctgtgacaagaacactggggactactatgaggac agctatgaggatatctctgcttatctgctgagcaagaataatgccattgagcccaggagc ttcagccagaacccccctgtgctgaagaggcaccagagggagatcactaggactaccctg cagtctgatcaggaggagattgactatgatgacaccatctctgtggagatgaagaaggag gactttgacatctatgatgaggatgagaaccagtcccccaggtctttccagaagaagacc aggcactacttcattgctgctgtggagaggctgtgggactatggcatgagctctagcccc catgtgctgaggaacagggctcagtctggctctgtgccccagttcaagaaggtggtcttc caggagttcactgatggctcttttacccagcctctgtacagaggggagctgaatgagcac ctgggcctgctgggcccctacatcagggctgaggtggaggataatatcatggtgaccttc agaaaccaggcctctaggccctacagcttctacagcagcctgatctcttatgaggaggat cagaggcagggggctgagcccaggaagaactttgtgaagcccaatgagaccaagacctac ttctggaaggtgcagcaccatatggcccctactaaggatgagtttgactgcaaggcctgg gcttatttttctgatgtggacctggagaaggatgtgcactctgggctgattggccccctg ctggtgtgccacaccaacaccctgaaccctgcccatggcaggcaggtgactgtgcaggag tttgccctgttcttcactatctttgatgagaccaagagctggtacttcactgagaacatg gagagaaattgtagggctccctgcaatatccagatggaggaccccaccttcaaagaaaat tacagattccatgccatcaatgggtacatcatggataccctgcctgggctggtgatggct caggaccagaggatcaggtggtacctgctgagcatggggtctaatgagaacatccactct atccatttctctggccatgtgttcactgtgagaaagaaggaggagtataagatggctctg tacaacctgtacccaggggtgtttgagactgtggaaatgctgcccagcaaagctgggatc tggagggtggagtgcctgattggggagcacctgcatgctggcatgtctaccctgttcctg gtgtacagcaacaagtgccagactcccctgggcatggcctctgggcacatcagggatttt cagatcactgcctctggccagtatggccagtgggcccccaagctggccaggctgcactac tctggcagcattaatgcttggagcactaaggagcccttcagctggatcaaggtggatctg ctggcccccatgatcatccatggcatcaagacccagggggccaggcagaagttctctagc ctgtacatttctcagttcatcatcatgtacagcctggatgggaagaagtggcagacctac agggggaacagcactgggaccctgatggtgttctttggcaatgtggatagctctggcatc aagcacaatatcttcaatccccccattattgccaggtacattaggctgcatcctactcac tactctattaggagcaccctgaggatggagctgatggggtgtgacctgaacagctgttct atgcccctgggcatggagtctaaggctatctctgatgcccagatcactgccagcagctac ttcactaatatgtttgccacctggagccctagcaaggccagactgcacctgcagggcagg agcaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggacttccag aagaccatgaaggtgactggggtgaccactcagggggtgaagagcctgctgaccagcatg tatgtgaaggagttcctgatcagcagcagccaggatggccaccagtggaccctgttcttc cagaatgggaaggtgaaggtgttccagggcaaccaggactctttcacccctgtggtgaac agcctggatcctcccctgctgaccaggtacctgaggatccacccccagagctgggtgcac cagattgctctgaggatggaagtgctgggctgtgaggcccaggatctgtactga FVIIIencodingCpGreducednucleicacidvariantX11 (SEQIDNO:11) atgcagattgagctgagcacctgcttcttcctgtgcctgctgaggttttgcttctctgct accaggaggtactacctgggggctgtggagctgagctgggactatatgcagtctgacctg ggggagctgcctgtggatgctaggttccctcccagggtgcccaagagcttcccctttaat acctctgtggtgtacaagaaaaccctgtttgtggagttcactgaccatctgttcaacatt gccaagcccaggcccccttggatgggcctgctgggccccaccattcaggctgaggtgtat gacactgtggtcattaccctgaagaacatggcttctcaccctgtgagcctgcatgctgtg ggggtgagctactggaaggcctctgagggggctgagtatgatgaccagaccagccagagg gagaaggaggatgataaggtgttccctgggggcagccacacctatgtgtggcaggtgctg aaggagaatggccccatggcctctgatcccctgtgcctgacctactcttatctgtctcat gtggacctggtgaaggacctgaactctggcctgattggggctctgctggtgtgcagggag ggctctctggccaaggagaagacccagaccctgcacaagtttattctgctgtttgctgtc tttgatgagggcaagagctggcattctgagaccaagaacagcctgatgcaggacagggat gctgcctctgccagggcctggcccaaaatgcacactgtgaatggctatgtgaacaggagc ctgcctggcctgattggctgccacaggaagtctgtgtactggcatgtgattggcatgggc accacccctgaggtgcacagcatcttcctggagggccacacctttctggtgaggaatcac aggcaggccagcctggagattagccccatcaccttcctgactgcccagaccctgctgatg gacctgggccagttcctgctgttctgccacatcagcagccaccagcatgatggcatggag gcctatgtgaaggtggatagctgccctgaggagccccagctgaggatgaaaaacaatgag gaggctgaggattatgatgatgacctgactgactctgagatggatgtggtgaggtttgat gatgacaatagccccagctttattcagattaggtctgtggctaagaagcaccccaagact tgggtgcactacattgctgctgaggaggaggattgggactatgcccctctggtcctggcc cctgatgataggtcttacaagagccagtatctgaacaatggcccccagaggattggcagg aagtacaagaaggtgaggttcatggcctacactgatgagacctttaagaccagggaggcc attcagcatgagtctgggatcctgggccccctgctgtatggggaggtgggggacactctg ctgatcatcttcaagaaccaggccagcaggccttataacatctaccctcatgggatcact gatgtgaggcccctgtactctagaaggctgcccaagggggtcaagcacctgaaggatttt cccatcctgcctggggagattttcaagtacaagtggactgtgactgtggaggatggcccc accaagtctgaccctaggtgcctgaccaggtactacagctcttttgtgaacatggagagg gacctggcctctggcctgattggccctctgctgatttgctacaaggagtctgtggaccag aggggcaaccagatcatgtctgacaagaggaatgtgatcctgttttctgtgtttgatgag aacaggtcttggtacctgactgagaacatccagaggttcctgcctaacccagctggggtg cagctggaggatcctgagttccaggccagcaatattatgcatagcattaatggctatgtg tttgatagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagc attggggcccagactgactttctgtctgtgttcttctctggctacaccttcaagcataag atggtgtatgaggacaccctgactctgttccctttttctggggagactgtgtttatgagc atggagaatcctggcctgtggatcctgggctgccataattctgacttcaggaacaggggc atgactgccctgctgaaagtgagcagctgtgacaagaatactggggactactatgaagac agctatgaggacatctctgcctacctgctgagcaagaacaatgccattgagcccaggagc ttcagccagaaccccccagtgctgaagaggcaccagagagagatcaccaggactaccctg cagtctgaccaggaggagattgactatgatgacaccatttctgtggagatgaagaaggag gactttgacatttatgatgaggatgagaatcagagccccaggagcttccagaagaagact aggcactattttattgctgctgtggagaggctgtgggactatggcatgagcagctctccc catgtgctgaggaatagggcccagtctggctctgtgcctcagttcaagaaggtggtgttc caggagttcactgatggcagctttacccagcccctgtataggggggagctgaatgagcac ctgggcctgctgggcccctatatcagggctgaggtggaggacaatattatggtgaccttt aggaaccaggccagcaggccctactctttctatagcagcctgatcagctatgaggaggac cagaggcagggggctgagcccaggaagaattttgtgaagcctaatgagaccaagacctac ttctggaaggtgcagcatcacatggcccccaccaaggatgagtttgactgcaaggcttgg gcctatttctctgatgtggacctggagaaggatgtgcactctggcctgattggccccctg ctggtgtgccacactaacactctgaatcctgcccatggcaggcaggtgactgtgcaggag tttgccctgttcttcaccatctttgatgagaccaagagctggtacttcactgagaacatg gagaggaactgcagggccccctgcaacatccagatggaggatcccaccttcaaggagaac tacaggtttcatgccatcaatggctacatcatggacactctgcctggcctggtgatggcc caggatcagaggatcaggtggtacctgctgagcatgggctctaatgagaatatccatagc atccacttctctggccatgtgttcactgtcaggaagaaggaggagtacaagatggctctg tataatctgtaccctggggtgtttgagactgtggagatgctgcccagcaaggctggcatc tggagggtggagtgcctgattggggagcacctgcatgctgggatgagcaccctgtttctg gtgtactctaacaagtgccagacccccctgggcatggcctctgggcacatcagggatttc cagatcactgcttctggccagtatggccagtgggcccccaagctggccaggctgcactac tctggcagcatcaatgcctggtctaccaaggagcccttttcttggattaaggtggacctg ctggcccccatgatcatccatggcatcaagacccagggggccaggcagaagttcagcagc ctgtacatcagccagttcatcatcatgtacagcctggatggcaaaaagtggcagacctac aggggcaatagcactgggactctgatggtgttctttggcaatgtggacagctctgggatc aagcacaatatcttcaaccctcccatcattgctaggtacatcaggctgcaccccacccac tatagcatcaggtctaccctgaggatggagctgatgggctgtgacctgaactcttgcagc atgcccctgggcatggagtccaaagctatctctgatgcccagattactgccagcagctac ttcaccaacatgtttgccacctggtctccctctaaggccaggctgcacctgcagggcagg agcaatgcctggaggccccaggtgaacaatcccaaggagtggctgcaggtggatttccag aaaactatgaaggtgactggggtgaccacccagggggtgaagtctctgctgaccagcatg tatgtgaaggagttcctgatctcttctagccaggatggccaccagtggactctgttcttc cagaatggcaaggtgaaggtgttccagggcaaccaggacagcttcacccctgtggtgaac tctctggatccccccctgctgaccaggtacctgaggattcatccccagagctgggtgcac cagattgctctgagaatggaggtgctggggtgtgaggctcaggacctgtattga FVIIIencodingCpGreducednucleicacidvariantX12 (SEQIDNO:12) atgcagattgagctgtctacttgtttttttctgtgcctgctgaggttctgcttctctgcc accaggaggtattacctgggggctgtggagctgagctgggattacatgcagtctgatctg ggggagctgcctgtggatgccaggttcccccccagggtgcccaagagcttccccttcaac acctctgtggtgtataagaagaccctgtttgtggagttcactgatcatctgtttaacatt gccaagcccaggcccccctggatgggcctgctgggcccaactatccaggctgaggtgtat gacactgtggtcatcaccctgaagaatatggccagccatcctgtgagcctgcatgctgtg ggggtgagctactggaaggcctctgagggggctgagtatgatgaccagaccagccagagg gagaaggaggatgacaaggtgttccctgggggcagccacacctatgtgtggcaggtgctg aaggagaatggccccatggcctctgaccccctgtgcctgacttatagctacctgtctcat gtggacctggtgaaggacctgaactctggcctgattggggccctgctggtctgtagggaa ggcagcctggccaaggagaagacccagaccctgcacaagtttattctgctgtttgctgtg tttgatgaaggcaagagctggcactctgagaccaagaattctctgatgcaggatagggat gctgcctctgccagggcctggcccaagatgcatactgtgaatggctatgtgaacagaagc ctgcctggcctgattggctgccataggaagtctgtgtattggcatgtgattgggatgggc actacccctgaagtgcacagcattttcctggagggccacactttcctggtgaggaaccac aggcaggcctctctggagatcagccccattactttcctgactgcccagaccctgctgatg gatctgggccagttcctgctgttctgccacatctctagccaccagcatgatggcatggag gcctatgtgaaggtggacagctgccctgaggagccccagctgaggatgaagaataatgag gaggctgaggattatgatgatgacctgactgactctgagatggatgtggtgaggtttgat gatgataatagccccagcttcatccagatcaggtctgtggccaagaagcatcccaagacc tgggtgcactatattgctgctgaagaggaggactgggactatgcccctctggtgctggct cctgatgacaggagctataagagccagtatctgaacaatgggccccagaggattgggagg aagtacaagaaggtgaggttcatggcctacactgatgagacctttaagaccagggaggcc atccagcatgagtctggcattctggggcccctgctgtatggggaggtgggggacactctg ctgatcattttcaagaaccaggccagcaggccctacaatatttacccccatggcatcact gatgtgaggcccctgtacagcaggaggctgcccaagggggtgaagcacctgaaggacttc cccatcctgcctggggagatcttcaagtacaagtggactgtgactgtggaggatggccct accaagtctgaccctaggtgtctgactaggtactacagcagctttgtgaacatggagaga gacctggcttctggcctgattggccccctgctgatctgctacaaggagtctgtggatcag aggggcaaccagattatgtctgataagaggaatgtcatcctgttctctgtgtttgatgag aacaggagctggtatctgactgagaacattcagaggttcctgcccaaccctgctggggtg cagctggaggaccctgagttccaggccagcaacatcatgcattctattaatggctatgtg tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtacatcctgagc attggggcccagactgactttctgtctgtgtttttctctgggtacaccttcaagcacaag atggtctatgaggacaccctgaccctgttccccttttctggggaaactgtgtttatgagc atggagaaccctgggctgtggatcctgggctgccacaactctgactttaggaataggggc atgactgccctgctgaaggtgagcagctgtgacaagaatactggggattactatgaggac agctatgaggatatctctgcctacctgctgagcaagaacaatgccattgagcctaggagc ttcagccagaacccccctgtgctgaagaggcaccagagggagatcaccaggaccaccctg cagtctgatcaggaggagattgactatgatgacaccatctctgtggagatgaagaaggag gactttgatatttatgatgaggatgagaaccagagccccaggagcttccagaagaagacc aggcactatttcattgctgctgtggagaggctgtgggactatggcatgagctctagcccc catgtgctgaggaacagggcccagtctggctctgtgccccagttcaagaaggtggtgttc caggaatttactgatggcagctttacccagcccctgtacagaggggagctgaatgagcac ctgggcctgctgggcccctacatcagggctgaggtggaggataatatcatggtgaccttt aggaaccaggcctctaggccctattctttttacagcagcctgatcagctatgaggaggac cagaggcagggggctgagcctaggaagaactttgtgaagcccaatgagaccaagacctac ttttggaaagtgcagcaccacatggcccccactaaggatgagtttgattgcaaggcctgg gcctatttctctgatgtggacctggagaaggatgtgcactctggcctgattggccccctg ctggtgtgccacaccaacactctgaaccctgcccatggcaggcaggtgactgtgcaggag tttgccctgttctttaccatctttgatgagactaagagctggtatttcactgagaacatg gagaggaactgcagagccccttgcaacatccagatggaggaccctaccttcaaggagaac tataggttccatgccatcaatgggtacatcatggataccctgcctggcctggtgatggct caggaccagaggatcaggtggtacctgctgagcatggggagcaatgagaacattcatagc atccacttctctgggcatgtgttcactgtgaggaagaaggaggagtataagatggccctg tacaacctgtaccctggggtgtttgagactgtggagatgctgcccagcaaggctggcatc tggagggtggagtgcctgattggggagcacctgcatgctggcatgagcactctgttcctg gtgtacagcaacaagtgccagacccccctgggcatggcctctggccacatcagggacttc cagattactgcctctgggcagtatgggcagtgggcccccaagctggccaggctgcactac tctgggtctatcaatgcttggagcaccaaggagcctttcagctggatcaaggtggatctg ctggcccccatgatcattcatgggatcaagacccagggggccaggcagaagttcagcagc ctgtatatttctcagttcatcatcatgtattctctggatggcaaaaagtggcagacctat agagggaacagcactgggaccctgatggtgttttttggcaatgtggatagctctggcatc aagcacaatatcttcaacccccccattattgccaggtacatcaggctgcaccccacccac tactctatcaggagcaccctgaggatggagctgatgggctgtgatctgaacagctgctct atgcctctggggatggaaagcaaggccatctctgatgcccagatcactgccagcagctat ttcaccaatatgtttgccacttggagccctagcaaggctaggctgcatctgcagggcagg tctaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggacttccag aagactatgaaagtgactggggtgaccacccagggggtgaaaagcctgctgaccagcatg tatgtgaaggagttcctgattagcagcagccaggatggccaccagtggaccctgttcttc cagaatgggaaggtgaaggtgtttcagggcaatcaggatagcttcaccccagtggtgaac agcctggacccccccctgctgaccaggtacctgaggatccacccccagagctgggtgcac cagattgccctgaggatggaggtgctgggctgtgaggcccaggatctgtactga FVIIIencodingCpGreducednucleicacidvariantX13 (SEQIDNO:13) atgcagattgagctgagcacctgctttttcctgtgcctgctgaggttctgcttctctgct accaggaggtactacctgggggctgtggagctgtcttgggattacatgcagtctgacctg ggggagctgcctgtggatgccaggtttccccccagggtgcccaagtctttcccctttaac acctctgtggtgtataagaagactctgtttgtggagttcactgatcacctgttcaatatt gccaagcccaggcccccttggatgggcctgctgggccccactatccaggctgaggtgtat gacactgtggtcatcaccctgaagaacatggccagccaccctgtgagcctgcatgctgtg ggggtgagctactggaaggcctctgagggggctgagtatgatgaccagaccagccagagg gagaaggaggatgacaaggtgttcccaggggggtctcacacttatgtgtggcaggtgctg aaggagaatgggcccatggcctctgaccctctgtgcctgacttatagctacctgtctcat gtggatctggtgaaggacctgaactctggcctgattggggccctgctggtgtgcagggag gggagcctggccaaggagaagacccagaccctgcacaagttcatcctgctgtttgctgtg tttgatgaggggaagagctggcactctgagaccaagaatagcctgatgcaggacagggat gctgcttctgctagggcctggcctaagatgcacactgtgaatggctatgtgaacaggagc ctgcctggcctgattgggtgtcacaggaagtctgtgtactggcatgtgattggcatgggg actactccagaagtgcacagcatcttcctggaggggcacaccttcctggtgaggaatcac aggcaggccagcctggagatttctcccatcactttcctgactgcccagaccctgctgatg gatctggggcagttcctgctgttctgccacatcagcagccatcagcatgatgggatggag gcctatgtgaaggtggacagctgccctgaggagcctcagctgaggatgaagaacaatgag gaggctgaggactatgatgatgatctgactgactctgagatggatgtggtgaggtttgat gatgacaactctcccagcttcatccagatcaggtctgtggccaagaagcaccccaagacc tgggtgcactacattgctgctgaggaggaggattgggattatgctcccctggtgctggct cctgatgataggagctacaagagccagtatctgaataatgggccccagaggattggcagg aagtataagaaggtgaggttcatggcctacactgatgagacctttaagaccagggaggct attcagcatgagtctggcatcctgggccccctgctgtatggggaggtgggggacaccctg ctgatcattttcaagaaccaggccagcaggccctataacatctatccccatgggatcact gatgtgaggcccctgtactctaggaggctgcccaagggggtcaagcacctgaaggacttc cccatcctgcctggggagatcttcaagtacaagtggactgtgactgtggaggatggcccc actaagtctgaccccaggtgcctgactaggtactacagcagctttgtgaacatggagaga gatctggcctctggcctgattggccccctgctgatctgctacaaagagtctgtggatcag aggggcaaccagatcatgtctgacaagaggaatgtgatcctgttctctgtgtttgatgag aacagaagctggtacctgactgagaacattcagaggtttctgcccaaccctgctggggtc cagctggaggaccctgagtttcaggccagcaacatcatgcacagcatcaatgggtatgtg tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcctactggtatatcctgagc attggggcccagactgatttcctgtctgtgttcttctctggctacactttcaagcacaag atggtgtatgaggataccctgaccctgttccctttctctggggaaactgtgttcatgagc atggagaaccctgggctgtggatcctggggtgccacaattctgatttcaggaacagaggc atgactgctctgctgaaggtgtctagctgtgacaagaacactggggactactatgaggac agctatgaggacatctctgcctacctgctgagcaagaacaatgctattgaacccaggtct ttcagccagaacccccctgtgctgaagaggcaccagagggagatcactaggaccaccctg cagtctgatcaggaggagattgactatgatgacaccatctctgtggagatgaagaaggag gactttgacatctatgatgaggatgagaatcagtctcccaggagcttccagaagaagact aggcattacttcattgctgctgtggagaggctgtgggactatggcatgagctctagccct catgtgctgaggaacagggcccagtctggctctgtgccccagttcaagaaggtggtgttt caggagttcactgatggcagcttcacccagcccctgtacaggggggagctgaatgagcat ctgggcctgctgggcccctacatcagggctgaggtggaggacaacatcatggtgaccttc agaaatcaggctagcaggccctacagcttctacagcagcctgatctcttatgaggaggac cagaggcagggggctgagcccaggaagaactttgtgaagcccaatgagaccaagacctat ttctggaaggtgcagcaccacatggcccccaccaaggatgagtttgattgcaaggcctgg gcctacttctctgatgtggacctggagaaggatgtgcattctgggctgattggccctctg ctggtgtgccacaccaacaccctgaatcctgcccatggcaggcaggtgactgtgcaggag tttgccctgttctttactatctttgatgagaccaagtcttggtattttactgagaacatg gagaggaactgcagggccccctgcaacatccagatggaggaccccaccttcaaggagaac tacagattccatgccatcaatggctacattatggacactctgcctggcctggtgatggcc caggaccagaggatcaggtggtacctgctgtctatgggcagcaatgagaacattcactct atccacttctctgggcatgtgttcactgtgaggaagaaggaggagtacaagatggccctg tacaacctgtaccctggggtgtttgagactgtggagatgctgcctagcaaggctgggatc tggagggtggagtgcctgattggggagcacctgcatgctggcatgtctaccctgttcctg gtgtacagcaacaagtgccagacccccctgggcatggcctctggccacatcagagatttt cagatcactgcctctggccagtatggccagtgggctcctaagctggccaggctgcactac tctggcagcatcaatgcctggagcaccaaggagccctttagctggatcaaggtggacctg ctggcccccatgatcatccatggcatcaagactcagggggccaggcagaagttctctagc ctgtacattagccagttcatcatcatgtatagcctggatggcaagaagtggcagacctac aggggcaacagcactgggaccctgatggtgttctttgggaatgtggacagctctgggatc aagcacaatatcttcaacccccccattattgccaggtatattaggctgcaccccactcac tacagcattaggagcaccctgaggatggagctgatgggctgtgatctgaacagctgcagc atgcccctgggcatggagtctaaggccatctctgatgcccagatcactgccagctcttac ttcaccaacatgtttgccacttggagccccagcaaggccaggctgcacctgcagggcagg agcaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggatttccag aagactatgaaggtgactggggtgaccactcagggggtgaagagcctgctgactagcatg tatgtgaaggagttcctgatcagctctagccaggatggccaccagtggaccctgttcttt cagaatggcaaggtgaaggtgttccagggcaaccaggactctttcacccctgtggtgaat tctctggaccctcccctgctgactaggtatctgaggattcatccccagagctgggtgcat cagattgccctgaggatggaggtgctgggctgtgaggcccaggacctgtattga FVIIIencodingCpGreducednucleicacidvariantX14 (SEQIDNO:14) atgcagattgagctgagcacctgcttcttcctgtgcctgctgaggttttgcttttctgcc actaggaggtactacctgggggctgtggagctgtcttgggattacatgcagtctgacctg ggggagctgccagtggatgccaggttccccccaagggtgcccaagtcttttcccttcaat acctctgtggtgtacaagaagaccctgtttgtggagtttactgatcatctgtttaacatt gccaagcccaggcccccctggatggggctgctgggccccaccatccaggctgaggtgtat gatactgtggtgattaccctgaagaatatggccagccatcctgtgtctctgcatgctgtg ggggtgtcttattggaaggcctctgagggggctgagtatgatgatcagaccagccagagg gagaaggaggatgataaggtgttccctgggggctctcacacctatgtgtggcaggtgctg aaggagaatgggcctatggcctctgacccactgtgcctgacttacagctatctgagccat gtggacctggtgaaggacctgaactctgggctgattggggccctgctggtgtgcagggag ggcagcctggccaaggagaagactcagaccctgcacaagttcatcctgctgtttgctgtg tttgatgagggcaagtcttggcactctgagaccaagaacagcctgatgcaggatagggat gctgcctctgccagggcctggcccaagatgcacactgtgaatggctatgtgaacaggtct ctgcctggcctgattggctgccacaggaagtctgtgtactggcatgtgattggcatgggc accacccctgaggtgcatagcattttcctggagggccacaccttcctggtgaggaaccac aggcaggctagcctggagatcagccccatcactttcctgactgcccagaccctgctgatg gacctgggccagttcctgctgttctgccacatctctagccaccagcatgatggcatggag gcctatgtgaaggtggactcttgtcctgaggagccccagctgaggatgaagaacaatgag gaggctgaggattatgatgatgatctgactgattctgagatggatgtggtgaggtttgat gatgacaacagcccctctttcatccagatcaggtctgtggccaagaagcaccccaagacc tgggtgcactacattgctgctgaggaggaggattgggattatgcccccctggtgctggcc cctgatgacaggagctataagtctcagtacctgaacaatggcccccagagaattggcagg aagtacaagaaggtgaggttcatggcctatactgatgagaccttcaaaaccagggaggcc attcagcatgagtctggcatcctggggcccctgctgtatggggaggtgggggacaccctg ctgatcatcttcaagaaccaggctagcaggccttacaacatctacccccatgggatcact gatgtgaggcccctgtacagcaggaggctgcctaagggggtgaagcacctgaaggacttt cccattctgcctggggagatcttcaagtataagtggactgtgactgtggaggatgggccc accaagtctgaccccaggtgcctgactaggtactactctagctttgtgaacatggagagg gacctggcctctgggctgattggccccctgctgatctgttacaaggagtctgtggaccag aggggcaaccagatcatgtctgataagaggaatgtgatcctgttctctgtgtttgatgag aacaggagctggtacctgactgagaacatccagagattcctgcccaaccctgctggggtg cagctggaggatcctgagttccaggccagcaacatcatgcattctatcaatgggtatgtg tttgatagcctgcagctgtctgtgtgtctgcatgaggtggcctactggtacattctgagc attggggcccagactgacttcctgtctgtgttcttctctggctacactttcaaacacaag atggtgtatgaggacaccctgaccctgttccccttctctggggagactgtgtttatgagc atggagaaccctgggctgtggattctgggctgccacaactctgacttcagaaacaggggc atgactgccctgctgaaggtgtcttcttgtgataagaacactggggactattatgaagac agctatgaggacatctctgcctacctgctgagcaagaataatgctattgagcccaggtct ttctctcagaacccccctgtgctgaagaggcaccagagggagatcaccaggaccaccctg cagtctgatcaggaggagattgactatgatgacactatttctgtggagatgaagaaggaa gactttgatatctatgatgaggatgagaaccagagccctaggagcttccagaagaagact aggcattacttcattgctgctgtggagaggctgtgggactatggcatgagcagcagcccc catgtgctgaggaatagggctcagtctggctctgtgcctcagttcaagaaggtggtgttc caggaattcactgatggcagcttcactcagcccctgtacaggggggagctgaatgagcac ctggggctgctgggcccttacatcagggctgaggtggaggacaatatcatggtgaccttt aggaaccaggcctctaggccttacagcttctactctagcctgatctcttatgaagaggac cagaggcagggggctgagcccaggaagaactttgtgaagcccaatgagactaagacttac ttctggaaggtgcagcaccacatggctcccaccaaggatgagtttgactgcaaggcttgg gcctacttctctgatgtggacctggagaaggatgtgcactctgggctgattgggcccctg ctggtgtgccacactaacactctgaatcctgcccatggcagacaggtgactgtgcaggag tttgccctgttttttaccatctttgatgagactaagtcttggtacttcactgagaacatg gagaggaactgcagggccccctgcaacatccagatggaggatcccaccttcaaggagaac tacaggtttcatgccatcaatggctacatcatggacaccctgcctggcctggtgatggct caggaccagaggattaggtggtatctgctgagcatgggcagcaatgagaatatccactct atccacttctctgggcatgtgttcactgtgaggaagaaggaggagtacaagatggccctg tataacctgtatcctggggtgtttgagactgtggagatgctgcccagcaaggctggcatc tggagagtggagtgcctgattggggagcacctgcatgctggcatgagcactctgtttctg gtgtatagcaacaagtgtcagacccctctgggcatggcctctgggcacattagggacttt cagatcactgcttctggccagtatgggcagtgggctcccaagctggccaggctgcactat tctggcagcattaatgcctggagcaccaaggagcctttcagctggatcaaggtggacctg ctggcccccatgatcatccatgggatcaagacccagggggctaggcagaagttcagcagc ctgtacatcagccagtttatcatcatgtattctctggatggcaagaagtggcagacctac aggggcaattctactggcactctgatggtgttctttgggaatgtggatagctctgggatc aagcataatatcttcaatccccccattattgctaggtatatcaggctgcaccccacccac tatagcatcaggagcaccctgaggatggagctgatggggtgtgacctgaacagctgcagc atgcccctgggcatggagagcaaggctatttctgatgcccagatcactgccagcagctac tttactaatatgtttgccacctggagccccagcaaggccagactgcacctgcagggcagg tctaatgcctggaggcctcaggtgaataaccccaaggagtggctgcaggtggacttccag aaaaccatgaaggtgactggggtgactacccagggggtgaagtctctgctgaccagcatg tatgtgaaggagttcctgatctcttctagccaggatggccaccagtggaccctgttcttt cagaatgggaaggtgaaggtcttccagggcaaccaggatagcttcacccctgtggtgaat agcctggatcctcctctgctgaccaggtatctgaggatccacccccagagctgggtgcat cagattgccctgaggatggaggtgctgggctgtgaggctcaggacctgtactga FVIIIencodingCpGreducednucleicacidvariantX15 (SEQIDNO:15) atgcagattgagctgagcacctgtttcttcctgtgcctgctgaggttctgtttctctgcc actaggaggtactacctgggggctgtggagctgagctgggactatatgcagtctgacctg ggggagctgcctgtggatgccaggttcccccccagggtgcctaagagcttccccttcaat acttctgtggtgtacaagaagactctgtttgtggagtttactgaccacctgttcaacatt gctaagcccaggcctccctggatggggctgctgggccccaccatccaggctgaggtgtat gatactgtggtgattaccctgaagaacatggcctctcatccagtgagcctgcatgctgtg ggggtgagctactggaaggcctctgaaggggctgagtatgatgaccagaccagccagagg gagaaggaggatgacaaggtgttccctgggggcagccacacctatgtgtggcaggtgctg aaggagaatggcccaatggcctctgaccccctgtgcctgacttatagctacctgagccat gtggatctggtgaaggacctgaattctggcctgattggggccctgctggtgtgcagagag ggctctctggctaaggagaagacccagactctgcacaagttcatcctgctgtttgctgtg tttgatgagggcaagagctggcactctgagactaagaatagcctgatgcaggacagggat gctgcttctgccagggcctggcccaagatgcatactgtgaatggctatgtgaacaggagc ctgcctggcctgattggctgtcacaggaaatctgtctactggcatgtgattgggatgggc actacccctgaggtgcactctatcttcctggagggccataccttcctggtgaggaaccac aggcaggccagcctggagatctctcccattaccttcctgactgcccagaccctgctgatg gatctgggccagttcctgctgttctgccacatcagcagccaccagcatgatgggatggag gcttatgtgaaggtggatagctgccctgaggagccccagctgaggatgaagaacaatgag gaggctgaggactatgatgatgacctgactgactctgagatggatgtggtgaggtttgat gatgacaactctcccagctttattcagatcaggtctgtggctaagaagcaccccaagact tgggtgcactacattgctgctgaggaggaggactgggactatgcccctctggtgctggct cctgatgacaggtcttacaagtctcagtacctgaataatggccctcagaggattggcagg aagtacaagaaggtgaggttcatggcctacactgatgagaccttcaagaccagggaggcc atccagcatgagtctggcatcctgggccccctgctgtatggggaggtgggggataccctg ctgatcatcttcaagaatcaggccagcaggccctacaacatctacccccatggcatcact gatgtgaggccactgtacagcaggaggctgcccaagggggtgaagcatctgaaggacttc cccattctgcctggggagatcttcaagtacaaatggactgtgactgtggaggatggccct accaagtctgaccccaggtgtctgaccaggtactacagcagctttgtgaatatggagagg gacctggcctctggcctgattggccccctgctgatctgctacaaggagtctgtggaccag aggggcaatcagatcatgtctgataagaggaatgtgattctgttctctgtgtttgatgag aacaggagctggtacctgactgagaacatccagaggttcctgcccaatcctgctggggtg cagctggaggaccctgagttccaggccagcaatatcatgcacagcatcaatggctatgtc tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcttactggtatattctgagc attggggcccagactgatttcctgtctgtgttcttttctggctatacctttaagcacaag atggtgtatgaggacaccctgaccctgttccccttctctggggagactgtgttcatgtct atggagaaccctgggctgtggatcctgggctgccacaactctgacttcaggaacaggggg atgactgccctgctgaaggtgtctagctgtgataagaacactggggactattatgaggac agctatgaggacatctctgcttacctgctgagcaagaacaatgccattgagcccaggtct ttcagccagaatccccctgtgctgaagaggcatcagagggagatcaccaggaccaccctg cagtctgatcaggaggagattgattatgatgacactatctctgtggaaatgaagaaggag gactttgacatctatgatgaggatgagaaccagagccccaggagcttccagaagaagacc aggcactacttcattgctgctgtggagaggctgtgggattatggcatgagcagctctccc catgtgctgaggaacagagcccagtctggctctgtgcctcagttcaagaaggtggtcttc caggagttcactgatggctctttcacccagcccctgtacaggggggagctgaatgagcac ctgggcctgctggggccctacattagggctgaggtggaggataacatcatggtgactttc agaaaccaggccagcaggccttacagcttttactcttctctgattagctatgaggaggat cagaggcagggggctgagcctaggaagaactttgtgaagcccaatgagaccaagacctat ttctggaaggtgcagcaccacatggctcccactaaggatgagtttgactgcaaggcttgg gcctacttctctgatgtggacctggagaaggatgtgcactctggcctgattgggcccctg ctggtgtgccacaccaacaccctgaaccctgcccatggcaggcaggtgactgtgcaggag tttgccctgttcttcaccatctttgatgagactaagagctggtacttcactgagaacatg gagaggaactgcagggccccctgcaacatccagatggaggaccccaccttcaaggagaat tacaggttccatgccatcaatggctacattatggacaccctgcctggcctggtgatggcc caggatcagaggatcaggtggtatctgctgagcatgggctctaatgagaacatccacagc atccacttctctggccatgtgtttactgtgaggaagaaggaggaatacaagatggctctg tataacctgtaccctggggtgtttgagactgtggagatgctgcccagcaaggctgggatc tggagggtggagtgcctgattggggagcacctgcatgctgggatgagcaccctgttcctg gtgtatagcaataagtgccagacccccctgggcatggcttctggccacatcagggatttc cagatcactgcttctggccagtatggccagtgggctcccaagctggctaggctgcattac tctgggtctatcaatgcctggagcactaaggagcccttcagctggatcaaggtggacctg ctggcccccatgatcattcatggcatcaagacccagggggctaggcagaagttcagcagc ctgtacatcagccagttcatcattatgtacagcctggatggcaagaagtggcagacttac aggggcaatagcactgggactctgatggtgttctttggcaatgtggactcttctggcatc aagcacaacatcttcaaccctcccatcattgccaggtacattaggctgcaccctacccac tactctatcaggagcaccctgaggatggagctgatggggtgtgatctgaactcttgcagc atgcctctgggcatggaaagcaaagccatctctgatgcccagatcactgcctctagctat ttcaccaatatgtttgccacctggagccctagcaaggccaggctgcacctgcagggcaga tctaatgcctggaggccccaggtgaacaatcccaaggagtggctgcaggtggacttccag aagaccatgaaggtgactggggtgaccactcagggggtgaagagcctgctgactagcatg tatgtgaaggagttcctgatctcttctagccaggatggccaccagtggaccctgttcttc cagaatggcaaggtgaaagtgttccagggcaaccaggatagcttcactcctgtggtgaac tctctggaccctcccctgctgactaggtacctgaggattcatccccagagctgggtgcac cagattgccctgaggatggaggtgctgggctgtgaggcccaggatctgtactga FVIIIencodingCpGreducednucleicacidvariantX16 (SEQIDNO:16) atgcagattgagctgagcacctgcttcttcctgtgcctgctgaggttctgcttctctgcc accaggaggtactacctgggggctgtggagctgtcttgggactatatgcagtctgacctg ggggagctgccagtggatgccaggttcccccccagggtgcccaagagctttcctttcaac acttctgtggtgtacaagaagaccctgtttgtggagttcactgaccacctgttcaatatt gctaagcccaggccaccctggatgggcctgctgggccctaccattcaggctgaggtgtat gacactgtggtgattactctgaagaatatggccagccaccctgtgagcctgcatgctgtg ggggtgtcttactggaaggcctctgagggggctgagtatgatgatcagacttctcagagg gagaaggaggatgataaggtgttccctgggggctctcacacttatgtgtggcaggtgctg aaggagaatggccccatggcttctgatccactgtgcctgacctactcttacctgagccat gtggacctggtgaaggacctgaactctggcctgattggggccctgctggtgtgcagggag ggcagcctggccaaggagaagacccagaccctgcataagttcatcctgctgtttgctgtg tttgatgaggggaagagctggcactctgagaccaagaattctctgatgcaggacagggat gctgcctctgccagggcctggcctaagatgcacactgtgaatggctatgtgaacaggtct ctgcctggcctgattggctgccacaggaagtctgtgtactggcatgtgattggcatgggc actacccctgaggtgcacagcattttcctggagggccacaccttcctggtcaggaaccat aggcaggcctctctggagatcagccccatcactttcctgactgcccagaccctgctgatg gacctgggccagttcctgctgttctgccacattagcagccaccagcatgatggcatggag gcctatgtgaaggtggactcttgccctgaggagccccagctgaggatgaagaacaatgag gaagctgaggattatgatgatgacctgactgactctgagatggatgtggtgaggtttgat gatgacaacagccccagcttcatccagatcaggtctgtggccaagaagcaccccaagacc tgggtgcactacattgctgctgaggaggaggattgggactatgctcccctggtgctggct cctgatgataggagctacaagtctcagtacctgaataatggcccccagaggattggcagg aagtacaagaaggtgaggttcatggcctacactgatgagaccttcaagaccagagaggct atccagcatgagtctgggatcctggggcccctgctgtatggggaggtgggggacaccctg ctgatcatcttcaagaaccaggccagcagaccctacaacatctacccccatgggatcact gatgtgaggcccctgtacagcaggaggctgcctaagggggtgaagcacctgaaggacttc cccatcctgcctggggagatcttcaagtataagtggactgtgactgtggaggatgggccc accaagtctgaccctaggtgcctgactaggtactactctagctttgtgaacatggagagg gacctggcctctggcctgattggccccctgctgatttgctacaaggagtctgtggatcag aggggcaatcagatcatgtctgacaagaggaatgtgatcctgttctctgtgtttgatgag aataggtcttggtacctgactgagaacatccagaggttcctgcctaatcctgctggggtg cagctggaggaccctgagtttcaggccagcaacatcatgcacagcatcaatggctatgtg tttgactctctgcagctgtctgtgtgcctgcatgaggtggcttactggtatatcctgagc attggggctcagactgacttcctgtctgtgttcttttctggctacacttttaagcacaag atggtgtatgaggacaccctgaccctgttccccttttctggggagactgtgttcatgtct atggagaaccctgggctgtggattctgggctgtcacaactctgacttcagaaacaggggc atgactgccctgctgaaggtgtctagctgtgacaagaatactggggactactatgaggac agctatgaggacatttctgcctatctgctgagcaagaacaatgccattgagcccaggagc ttttctcagaatccccctgtgctgaagaggcaccagagagagatcaccaggaccactctg cagtctgatcaggaggagattgattatgatgacactatctctgtggagatgaagaaagag gactttgatatctatgatgaggatgagaatcagtctcccaggagcttccagaagaagact agacactacttcattgctgctgtggagaggctgtgggactatggcatgagctctagccct catgtgctgaggaacagggcccagtctgggtctgtgccccagttcaagaaggtggtgttc caggagttcactgatggcagctttacccagcccctgtataggggggagctgaatgagcat ctgggcctgctgggcccctatattagggctgaagtggaggacaacatcatggtgaccttt aggaaccaggccagcaggccctacagcttttacagcagcctgattagctatgaggaggat cagagacagggggctgagcccaggaagaactttgtgaagcccaatgagaccaagacctac ttctggaaggtgcagcaccacatggcccctaccaaggatgagtttgactgcaaggcctgg gcttacttctctgatgtggacctggagaaagatgtgcactctggcctgattgggcccctg ctggtgtgccacaccaacaccctgaaccctgcccatgggaggcaggtgactgtgcaggag tttgccctgtttttcaccatctttgatgagaccaagagctggtacttcactgagaacatg gagaggaactgcagggccccctgtaacatccagatggaggatcctactttcaaggagaac tacaggttccatgccattaatgggtacatcatggacaccctgcctgggctggtgatggcc caggatcagaggattaggtggtatctgctgtctatgggctctaatgagaacatccactct atccacttctctggccatgtgttcactgtgaggaagaaggaggagtacaagatggccctg tacaacctgtaccctggggtgtttgaaactgtggagatgctgccctctaaagctgggatc tggagggtggagtgcctgattggggagcacctgcatgctggcatgagcaccctgttcctg gtgtacagcaataagtgccagactcccctgggcatggcttctgggcacatcagggatttc cagatcactgcctctggccagtatggccagtgggcccccaagctggctaggctgcactac tctggcagcatcaatgcctggagcaccaaggagcccttctcttggattaaggtggacctg ctggctcccatgatcattcatggcatcaagacccagggggccaggcagaagttttctagc ctgtatattagccagttcatcatcatgtatagcctggatgggaagaagtggcagacctac agggggaatagcactggcaccctgatggtgttttttggcaatgtggattcttctggcatc aagcataacatcttcaatccccctatcattgccaggtacattaggctgcatcccacccat tactctatcaggagcaccctgaggatggagctgatggggtgtgatctgaacagctgtagc atgcccctgggcatggagtccaaggctatctctgatgcccagatcactgccagcagctac ttcaccaacatgtttgccacctggagccccagcaaggccaggctgcacctgcagggcagg tctaatgcctggaggccccaggtgaacaatcccaaggagtggctgcaggtggacttccag aagactatgaaggtgactggggtgaccactcagggggtgaagagcctgctgaccagcatg tatgtgaaggagttcctgatctcttctagccaggatgggcatcagtggaccctgtttttt cagaatggcaaagtgaaggtgtttcaggggaatcaggacagctttacccctgtggtgaac agcctggatcctcctctgctgactagatacctgaggatccacccccagagctgggtccac cagattgctctgaggatggaggtgctggggtgtgaggctcaggacctgtactga FVIIIencodingCpGreducednucleicacidvariantX17 (SEQIDNO:17) atgcagattgagctgagcacctgcttctttctgtgcctgctgaggttctgcttctctgcc accaggaggtactacctgggggctgtggaactgagctgggactatatgcagtctgacctg ggggagctgcctgtggatgccaggttcccccccagggtgcccaagtctttcccctttaac acttctgtggtgtacaagaagaccctgtttgtggagtttactgaccacctgttcaatatt gccaagcccaggcccccctggatgggcctgctgggcccaaccatccaggctgaggtgtat gatactgtggtgatcaccctgaagaacatggccagccaccctgtgagcctgcatgctgtg ggggtgagctattggaaggcttctgagggggctgagtatgatgaccagactagccagagg gagaaggaggatgacaaggtgttccctggggggtctcatacctatgtgtggcaggtgctg aaggagaatggccccatggcctctgaccccctgtgcctgacctattcttacctgagccat gtggacctggtcaaggacctgaactctggcctgattggggctctgctggtgtgcagggag ggcagcctggccaaggagaagactcagactctgcataagttcatcctgctgtttgctgtg tttgatgagggcaagagctggcactctgagaccaagaactctctgatgcaggatagggat gctgcctctgccagggcctggcccaagatgcacactgtgaatggctatgtgaataggtct ctgcctggcctgattggctgccataggaagtctgtgtactggcatgtgattggcatgggc actacccctgaggtgcactctatcttcctggaggggcacaccttcctggtgaggaaccac aggcaggccagcctggagatctctcccatcaccttcctgactgcccagactctgctgatg gacctgggccagttcctgctgttctgccatatcagcagccaccagcatgatggcatggag gcctatgtgaaggtggacagctgcccagaggaaccccagctgaggatgaagaacaatgag gaggctgaggactatgatgatgacctgactgactctgagatggatgtggtgaggtttgat gatgacaacagccccagctttattcagatcaggtctgtggccaagaagcaccccaagacc tgggtgcactacattgctgctgaggaggaggactgggattatgcccccctggtgctggcc cctgatgacaggtcttacaagtctcagtacctgaacaatggcccccagaggattgggagg aagtacaagaaggtgaggttcatggcctacactgatgagaccttcaagaccagggaggcc atccagcatgagtctggcatcctggggcccctgctgtatggggaggtgggggataccctg ctgattatcttcaagaaccaggctagcaggccctataacatctacccccatggcattact gatgtgaggcccctgtactctaggagactgcccaagggggtgaagcacctgaaagacttc cccatcctgcctggggagatcttcaagtataagtggactgtgactgtggaggatggcccc actaagtctgaccccaggtgcctgaccaggtattacagcagctttgtgaatatggagagg gatctggcttctggcctgattgggcctctgctgatttgctacaaggagtctgtggatcag agggggaaccagattatgtctgacaagaggaatgtgattctgttctctgtgtttgatgag aacaggagctggtacctgactgagaatatccagaggttcctgcctaatcctgctggggtg cagctggaggaccctgagttccaggctagcaacattatgcacagcatcaatggctatgtg tttgacagcctgcagctgtctgtgtgcctgcatgaggtggcttactggtacattctgtct attggggcccagactgacttcctgtctgtgttcttctctggctacaccttcaagcacaag atggtgtatgaggacactctgaccctgttccccttctctggggagactgtgttcatgagc atggagaatcctgggctgtggattctggggtgccacaactctgatttcaggaacaggggc atgactgccctgctgaaggtgagcagctgtgacaagaacactggggattattatgaggac agctatgaggacatttctgcctacctgctgagcaagaacaatgccattgagcctaggagc ttcagccagaatccccctgtgctgaagagacaccagagggagatcactaggaccactctg cagtctgatcaggaggagattgactatgatgacaccatttctgtggagatgaagaaggag gactttgatatttatgatgaggatgagaaccagagccccagaagcttccagaagaagacc aggcactacttcattgctgctgtggagaggctgtgggattatggcatgtcttctagcccc catgtgctgaggaacagggctcagtctggctctgtgcctcagttcaagaaggtggtgttc caggagttcactgatgggagcttcacccagcctctgtacaggggggagctgaatgaacat ctgggcctgctggggccctacatcagggctgaggtggaggataatatcatggtgactttc aggaatcaggcctctaggccctacagcttctactctagcctgatcagctatgaggaggac cagaggcagggggctgagcctaggaagaattttgtgaaacccaatgagaccaagacctac ttttggaaggtgcagcaccacatggcccctaccaaggatgagtttgactgtaaggcctgg gcctacttctctgatgtggacctggagaaggatgtgcattctgggctgattggccccctg ctggtgtgccacaccaacaccctgaaccctgcccatggcaggcaggtgactgtgcaggag tttgccctgttcttcaccatctttgatgagactaagagctggtatttcactgagaacatg gagaggaactgtagggctccctgcaacatccagatggaggatccaactttcaaggagaac tacaggttccatgccatcaatggctacatcatggacaccctgcctggcctggtgatggcc caggaccagaggattaggtggtacctgctgagcatgggctctaatgagaacatccactct atccacttctctggccatgtgtttactgtgaggaagaaggaggagtacaagatggctctg tacaacctgtaccctggggtgtttgagactgtggagatgctgcctagcaaggctggcatt tggagagtggagtgtctgattggggagcacctgcatgctgggatgtctaccctgttcctg gtgtactctaacaagtgccagacccccctggggatggcttctgggcacatcagagatttt cagattactgcttctgggcagtatggccagtgggctcccaagctggccagactgcattac tctggctctattaatgcttggagcaccaaggagcctttcagctggatcaaggtggacctg ctggctcccatgatcatccatggcattaagactcagggggctaggcagaagttcagcagc ctgtatatttctcagtttattatcatgtattctctggatggcaagaagtggcagacttac aggggcaacagcactggcaccctgatggtgttctttggcaatgtggacagctctgggatc aagcataacatcttcaacccccccattattgccaggtacatcaggctgcaccccacccac tattctatcaggagcactctgaggatggagctgatggggtgtgacctgaacagctgctct atgcccctgggcatggagagcaaggccatctctgatgcccagatcactgccagctcttat ttcaccaacatgtttgccacctggagccccagcaaggccaggctgcacctgcagggcaga agcaatgcctggaggccccaggtgaacaatcctaaggagtggctgcaggtggacttccag aagactatgaaggtgactggggtgactacccagggggtgaagagcctgctgaccagcatg tatgtgaaggagttcctgattagcagcagccaggatgggcatcagtggaccctgttcttc cagaatgggaaggtgaaggtgttccagggcaatcaggacagcttcacccctgtggtgaac agcctggacccccccctgctgaccaggtacctgaggatccatccccagagctgggtgcac cagattgctctgagaatggaggtgctgggctgtgaggcccaggacctgtattga FVIIIencodingCpGreducednucleicacidvariantX18 (SEQIDNO:18) atgcagattgagctgtctacctgtttttttctgtgcctgctgaggttctgcttctctgct accaggaggtattatctgggggctgtggagctgagctgggactacatgcagtctgacctg ggggagctgcctgtggatgccaggtttcctcccagggtgcctaagagcttccccttcaac acctctgtggtgtacaagaagactctgtttgtggagttcactgaccacctgttcaacatt gccaagcccaggcccccctggatggggctgctgggccccactatccaggctgaggtgtat gatactgtggtgattaccctgaagaacatggcctctcaccctgtgtctctgcatgctgtg ggggtgagctactggaaggcttctgagggggctgaatatgatgatcagacctctcagagg gagaaggaggatgacaaggtgtttcctgggggcagccacacctatgtgtggcaggtgctg aaggagaatgggcccatggcctctgatcccctgtgcctgacctacagctacctgagccat gtggacctggtgaaggacctgaactctggcctgattggggccctgctggtgtgcagggag ggcagcctggccaaggaaaagacccagaccctgcataagttcatcctgctgtttgctgtg tttgatgagggcaagtcttggcactctgagaccaagaacagcctgatgcaggacagggat gctgcctctgctagggcctggcccaagatgcacactgtgaatgggtatgtgaacagatct ctgcctggcctgattggctgccacaggaagtctgtgtactggcatgtgattggcatgggg accacccctgaggtgcatagcatcttcctggaggggcacaccttcctggtgagaaatcat aggcaggccagcctggagattagccccatcaccttcctgactgcccagaccctgctgatg gacctgggccagttcctgctgttctgccacatttctagccaccagcatgatggcatggag gcctatgtgaaggtggatagctgccctgaagagccccagctgaggatgaagaacaatgag gaggctgaggattatgatgatgatctgactgactctgagatggatgtggtgaggtttgat gatgacaacagccccagcttcatccagatcaggtctgtggccaagaagcaccctaagacc tgggtgcactacattgctgctgaagaggaggactgggactatgcccccctggtgctggcc ccagatgacaggtcttacaagagccagtacctgaataatggcccccagaggattgggagg aagtataagaaagtgaggttcatggcttacactgatgagacctttaagactagggaggcc attcagcatgagtctgggattctgggccctctgctgtatggggaggtgggggacaccctg ctgatcattttcaagaaccaggccagcaggccctataatatttatccccatgggattact gatgtcaggcccctgtacagcaggaggctgcctaagggggtgaagcacctgaaggacttc cccattctgcctggggagatcttcaagtataagtggactgtgactgtggaggatggcccc accaagtctgatcctaggtgcctgaccaggtactatagcagctttgtgaacatggagagg gacctggcttctggcctgattggccccctgctgatctgctacaaggaatctgtggaccag aggggcaaccagattatgtctgacaagaggaatgtgatcctgttttctgtgtttgatgag aataggagctggtatctgactgagaacatccagaggttcctgcccaatcctgctggggtg cagctggaggaccctgagttccaggcttctaacatcatgcatagcatcaatgggtatgtg tttgactctctgcagctgtctgtgtgcctgcatgaggtggcctattggtacatcctgagc attggggcccagactgacttcctgtctgtgttcttctctggctacaccttcaagcacaag atggtgtatgaggacaccctgaccctgttccctttctctggggagactgtgttcatgagc atggagaaccctggcctgtggattctgggctgccataattctgacttcagaaacaggggc atgactgctctgctgaaggtgagcagctgtgacaagaatactggggactactatgaggac tcttatgaggatatttctgcctacctgctgagcaagaacaatgctattgagcccaggagc ttcagccagaacccccctgtcctgaagaggcatcagagggagatcactaggaccaccctg cagtctgatcaggaggagattgactatgatgacactatctctgtggaaatgaagaaggag gactttgatatctatgatgaggatgagaaccagagccccaggtctttccagaagaagacc aggcactacttcattgctgctgtggagaggctgtgggactatggcatgtctagcagcccc catgtgctgaggaacagagcccagtctggctctgtgccccagttcaagaaggtggtgttt caggagttcactgatgggagcttcactcagcccctgtataggggggagctgaatgagcat ctgggcctgctggggccctacatcagggctgaggtggaggataacatcatggtgaccttc aggaaccaggccagcaggccctactctttctactcttctctgatcagctatgaggaggat cagaggcagggggctgagcctaggaagaactttgtcaagcctaatgagactaagacctac ttttggaaggtgcagcaccacatggctcccactaaggatgagtttgattgcaaggcctgg gcctacttctctgatgtggacctggagaaggatgtgcactctggcctgattggccccctg ctggtgtgtcacaccaataccctgaaccctgcccatggcaggcaggtcactgtgcaggag tttgccctgtttttcactatctttgatgagactaagtcttggtacttcactgagaacatg gaaaggaattgcagggctccctgcaacatccagatggaggaccccaccttcaaggagaac tacaggtttcatgccatcaatggctacatcatggacaccctgcctggcctggtgatggct caggatcagaggattaggtggtatctgctgagcatgggcagcaatgagaacatccacagc atccacttttctggccatgtgttcactgtgaggaagaaggaggagtacaagatggctctg tacaatctgtaccctggggtgtttgagactgtggagatgctgcccagcaaggctgggatc tggagggtggagtgcctgattggggaacacctgcatgctggcatgtctaccctgttcctg gtgtactctaacaagtgccagactcccctgggcatggcctctgggcacatcagggacttc cagatcactgcctctgggcagtatggccagtgggcccctaagctggctaggctgcattac tctggcagcatcaatgcctggagcaccaaggagcccttcagctggatcaaggtggacctg ctggcccctatgatcatccatggcatcaagacccagggggccagacagaagttctcttct ctgtacatctctcagttcatcatcatgtactctctggatggcaagaagtggcagacctac agggggaattctactggcactctgatggtgttctttgggaatgtggatagctctgggatc aagcataatattttcaacccccccattattgctaggtacatcaggctgcacccaacccac tactctattaggtctaccctgaggatggagctgatgggctgtgacctgaactcttgtagc atgcccctgggcatggagagcaaggctatctctgatgcccagatcactgccagcagctac tttaccaacatgtttgctacttggagccccagcaaggccaggctgcacctgcagggcagg agcaatgcctggaggccccaggtgaacaaccccaaggagtggctgcaggtggattttcag aagaccatgaaggtgactggggtgaccactcagggggtgaaaagcctgctgactagcatg tatgtgaaggagtttctgatcagcagctctcaggatggccatcagtggaccctgttcttc cagaatggcaaggtgaaggtgttccagggcaaccaggatagcttcacccctgtggtgaat agcctggacccccccctgctgaccaggtacctgaggatccatccccagagctgggtgcac cagattgccctgaggatggaggtgctgggctgtgaagcccaggacctgtactga Wild-typefactorVIII-BDDcDNA (SEQIDNO:19) ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCC ACCAGAAGATACTACCTGGGTGCAGTGGAACTGTCATGGGACTATATGCAAAGTGATCTC GGTGAGCTGCCTGTGGACGCAAGATTTCCTCCTAGAGTGCCAAAATCTTTTCCATTCAAC ACCTCAGTCGTGTACAAAAAGACTCTGTTTGTAGAATTCACGGATCACCTTTTCAACATC GCTAAGCCAAGGCCACCCTGGATGGGTCTGCTAGGTCCTACCATCCAGGCTGAGGTTTAT GATACAGTGGTCATTACACTTAAGAACATGGCTTCCCATCCTGTCAGTCTTCATGCTGTT GGTGTATCCTACTGGAAAGCTTCTGAGGGAGCTGAATATGATGATCAGACCAGTCAAAGG GAGAAAGAAGATGATAAAGTCTTCCCTGGTGGAAGCCATACATATGTCTGGCAGGTCCTG AAAGAGAATGGTCCAATGGCCTCTGACCCACTGTGCCTTACCTACTCATATCTTTCTCAT GTGGACCTGGTAAAAGACTTGAATTCAGGCCTCATTGGAGCCCTACTAGTATGTAGAGAA GGGAGTCTGGCCAAGGAAAAGACACAGACCTTGCACAAATTTATACTACTTTTTGCTGTA TTTGATGAAGGGAAAAGTTGGCACTCAGAAACAAAGAACTCCTTGATGCAGGATAGGGAT GCTGCATCTGCTCGGGCCTGGCCTAAAATGCACACAGTCAATGGTTATGTAAACAGGTCT CTGCCAGGTCTGATTGGATGCCACAGGAAATCAGTCTATTGGCATGTGATTGGAATGGGC ACCACTCCTGAAGTGCACTCAATATTCCTCGAAGGTCACACATTTCTTGTGAGGAACCAT CGCCAGGCGTCCTTGGAAATCTCGCCAATAACTTTCCTTACTGCTCAAACACTCTTGATG GACCTTGGACAGTTTCTACTGTTTTGTCATATCTCTTCCCACCAACATGATGGCATGGAA GCTTATGTCAAAGTAGACAGCTGTCCAGAGGAACCCCAACTACGAATGAAAAATAATGAA GAAGCGGAAGACTATGATGATGATCTTACTGATTCTGAAATGGATGTGGTCAGGTTTGAT GATGACAACTCTCCTTCCTTTATCCAAATTCGCTCAGTTGCCAAGAAGCATCCTAAAACT TGGGTACATTACATTGCTGCTGAAGAGGAGGACTGGGACTATGCTCCCTTAGTCCTCGCC CCCGATGACAGAAGTTATAAAAGTCAATATTTGAACAATGGCCCTCAGCGGATTGGTAGG AAGTACAAAAAAGTCCGATTTATGGCATACACAGATGAAACCTTTAAGACTCGTGAAGCT ATTCAGCATGAATCAGGAATCTTGGGACCTTTACTTTATGGGGAAGTTGGAGACACACTG TTGATTATATTTAAGAATCAAGCAAGCAGACCATATAACATCTACCCTCACGGAATCACT GATGTCCGTCCTTTGTATTCAAGGAGATTACCAAAAGGTGTAAAACATTTGAAGGATTTT CCAATTCTGCCAGGAGAAATATTCAAATATAAATGGACAGTGACTGTAGAAGATGGGCCA ACTAAATCAGATCCTCGGTGCCTGACCCGCTATTACTCTAGTTTCGTTAATATGGAGAGA GATCTAGCTTCAGGACTCATTGGCCCTCTCCTCATCTGCTACAAAGAATCTGTAGATCAA AGAGGAAACCAGATAATGTCAGACAAGAGGAATGTCATCCTGTTTTCTGTATTTGATGAG AACCGAAGCTGGTACCTCACAGAGAATATACAACGCTTTCTCCCCAATCCAGCTGGAGTG CAGCTTGAGGATCCAGAGTTCCAAGCCTCCAACATCATGCACAGCATCAATGGCTATGTT TTTGATAGTTTGCAGTTGTCAGTTTGTTTGCATGAGGTGGCATACTGGTACATTCTAAGC ATTGGAGCACAGACTGACTTCCTTTCTGTCTTCTTCTCTGGATATACCTTCAAACACAAA ATGGTCTATGAAGACACACTCACCCTATTCCCATTCTCAGGAGAAACTGTCTTCATGTCG ATGGAAAACCCAGGTCTATGGATTCTGGGGTGCCACAACTCAGACTTTCGGAACAGAGGC ATGACCGCCTTACTGAAGGTTTCTAGTTGTGACAAGAACACTGGTGATTATTACGAGGAC AGTTATGAAGATATTTCAGCATACTTGCTGAGTAAAAACAATGCCATTGAACCAAGAAGC TTCTCCCAAAACCCACCAGTCTTGAAACGCCATCAACGGGAAATAACTCGTACTACTCTT CAGTCAGATCAAGAGGAAATTGACTATGATGATACCATATCAGTTGAAATGAAGAAGGAA GATTTTGACATTTATGATGAGGATGAAAATCAGAGCCCCCGCAGCTTTCAAAAGAAAACA CGACACTATTTTATTGCTGCAGTGGAGAGGCTCTGGGATTATGGGATGAGTAGCTCCCCA CATGTTCTAAGAAACAGGGCTCAGAGTGGCAGTGTCCCTCAGTTCAAGAAAGTTGTTTTC CAGGAATTTACTGATGGCTCCTTTACTCAGCCCTTATACCGTGGAGAACTAAATGAACAT TTGGGACTCCTGGGGCCATATATAAGAGCAGAAGTTGAAGATAATATCATGGTAACTTTC AGAAATCAGGCCTCTCGTCCCTATTCCTTCTATTCTAGCCTTATTTCTTATGAGGAAGAT CAGAGGCAAGGAGCAGAACCTAGAAAAAACTTTGTCAAGCCTAATGAAACCAAAACTTAC TTTTGGAAAGTGCAACATCATATGGCACCCACTAAAGATGAGTTTGACTGCAAAGCCTGG GCTTATTTCTCTGATGTTGACCTGGAAAAAGATGTGCACTCAGGCCTGATTGGACCCCTT CTGGTCTGCCACACTAACACACTGAACCCTGCTCATGGGAGACAAGTGACAGTACAGGAA TTTGCTCTGTTTTTCACCATCTTTGATGAGACCAAAAGCTGGTACTTCACTGAAAATATG GAAAGAAACTGCAGGGCTCCCTGCAATATCCAGATGGAAGATCCCACTTTTAAAGAGAAT TATCGCTTCCATGCAATCAATGGCTACATAATGGATACACTACCTGGCTTAGTAATGGCT CAGGATCAAAGGATTCGATGGTATCTGCTCAGCATGGGCAGCAATGAAAACATCCATTCT ATTCATTTCAGTGGACATGTGTTCACCGTACGAAAAAAAGAGGAGTATAAAATGGCACTG TACAATCTCTATCCAGGTGTTTTTGAGACAGTGGAAATGTTACCATCCAAAGCTGGAATT TGGCGGGTGGAATGCCTTATTGGCGAGCATCTACATGCTGGGATGAGCACACTTTTTCTG GTGTACAGCAATAAGTGTCAGACTCCCCTGGGAATGGCTTCTGGACACATTAGAGATTTT CAGATTACAGCTTCAGGACAATATGGACAGTGGGCCCCAAAGCTGGCCAGACTTCATTAT TCCGGATCAATCAATGCCTGGAGCACCAAGGAGCCCTTTTCTTGGATCAAGGTGGATCTG TTGGCACCAATGATTATTCACGGCATCAAGACCCAGGGTGCCCGTCAGAAGTTCTCCAGC CTCTACATCTCTCAGTTTATCATCATGTATAGTCTTGATGGGAAGAAGTGGCAGACTTAT CGAGGAAATTCCACTGGAACCTTAATGGTCTTCTTTGGCAATGTGGATTCATCTGGGATA AAACACAATATTTTTAACCCTCCAATTATTGCTCGATACATCCGTTTGCACCCAACTCAT TATAGCATTCGCAGCACTCTTCGCATGGAGTTGATGGGCTGTGATTTAAATAGTTGCAGC ATGCCATTGGGAATGGAGAGTAAAGCAATATCAGATGCACAGATTACTGCTTCATCCTAC TTTACCAATATGTTTGCCACCTGGTCTCCTTCAAAAGCTCGACTTCACCTCCAAGGGAGG AGTAATGCCTGGAGACCTCAGGTGAATAATCCAAAAGAGTGGCTGCAAGTGGACTTCCAG AAGACAATGAAAGTCACAGGAGTAACTACTCAGGGAGTAAAATCTCTGCTTACCAGCATG TATGTGAAGGAGTTCCTCATCTCCAGCAGTCAAGATGGCCATCAGTGGACTCTCTTTTTT CAGAATGGCAAAGTAAAGGTTTTTCAGGGAAATCAAGACTCCTTCACACCTGTGGTGAAC TCTCTAGACCCACCGTTACTGACTCGCTACCTTCGAATTCACCCCCAGAGTTGGGTGCAC CAGATTGCCCTGAGGATGGAGGTTCTGGGCTGCGAGGCACAGGACCTCTACTGA V3factorVIIIcDNA (SEQIDNO:20) ATGCAGATTGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAG ATACTACCTGGGGGCTGTGGAGCTGAGCTGGGACTACATGCAGTCTGACCTGGGGGAGCTGCCTGTGG ATGCCAGGTTCCCCCCCAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCTGTGGTGTACAAGAAGACC CTGTTTGTGGAGTTCACTGACCACCTGTTCAACATTGCCAAGCCCAGGCCCCCCTGGATGGGCCTGCT GGGCCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACCCTGAAGAACATGGCCAGCCACC CTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGGGCTGAGTATGATGACCAG ACCAGCCAGAGGGAGAAGGAGGATGACAAGGTGTTCCCTGGGGGCAGCCACACCTATGTGTGGCAGGT GCTGAAGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCATGTGG ACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGCCCTGCTGGTGTGCAGGGAGGGCAGCCTGGCC AAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTG GCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGATGCTGCCTCTGCCAGGGCCTGGCCCAAGA TGCACACTGTGAATGGCTATGTGAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTG TACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTT CCTGGTCAGGAACCACAGGCAGGCCAGCCTGGAGATCAGCCCCATCACCTTCCTGACTGCCCAGACCC TGCTGATGGACCTGGGCCAGTTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCATGATGGCATGGAG GCCTATGTGAAGGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAATGAGGAGGCTGA GGACTATGATGATGACCTGACTGACTCTGAGATGGATGTGGTGAGGTTTGATGATGACAACAGCCCCA GCTTCATCCAGATCAGGTCTGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCTGCTGAG GAGGAGGACTGGGACTATGCCCCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCT GAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTCAGGTTCATGGCCTACACTGATGAAA CCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCCTGCTGTATGGGGAGGTG GGGGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCAGGCCCTACAACATCTACCCCCATGGCAT CACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAGGACTTCCCCA TCCTGCCTGGGGAGATCTTCAAGTACAAGTGGACTGTGACTGTGGAGGATGGCCCCACCAAGTCTGAC CCCAGGTGCCTGACCAGATACTACAGCAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGAT TGGCCCCCTGCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGA GGAATGTGATCCTGTTCTCTGTGTTTGATGAGAACAGGAGCTGGTACCTGACTGAGAACATCCAGAGG TTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCTGAGTTCCAGGCCAGCAACATCATGCACAG CATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGAGGTGGCCTACTGGTACA TCCTGAGCATTGGGGCCCAGACTGACTTCCTGTCTGTGTTCTTCTCTGGCTACACCTTCAAGCACAAG ATGGTGTATGAGGACACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAA CCCTGGCCTGTGGATTCTGGGCTGCCACAACTCTGACTTCAGGAACAGGGGCATGACTGCCCTGCTGA AAGTCTCCAGCTGTGACAAGAACACTGGGGACTACTATGAGGACAGCTATGAGGACATCTCTGCCTAC CTGCTGAGCAAGAACAATGCCATTGAGCCCAGGAGCTTCAGCCAGAACAGCAGGCACCCCAGCACCAG GCAGAAGCAGTTCAATGCCACCACCATCCCTGAGAATGACATAGAGAAGACAGACCCATGGTTTGCCC ACCGGACCCCCATGCCCAAGATCCAGAATGTGAGCAGCTCTGACCTGCTGATGCTGCTGAGGCAGAGC CCCACCCCCCATGGCCTGAGCCTGTCTGACCTGCAGGAGGCCAAGTATGAAACCTTCTCTGATGACCC CAGCCCTGGGGCCATTGACAGCAACAACAGCCTGTCTGAGATGACCCACTTCAGGCCCCAGCTGCACC ACTCTGGGGACATGGTGTTCACCCCTGAGTCTGGCCTGCAGCTGAGGCTGAATGAGAAGCTGGGCACC ACTGCTGCCACTGAGCTGAAGAAGCTGGACTTCAAAGTCTCCAGCACCAGCAACAACCTGATCAGCAC CATCCCCTCTGACAACCTGGCTGCTGGCACTGACAACACCAGCAGCCTGGGCCCCCCCAGCATGCCTG TGCACTATGACAGCCAGCTGGACACCACCCTGTTTGGCAAGAAGAGCAGCCCCCTGACTGAGTCTGGG GGCCCCCTGAGCCTGTCTGAGGAGAACAATGACAGCAAGCTGCTGGAGTCTGGCCTGATGAACAGCCA GGAGAGCAGCTGGGGCAAGAATGTGAGCACCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTG CTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAG TCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACTGATGGCAGCTTCACCCAGCC CCTGTACAGAGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGCCCCTACATCAGGGCTGAGGTGGAGG ACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGCCTGATCAGC TATGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAGAACTTTGTGAAGCCCAATGAAACCAAGAC CTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCT ACTTCTCTGATGTGGACCTGGAGAAGGATGTGCACTCTGGCCTGATTGGCCCCCTGCTGGTGTGCCAC ACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTGACTGTGCAGGAGTTTGCCCTGTTCTTCACCAT CTTTGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTGCAGGGCCCCCTGCAACA TCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGGAC ACCCTGCCTGGCCTGGTGATGGCCCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCAGCAA TGAGAACATCCACAGCATCCACTTCTCTGGCCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGA TGGCCCTGTACAACCTGTACCCTGGGGTGTTTGAGACTGTGGAGATGCTGCCCAGCAAGGCTGGCATC TGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCATGCTGGCATGAGCACCCTGTTCCTGGTGTACAG CAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTG GCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCTGGCAGCATCAATGCCTGGAGC ACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCATGGCATCAAGAC CCAGGGGGCCAGGCAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGATG GCAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGCAATGTGGAC AGCTCTGGCATCAAGCACAACATCTTCAACCCCCCCATCATTGCCAGATACATCAGGCTGCACCCCAC CCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCATGC CCCTGGGCATGGAGAGCAAGGCCATCTCTGATGCCCAGATCACTGCCAGCAGCTACTTCACCAACATG TTTGCCACCTGGAGCCCCAGCAAGGCCAGGCTGCACCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCA GGTCAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACTGGGGTGACCA CCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCTGATCAGCAGCAGCCAGGAT GGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTT CACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATTCACCCCCAGAGCT GGGTGCACCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGA CO3factorVIIIcDNA (SEQIDNO21) atgcagattgagctgtcaacttgctttttcctgtgcctgctgagattttgtttttccgct actagaagatactacctgggggctgtggaactgtcttgggattacatgcagagtgacctg ggagagctgccagtggacgcacgatttccacctagagtccctaaatcattccccttcaac accagcgtggtctataagaaaacactgttcgtggagtttactgatcacctgttcaacatc gctaagcctcggccaccctggatgggactgctgggaccaacaatccaggcagaggtgtac gacaccgtggtcattacactgaaaaacatggcctcacaccccgtgagcctgcatgctgtg ggcgtcagctactggaaggcttccgaaggggcagagtatgacgatcagacttcccagaga gaaaaagaggacgataaggtgtttcctggcgggtctcatacctatgtgtggcaggtcctg aaagagaatggccccatggcttccgaccctctgtgcctgacctactcttatctgagtcac gtggacctggtcaaggatctgaacagcggactgatcggagcactgctggtgtgtagggaa gggagcctggctaaggagaaaacccagacactgcataagttcattctgctgttcgccgtg tttgacgaaggaaaatcatggcacagcgagacaaagaatagtctgatgcaggaccgggat gccgcttcagccagagcttggcccaaaatgcacactgtgaacggctacgtcaatcgctca ctgcctggactgatcggctgccaccgaaagagcgtgtattggcatgtcatcggaatgggc accacacctgaagtgcactccattttcctggaggggcatacctttctggtccgcaaccac cgacaggcctccctggagatctctccaattaccttcctgacagctcagactctgctgatg gatctgggacagttcctgctgttttgccacatcagctcccaccagcatgatggcatggag gcctacgtgaaagtggacagctgtcccgaggaacctcagctgaggatgaagaacaatgag gaagctgaagactatgacgatgacctgaccgactccgagatggatgtggtccgattcgat gacgataacagcccctcctttatccagattagatctgtggccaagaaacaccctaagaca tgggtccattacatcgcagccgaggaagaggactgggattatgcaccactggtgctggca ccagacgatcgatcctacaaatctcagtatctgaacaatggaccacagcggattggcaga aagtacaagaaagtgaggttcatggcttataccgatgaaaccttcaagactcgcgaagca atccagcacgagagcgggattctgggaccactgctgtacggagaagtgggggacaccctg ctgatcatttttaagaaccaggccagcaggccttacaatatctatccacatggaattaca gatgtgcgccctctgtacagccggagactgccaaagggcgtcaaacacctgaaggacttc ccaatcctgcccggggaaatttttaagtataaatggactgtcaccgtcgaggatggcccc actaagagcgaccctaggtgcctgacccgctactattctagtttcgtgaatatggaaagg gatctggccagcggactgatcggcccactgctgatttgttacaaagagagcgtggatcag agaggcaaccagatcatgtccgacaagaggaatgtgattctgttcagtgtctttgacgaa aaccggtcatggtatctgaccgagaacatccagagattcctgcctaatccagccggagtg cagctggaagatcctgagtttcaggcttctaacatcatgcatagtattaatggctacgtg ttcgacagtctgcagctgtcagtgtgtctgcacgaggtcgcttactggtatatcctgagc attggagcacagacagatttcctgagcgtgttcttttccggctacacttttaagcataaa atggtgtatgaggacacactgactctgttccccttcagcggcgaaaccgtgtttatgtcc atggagaatcccgggctgtggatcctgggatgccacaacagcgatttcaggaatcgcggg atgactgccctgctgaaagtgtcaagctgtgacaagaacaccggagactactatgaagat tcatacgaggacatcagcgcatatctgctgtccaaaaacaatgccattgaacccaggtct tttagtcagaatcctccagtgctgaagaggcaccagcgcgagatcacccgcactaccctg cagagtgatcaggaagagatcgactacgacgatacaatttctgtggaaatgaagaaagag gacttcgatatctatgacgaagatgagaaccagagtcctcgatcattccagaagaaaacc cggcattactttattgctgcagtggagcgcctgtgggattatggcatgtcctctagtcct cacgtgctgcgaaatcgggcccagtcagggagcgtcccacagttcaagaaagtggtcttc caggagtttacagacggatcctttactcagccactgtaccggggcgaactgaacgagcac ctggggctgctgggaccctatatcagagctgaagtggaggataacattatggtcaccttc agaaatcaggcatctaggccttacagtttttattcaagcctgatctcttacgaagaggac cagaggcagggagcagaaccacgaaaaaacttcgtgaagcctaatgagaccaaaacatac ttttggaaggtgcagcaccatatggccccaacaaaagacgaattcgattgcaaggcatgg gcctatttttctgacgtggatctggagaaggacgtccacagtggcctgatcgggccactg ctggtgtgtcatactaacaccctgaatcccgcacacggcaggcaggtcactgtccaggaa ttcgccctgttctttaccatctttgatgagacaaaaagctggtacttcaccgaaaacatg gagcgaaattgccgggctccatgtaatattcagatggaagaccccacattcaaggagaac taccgctttcatgccatcaatgggtatattatggatactctgcccggactggtcatggct caggaccagagaatcaggtggtacctgctgagcatggggtccaacgagaatatccactca attcatttcagcggacacgtgtttactgtccggaagaaagaagagtataaaatggccctg tacaacctgtatcccggcgtgttcgaaaccgtcgagatgctgcctagcaaggcagggatc tggagagtggaatgcctgattggggagcacctgcatgccggaatgtctaccctgtttctg gtgtacagtaataagtgtcagacacccctggggatggcttccggacatatccgggatttc cagattaccgcatctggacagtacggccagtgggcccctaagctggctagactgcactat tccgggtctatcaacgcttggtccacaaaagagcctttctcttggattaaggtggacctg ctggcaccaatgatcattcatggcatcaaaactcagggggccaggcagaagttctcctct ctgtacatctcacagtttatcatcatgtacagcctggatggcaagaaatggcagacatac cgcggcaatagcacagggactctgatggtgttctttggcaacgtggacagttcagggatc aagcacaacattttcaatccccctatcattgctagatacatcaggctgcacccaacccat tattctattcgaagtacactgcggatggaactgatggggtgcgatctgaacagttgttca atgcccctgggaatggagtccaaggcaatctctgacgcccagattaccgctagctcctac ttcactaatatgtttgctacctggagcccctccaaagcacgactgcatctgcagggacga agcaacgcatggcgaccacaggtgaacaatcccaaggagtggctgcaggtcgattttcag aaaactatgaaggtgaccggagtcacaactcagggcgtgaaaagtctgctgacctcaatg tacgtcaaggagttcctgatctctagttcacaggacggccaccagtggacactgttcttt cagaacggaaaggtgaaagtcttccagggcaatcaggattcctttacacctgtggtcaac tctctggacccacccctgctgactcgctacctgcgaatccacccacagtcctgggtgcat cagattgcactgagaatggaagtcctgggctgcgaggcccaggacctgtattga FulllengthcassetteincludingmutatedTTRpromoter(TTRmut), syntheticintron,CpGreducedfactorVIIIcDNA,polyAandITRs (SEQIDNO:23) cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtc gggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggcca actccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgttcc gatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgt tgactaagtcaataatcagaatcagcaggtttggagtcagcttggcagggatcagcagcc tgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatcca caagctcctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgg gttatggcccttgcgtgccttgaattactgacactgacatccactttttctttttctcca caggtttaaacgccaccatgcagattgagctgagcacctgcttcttcctgtgtctgctga ggttctgcttctctgccaccaggaggtattacctgggggctgtggagctgagctgggact atatgcagtctgacctgggggagctgcctgtggatgctaggttcccccccagggtgccca agagcttcccctttaacacttctgtggtgtacaagaagaccctgtttgtggagttcactg accacctgttcaacattgccaagcccaggcccccctggatggggctgctggggcccacca tccaggctgaggtgtatgacactgtggtgatcaccctgaagaacatggccagccaccctg tgagcctgcatgctgtgggggtgagctactggaaggcttctgagggggctgagtatgatg accagactagccagagggagaaggaggatgacaaggtgtttcctgggggcagccatacct atgtgtggcaggtgctgaaggagaatggccccatggcctctgaccccctgtgcctgacct acagctacctgtctcatgtggacctggtgaaggacctgaactctggcctgattggggctc tgctggtgtgtagggagggcagcctggctaaggaaaagacccagaccctgcataagttta tcctgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaacagcc tgatgcaggatagggatgctgcctctgccagggcttggcctaagatgcacactgtgaatg ggtatgtgaataggagcctgcctggcctgattggctgccacaggaagtctgtgtactggc atgtgattgggatgggcaccacccctgaggtccatagcatcttcctggagggccacactt tcctggtgaggaaccacagacaggcctctctggagatctctcccatcaccttcctgactg ctcagactctgctgatggacctgggccagttcctgctgttttgccatattagcagccacc agcatgatgggatggaggcctatgtgaaggtggatagctgccctgaggagcctcagctga ggatgaagaacaatgaggaggctgaagactatgatgatgacctgactgattctgagatgg atgtggtgaggtttgatgatgacaatagccccagcttcattcagatcaggtctgtggcca agaaacaccccaagacctgggtgcactacattgctgctgaggaagaggactgggactatg ctcccctggtgctggcccctgatgataggtcttataagagccagtacctgaacaatgggc cccagaggattggcaggaagtacaagaaggtgaggttcatggcctacactgatgaaacct tcaaaaccagggaggccattcagcatgagtctggcatcctgggccctctgctgtatgggg aggtgggggacaccctgctgatcatcttcaagaaccaggccagcaggccctacaacatct atcctcatggcatcactgatgtgaggcccctgtacagcaggaggctgcccaagggggtga agcacctgaaagacttccccatcctgcctggggagatctttaagtataagtggactgtga ctgtggaggatggccctaccaagtctgaccccaggtgtctgaccaggtactattctagct ttgtgaacatggagagggacctggcctctggcctgattgggcccctgctgatctgctaca aggagtctgtggaccagaggggcaaccagatcatgtctgacaagaggaatgtgatcctgt tttctgtgtttgatgagaataggagctggtacctgactgagaacatccagaggtttctgc ccaatcctgctggggtgcagctggaggatcctgagttccaggccagcaatatcatgcata gcatcaatggctatgtgtttgacagcctgcagctgtctgtgtgcctgcatgaggtggcct actggtacatcctgagcattggggcccagactgactttctgtctgtgttcttttctggct ataccttcaagcacaagatggtgtatgaggataccctgaccctgttccccttctctgggg agactgtgttcatgagcatggagaatcctgggctgtggatcctggggtgccacaactctg attttaggaacagggggatgactgccctgctgaaggtgtctagctgtgataagaacactg gggactactatgaggacagctatgaggacatttctgcttatctgctgtctaagaataatg ccattgagcccagaagcttcagccagaatccccctgtgctgaagagacatcagagggaga tcaccagaactaccctgcagtctgatcaggaggagattgactatgatgacactatctctg tggagatgaagaaggaggactttgacatctatgatgaggatgagaatcagtctcccagga gctttcagaagaagaccagacattacttcattgctgctgtggagaggctgtgggactatg gcatgagctctagccctcatgtgctgaggaacagggcccagtctggctctgtgccccagt tcaagaaggtggtgttccaggaattcactgatggcagcttcacccagcccctgtacaggg gggagctgaatgagcacctgggcctgctggggccttatatcagggctgaggtggaggata atattatggtgactttcaggaaccaggccagcaggccctactctttctatagcagcctga tctcttatgaggaggatcagaggcagggggctgagcctaggaagaactttgtgaagccca atgagactaagacctacttctggaaggtccagcaccacatggcccctaccaaggatgagt ttgactgcaaggcctgggcctatttctctgatgtggatctggagaaggatgtccattctg ggctgattggccccctgctggtgtgccacactaacactctgaatcctgcccatggcaggc aggtgactgtccaggagtttgccctgttcttcactatctttgatgagaccaagagctggt actttactgagaacatggagaggaactgcagagctccttgcaatattcagatggaggacc ccaccttcaaggagaattacaggttccatgccattaatgggtacatcatggacaccctgc ctggcctggtgatggctcaggaccagaggatcaggtggtacctgctgagcatgggctcta atgagaatatccacagcatccacttctctgggcatgtgttcactgtgaggaagaaggagg agtacaagatggctctgtataatctgtaccctggggtgtttgaaactgtggagatgctgc cctctaaggctggcatctggagggtggagtgcctgattggggagcacctgcatgctggca tgagcaccctgttcctggtgtacagcaacaagtgccagacccccctgggcatggcctctg gccacatcagggacttccagatcactgcctctggccagtatggccagtgggcccccaagc tggccaggctgcactattctggcagcatcaatgcctggagcaccaaggagcccttcagct ggatcaaggtggacctgctggcccccatgatcattcatggcatcaagacccagggggcca ggcagaagttcagctctctgtacatctctcagttcatcatcatgtactctctggatggga agaagtggcagacctacaggggcaacagcactggcaccctgatggtgttctttgggaatg tggactcttctggcatcaagcacaacatcttcaatccccccatcattgctaggtatatta ggctgcatcccacccactacagcatcaggtctaccctgaggatggagctgatgggctgtg acctgaactcttgcagcatgcccctgggcatggagtctaaggccatctctgatgcccaga ttactgccagcagctacttcaccaacatgtttgccacctggagcccctctaaggccaggc tgcatctgcaggggaggagcaatgcctggaggcctcaggtgaacaaccccaaggagtggc tgcaggtggatttccagaagaccatgaaggtgactggggtgaccacccagggggtcaaga gcctgctgaccagcatgtatgtgaaggagttcctgatcagcagcagccaggatggccacc agtggactctgttctttcagaatgggaaggtgaaggtgtttcagggcaatcaggactctt tcacccctgtggtgaacagcctggacccccccctgctgaccagatacctgaggatccacc cccagtcttgggtgcatcagattgccctgaggatggaggtgctgggctgtgaggctcagg atctgtactgagcggccgcaataaaagatcagagctctagagatctgtgtgttggttttt tgtgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcac tgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgag cgagcgagcgcgcagctgcctgcagg FulllengthplasmidincludingmutatedTTRpromoter(TTRmut), syntheticintron,CpGreducedfactorVIIIcDNA,polyAandITRs (SEQIDNO:24) cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtc gggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggcca actccatcactaggggttcctacgcgtgtctgtctgcacatttcgtagagcgagtgttcc gatactctaatctccctaggcaaggttcatattgacttaggttacttattctccttttgt tgactaagtcaataatcagaatcagcaggtttggagtcagcttggcagggatcagcagcc tgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatcca caagctcctgctagcaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgg gttatggcccttgcgtgccttgaattactgacactgacatccactttttctttttctcca caggtttaaacgccaccatgcagattgagctgagcacctgcttcttcctgtgtctgctga ggttctgcttctctgccaccaggaggtattacctgggggctgtggagctgagctgggact atatgcagtctgacctgggggagctgcctgtggatgctaggttcccccccagggtgccca agagcttcccctttaacacttctgtggtgtacaagaagaccctgtttgtggagttcactg accacctgttcaacattgccaagcccaggcccccctggatggggctgctggggcccacca tccaggctgaggtgtatgacactgtggtgatcaccctgaagaacatggccagccaccctg tgagcctgcatgctgtgggggtgagctactggaaggcttctgagggggctgagtatgatg accagactagccagagggagaaggaggatgacaaggtgtttcctgggggcagccatacct atgtgtggcaggtgctgaaggagaatggccccatggcctctgaccccctgtgcctgacct acagctacctgtctcatgtggacctggtgaaggacctgaactctggcctgattggggctc tgctggtgtgtagggagggcagcctggctaaggaaaagacccagaccctgcataagttta tcctgctgtttgctgtgtttgatgagggcaagagctggcactctgagaccaagaacagcc tgatgcaggatagggatgctgcctctgccagggcttggcctaagatgcacactgtgaatg ggtatgtgaataggagcctgcctggcctgattggctgccacaggaagtctgtgtactggc atgtgattgggatgggcaccacccctgaggtccatagcatcttcctggagggccacactt tcctggtgaggaaccacagacaggcctctctggagatctctcccatcaccttcctgactg ctcagactctgctgatggacctgggccagttcctgctgttttgccatattagcagccacc agcatgatgggatggaggcctatgtgaaggtggatagctgccctgaggagcctcagctga ggatgaagaacaatgaggaggctgaagactatgatgatgacctgactgattctgagatgg atgtggtgaggtttgatgatgacaatagccccagcttcattcagatcaggtctgtggcca agaaacaccccaagacctgggtgcactacattgctgctgaggaagaggactgggactatg ctcccctggtgctggcccctgatgataggtcttataagagccagtacctgaacaatgggc cccagaggattggcaggaagtacaagaaggtgaggttcatggcctacactgatgaaacct tcaaaaccagggaggccattcagcatgagtctggcatcctgggccctctgctgtatgggg aggtgggggacaccctgctgatcatcttcaagaaccaggccagcaggccctacaacatct atcctcatggcatcactgatgtgaggcccctgtacagcaggaggctgcccaagggggtga agcacctgaaagacttccccatcctgcctggggagatctttaagtataagtggactgtga ctgtggaggatggccctaccaagtctgaccccaggtgtctgaccaggtactattctagct ttgtgaacatggagagggacctggcctctggcctgattgggcccctgctgatctgctaca aggagtctgtggaccagaggggcaaccagatcatgtctgacaagaggaatgtgatcctgt tttctgtgtttgatgagaataggagctggtacctgactgagaacatccagaggtttctgc ccaatcctgctggggtgcagctggaggatcctgagttccaggccagcaatatcatgcata gcatcaatggctatgtgtttgacagcctgcagctgtctgtgtgcctgcatgaggtggcct actggtacatcctgagcattggggcccagactgactttctgtctgtgttcttttctggct ataccttcaagcacaagatggtgtatgaggataccctgaccctgttccccttctctgggg agactgtgttcatgagcatggagaatcctgggctgtggatcctggggtgccacaactctg attttaggaacagggggatgactgccctgctgaaggtgtctagctgtgataagaacactg gggactactatgaggacagctatgaggacatttctgcttatctgctgtctaagaataatg ccattgagcccagaagcttcagccagaatccccctgtgctgaagagacatcagagggaga tcaccagaactaccctgcagtctgatcaggaggagattgactatgatgacactatctctg tggagatgaagaaggaggactttgacatctatgatgaggatgagaatcagtctcccagga gctttcagaagaagaccagacattacttcattgctgctgtggagaggctgtgggactatg gcatgagctctagccctcatgtgctgaggaacagggcccagtctggctctgtgccccagt tcaagaaggtggtgttccaggaattcactgatggcagcttcacccagcccctgtacaggg gggagctgaatgagcacctgggcctgctggggccttatatcagggctgaggtggaggata atattatggtgactttcaggaaccaggccagcaggccctactctttctatagcagcctga tctcttatgaggaggatcagaggcagggggctgagcctaggaagaactttgtgaagccca atgagactaagacctacttctggaaggtccagcaccacatggcccctaccaaggatgagt ttgactgcaaggcctgggcctatttctctgatgtggatctggagaaggatgtccattctg ggctgattggccccctgctggtgtgccacactaacactctgaatcctgcccatggcaggc aggtgactgtccaggagtttgccctgttcttcactatctttgatgagaccaagagctggt actttactgagaacatggagaggaactgcagagctccttgcaatattcagatggaggacc ccaccttcaaggagaattacaggttccatgccattaatgggtacatcatggacaccctgc ctggcctggtgatggctcaggaccagaggatcaggtggtacctgctgagcatgggctcta atgagaatatccacagcatccacttctctgggcatgtgttcactgtgaggaagaaggagg agtacaagatggctctgtataatctgtaccctggggtgtttgaaactgtggagatgctgc cctctaaggctggcatctggagggtggagtgcctgattggggagcacctgcatgctggca tgagcaccctgttcctggtgtacagcaacaagtgccagacccccctgggcatggcctctg gccacatcagggacttccagatcactgcctctggccagtatggccagtgggcccccaagc tggccaggctgcactattctggcagcatcaatgcctggagcaccaaggagcccttcagct ggatcaaggtggacctgctggcccccatgatcattcatggcatcaagacccagggggcca ggcagaagttcagctctctgtacatctctcagttcatcatcatgtactctctggatggga agaagtggcagacctacaggggcaacagcactggcaccctgatggtgttctttgggaatg tggactcttctggcatcaagcacaacatcttcaatccccccatcattgctaggtatatta ggctgcatcccacccactacagcatcaggtctaccctgaggatggagctgatgggctgtg acctgaactcttgcagcatgcccctgggcatggagtctaaggccatctctgatgcccaga ttactgccagcagctacttcaccaacatgtttgccacctggagcccctctaaggccaggc tgcatctgcaggggaggagcaatgcctggaggcctcaggtgaacaaccccaaggagtggc tgcaggtggatttccagaagaccatgaaggtgactggggtgaccacccagggggtcaaga gcctgctgaccagcatgtatgtgaaggagttcctgatcagcagcagccaggatggccacc agtggactctgttctttcagaatgggaaggtgaaggtgtttcagggcaatcaggactctt tcacccctgtggtgaacagcctggacccccccctgctgaccagatacctgaggatccacc cccagtcttgggtgcatcagattgccctgaggatggaggtgctgggctgtgaggctcagg atctgtactgagcggccgcaataaaagatcagagctctagagatctgtgtgttggttttt tgtgtaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcac tgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgag cgagcgagcgcgcagctgcctgcaggggcagcttgaaggaaatactaaggcaaaggtact gcaagtgctcgcaacattcgcttatgcggattattgccgtagtgccgcgacgccgggggc aagatgcagagattgccatggtacaggccgtgcggttgatattgccaaaacagagctgtg ggggagagttgtcgagaaagagtgcggaagatgcaaaggcgtcggctattcaaggatgcc agcaagcgcagcatatcgcgctgtgacgatgctaatcccaaaccttacccaacccacctg gtcacgcactgttaagccgctgtatgacgctctggtggtgcaatgccacaaagaagagtc aatcgcagacaacattttgaatgcggtcacacgttagcagcatgattgccacggatggca acatattaacggcatgatattgacttattgaataaaattgggtaaatttgactcaacgat gggttaattcgctcgttgtggtagtgagatgaaaagaggcggcgcttactaccgattccg cctagttggtcacttcgacgtatcgtctggaactccaaccatcgcaggcagagaggtctg caaaatgcaatcccgaaacagttcgcaggtaatagttagagcctgcataacggtttcggg attttttatatctgcacaacaggtaagagcattgagtcgataatcgtgaagagtcggcga gcctggttagccagtgctctttccgttgtgctgaattaagcgaataccggaagcagaacc ggatcaccaaatgcgtacaggcgtcatcgccgcccagcaacagcacaacccaaactgagc cgtagccactgtctgtcctgaattcattagtaatagttacgctgcggccttttacacatg accttcgtgaaagcgggtggcaggaggtcgcgctaacaacctcctgccgttttgcccgtg catatcggtcacgaacaaatctgattactaaacacagtagcctggatttgttctatcagt aatcgaccttattcctaattaaatagagcaaatccccttattgggggtaagacatgaaga tgccagaaaaacatgacctgttggccgccattctcgcggcaaaggaacaaggcatcgggg caatccttgcgtttgcaatggcgtaccttcgcggcagatataatggcggtgcgtttacaa aaacagtaatcgacgcaacgatgtgcgccattatcgcctagttcattcgtgaccttctcg acttcgccggactaagtagcaatctcgcttatataacgagcgtgtttatcggctacatcg gtactgactcgattggttcgcttatcaaacgcttcgctgctaaaaaagccggagtagaag atggtagaaatcaataatcaacgtaaggcgttcctcgatatgctggcgtggtcggaggga actgataacggacgtcagaaaaccagaaatcatggttatgacgtcattgtaggcggagag ctatttactgattactccgatcaccctcgcaaacttgtcacgctaaacccaaaactcaaa tcaacaggcgccggacgctaccagcttctttcccgttggtgggatgcctaccgcaagcag cttggcctgaaagacttctctccgaaaagtcaggacgctgtggcattgcagcagattaag gagcgtggcgctttacctatgattgatcgtggtgatatccgtcaggcaatcgaccgttgc agcaatatctgggcttcactgccgggcgctggttatggtcagttcgagcataaggctgac agcctgattgcaaaattcaaagaagcgggcggaacggtcagagagattgatgtatgagca gagtcaccgcgattatctccgctctggttatctgcatcatcgtctgcctgtcatgggctg ttaatcattaccgtgataacgccattacctacaaagcccagcgcgacaaaaatgccagag aactgaagctggcgaacgcggcaattactgacatgcagatgcgtcagcgtgatgttgctg cgctcgatgcaaaatacacgaaggagttagctgatgctaaagctgaaaatgatgctctgc gtgatgatgttgccgctggtcgtcgtcggttgcacatcaaagcagtctgtcagtcagtgc gtgaagccaccaccgcctccggcgtggataatgcagcctccccccgactggcagacaccg ctgaacgggattatttcaccctcagagagaggctgatcactatgcaaaaacaactggaag gaacccagaagtatattaatgagcagtgcagatagagttgcccatatcgatgggcaactc atgcaattattgtgagcaatacacacgcgcttccagcggagtataaatgcctaaagtaat aaaaccgagcaatccatttacgaatgtttgctgggtttctgttttaacaacattttctgc gccgccacaaattttggctgcatcgacagttttcttctgcccaattccagaaacgaagaa atgatgggtgatggtttcctttggtgctactgctgccggtttgttttgaacagtaaacgt ctgttgagcacatcctgtaataagcagggccagcgcagtagcgagtagcatttttttcat ggtgttattcccgatgctttttgaagttcgcagaatcgtatgtgtagaaaattaaacaaa ccctaaacaatgagttgaaatttcatattgttaatatttattaatgtatgtcaggtgcga tgaatcgtcattgtattcccggattaactatgtccacagccctgacggggaacttctctg cgggagtgtccgggaataattaaaacgatgcacacagggtttagcgcgtacacgtattgc attatgccaacgccccggtgctgacacggaagaaaccggacgttatgatttagcgtggaa agatttgtgtagtgttctgaatgctctcagtaaatagtaatgaattatcaaaggtatagt aatatcttttatgttcatggatatttgtaacccatcggaaaactcctgctttagcaagat tttccctgtattgctgaaatgtgatttctcttgatttcaacctatcataggacgtttcta taagatgcgtgtttcttgagaatttaacatttacaacctttttaagtccttttattaaca cggtgttatcgttttctaacacgatgtgaatattatctgtggctagatagtaaatataat gtgagacgttgtgacgttttagttcagaataaaacaattcacagtctaaatcttttcgca cttgatcgaatatttctttaaaaatggcaacctgagccattggtaaaaccttccatgtga tacgagggcgcgtagtttgcattatcgtttttatcgtttcaatctggtctgacctccttg tgttttgttgatgatttatgtcaaatattaggaatgttttcacttaatagtattggttgc gtaacaaagtgcggtcctgctggcattctggagggaaatacaaccgacagatgtatgtaa ggccaacgtgctcaaatcttcatacagaaagatttgaagtaatattttaaccgctagatg aagagcaagcgcatggagcgacaaaatgaataaagaacaatctgctgatgatccctccgt ggatctgattcgtgtaaaaaatatgcttaatagcaccatttctatgagttaccctgatgt tgtaattgcatgtatagaacataaggtgtctctggaagcattcagagcaattgaggcagc gttggtgaagcacgataataatatgaaggattattccctggtggttgactgatcaccata actgctaatcattcaaactatttagtctgtgacagagccaacacgcagtctgtcactgtc aggaaagtggtaaaactgcaactcaattactgcaatgccctcgtaattaagtgaatttac aatatcgtcctgttcggagggaagaacgcgggatgttcattcttcatcacttttaattga tgtatatgctctcttttctgacgttagtctccgacggcaggcttcaatgacccaggctga gaaattcccggaccctttttgctcaagagcgatgttaatttgttcaatcatttggttagg aaagcggatgttgcgggttgttgttctgcgggttctgttcttcgttgacatgaggttgcc ccgtattcagtgtcgctgatttgtattgtctgaagttgtttttacgttaagttgatgcag atcaattaatacgatacctgcgtcataattgattatttgacgtggtttgatggcctccac gcacgttgtgatatgtagatgataatcattatcactttacgggtcctttccggtgatccg acaggttacggggcggcgacctgcctgatgcggtattttctccttacgcatctgtgcggt atttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcg cggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccg ctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctc taaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaa aacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgcc ctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacac tcaactctatctcgggctattcttttgatttagacctgcaggcatgcaagcttggcactg gccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgcctt gcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccct tcccaacagttgcgcagcctgaatggcgaatgcgatttattcaacaaagccgccgtcccg tcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaa ctcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatattt ttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggc aagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaattt cccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccgg tgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacg ctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagc gagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccg gcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaa tacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagt acggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgac catctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctgg cgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcg agcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcttcgagca agacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcaga cagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttg agacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcac gcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactg gtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatgg ggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcagacctctcgacgg agttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttttt ttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtt tgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcaga taccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtag caccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgata agtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgg gctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactga gatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggaca ggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaa acgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttt tgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttac ggttcctggccttttgctggccttttgctcacatgt FVIII-BDDencodedbyX01-X18nucleicacidsequences.SQsequence bold/underlined (SEQIDNO:25) MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQ TSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLA KEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSV YWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGME AYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAE EEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEV GDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSD PRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAY LLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSF QKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLL GPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTK DEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVIVQEFALFFTIFDETKSWYFTENM ERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVF TVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGH IRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYIS QFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELM GCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQ KTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLT RYLRIHPQSWVHQIALRMEVLGCEAQDLY Wild-typeFVIIIwithBDD(SEQIDNO:26) MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT LFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQ TSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLA KEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSV YWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGME AYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAE EEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEV GDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSD PRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQR FLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHK MVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAY LLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQS PTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGT TAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESG GPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTN KTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTT SSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKS VEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLI QENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGE EENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWS KNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLF QDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVEN TVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVP FLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAI NEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYD EDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYR GELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFW KVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDE TKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENI HSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKC QTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGA RQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYS IRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNN PKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPV VNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY AAV-LK03VP1Capsid (SEQIDNO:27) MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAA DAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAA KTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMA SGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHY FGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTV QVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNN FQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQAR NWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFG KEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDV YLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVS VEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL AAV-SPKVP1Capsid(SEQIDNO:28)usedinAAV-SPK-8005andAAV-SPK-hFIX MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAA DAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPV KTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAAPSGVGPNTM AAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTND NTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLT STIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAV GRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTA GTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAM ATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPI VGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPT TFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIG TRYLTRNL

TABLE-US-00010 Percent Identity Matrix of hFVIII Vectors (WT, CO3, x09, X02, X06, X08, X15, X05, X18, X14, X01, X12, X04, X11, X07, X03, X16, X13, X17 and X10) hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII WT CO3 X09 X02 X06 X08 X15 X05 X18 X14 hFVIII 77.2 79.5 79.1 79.3 79.2 79.3 79.1 79 79.6 WT hFVIII 77.2 81.9 81.9 81.5 81.3 81.6 81.6 81.2 81.4 CO3 hFVIII 79.5 81.9 91.5 91.4 91.8 92 91.8 91 91.4 X09 hFVIII 79.1 81.9 91.5 91.4 91.3 92 92.1 92.2 91.7 X02 hFVIII 79.3 81.5 91.4 91.4 91.8 91.9 91.8 91.5 91.8 X06 hFVIII 79.2 81.3 91.8 91.3 91.8 91.8 91.5 91.5 91.8 X08 hFVIII 79.3 81.6 92 92 91.9 91.8 92.2 91.6 91.7 X15 hFVIII 79.1 81.6 91.8 92.1 91.8 91.5 92.2 92.5 91.9 X05 hFVIII 79 81.2 91 92.2 91.5 91.5 91.6 92.5 91.6 X18 hFVIII 79.6 81.4 91.4 91.7 91.8 91.8 91.7 91.9 91.6 X14 hFVIII 79.6 81.1 91.5 92 92.3 92.2 92.3 92.7 93 93 X01 hFVIII 79.4 81.1 91.5 91.9 91.7 91.5 92.1 92.4 92.1 92 X12 hFVIII 79.4 81.3 91.7 91.9 91.8 92.3 92.2 92.1 91.5 91.6 X04 hFVIII 79.4 81.7 91.7 92 92 92.5 92.5 91.5 91.8 91.8 X11 hFVIII 79.2 81.8 92.2 91.5 91.5 92 92 92.1 91.7 91.3 X07 hFVIII 79.4 81.6 91.5 91 91.4 91.7 92.1 91.6 91.4 91.8 X03 hFVIII 79.1 81.9 92.1 91.5 91.7 91.4 92.2 91.7 91.1 92.3 X16 hFVIII 79 81.8 91.8 92.3 92.4 92.3 92.3 92.3 91.8 92.2 X13 hFVIII 79.6 82.1 91.1 91.9 91.6 91.6 92.5 91.9 91.8 91.8 X17 hFVIII 79.3 82.2 91.6 92.1 91.8 91.9 92 92 92 92 X10 hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII hFVIII X01 X12 X04 X11 X07 X03 X16 X13 X17 X10 hFVIII 79.6 79.4 79.4 79.4 79.2 79.4 79.1 79 79.6 79.3 WT hFVIII 81.1 81.1 81.3 81.7 81.8 81.6 81.9 81.8 82.1 82.2 CO3 hFVIII 91.5 91.5 91.7 91.7 92.2 91.5 92.1 91.8 91.1 91.6 X09 hFVIII 92 91.9 91.9 92 91.5 91 91.5 92.3 91.9 92.1 X02 hFVIII 92.3 91.7 91.8 92 91.5 91.4 91.7 92.4 91.6 91.8 X06 hFVIII 92.2 91.5 92.3 92.5 92 91.7 91.4 92.3 91.6 91.9 X08 hFVIII 92.3 92.1 92.2 92.5 92 92.1 92.2 92.3 92.5 92 X15 hFVIII 92.7 92.4 92.1 91.5 92.1 91.6 91.7 92.3 91.9 92 X05 hFVIII 93 92.1 91.5 91.8 91.7 91.4 91.1 91.8 91.8 92 X18 hFVIII 93 92 91.6 91.8 91.3 91.8 92.3 92.2 91.8 92 X14 hFVIII 93.4 92.3 92.5 92.6 92.5 92.2 92.6 92.4 92.1 X01 hFVIII 93.4 92 92 92.4 92.4 91.7 92.4 92.6 92.6 X12 hFVIII 92.3 92 92.6 92 91.5 91.5 92 91.9 92.5 X04 hFVIII 92.5 92 92.6 92.6 92 91.9 92.3 91.8 91.9 X11 hFVIII 92.6 92.4 92 92.6 92.1 92 92.4 91.9 92.7 X07 hFVIII 92.5 92.4 91.5 92 92.1 92 92.7 92.1 91.6 X03 hFVIII 92.2 91.7 91.5 91.9 92 92 92.4 92 92.8 X16 hFVIII 92.6 92.4 92 92.3 92.4 92.7 92.4 92.4 92.8 X13 hFVIII 92.4 92.6 91.9 91.8 91.9 92.1 92 92.4 92.9 X17 hFVIII 92.1 92.6 92.5 91.9 92.7 91.6 92.8 92.8 92.9 X10

Certain Definitions/Abbreviations Used

[0293] BDD: all or at least part of B domain (BD) deleted
FVIII-BDD: FVIII with B domain deletion

SQ: SFSQNPPVLKRHQR (SEQ ID NO:29)

[0294] FVIII/SQ: FVIII with SQ
FVIIIX01-X18: CpG reduced FVIII encoding nucleic acid variants, set forth as SEQ ID Nos: 1-18, respectively.
TTRmut: TTR promoter with 4 mutations, from TAmGTGTAG to TATTGACTTAG
CO3: codon optimized FVIII nucleic acid variant, set forth as SEQ ID NO:21
NHP: Non human primate
ALT: Alanine aminotransferase
D-dimer: A protein fragment from the break down of a blood clot
SPK-8005: AAV capsid (SEQ ID NO:28)+TTRmut-hFVIII-X07; also referred to as AAV-SPK-8005
SPK-8011: AAV LK03 capsid (SEQ ID NO:27)+TTRmut-hFVIII-X07; also referred to as AAV-SPK-8011

[0295] While certain of the embodiments of the invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the invention, as set forth in the following claims.