RU2808274C2 - Methods of genotherapy using factor viii (fviii) gene - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions including a method of treating a person suffering from hemophilia A (variants) is described. In one embodiment, the method includes administering to a person having hemophilia A recombinant adeno-associated virus (rAAV) vector, wherein the vector genome includes a nucleic acid variant encoding factor VIII (FVIII) having a B-domain deletion (hFVIII-BDD), wherein the nucleic acid variant has 95% or greater identity with SEQ ID NO:7.

EFFECT: invention expands the arsenal of methods of treating hemophilia A leading to the absence of spontaneous bleeding in a person for at least 1 year.

66 cl, 28 dwg, 6 tbl, 10 ex

Description

Related applications

[0001] This patent application claims priority to U.S. Provisional Patent Application 62/540,053, filed August 1, 2017; US Provisional Patent Application 62/583,890, filed November 9, 2017; US Provisional Patent Application 62/596,535, filed December 8, 2017; and US Provisional Patent Application 62/596,670, filed December 8, 2017. The entire contents of the prior applications are incorporated herein by reference, including all text, tables, and drawings.

Field of technology to which the invention relates

[0002] The present invention relates to the field of production of recombinant blood coagulation factor and treatment of diseases associated with impaired hemostasis. In particular, the invention provides methods for administering a nucleic acid encoding factor VIII (FVIII) protein and methods for treating hemophilia A.

Introduction

[0003] Several publications and patent documents are cited throughout the application to describe the prior art to which the present invention relates. Each of these references is incorporated herein by reference as if set forth in their entirety.

[0004] Hemophilia is an X-linked bleeding disorder present in 1 in 5,000 men worldwide. Therapies aimed at increasing clotting factor levels to just above 1% of normal are associated with significant improvement in the severe disease phenotype. Recent clinical studies of AAV-mediated gene transfer in the treatment of hemophilia B (HB) have demonstrated consistent long-term expression of therapeutic levels of factor IX (FIX), but have found that the dose of the AAV vector may be dose limiting due to immune responses directed against the AAV capsid . While these data apply to hemophilia B, 80% of all hemophilia cases are caused by FVIII deficiency and are classified as hemophilia A (HA).

[0005] The current treatment for this disease is protein replacement therapy, which requires frequent infusions of factor VIII protein. There is an urgent need to achieve sustained therapeutic levels of factor VIII expression so that patients no longer require such frequent protein treatments. In fact, persistent expression of factor VIII could prevent bleeding episodes and ensure the establishment of immune tolerance to this protein.

The essence of the invention

[0006] The invention relates to methods of treating a person suffering from hemophilia A or requiring factor VIII (FVIII). In one embodiment, the method includes introducing a recombinant adeno-associated virus (rAAV) vector, wherein the vector genome includes a nucleic acid variant encoding factor VIII (FVIII) having a B domain deletion (hFVIII-BDD), wherein the nucleic acid variant has 95 % or greater identity with SEQ ID NO:7. In another embodiment, the method includes introducing a recombinant adeno-associated virus (rAAV) vector, wherein the vector genome includes a nucleic acid variant encoding factor VIII (FVIII) having a B domain deletion (hFVIII-BDD), wherein the nucleic acid variant contains no more than 2 cytosine-guanine dinucleotides (CpG).

[0007] In another embodiment, a method of treating a person suffering from hemophilia A or requiring Factor VIII (FVIII) includes administering a recombinant adeno-associated virus (rAAV) vector, wherein the vector genome includes a nucleic acid encoding factor VIII (FVIII) or encoding factor VIII (FVIII) having a B-domain deletion (hFVIII-BDD), where the dose of rAAV vector administered to a person is less than 6 x 10 ¹² vector genomes per kilogram (vg/kg).

[0008] Embodiments of the methods and uses include administering to a human a dose of the rAAV vector from about 1×10 ⁹ to about 1×10 ¹⁴ vg/kg, inclusive.

[0009] Embodiments of the methods and uses include administering to a human a dose of the rAAV vector from about 1×10 ¹⁰ to about 6×10 ¹³ vg/kg, inclusive.

[0010] Embodiments of the methods and uses include administering to a human a dose of the rAAV vector from about 1×10 ¹⁰ to about 1×10 ¹³ vg/kg, inclusive.

[0011] Embodiments of the methods and uses include administering to a human a dose of the rAAV vector from about 1×10 ¹⁰ to about 6×10 ¹² vg/kg, inclusive.

[0012] Embodiments of the methods and uses include administering to a human a dose of the rAAV vector from about 1×10 ¹⁰ to about 5×10 ¹² vg/kg, inclusive.

[0013] The method according to any of claims 1-3, wherein the dose of the rAAV vector administered to a human is from about 1×10 ¹¹ to about 1×10 ¹² vg/kg, inclusive.

[0014] Embodiments of the methods and uses include administering to a human a dose of the rAAV vector from about 2×10 ¹¹ to about 9×10 ¹¹ vg/kg, inclusive.

[0015] Embodiments of the methods and uses include administering to a human a dose of the rAAV vector from about 3×10 ¹¹ to about 8×10 ¹² vg/kg, inclusive.

[0016] 12. The method of any one of claims 1-3, wherein the dose of the rAAV vector administered to a human is from about 3×10 ¹¹ to about 7×10 ¹² vg/kg, inclusive.

[0017] Embodiments of the methods and uses include administering to a human a dose of the rAAV vector from about 3×10 ¹¹ to about 6×10 ¹² vg/kg, inclusive.

[0018] Embodiments of the methods and uses include administering to a human a dose of the rAAV vector from about 4×10 ¹¹ to about 6×10 ¹² vg/kg, inclusive.

[0019] Embodiments of the methods and uses include administering to a human a dose of the rAAV vector of approximately 5×10 ¹¹ vg/kg or approximately 1×10 ¹² vg/kg.

[0020] Embodiments of the methods and uses include providing greater than expected amounts of FVIII or hFVIII-BDD to humans based on data obtained from studies in non-human primates administered the rAAV vector. The amounts of FVIII or hFVIII-BDD expressed in humans, as reflected in coagulation activity, for example, may be greater than predicted based on the linear regression curve obtained from studies in non-human primates injected with the rAAV vector.

[0021] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a human, as reflected by clotting activity, is greater than predicted based on data obtained from studies in non-human primates administered the rAAV vector.

[0022] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a human, as reflected in coagulation activity, is 1-4 times greater than the expression predicted based on a linear regression curve obtained from studies in non-human primates , which were injected with the rAAV vector.

[0023] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a human, as reflected in coagulation activity, is 2-4 times greater than predicted based on a linear regression curve obtained from studies in non-human primates administered rAAV vector.

[0024] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a human, as reflected by coagulation activity, is 2-3 times greater than predicted based on a linear regression curve obtained from studies in non-human primates administered rAAV vector.

[0025] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a human, as reflected in coagulation activity, is 1-2 greater than predicted based on a linear regression curve obtained from studies in non-human primates administered the vector rAAV.

[0026] Non-human primates include the genus Macaca . In a specific embodiment, the non-human primate is a cynomolgus macaque ( Macaca fascicularis ).

[0027] In some embodiments, FVIII or hFVIII-BDD is expressed for a period of time that provides short-term, medium-term, or longer-term improvement in hemostasis. In some embodiments, the time period is such that no additional FVIII protein or recombinant FVIII protein needs to be administered to the subject to maintain hemostasis.

[0028] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 14 days after administration of the rAAV vector.

[0029] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 21 days after administration of the rAAV vector.

[0030] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 28 days after administration of the rAAV vector.

[0031] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 35 days after administration of the rAAV vector.

[0032] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 42 days after administration of the rAAV vector.

[0033] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 49 days after administration of the rAAV vector.

[0034] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 56 days after administration of the rAAV vector.

[0035] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 63 days after administration of the rAAV vector.

[0036] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 70 days after administration of the rAAV vector.

[0037] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 77 days after administration of the rAAV vector.

[0038] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 84 days after administration of the rAAV vector.

[0039] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 91 days after administration of the rAAV vector.

[0040] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 98 days after administration of the rAAV vector.

[0041] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 105 days after administration of the rAAV vector.

[0042] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 112 days after administration of the rAAV vector.

[0043] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 4 months after administration of the rAAV vector.

[0044] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 154 days.

[0045] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 210 days.

[0046] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 6 months after administration of the rAAV vector.

[0047] In some embodiments, FVIII or hFVIII-BDD is expressed for at least about 12 months after administration of the rAAV vector.

[0048] FVIII or hFVIII-BDD may be expressed in some amounts for some period of time after administration of the rAAV vector. In some embodiments, the amount is such that detectable FVIII or hFVIII-BDD is present, or an amount of FVIII or hFVIII-BDD that provides a therapeutic effect.

[0049] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a human, as reflected by clotting activity, is about 3% or more 14 or more days after administration of the rAAV vector, about 4% or more after 21 or more days after administration of the rAAV vector, approximately 5% or more at 21 or more days after administration of the rAAV vector, approximately 6% or more at 21 or more days after administration of the rAAV vector, approximately 7% or more at 21 or more days after administration of the rAAV vector, approximately 8% or more 28 or more days after administration of the rAAV vector, approximately 9% or more 28 or more days after administration of the rAAV vector, approximately 10% or more 35 or more days after administration of the rAAV vector, approximately 11% or more 35 or more days after administration of the rAAV vector, approximately 12% or more 35 or more days after administration of the rAAV vector.

[0050] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a person, as reflected by clotting activity, over a continuous 14-day period averages about 10% or more.

[0051] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a person, as reflected by clotting activity, over a continuous 4-week period averages about 10% or more.

[0052] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a person, as reflected by clotting activity, over a continuous 8-week period averages about 10% or more.

[0053] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a person, as reflected by clotting activity, over a continuous 12-week period averages about 10% or more.

[0054] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a person, as reflected by clotting activity, over a continuous 16-week period averages about 10% or more.

[0055] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a person, as reflected by clotting activity, over a continuous 6-month period averages about 10% or more.

[0056] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a person, as reflected by clotting activity, over a continuous 7-month period averages about 10% or more.

[0057] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a person, as reflected by clotting activity, over a continuous 14-day period averages about 12% or more.

[0058] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a person, as reflected by clotting activity, averages from about 12% to about 100% over a continuous 4-week period, over a continuous 8-week period , for a continuous 12-week period, for a continuous 16-week period, for a continuous 6-month period, for a continuous 7-month period, or for a continuous 1-year period.

[0059] In some embodiments, the amount of FVIII or hFVIII-BDD expressed in a human, as reflected by clotting activity, averages from about 20% to about 80% over a continuous 4-week period, over a continuous 8-week period , over a continuous 12-week period, over a continuous 16-week period, over a continuous 6-month period, or over a continuous 1-year period.

[0060] Steady-state FVIII expression can also be achieved after a period of time, for example 4-6, 6-8 or 6-12 weeks or more, for example 6-12 months or even years after administration of the rAAV vector.

[0061] In some embodiments, FVIII or hFVIII-BDD is produced in a person at a steady state where FVIII activity does not change by more than 5-50% over a period of 4, 6, 8, or 12 weeks or months.

[0062] In some embodiments, FVIII or hFVIII-BDD is produced in a person at a steady state where FVIII activity does not change by more than 25-100% over a period of 4, 6, 8, or 12 weeks or months.

[0063] The rAAV vector can be administered in doses that are expected to produce FVIII expression in certain amounts and for certain periods of time, with stable expression following administration.

[0064] In some embodiments, the rAAV vector is administered to a human at a dose of from about 1 x 10 ⁹ to about 1 x 10 ¹⁴ vg/kg, inclusive, and FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to approximately 100% activity, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months after administration of the rAAV vector.

[0065] In some embodiments, the rAAV vector is administered to a human at a dose of from about 5 x 10 ⁹ to about 6 x 10 ¹³ vg/kg, inclusive, and FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to approximately 100% activity, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months after administration of the rAAV vector.

[0066] In some embodiments, the rAAV vector is administered to a human at a dose of from about 1 x 10 ¹⁰ to about 6 x 10 ¹³ vg/kg, inclusive, and FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to approximately 100% activity, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months after administration of the rAAV vector.

[0067] In some embodiments, the rAAV vector is administered to a human at a dose of from about 1 x 10 ¹⁰ to about 1 x 10 13 vg/kg, inclusive, and FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to approximately 100% activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months after administration of the rAAV vector.

[0068] In some embodiments, the rAAV vector is administered to a human at a dose of from about 1 x 10 ¹⁰ to about 6 x 10 ¹² vg/kg, inclusive, and FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to approximately 100% activity, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months after administration of the rAAV vector.

[0069] In some embodiments, the rAAV vector is administered to a human at a dose of less than 6 x 10 ¹² vg/kg and FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to about 100% activity for at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 continuous days, weeks, or months after administration of the rAAV vector.

[0070] In some embodiments, the rAAV vector is administered to a human at a dose of from about 1 x 10 ¹⁰ to about 5 x 10 ¹² vg/kg, inclusive, and FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to approximately 100% activity, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months after administration of the rAAV vector.

[0071] In some embodiments, the rAAV vector is administered to a human at a dose of from about 1 x 10 ¹¹ to about 1 x 10 ¹² vg/kg, inclusive, and FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to approximately 100% activity, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months after administration of the rAAV vector.

[0072] In some embodiments, the rAAV vector is administered to a human at a dose of from about 2 x 10 ¹¹ to about 9 x 10 ¹¹ vg/kg, inclusive, and FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to approximately 100% activity, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months after administration of the rAAV vector.

[0073] In some embodiments, the rAAV vector is administered to a human at a dose of from about 3 x 10 ¹¹ to about 8 x 10 ¹² vg/kg, inclusive, and FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to approximately 100% activity, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months after administration of the rAAV vector.

[0074] In some embodiments, the rAAV vector is administered to a human at a dose of from about 3 x 10 ¹¹ to about 7 x 10 ¹² vg/kg, inclusive, and FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to approximately 100% activity, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months after administration of the rAAV vector.

[0075] In some embodiments, the rAAV vector is administered to a human at a dose of from about 3 x 10 ¹¹ to about 6 x 10 ¹² vg/kg, inclusive, and FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to approximately 100% activity, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months after administration of the rAAV vector.

[0076] In some embodiments, the rAAV vector is administered to a human at a dose of from about 4 x 10 ¹¹ to about 6 x 10 ¹² vg/kg, inclusive, and FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to approximately 100% activity, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months after administration of the rAAV vector.

[0077] In some embodiments, the rAAV vector is administered at a dose of about 5×10 ¹¹ vg/kg or about 1×10 ¹² vg/kg, and FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to approximately 100% activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months after administration of the rAAV vector.

[0078] Humans according to the methods and uses include people who are seronegative for anti-AAV antibodies or have no detectable anti-AAV antibodies.

[0079] In some embodiments, anti-AAV antibodies are not detected in a person until administration of the rAAV vector, wherein the person is seronegative for AAV.

[0080] In some embodiments, anti-AAV antibodies against FVIII or hFVIII-BDD are not detected for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or months or more after introduction of the rAAV vector.

[0081] In some embodiments, anti-AAV antibodies against the rAAV vector are not detected for at least about 14 days, or for at least about 21 days, or for at least about 28 days, or for at least about 35 days, or for at least about 42 days, or for at least about 49 days, or for at least about 56 days, or for at least about 63 days, or for at least about 70 days, or for at least about 77 days, or for at least about 84 days, or for at least about 91 days, or for at least about 98 days, or for at least about 105 days, or for at least about 112 days after administration of the rAAV vector.

[0082] Humans according to the methods and applications include humans who have detectable antibodies to AAV.

[0083] In some embodiments, anti-AAV antibodies are present in humans at a level of about 1:5 or less prior to administration of the rAAV vector.

[0084] In some embodiments, anti-AAV antibodies are present in humans at a level of about 1:3 or less prior to administration of the rAAV vector.

[0085] In some methods and applications, a person who has been administered the rAAV vector does not develop a cell-mediated immune response against the rAAV vector.

[0086] In some embodiments, a person who has been administered the rAAV vector does not develop a cell-mediated immune response against the rAAV vector for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, or 14 continuous weeks or months after administration of the rAAV vector.

[0087] In some embodiments, a person who has been administered the rAAV vector does not develop a humoral immune response against the rAAV vector sufficient to reduce or block the therapeutic effect of FVIII or hFVIII-BDD.

[0088] In some embodiments, a person who has been administered the rAAV vector does not develop detectable antibodies against the rAAV vector for at least about 1, 2, 3, 4, 5, or 6 months after administration of the rAAV vector.

[0089] In some embodiments, the person who has been administered the rAAV vector is not administered an immunosuppressive agent before, during, and/or after administration of the rAAV vector.

[0090] In some embodiments, FVIII or hFVIII-BDD expression is achieved in a human who has been administered the rAAV vector without administration of an immunosuppressive agent.

[0091] If an immune response pre-exists or develops after administration of the rAAV vector, an immunosuppressive agent may be administered to the individual before or after administration of the rAAV vector.

[0092] In some embodiments, the method or use includes administering an immunosuppressive agent prior to administration of the rAAV vector.

[0093] In some embodiments, the method or use includes administering an immunosuppressive agent after administration of the rAAV vector.

[0094] In some embodiments, the immunosuppressive agent is administered from a period of 1 hour to 45 days after administration of the rAAV vector.

[0095] In some embodiments, the immunosuppressive agent includes a steroid, cyclosporine (eg, cyclosporine A), mycophenolate, rituximab, or a derivative thereof.

[0096] In some embodiments, the nucleic acid variants have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or greater sequence identity with any of SEQ ID NO:1-18. In some embodiments, the nucleic acid variants have 90-95% sequence identity to any of SEQ ID NO: 1-18. In some embodiments, the nucleic acid variants have 95-100% sequence identity to any of SEQ ID NO: 1-18.

[0097] In some embodiments, the variant nucleic acid encoding FVIII or hFVIII-BDD has reduced CpG content compared to the wild-type nucleic acid encoding FVIII. In some embodiments, the variant nucleic acid contains at least 20 fewer CpGs than the wild-type FVIII-encoding nucleic acid (SEQ ID NO:19). In some embodiments, the nucleic acid variant contains no more than 10 CpGs, contains no more than 9 CpGs, contains no more than 8 CpGs, contains no more than 7 CpGs, contains no more than 6 CpGs, contains no more than 5 CpGs, contains no more than 4 CpGs; contains no more than 3 CpG; contains no more than 2 CpG; or contains no more than 1 CpG. In some embodiments, the nucleic acid variant contains no more than 4 CpGs; 3 CpG; 2 CpG; or 1 CpG. In some embodiments, the nucleic acid variant does not contain a CpG.

[0098] In some embodiments, the nucleic acid variant encoding FVIII or hFVIII-BDD has reduced CpG content compared to the wild-type nucleic acid encoding FVIII, and such reduced CpG nucleic acid variants have 90% or greater sequence identity with any of SEQ ID NO:1-18. In some embodiments, the CpG reduced nucleic acid variants have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or greater sequence identity to any of SEQ ID NO:1-18. In some embodiments, the CpG reduced nucleic acid variants have 90-95% sequence identity to any of SEQ ID NO: 1-18. In some embodiments, the CpG reduced nucleic acid variants have 95-100% sequence identity to any of SEQ ID NO: 1-18. In some embodiments, FVIII-encoding CpG-reduced nucleic acid variants are provided in any of SEQ ID NO: 1-18.

[0099] In some embodiments, the nucleic acid variants encoding the FVIII or hFVIII-BDD protein are at least 75% identical to wild-type human FVIII nucleic acid or wild-type human FVIII nucleic acid including a B-domain deletion. In some embodiments, the nucleic acid variants encoding the FVIII protein are about 75-95% identical (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83% , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% identical) to wild-type human FVIII nucleic acid or wild-type human FVIII nucleic acid type, including deletion of the B-domain.

[0100] In some embodiments, the nucleic acids and variants encoding the FVIII protein are nucleic acids from a mammal, such as a human. Such nucleic acids and variants of mammalian nucleic acids encoding the FVIII protein include human forms, which may be based on wild-type human FVIII or wild-type human FVIII including a deletion of the B domain.

[0101] In some embodiments, the recombinant adeno-associated virus (sAAV) vector includes an AAV vector, includes an AAV serotype or a pseudotype of AAV, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , Rh10, Rh74 or AAV-2i8 AAV. In some embodiments, the rAAV vector includes any of SEQ ID NO:1-18 or includes SEQ ID NO:23 or 24.

[0102] In some embodiments, the expression control element includes a constitutive or regulated control element, or a tissue-specific expression control element or promoter. In some embodiments, the expression control element includes a liver expression element. In some embodiments, the expression control element includes a TTR promoter or a mutant TTR promoter, such as SEQ ID NO:22. In other specific aspects, the expression control element includes the promoter presented in PCT publication WO 2016/168728 (USSN 62/148,696; 62/202,133; and 62/212,634), which are incorporated herein by reference in their entirety.

[0103] In some embodiments, the rAAV vector includes an AAV serotype or an AAV pseudotype including an AAV capsid serotype other than the ITR serotype. In additional embodiments, the rAAV vector includes a VP1, VP2, and/or VP3 capsid sequence having 75% or greater sequence identity (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, etc.) with any of the serotypes AAV AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8.

[0104] In some embodiments, the rAAV vector includes a VP1, VP2, and/or VP3 capsid sequence having 75% or greater sequence identity (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, etc.) with any of SEQ ID NO:27 or SEQ ID NO:28. In some embodiments, the rAAV vector includes a VP1, VP2, and/or VP3 capsid that is 100% identical to SEQ ID NO:27 or SEQ ID NO:28.

[0105] In some embodiments, the rAAV vector further includes an intron, an expression control element, one or more AAV inverted terminal repeats (ITRs) (e.g., any of the AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 or a combination thereof), filler polynucleotide sequence and/or poly-A signal.

[0106] In some embodiments, the intron is located within or flanks a nucleic acid or nucleic acid variant encoding FVIII or hFVIII-BDD, and/or an expression control element is operably linked to a nucleic acid or nucleic acid variant encoding FVIII or hFVIII-BDD, and /or AAV ITR repeat(s) flanks the 5' or 3' end of a nucleic acid or nucleic acid variant encoding FVIII, and/or a filler polynucleotide sequence flanks the 5' or 3' end of a nucleic acid or nucleic acid variant encoding FVIII or hFVIII-BDD.

[0107] In particular embodiments, the expression control element includes a constitutive or regulated control element, or a tissue-specific expression control element or promoter. In some embodiments, the expression control element includes a liver expression element (eg, a TTR promoter or a mutant TTR promoter).

[0108] In some embodiments, the rAAV includes a pharmaceutical composition. Such pharmaceutical compositions optionally include an empty AAV capsid (eg, a partial vector genome comprising a nucleic acid or nucleic acid variant encoding FVIII or hFVIII-BDD).

[0109] In some embodiments, the nucleic acid or nucleic acid variant encoding FVIII or hFVIII-BDD protein, vectors, expression vectors or virus, or AAV vectors are encapsulated in a liposome or mixed with phospholipids or micelles.

[0110] The methods of the invention also include treating mammals (e.g., humans), such as people who are FVIII-deficient (a person who produces insufficient FVIII protein or a defective or aberrant FVIII protein) or who have hemophilia A.

[0111] In one embodiment, the individual produces an insufficient amount of FVIII protein or a defective or aberrant FVIII protein. In another embodiment, the person has mild, moderate, or severe hemophilia A.

[0112] In some embodiments, the FVIII or hFVIII-BDD expressed by the administered rAAV vector is expressed at levels that have a beneficial or therapeutic effect in the mammal.

[0113] Candidate patients (e.g., a patient) and mammals (e.g., humans) for administration (e.g., delivery) of an rAAV comprising a nucleic acid or nucleic acid variant encoding FVIII or hFVIII-BDD include patients who have or are at risk of development of a disorder such as hemophilia A, von Willebrand disease and bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC), or a disorder caused by overdose of anticoagulation therapy.

[0114] Candidate patients (e.g., a patient) and mammals (e.g., humans) for administration (e.g., delivery) of a nucleic acid or nucleic acid variant encoding FVIII include patients who are seronegative for anti-AAV antibodies, as well as patients who have (are seropositive) or are at risk of developing antibodies to AAV. Such patients (eg, patient) and mammals (eg, humans) may be seronegative or seropositive for serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV-Rh10, or AAV-Rh74 .

[0115] In some embodiments, an empty capsid of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV-12, AAV-Rh10, and/or AAV-Rh74 is further administered to a mammal or patient alone or in combination with an rAAV vector comprising a nucleic acid or nucleic acid variant encoding FVIII.

[0116] Methods of administration (eg, delivery) in accordance with the invention include any mode of contact or delivery, ex vivo or in vivo . In certain embodiments, administration (eg, delivery): intravenous, intra-arterial, intramuscular, subcutaneous, intracavitary, by intubation, or by catheter.

[0117] In some embodiments, FVIII or hFVIII-BDD is expressed at levels without significantly increasing the risk of thrombosis.

[0118] In some embodiments, the risk of thrombosis is determined by measuring fibrin breakdown products.

[0119] In some embodiments, FVIII or hFVIII-BDD activity in a person 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.

[0120] In some embodiments, the individual is further tested or monitored for the presence of one or more of the following factors: the presence or amount of anti-AAV antibodies, anti-AAV immune response, anti-FVIII or hFVIII-BDD antibodies, anti-FVIII or hFVIII-BDD immune response, amount FVIII or hFVIII-BDD, the activity of FVIII or hFVIII-BDD, the amount or levels of one or more liver enzymes, or the frequency and/or severity or duration of bleeding episodes.

Description of drawings

[0121] Figure 1 shows the design of the NCP study.

[0122] Figures 2A-2C show hFVIII antigen levels in NHPs following intravenous administration of 2 x 10 ¹² (A), 5 x 10 ¹² (B) or 1 x 10 ¹³ vg/kg (C) AAV-SPK-8005. Lines represent individual animals. Plasma levels of human FVIII were examined by ELISA and represent multiple measurements obtained from sequential blood samples taken from the same group of animals throughout the study (n=2-3 animals per cohort). Levels of human FVIII measured in vehicle-treated animals are indicated by open squares in all three graphs.

ε=Production of inhibitors against FVIII.

[0123] Figures 3A-3C show ALT levels in NHPs when administered 2 x 10 ¹² (A), 5 x 10 ¹² (B), or 1 x 10 ¹³ vg/kg (C) AAV-SPK-8005.

[0124] Figures 4A-4C show D-dimer levels in NNPs. Concentration of D-dimer antigen in NHP plasma after intravenous administration of 2 × 10 ¹² (A), 5 × 10 ¹² (B) or 1 × 10 ¹³ vg/kg (C) AAV-SPK-8005. The dotted line represents 500 ng/mL, the upper limit of normal values for D-dimer in humans.

[0125] Figure 5 shows summary data for FVIII levels at three doses of AAV-SPK-8005.

[0126] Figures 6A-6D show hFVIII plasma levels in cynomolgus monkeys following intravenous administration of 2 x 10 ¹² (A), 6 x 10 ¹² (B) or 2 x 10 ¹³ (vg/kg) (C) AAV-SPK- 8011(LK03 capsid)-hFVIII (preliminary study). Lines represent individual animals. Plasma hFVIII levels were examined by ELISA and are multiple measurements obtained from sequential blood samples taken from the same group of animals during the study (n=3 animals per cohort). Human FVIII levels measured in vehicle-treated animals are indicated by open squares (n=2). ε=Time at which production of anti-FVIII inhibitors was detected in each individual animal.

[0127] Figure 7 shows the expression levels of human FVIII in cynomolgus monkeys after administration of SPK-8011. Preliminary study (squares) and GLP standard study (circles).

[0128] Figure 8 shows a comparison of the levels of FVIII achieved with AAV-SPK-8011 (LK03 capsid)-hFVIII with published levels of FVIII delivered using AAV vectors with AAV5 and AAV8 capsids. 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.

[0129] Figure 9 shows the tissue biodistribution of AAV-SPK (SEQ ID NO:28) and AAV-LK03 (SEQ ID NO:27) in non-human primates (NHP), mainly in the kidney, spleen and liver (3 th column for each fabric).

[0130] Figure 10 shows FVIII expression in the liver and spleen after systemic administration of AAV-SPK-8005 to mice.

[0131] Figure 11 shows the AAV-LK03 capsid transduction efficiency tested in vitro . X-axis, cynomolgus macaque (left vertical column), human (right vertical column).

[0132] Figure 12 shows the expression levels of human FVIII in cynomolgus monkeys after administration of SPK-8011 according to a linear dose-response relationship. Panels A and B show SPK-8011 doses on a linear scale, whereas panels C and D use a logarithmic x-axis.

[0133] Figure 13 shows a linear regression analysis using only data from the low and medium dose cohorts. Panels A and B show SPK-8011 doses on a linear scale, whereas panels C and D use a logarithmic x-axis.

[0134] Figure 14 shows FVIII activity in 3 human patients who were infused with the AAV-LK03 (FVIII) vector. Patients 1 and 2 (diamond, circle) received an infusion of 5×10 ¹¹ vg/kg AAV-LK03 (FVIII) vector. Patient 3 (triangle) received an infusion of 1 x 10 ¹² vg/kg AAV-LK03 (FVIII) vector.

[0135] Figure 15 shows the long-term expression of FVIII activity at therapeutic levels in the same human patients (Patients 1 and 2, Figure 14) receiving infusion of the AAV-LK03 (FVIII) vector. Patients 1 and 2 (circle, square) received an infusion of 5×10 ¹¹ vg/kg AAV-LK03 (FVIII) vector.

[0136] Figure 16 shows 10 human patients (Patients 1-10) demonstrating therapeutic levels of FVIII. Patient 1 received an FVIII infusion following an emergency tooth extraction at week 6 post-infusion. Shortly thereafter, a 19% level of FVIII activity was recorded; excluded from this diagram due to proximity of FVIII infusion. FVIII activity refers to FVIII:C values from local laboratories.

[0137] Figure 17 shows therapeutic FVIII levels in Patient 1 receiving an infusion of 5×10 ¹¹ vg/kg AAV-LK03 vector (FVIII). The bottom panel shows the results of the interferon-γ enzyme-linked immunosorbent spot (ELISPOT) assay for the patient's peripheral blood mononuclear cell (PBMC) response to AAV capsid peptides (filled bar) and FVIII peptides (open circle). Results are shown as spot-forming units (SFU) per million PBMC; values that exceed 50 PFU or three times higher than the control medium (dashed line) are considered positive.

[0138] Figure 18 shows therapeutic FVIII levels in Patient 2 receiving a 5 x 10 ¹¹ vg/kg infusion of AAV-LK03 vector (FVIII). The bottom graph shows the results of the interferon-γ ELISPOT assay for the response of patient PBMCs to AAV capsid peptides (filled bar) and FVIII peptides (open circle). Results are shown as number of PFU per 1 million PBMCs; values that exceed 50 PFU or that are three times higher than the control medium (dashed line) are considered positive.

[0139] Figure 19 shows therapeutic FVIII levels in Patient 3 receiving an infusion of 1 x 10 ¹² vg/kg AAV-LK03 vector (FVIII). The bottom graph shows the results of the interferon-γ ELISPOT assay for the response of patient PBMCs to AAV capsid peptides (filled bar) and FVIII peptides (open circle). Results are shown as number of PFU per 1 million PBMCs; values that exceed 50 PFU or that are three times higher than the control medium (dashed line) are considered positive.

[0140] Figure 20 shows therapeutic FVIII levels in Patient 4 receiving an infusion of 1 x 10 ¹² vg/kg AAV-LK03 vector (FVIII). The bottom panel shows the results of the interferon-γ ELISPOT assay for the response of patient PBMCs to AAV capsid peptides (filled bar) and FVIII peptides (open circle). Results are shown as number of PFU per 1 million PBMCs; values that exceed 50 PFU or that are three times higher than the control medium (dashed line) are considered positive.

[0141] Figure 21 shows therapeutic FVIII levels in Patient 5 receiving an infusion of 2×10 ¹² vg/kg AAV-LK03 vector (FVIII). The bottom graph shows the results of the interferon-γ ELISPOT assay for the response of patient PBMCs to AAV capsid peptides (filled bar) and FVIII peptides (open circle). Results are shown as number of PFU per 1 million PBMCs; values that exceed 50 PFU or that are three times higher than the control medium (dashed line) are considered positive.

[0142] Figure 22 shows therapeutic FVIII levels in Patient 6 receiving an infusion of 1 x 10 ¹² vg/kg AAV-LK03 vector (FVIII). The bottom graph shows the results of the interferon-γ ELISPOT assay for the response of patient PBMCs to AAV capsid peptides (filled bar) and FVIII peptides (open circle). Results are shown as number of PFU per 1 million PBMCs; values that exceed 50 PFU or that are three times higher than the control medium (dashed line) are considered positive.

[0143] Figure 23 shows therapeutic FVIII levels in Patient 7 receiving an infusion of 2×10 ¹² vg/kg AAV-LK03 vector (FVIII). The bottom panel shows the results of the interferon-γ ELISPOT assay for the response of patient PBMCs to AAV capsid peptides (filled bar) and FVIII peptides (open circle). Results are shown as number of PFU per 1 million PBMCs; values that exceed 50 PFU or that are three times higher than the control medium (dashed line) are considered positive.

[0144] Figure 24 shows therapeutic FVIII levels in Patient 8 receiving an infusion of 2×10 ¹² vg/kg AAV-LK03 vector (FVIII). The bottom graph shows the results of the interferon-γ ELISPOT assay regarding the response of patient PBMCs to AAV capsid peptides (filled bar) and FVIII peptides (open circle). Results are shown as number of PFU per 1 million PBMCs; values that exceed 50 PFU or that are three times higher than the control medium (dashed line) are considered positive.

[0145] Figure 25 shows therapeutic FVIII levels in Patient 9 receiving an infusion of 2×10 ¹² vg/kg AAV-LK03 vector (FVIII). The bottom graph shows the results of the interferon-γ ELISPOT assay for the response of patient PBMCs to AAV capsid peptides (filled bar) and FVIII peptides (open circle). Results are shown as number of PFU per 1 million PBMCs; values that exceed 50 PFU or that are three times higher than the control medium (dashed line) are considered positive.

[0146] Figure 26 shows therapeutic FVIII levels in Patient 10 receiving a 2 x 10 ¹² vg/kg infusion of AAV-LK03 (FVIII) vector. The bottom graph shows the results of the interferon-γ ELISPOT assay for the response of patient PBMCs to AAV capsid peptides (filled bar) and FVIII peptides (open circle). Results are shown as number of PFU per 1 million PBMCs; values that exceed 50 PFU or that are three times higher than the control medium (dashed line) are considered positive.

[0147] Figure 27 shows therapeutic FVIII levels in Patient 11 receiving an infusion of 2×10 ¹² vg/kg AAV-LK03 vector (FVIII). The bottom graph shows the results of the interferon-γ ELISPOT assay for the response of patient PBMCs to AAV capsid peptides (filled bar) and FVIII peptides (open circle). Results are shown as number of PFU per 1 million PBMCs; values that are greater than 50 PFU or that are three times higher than the control medium (dashed line) are considered positive.

[0148] Figure 28 shows therapeutic FVIII levels in Patient 12 receiving an infusion of 2×10 ¹² vg/kg AAV-LK03 vector (FVIII). The bottom graph shows the results of the interferon-γ ELISPOT assay for the response of patient PBMCs to AAV capsid peptides (filled bar) and FVIII peptides (open circle). Results are shown as number of PFU per 1 million PBMCs; values that exceed 50 PFU or that are three times higher than the control medium (dashed line) are considered positive.

Detailed description

[0149] The invention relates to methods of treating a person suffering from hemophilia A or requiring Factor VIII (FVIII). Such methods can be carried out using rAAV vectors with a genome including nucleic acid or nucleic acid variants encoding FVIII or hFVIII-BDD, which can be expressed in cells and/or humans, which in turn can provide increased levels of FVIII protein or hFVIII-BDD in vivo . Examples of nucleic acid variants encoding FVIII or hFVIII-BDD may have reduced CpG content compared to a wild-type mammalian (e.g., human) reference FVIII or hFVIII-BDD and/or have less than 100% sequence identity to the reference FVIII or hFVIII-BDD Wild-type mammalian (eg human) BDD. Such methods can also be carried out by administering a dose of rAAV vector in an amount of less than 6×10 ¹² vrAAV vector genomes per kilogram (vg/kg). rAAV vectors administered at a dose of less than 6×10 ¹² vrAAV vector genomes per kilogram (vg/kg) may include a vector genome including a nucleic acid or nucleic acid variant encoding FVIII or hFVIII-BDD.

[0150] The terms "polynucleotide" and "nucleic acid" are used interchangeably in the present invention 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, as well as 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 (mi) )RNA, trans-spliced RNA or antisense RNA). Polynucleotides include natural, synthetic, and intentionally modified or altered polynucleotides (eg, variant nucleic acid). Polynucleotides can be single, double or triple, linear or circular, and can be of any length. When discussing polynucleotides, the sequence or structure of a particular polynucleotide can be described in the present invention in accordance with the rule of providing the sequence in the 5'→3' direction.

[0151] As used herein, the terms “modify” or “variant” and their grammatical variations mean that a nucleic acid, polypeptide, or subsequence thereof deviates from the reference sequence. Thus, modified and variant sequences may have substantially the same, greater or lesser expression, activity or function than the reference sequence, but at least retain partial activity or function of the reference sequence. A specific example of a modification or variant is a CpG-reduced nucleic acid variant encoding FVIII.

As used herein, the terms “modify” or “variant” and their grammatical variations mean that the nucleic acid, polypeptide, or subsequence thereof differs from the reference sequence. Modified and variant sequences may therefore have substantially the same, greater or lesser expression, activity or function than the reference sequence while retaining at least partial activity or function of the reference sequence. A specific example of a modification or variant is a CpG reduced nucleic acid variant encoding FVIII.

[0152] A "nucleic acid" or "polynucleotide" variant refers to a modified sequence that has been genetically altered from the wild type sequence. The sequence can be genetically modified without changing the sequence of the encoded protein. Alternatively, the sequence may be genetically modified to encode a protein variant. A variant nucleic acid or polynucleotide may also refer to a combination sequence that has been subjected to codon modification to encode a protein that still retains at least partial sequence identity to a reference sequence, such as a wild-type protein sequence, and has also been subject to codon modification to encode a variant protein. For example, some codons of such a nucleic acid variant will be changed without changing the protein (FVIII) amino acids they encode, and some codons of a nucleic acid variant will be changed, which in turn will cause a change in the protein (FVIII) amino acids they encode.

[0153] The term “variant Factor VIII (FVIII)” refers to modified FVIII, genetically altered from unmodified wild-type FVIII (eg, SEQ ID NO:19) or FVIII-BDD. Such a variant may be referred to as a “Factor VIII (FVIII)-encoding nucleic acid variant.” A specific example of a variant is a CpG-reduced nucleic acid encoding a FVIII or FVIII-BDD protein. The term "variant" need not appear in every instance when referring to a CpG-reduced nucleic acid encoding FVIII. Likewise, the term "CpG-reduced nucleic acid" and the like. may not contain the term "variant", but it is intended that reference to "CpG reduced nucleic acid" includes variants at the genetic level.

[0154] CpG-reduced FVIII and hFVIII-BDD constructs can demonstrate improvements over wild-type FVIII or FVIII-BDD that are not CpG-reduced, and do so without nucleic acid modifications that result in changes to the encoded FVIII protein or FVIII-BDD at the amino acid level. When comparing expression, if a CpG-reduced nucleic acid encodes an FVIII protein that retains the B domain, it is preferable to compare it with wild-type FVIII expression; and if the CpG-reduced nucleic acid encodes an FVIII protein without the B domain, it is compared to the expression of wild-type FVIII, which also has a B domain deletion.

[0155] “Variant Factor VIII (FVIII)” can also mean a modified FVIII protein, wherein the modified protein has an amino acid change compared to wild-type FVIII. Again, when comparing activity and/or stability, if the encoded variant FVIII protein retains the B domain, they are preferably compared to wild-type FVIII; and if the encoded FVIII variant protein has a B-domain deletion, it is compared to wild-type FVIII, which also has a B-domain deletion.

[0156] The variant FVIII may include part of the B domain. Thus, FVIII-BDD includes part of the B domain. Typically, FVIII-BDD has most of the B domain removed.

[0157] The variant FVIII may include an "SQ" sequence, represented as SFSQNPPVLKRHQR (SEQ ID NO:29). Typically, such a variant FVIII with SQ (FVIII/SQ) has a BDD, for example, at least all or part of the BD has been removed. A variant FVIII, such as FVIII-BDD, may contain all or part of the "SQ" sequence, i.e. all or parts of SEQ ID NO:29. Thus, for example, a variant FVIII-BDD with an SQ sequence (SFSQNPPVLKRHQR, SEQ ID NO:29) may contain all or only a portion of the amino acid sequence SFSQNPPVLKRHQR. For example, FVIII-BDD may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acid residues SFSQNPPVLKRHQR in its composition. Thus, SFSQNPPVLKRHQR with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 internal deletions is the same as with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 N- or C-terminal deletions, is included in the variant FVIII proteins provided in the present invention.

[0158] "Polypeptides", "proteins" and "peptides" encoded by "nucleic acid" sequences or "polynucleotide" sequences include full-length native sequences (FVIII), both with naturally occurring wild-type proteins and functional subsequences, modified forms or sequence variants, provided that the subsequence, modified form, or variant retains some degree of functionality of the native full-length protein. For example, a CpG-reduced nucleic acid encoding an FVIII or hFVIII-BDD protein may have a B domain deletion as provided herein and retain blood clotting function. In the methods and applications of the invention, such polypeptides, proteins and peptides encoded by nucleic acid sequences can be, but need not be, identical to an endogenous protein that is defective, or deficient in expression, or that is deficient in the mammal being treated.

[0159] Non-limiting examples of modifications include substitution of one or more nucleotides or amino acids (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 a decrease in CpG content. In some embodiments, the CpG-reduced FVIII-encoding nucleic acid, such as human FVIII protein, contains 10 or fewer CpGs compared to the wild-type human FVIII-encoding sequence; or contains 5 or fewer CpGs compared to the wild-type sequence encoding human factor FVIII; or the CpG-reduced nucleic acid encoding FVIII contains no more than 5 CpGs.

[0160] An example of an amino acid modification is a conservative amino acid substitution or deletion (eg, subsequences or fragments) of a reference sequence, for example, FVIII, such as FVIII with B-domain deletion. In certain embodiments, the modified or variant sequence retains at least a portion of the function or activity of the unmodified sequence.

[0161] All forms of mammalian and non-mammalian protein-encoding nucleic acids, including other CpG-reduced forms of mammalian nucleic acids encoding FVIII and hFVIII-BDD disclosed in the present invention are expressly included, whether known or unknown. Thus, the invention includes non-mammalian, non-human and human mammalian genes and proteins, such genes and proteins functioning substantially like the FVIII (eg human) genes and proteins described in the present invention.

[0162] The term “vector” refers to a small nucleic acid carrier molecule, plasmid, virus (eg, AAV vector), or other carrier that can be manipulated to insert or incorporate a nucleic acid. Such vectors can be used for genetic manipulation (ie, "cloning vectors"), for the introduction/transfer of polynucleotides into cells, and for transcription or translation of 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 required for expression in a host cell. The vector nucleic acid sequence typically contains at least an origin of replication for propagation in the cell and optionally additional elements such as a heterologous polynucleotide sequence, an expression control element (e.g., promoter, enhancer), an intron, ITR repeat(s), selectable marker (eg, antibiotic resistance), polyadenylation signal.

[0163] A viral vector is derived from or based on one or more nucleic acid elements that are contained in the genome of a virus. Specific viral vectors include lentiviral, pseudotyped lentiviral and parvoviral vectors, such as adeno-associated virus (AAV) vectors.

[0164] The term "recombinant", as a definition of a vector, such as a recombinant viral, for example, lenti- or parvovirus (eg, AAV) vector, as well as a definition of sequences, such as recombinant polynucleotides and polypeptides, means that the compositions have been manipulated (i.e. genetically modified) in a way not typically found in nature. A specific example of a recombinant vector, such as an AAV vector, would be one in which a polynucleotide not normally present in the genome of a wild-type virus (eg, AAV) is inserted into the viral genome. An example of a recombinant polynucleotide would be one in which a CpG-reduced nucleic acid encoding a FVIII or hFVIII-BDD protein is cloned into a vector, with or without the 5' and/or 3' intronic regions with which the gene is normally associated in a virus (eg , AAV) genome. Although the term "recombinant" is not always used in the present invention to refer to vectors such as viral and AAV vectors, as well as sequences such as polynucleotides, recombinant forms, including polynucleotides, are expressly included notwithstanding any such exclusion .

[0165] A recombinant viral “vector” or “AAV vector” is derived from the genome of a wild-type virus, such as AAV, using molecular techniques to remove the wild-type genome from the virus (e.g., AAV) and replace it with a non-native nucleic acid, such as nucleic acid with reduced CpG content, encoding FVIII. Typically, in the case of AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the AAV vector. A "recombinant" viral vector (eg, AAV) is different from a viral (eg, AAV) genome because the viral genome has been replaced in whole or in part by a non-native sequence relative to a viral (eg, AAV) genomic nucleic acid, such as a reduced nucleic acid content of CpG encoding FVIII or hFVIII-BDD. The inclusion of a non-native sequence thus defines the viral vector (eg, AAV) as a “recombinant” vector, which in the case of AAV may be referred to as a “rAAV vector.”

[0166] The recombinant vector sequence (eg, lenti-, parvo-, AAV) may be packaged, and may be referred to in the present invention as a "particle", for subsequent infection (transduction) of a cell ex vivo , in vitro or in vivo . In the case where the recombinant vector sequence is encapsulated or packaged into an AAV particle, the particle may also be referred to as "rAAV". Such particles include proteins that encapsulate or package the vector genome. Specific examples include viral envelope proteins and, in the case of AAV, capsid proteins.

[0167] A vector “genome” refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsulated to produce a viral (eg, AAV) particle. In cases where recombinant plasmids are used for the design or production of recombinant vectors, the vector genome does not contain a portion of the “plasmid” that does not match the vector genome sequence of the recombinant plasmid. This non-genomic vector 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 necessary for propagation and production of the recombinant virus, but is not itself packaged or encapsulated to form viral (e.g., AAV ) particles. Thus, a vector "genome" refers to the nucleic acid packaged or encapsulated by a virus (eg, AAV).

[0168] "Transgene" is used in the present invention to appropriately designate a nucleic acid to be introduced or introduced into a cell or organism. Transgenes include any nucleic acid, such as a gene encoding a polypeptide or protein (eg, a CpG-reduced nucleic acid encoding FVIII or hFVIII-BDD).

[0169] In a cell having a transgene, the transgene has been introduced/transferred via a vector, such as AAV, as a result of "transduction" or "transfection" of the cell. The terms "transduce" and "transfect" refer to the introduction of a molecule, such as a nucleic acid, into a cell or host organism. The transgene may or may not integrate into the genomic nucleic acid of the recipient cell. If the introduced nucleic acid is integrated into the nucleic acid (genomic DNA) of a recipient cell or organism, it can be stably maintained in that cell or organism and then transmitted or inherited by daughter cells or organisms that are derived from the recipient cell or organism. Finally, the introduced nucleic acid may exist extrachromosomally or only transiently in the recipient cell or host organism.

[0170] A "transduced cell" is a cell into which a transgene has been introduced. Thus, a "transduced" cell (eg, in a mammal, such as a cell or tissue or organ cell) means a genetic change in the cell following the incorporation of an exogenous molecule, such as a nucleic acid (eg, a transgene) into the cell. Thus, a "transduced" cell is a cell or its progeny into which exogenous nucleic acid has been introduced. The cell(s) can be expanded and the introduced protein can be expressed or the nucleic acid can be transcribed. In the case of gene therapy applications and methods, the transduced cell may be present in the patient's body.

[0171] “Expression control element” refers to nucleic acid sequence(s) that influence the expression of an operably linked nucleic acid. Control elements, including expression control elements as defined in the present invention, such as promoters and enhancers, vector sequences, including AAV vectors, may include one or more “expression control elements”. Typically, such elements are included to facilitate proper transcription of the heterologous polynucleotide and proper translation (e.g., promoter, enhancer, intronic splicing signal, maintaining the correct gene reading frame to allow in-frame translation of the mRNA, as well as stop codons, etc.) . Such elements typically act in a cis configuration and are called a “cis-acting” element, but can also act in a trans configuration.

[0172] Expression control may be present at the level of transcription, translation, splicing, stability of mRNA transcripts, etc. Typically, an expression control element that modulates transcription is placed near the 5' end (ie, "upstream") of the nucleic acid being transcribed. Expression control elements may also be located at the 3' end (ie, "downstream") of the transcribed sequence or within the transcript (eg, in an intron). Expression control elements may be adjacent to or located remotely from the transcribed sequence (eg, 1-10, 10-25, 25-50, 50-100, 100-500 or more nucleotides of a polynucleotide), even at significant distances. However, given the length limitations of some vectors, such as AAV vectors, expression control elements will typically be located within 1-1000 nucleotides of the transcribed nucleic acid.

[0173] At the functional level, the expression of an operably linked nucleic acid is, at least in part, regulated by an element (eg, a promoter), wherein the element modulates the transcription of the nucleic acid and, if necessary, the translation of the transcript. A specific example of an expression control element is a promoter, which is typically located 5' of a transcribed sequence, for example, a CpG-reduced nucleic acid encoding FVIII or hFVIII-BDD. A promoter generally increases the amount expressed from an operably linked nucleic acid compared to the amount expressed in the absence of a promoter.

[0174] As used herein, "enhancer" may refer to a sequence that is located adjacent to a heterologous polynucleotide. Enhancer elements are typically located upstream of a promoter element, but also function and can be located downstream or within a sequence (eg, a CpG-reduced nucleic acid encoding FVIII or hFVIII-BDD). Thus, the enhancer element may be located 100 base pairs, 200 base pairs, or 300 base pairs or more upstream or downstream of the CpG-reduced nucleic acid encoding FVIII. Enhancer elements typically increase the expression of an operably linked nucleic acid above the level of expression provided by the promoter element.

[0175] An expression construct may include regulatory elements that serve to direct expression in a particular cell or tissue type. Expression control elements (eg, promoters) include elements active in a particular tissue or cell type, referred to herein as “tissue-specific expression control elements/promoters.” Tissue-specific expression control elements are usually active in a specific cell or tissue (eg, liver). Expression control elements are typically active in specific cells, tissues, or organs and are therefore recognized by transcriptional activator proteins or other transcriptional regulators that are specific to a particular cell, tissue, or organ type. Such regulatory elements are known to those skilled in the art (see, for example, Sambrook et al. (1989) and Ausubel et al. (1992)).

[0176] The introduction of tissue-specific regulatory elements into the expression constructs of the invention provides at least partial tissue tropism for the expression of a CpG-reduced nucleic acid encoding FVIII or hFVIII-BDD. Examples of promoters that are active in the liver include the TTR promoter, the human alpha-1 antitrypsin (hAAT) promoter; albumin, Miyatake, et al. J. Virol., 71:5124-32 (1997); hepatitis B virus core protein promoter, Sandig, et al., Gene Ther. 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot, et al., Hum. Gene. Ther., 7:1503-14 (1996)], and others. An example of an enhancer active in the liver is apolipoprotein E (apoE) HCR-1 and HCR-2 (Allan et al., J. Biol. Chem., 272:29113-19 (1997)).

[0177] Expression control elements also include ubiquitous or universal promoters/enhancers that are capable of directing expression of a polynucleotide in many different cell types. Such elements include, but are not limited to, cytomegalovirus (CMV) immediate early promoter/enhancer sequences, Rous sarcoma virus (RSV) promoter/enhancer sequences, and other viral promoters/enhancers active in a variety of mammalian cell types, or synthetic elements not naturally occurring ( see, for example, Boshart et al, Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic β-actin promoter and the phosphoglycerol kinase (PGK) promoter.

[0178] Expression control elements can also provide expression in a regulated manner, that is, act as a signal or stimulus to increase or decrease the expression of an operably linked heterologous polynucleotide. A regulated element that increases the expression of an operably linked polynucleotide in response to a signal or stimulus is also referred to as an “inducible element” (ie, induced by a signal). Specific examples include, but are not limited to, a hormone (eg, steroid) inducible promoter. Typically, the amount of increase or decrease imparted by such elements is proportional to the magnitude of the signal or stimulus present; the greater the magnitude of the signal or stimuli, the greater the increase or decrease in expression. Specific non-limiting examples include the zinc-inducible ovine metallothionein (MT) promoter; steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; T7 polymerase promoter system (WO 98/10088); tetracycline-repressible system (Gossen, et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992); tetracycline-inducible system (Gossen, et al., Science. 268:1766-1769 (1995 ); also see Harvey, et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)); 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 regulated control elements that may be useful in this context are those that are regulated by a particular physiological state, eg temperature, acute phase, development.

[0179] Expression control elements also include element(s) native to the heterologous polynucleotide. A native control element (eg, a promoter) can be used if expression of a heterologous polynucleotide is desired to mimic native expression. A native element can be used if the expression of a heterologous polynucleotide needs to be regulated depending on time or phase of the developmental cycle, or tissue-specifically, or in response to certain transcriptional stimuli. Other native expression control elements such as introns, polyadenylation sites, or Kozak consensus sequences may also be used.

[0180] The term "operably linked" means that the regulatory sequences necessary for expression of the coding sequence are placed at appropriate positions relative to the coding sequence to allow expression of the coding sequence. The same definition is sometimes applied to the location of coding sequences and transcriptional control elements (eg, promoters, enhancers, and termination elements) in an expression vector. This definition also sometimes refers to the arrangement of the nucleic acid sequences of the first and second nucleic acid molecules when forming a hybrid nucleic acid molecule.

[0181] In an example of an expression control element operably linked to a nucleic acid, their relationship is such that the control element modulates the expression of the nucleic acid. In particular, for example, two DNA sequences are operably linked, which means that the two DNAs are arranged (cis or trans) in such a relationship that at least one of the DNA sequences is capable of exerting a physiological effect on the other sequence.

[0182] Thus, additional vector elements include, but are not limited to, an expression control element (e.g., a promoter/enhancer), a transcription termination signal or stop codon, 5' or 3' untranslated regions (e.g., polyadenylation sequences (poly(A) ))) that flank a sequence, such as one or more copies of an AAV ITR sequence, or an intron.

[0183] Other elements include, for example, filler or padding polynucleotide sequences, for example, to improve packaging and reduce the presence of contaminating nucleic acid. AAV vectors typically allow for the insertion of DNA having a length ranging from about 4 to about 5.2 kb or slightly longer. Thus, in the case of shorter sequences, the inclusion of padding or filler is necessary to adjust the length to a normal or near normal size of the viral genomic sequence suitable for packaging the AAV vector into a viral particle. In various embodiments, the filler/filler nucleic acid sequence is a non-translated (non-protein coding) segment of the nucleic acid. In the case of a nucleic acid sequence less than 4.7 kb, the filler or filler polynucleotide sequence has a length that, when combined (eg, inserted into a vector) with the sequence, has a total length of about 3.0-5.5 kb or about 4.0-5 .0 kb, or approximately 4.3-4.8 kb.

[0184] The intron can also act as a filler or padding polynucleotide sequence to provide the desired length for packaging the AAV vector into a viral particle. Introns and fragments of introns that function as filler or filler polynucleotide sequences can also enhance expression.

[0185] The phrase "hemostasis-related disorder" refers to coagulation disorders such as hemophilia A, hemophilia A in patients with inhibitory antibodies, deficiency of coagulation factors VII, VIII, IX and X, XI, V, XII, II, factor von Willebrand, 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); excess doses during anticoagulation therapy with heparin, low molecular weight heparin, pentasaccharide, warfarin, low molecular weight antithrombotic agents (i.e. FXa inhibitors); and platelet disorders such as Bernard-Soulier syndrome, Glanzmann thrombasthenia, and storage pool deficiency.

[0186] The term “separated,” when used to define a composition, means that the compositions are man-made or separated, in whole or at least in part, from their naturally occurring in vivo environment. Typically, isolated compositions are substantially free of one or more materials with which they are typically associated in nature, eg, one or more proteins, nucleic acids, lipids, carbohydrates, cell membranes.

[0187] With respect to nucleic acids according to the invention, the term "isolated" refers to a nucleic acid molecule that is separated from one or more sequences to which it is immediately adjacent (in the 5' and 3' directions) in the natural genome (genomic DNA) of an organism , from which it comes. For example, an "isolated nucleic acid" may include a DNA or cDNA molecule inserted into a vector, such as a plasmid or viral vector, or integrated into prokaryotic or eukaryotic DNA.

[0188] With respect to RNA molecules according to the invention, the term “isolated” primarily refers to the RNA molecule encoded by the isolated DNA molecule, as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from the RNA molecules with which it might be associated in its natural state (i.e., in cells or tissues) and exists in an "essentially pure" form (the term "substantially pure" is defined below).

[0189] When referring to a protein, the term "isolated protein" or "isolated and purified protein" is sometimes used in the present invention. The term refers primarily to a protein synthesized as a result of the expression of an isolated nucleic acid molecule. The term may also refer to a protein that is sufficiently separated from other proteins with which it is naturally associated and exists in an "essentially pure" form.

[0190] The term “isolated” does not exclude human-made combinations, such as a recombinant vector sequence (eg, rAAV) or a viral particle that packages or encapsulates the vector genome, and a pharmaceutical composition. The term “isolated” also does not exclude alternative physical forms of the composition, such as hybrids/chimeras, multimers/oligomers, modifications (eg, phosphorylation, glycosylation, lipidation), or derivatized forms or forms expressed in human host cells.

[0191] The term “substantially pure” refers to a preparation comprising at least 50-60% by weight of the compound of interest (eg, nucleic acid, oligonucleotide, protein, etc.). The formulation may comprise at least 75% by weight, or approximately 90-99% by weight, of the compound of interest. Purity is measured using methods appropriate to the compound of interest (eg, chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0192] The phrase "consisting essentially of" with respect to a particular nucleotide sequence or amino acid sequence means that the sequence has the properties of the given SEQ ID NO. For example, when used in relation to an amino acid sequence, the phrase includes the sequence itself and molecular properties that will not affect the basic and novel features of the sequence.

[0193] The term "oligonucleotide" as used herein refers to primers and probes and is defined as a nucleic acid molecule consisting 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 specific application for which the oligonucleotide is used.

[0194] The term “probe” as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, RNA or DNA, whether naturally occurring, for example as a purified restriction digest, or produced synthetically, that is capable of annealing to or specifically hybridizing to a nucleic acid. an acid having sequences complementary to the probe. The probe can be single-stranded or double-stranded. The exact probe length will depend on many factors, including temperature, probe source, and application. 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.

[0195] The probes of the present invention are selected to be “substantially” complementary to various strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary to be able to “specifically hybridize” or anneal to the strands of their respective targets under some given conditions. Thus, the probe sequence does not need to 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 may be distributed in the probe, provided that the probe sequence has sufficient complementarity to the target nucleic acid sequence to specifically anneal to it.

[0196] The term “specifically hybridizes” refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under specified conditions commonly used in the art (sometimes referred to as “substantially complementary”). In particular, the term refers to the hybridization of an oligonucleotide to a substantially complementary sequence contained in a single-stranded DNA or RNA molecule of the invention, and essentially to the avoidance of hybridization of the oligonucleotide to single-stranded nucleic acids with a non-complementary sequence.

[0197] The term "primer" as used herein refers to an oligonucleotide, RNA or DNA, single-stranded or double-stranded, derived from a biological system, created by restriction enzyme digestion, or produced artificially, which, when placed in an appropriate environment, is capable of acting functionally as an initiator of template-dependent synthesis nucleic acid. Given a suitable nucleic acid as a template, suitable nucleoside triphosphate nucleic acid precursors, a polymerase enzyme, suitable cofactors and conditions such as suitable temperature and pH, the primer can be extended at the 3' end by the addition of nucleotides by polymerase or similar activity to produce primer completion product.

[0198] The primer can have different lengths depending on the specific conditions and requirements of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 nucleotides or longer in length. The primer must have sufficient complementarity with the desired template to initiate synthesis of the desired extension product, that is, be capable of annealing to the desired template strand in a manner sufficient to ensure the correct location of the primer's 3'-hydroxyl group for use in initiating synthesis by a polymerase or similar enzyme. The primer sequence is not required to be the exact complement of the desired template. For example, a non-complementary nucleotide sequence may be added to the 5' end of an otherwise complementary primer. Alternatively, non-complementary bases may be distributed throughout the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to form a functional template-primer complex and synthesize an extension product.

[0199] The terms "identity", "homology" and their grammatical variations mean that two or more units in question are the same if 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 region or portion in question. When two polynucleotide sequences are identical, they have the same polynucleotide sequence at least within the boundaries of the region or portion in question. An identity may be present in a specific part (region or domain) of the sequence. An "area" or "domain" with identity refers to the portion of two or more units in question that are the same. Thus, when two protein or nucleic acid sequences are identical in one or more portions or regions of the sequence, they have identity within the boundaries of that region. An "aligned" sequence refers to a plurality of polynucleotide or protein (amino acid) sequences, often containing amendments caused by the absence or presence of additional bases or amino acids (gaps) compared to the reference sequence.

[0200] The identity may extend over the entire length or part of the sequence. In some embodiments, the length of the sequence having some percentage identity is 2, 3, 4, 5 or more consecutive nucleic acids or amino acids, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. consecutive nucleic acids or amino acids. In further embodiments, the length of the sequence having the identity is 21 or more consecutive nucleic acids or amino acids, for example, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, etc. consecutive nucleic acids or amino acids. In other embodiments, the length of the sequence having the identity is 41 or more consecutive nucleic acids or amino acids, for example, 42, 43, 44, 45, 45, 47, 48, 49, 50, etc. consecutive nucleic acids or amino acids. In other embodiments, the length of the sequence having identity is 50 or more consecutive nucleic acids or amino acids, for example, 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, 500-1000, etc. consecutive nucleic acids or amino acids.

[0201] As provided herein, nucleic acid variants, such as CpG-reduced variants encoding FVIII or hFVIII-BDD, will differ from the wild type, but may exhibit sequence identity to the wild-type FVIII protein with or without the B domain him. In CpG reduced nucleic acid variants encoding FVIII or hFVIII-BDD, at the nucleotide sequence level, the CpG reduced nucleic acid encoding FVIII or hFVIII-BDD will generally be at least about 70% identical, more typically about 75% identical, even more typically about 80-85% identical to the nucleic acid encoding wild-type FVIII. Thus, for example, a CpG-reduced nucleic acid encoding FVIII or hFVIII-BDD may have 75-85% identity with the gene encoding wild-type FVIII or with each other, i.e. X01 with X02, X03 with X04, etc., as specified in the present invention.

[0202] At the amino acid sequence level, a variant, such as a FVIII or hFVIII-BDD protein variant, will be at least about 70% identical, more typically about 75% identical or 80% identical, even more typically about 85 identical, or 90% identical or more. In other embodiments, the variant, such as a FVIII or hFVIII-BDD protein variant, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to a reference sequence, for example wild-type FVIII protein with or without the B domain.

[0203] To determine identity, if FVIII (eg, a CpG-reduced nucleic acid encoding FVIII) retains the B domain, identity is preferably compared to wild-type FVIII. If FVIII (eg, a CpG-reduced nucleic acid encoding hFVIII-BDD) has a B-domain deletion, identity is preferably compared to wild-type FVIII, which also has a B-domain deletion.

[0204] The terms "homologous" or "homology" mean that two or more units in question have at least partial identity in a given region or part. "Parts, regions or domains" with homology or identity means that part of two or more units in question have homology or are the same. Thus, when two sequences are identical in one or more sequence regions, they have identity in those regions. "Substantial homology" means that the molecule is structurally or functionally conserved such that it has, or is predicted to have, at least in part, the structure or function of one or more structures or functions (e.g., biological function or activity) of reference molecule or the relevant/corresponding region or part of a reference molecule with which it has homology.

[0205] The degree of identity (homology) or "percentage identity" between two sequences can be determined using a computer program and/or a mathematical algorithm. In the context of the present invention, comparisons of nucleic acid sequences are performed using the GCG Wisconsin Package version 9.1 released by Genetics Computer Group, Madison, Wisconsin. For convenience, the default parameters (gap penalty=12, skip continuation penalty=4) specified in this program are intended to be used in the present invention for comparing sequence identities. An alternative is the Blastn 2.0 program provided by the US National Center for Biotechnology Information (available on the Internet at ncbi.nlm.nih.gov/blast/; Altschul et al., 1990, J Mol Biol 215:403-410), which uses alignment with blanks, with default parameters, can be used to determine the level of identity and similarity between nucleic acid sequences and amino acid sequences. Polypeptide sequence comparisons typically use the BLASTP algorithm in combination with a substitution matrix such as PAM100, PAM 250, BLOSUM 62, or BLOSUM 50. The sequence comparison programs FASTA (e.g., FASTA2 and FASTA3) and SSEARCH are also used to quantify the degree 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 have also been developed to quantify the structural similarity of proteins using topological mapping based on the Delaunay method (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

[0206] Nucleic acid molecules, expression vectors (eg, vector genomes), plasmids, including nucleic acids and nucleic acid variants encoding FVIII or hFVIII-BDD according to the invention, can be produced using recombinant DNA technology methods. The availability of nucleotide sequence information makes it possible to obtain isolated nucleic acid molecules according to the invention by various methods. For example, CpG-reduced nucleic acid variants encoding FVIII or hFVIII-BDD can be produced using various standard cloning techniques, recombinant DNA technology, cell expression, or in vitro translation and chemical synthesis techniques. The purity of polynucleotides can be determined by sequencing, gel electrophoresis, etc. For example, nucleic acids can be isolated using hybridization techniques or computer database screening. Such methods include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) screening antibodies to detect polypeptides that have common structural features, for example, using an expression library; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the nucleic acid sequence of interest; (4) computer search for related sequences in sequence databases; and (5) differential library screening with nucleic acid subtraction.

[0207] The nucleic acids of the invention may be maintained as DNA in any convenient cloning vector. In one embodiment, the clones are maintained in a plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, CA), which is propagated in a suitable E. coli host cell. Also, the nucleic acids may be supported in a vector suitable for expression in mammalian cells. In cases where post-translational modification affects coagulation function, the nucleic acid molecule may be expressed in mammalian cells.

[0208] Nucleic acids and nucleic acid variants encoding FVIII or hFVIII-BDD include cDNA, genomic DNA, RNA, and fragments thereof, which may be single or double stranded. Thus, the present invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one nucleic acid sequence according to the invention. Such oligonucleotides can be used as probes to detect the expression of FVIII or hFVIII-BDD.

[0209] Vectors, such as the vectors described herein (rAAV), optionally include regulatory elements necessary for DNA expression in a host cell, arranged to allow 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 defined herein and known to one skilled in the art.

[0210] The methods and applications of the invention include delivery (transduction) of a nucleic acid (transgene) into host cells, including dividing and/or non-dividing cells. The nucleic acids, rAAV vector, methods, uses and pharmaceutical compositions of the invention can also be used in a method of delivering, administering or providing FVIII or hFVIII-BDD to a patient, as a method of treatment. In this way, the nucleic acid is transcribed and the protein can be synthesized in vivo in the patient. A patient may benefit from or require FVIII or hFVIII-BDD because the patient has FVIII deficiency, or because production of FVIII in the patient may produce some therapeutic effect as a treatment or otherwise.

[0211] rAAV vectors comprising a genome with a nucleic acid or nucleic acid variant encoding FVIII or hFVIII-BDD provide treatment for inherited diseases, such as FVIII deficiency. In deficiency diseases, gene transfer can be used to introduce a normal gene into damaged tissues for replacement therapy, as well as to create animal models of the disease using antisense mutations. In the case of imbalanced pathological conditions, gene transfer can be used to create the pathological condition in a model system, which can then be used in attempts to counteract the pathological condition. It is also possible to use site-specific integration of nucleic acid sequences to correct disorders.

[0212] In certain embodiments, rAAV vectors comprising a genome with a nucleic acid or nucleic acid variant encoding FVIII or hFVIII-BDD can be used, for example, as therapeutic and/or prophylactic agents (protein or nucleic acid) that modulate the cascade blood clotting, or as a transgene in a gene. For example, the encoded FVIII or hFVIII-BDD may have similar coagulating activity as wild-type FVIII or altered coagulating activity compared to wild-type FVII. Cell-based strategies allow continuous expression of FVIII or hFVIII-BDD in hemophilia patients. As disclosed in the present invention, certain modifications of FVIII molecules (nucleic acid and protein) result in increased expression at the nucleic acid level, increasing coagulating activity, thereby effectively improving hemostasis.

[0213] Administration of rAAV vectors encoding FVIII or hFVIII-BDD to a patient results in expression of the FVIII or hFVIII-BDD protein, which serves to alter the coagulation cascade. In accordance with the invention, expression of the FVIII or hFVIII-BDD protein as described in the present invention, or a functional fragment, enhances hemostasis.

[0214] rAAV vectors can be administered alone or in combination with other molecules useful for correcting hemostasis. According to the invention, rAAV vectors or a combination of therapeutic agents can be administered to a patient alone or in pharmaceutically acceptable or biocompatible compositions.

[0215] Adeno-associated viruses (AAVs) are members of the parvovirus family. AAVs are viruses that can be used as vectors for gene therapy because they can enter cells and introduce nucleic acid/genetic material, and the nucleic acid/genetic material can be stably maintained in the cells. In addition, these viruses can introduce nucleic acid/genetic material, for example, into certain areas. Because AAVs are not associated with pathogenic disease in humans, rAAV vectors are capable of delivering heterologous polynucleotide sequences (eg, therapeutic proteins and agents) to patients without causing significant AAV pathogenesis or disease.

[0216] rAAV vectors have many desirable properties for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with such vectors also did not demonstrate long-term toxicity, with minimal or undetectable immune responses. AAVs are known to infect a variety of cell types in vivo and in vitro via receptor-mediated endocytosis or transcytosis. Such vector systems have been studied in humans by targeting the retinal epithelium, liver, skeletal muscle, respiratory tract, brain, joints, and hematopoietic stem cells.

[0217] It may be desirable to introduce an rAAV vector that can provide, for example, multiple copies of a desired gene and therefore a larger amount of that gene product. Improved rAAV vectors and methods for preparing such 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), describing the technology used to obtain the vector for clinical use at the Children's Hospital of Philadelphia.

[0218] Thus, the invention provides methods for viral delivery of FVIII or hFVIII-BDD via the rAAV vector. For example, a recombinant AAV vector may include a nucleic acid variant encoding FVIII, wherein the encoded FVIII protein optionally has a B domain deletion. Delivery or administration of the rAAV vector to a patient (eg, a mammal) provides FVIII in this manner to the patient, such as a mammal (eg, a human).

[0219] Direct delivery of vectors or ex vivo transduction of human cells followed by infusion into the body will induce expression of FVIII or hFVIII-BDD, thereby providing a beneficial therapeutic effect on hemostasis. Within the scope of the invention, such administration of Factor VIII described in the present invention improves the procoagulant activity.

[0220] AAV vectors generally do not include viral genes associated with pathogenesis. Such vectors typically delete one or more wild-type AAV genes, in whole or in part, such as the rep and/or cap genes, but retain at least one functional flanking ITR sequence necessary for rescue, replication, and packaging of the recombinant vector into AAV vector particle. For example, accordingly, only the main parts of the vector, such as ITR elements, are included. Therefore, the AAV vector genome will include sequences required in cis configuration for replication and packaging (e.g. functional ITR sequences)

[0221] The recombinant AAV vector, as well as methods and uses thereof, include any viral strain or serotype. As a non-limiting example, the recombinant AAV vector can be based on 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 may be based on the same strain or serotype (or subgroup or variant), or may differ from each other. As a non-limiting example, a recombinant AAV vector based on the genome of one serotype may be identical to one or more capsid proteins that package the vector. In addition, the recombinant AAV vector genome may be based on an AAV genome serotype (eg, AAV2) that is different from one or more AAV capsid proteins that package the vector. For example, the AAV vector genome may be based on AAV2, while at least one of the three capsid proteins may be AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV -2i8 or their variant, for example.

[0222] In certain 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 (eg, capsid variants such as insertions, additions, substitutions and deletions of amino acids), 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 (US patent 9,169,299, which discloses LK01, LK02, LK03, etc.).

[0223] AAV variants include capsid variants and chimeras AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, and AAV-2i8. Thus, AAV vectors and AAV variants (eg, capsid variants) that include (encapsulate or package) a nucleic acid or nucleic acid variant encoding FVIII or hFVIII-BDD.

[0224] The serotypes of AAV and AAV variants (e.g., capsid variants) (e.g., VP1, VP2, and/or VP3 sequences) may or may not be different from other AAV serotypes including, for example, AAV1-AAV12, Rh74, or Rh10 (e.g. , differ from the VP1, VP2 and/or VP3 sequences of any of the serotypes AAV1-AAV12, Rh74 or Rh10).

[0225] As used herein, the term “serotype” is a variant used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic difference is determined based on the lack of cross-reactivity between antibodies to one AAV versus another AAV. Such differences in cross-reactivity are usually due to differences in the capsid protein/antigenic determinant sequences (eg, due to differences in the VP1, VP2 and/or VP3 sequences of AAV serotypes). Although it is possible that AAV variants, including capsid variants, may not be serologically different from the reference AAV or another AAV serotype, they differ by at least one nucleotide or amino acid residue compared to the reference or other AAV serotype.

[0226] According to the traditional definition, a serotype means that the virus of interest has been tested against sera specific for all existing and characterized serotypes for neutralizing activity, and no antibodies have been detected that neutralize the virus of interest. As more natural virus isolates are discovered and/or viruses with mutant capsids are produced, serological differences may or may not be present with any of the currently extant serotypes. Thus, in cases where a new virus (eg, AAV) is not serologically distinct, the new virus (eg, AAV) will be a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity on mutant viruses with modifications to the capsid sequence must be performed to determine whether they are of a different serotype according to the traditional definition of serotype. Thus, for the sake of convenience and to avoid repetition, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) and viruses (e.g., AAV) that are not serologically distinct, which may be within a subgroup or be a variant of a given serotype.

[0227] Thus, AAV vectors include gene/protein sequences identical to gene/protein sequences characteristic of a particular serotype. As used herein, the term "AAV1-related AAV vector" refers to one or more AAV proteins (e.g., VP1, VP2 and/or VP3 sequences) that have substantial sequence identity with one or more polynucleotides or polypeptide sequences that include AAV1. Likewise, “AAV8-related AAV vector” refers to one or more AAV proteins (eg, VP1, VP2 and/or VP3 sequences) that have substantial sequence identity to one or more polynucleotides or polypeptide sequences that include AAV8. “AAV-Rh74-linked AAV vector” refers to one or more AAV proteins (eg, VP1, VP2 and/or VP3 sequences) that have substantial sequence identity with one or more polynucleotides or polypeptide sequences that contain AAV-Rh74. Such AAV vectors related to another serotype, for example AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8, may therefore contain one or more defined sequences from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 and AAV-2i8, but may show significant sequence identity to one or more genes and/or proteins, and/or have one or more functional features of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 (for example, such as tropism for cells/tissues). Exemplary non-limiting AAV variants include capsid variants of any of VP1, VP2 and/or VP3.

[0228] In various embodiments, the AAV vector associated with the reference serotype contains a polynucleotide, polypeptide, or a subsequence thereof comprising or consisting of a sequence identity that is at least 80% or greater (e.g., 85%, 90%, 95% identical) , 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 (eg, such as ITR or VP1, VP2 and/or VP3 sequences).

[0229] The compositions, methods and applications 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 different from and not identical to known AAV genes or proteins, such as the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 genes or proteins , AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8, etc. In one embodiment, an AAV polypeptide or subsequence thereof includes or consists of a sequence that is at least 75% identical or more, for example, 80% identical, 85% identical, 85% identical, 87% identical, 88% identical, 89% identical, 90% identical, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5 %, etc., up to 100%, of any AAV reference sequence or subsequence thereof, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV -2i8 (eg capsid VP1, VP2 and/or VP3 or ITR). In some embodiments, the AAV variant has 1, 2, 3, 4, 5, 5-10, 10-15, 15-20, or more amino acid substitutions.

[0230] 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 methods that are known to one skilled in the art to include one or more nucleic acid sequences (transgenes) flanked by one or more functional AAV ITR sequences.

[0231] In one embodiment, an rAAV vector comprising a nucleic acid or variant encoding FVIII or hFVIII-BDD can be administered to a patient by infusion in a biocompatible vehicle, for example, by intravenous injection. rAAV vectors may optionally be encapsulated in liposomes or mixed with other phospholipids or micelles to increase the stability of the molecule.

[0232] In accordance with the invention, rAAV vectors can be administered alone or in combination with other agents known to modulate hemostasis (eg, Factor V, Factor Va, or derivatives thereof).

[0233] Thus, rAAV vectors and other compositions, substances, drugs, biologicals (proteins) can be included in pharmaceutical compositions. Such pharmaceutical compositions may be used, among other things, for administration and delivery to a patient in vivo or ex vivo .

[0234] In certain embodiments, the 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 person receiving the composition and that can be administered without causing nonspecific toxicity.

[0235] As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" mean a biologically acceptable composition, gaseous, liquid or solid, or a mixture thereof, suitable for one or more routes of administration, delivery or in vivo contact. A "pharmaceutically acceptable" or "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, eg, such material can be administered to a patient without exhibiting significant adverse biological effects. Thus, such a pharmaceutical composition can be used, for example, when administering a nucleic acid, vector, viral particle or protein to a patient.

[0236] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, sodium chloride solution, glycerin, sugar and ethanol. Pharmaceutically acceptable salts may also be included therein, for example, salts of mineral acids such as hydrochlorides, hydrobromides, phosphates, sulfates and the like; and salts of organic acids such as acetates, propionates, malonates, benzoates and the like. In addition, excipients such as wetting or emulsifying agents, pH buffering agents, and the like may be present in such carriers.

[0237] The pharmaceutical composition may be in salt form and may be prepared with various acids including, but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts may have higher solubility in aqueous or other protic solvents than the corresponding free base forms. In other cases, the preparation may be a lyophilized powder that may contain any of the following: 1-50 mM histidine, 0.1-2% sucrose and 2-7% mannitol, at pH 4.5-5.5, which is combined with a buffer before use.

[0238] Pharmaceutical compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (for example, oil-in-water or water-in-oil), suspensions, syrups, tinctures, dispersion and suspension media, coatings, isotonic and enhancing or absorption 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), microspheres, powder, granules and crystals. Additional active compounds (eg, preservatives, antibacterial, antiviral and antifungal agents) may also be included in the compositions.

[0239] Pharmaceutical compositions may be formulated in a manner that is compatible with a particular route of administration or delivery as described herein or known to one of ordinary skill in the art. Thus, pharmaceutical compositions include carriers, diluents or excipients suitable for administration by various routes.

[0240] Compositions suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are generally sterile and may 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 that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

[0241] In addition, suspensions of the active compounds can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or carriers 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 substances that increase the solubility of the compounds to formulate highly concentrated solutions.

[0242] 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, polyethylene glycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylated alcohols and polyoxyethylated fatty acid esters. Adjuvants include, for example, surfactants such as soy lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

[0243] Once the pharmaceutical compositions are received, they may be placed in a suitable container and labeled with medical information. Such labeling may include the amount, frequency, and route of administration.

[0244] Pharmaceutical compositions and delivery systems suitable for the compositions, methods and uses of the invention are known in the art (see, for example, Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton , PA; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano , ed., Oxford, N.Y., pp. 253-315).

[0245] "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 immunosuppressive agents, such as a drug), treatments, protocols or therapeutic regimens, a detectable response of any duration (long-term or short-term), an expected or desired treatment outcome, or a beneficial effect in a patient to any measurable or detectable extent or for any duration (e.g., minutes, hours , days, months, years or complete cure).

[0246] Dosages may vary and depend on the type, onset, progression, severity, frequency, duration or likelihood of occurrence of the disease being treated, the desired clinical outcome, previous or concurrent treatment, general health status, age, sex, race or the immunological status of the patient, as well as other factors that will be taken into account by the specialist. The dosage amount, number, frequency or duration may be proportionally increased or decreased depending on any unwanted side effects, complications or other risk factors of the treatment or therapy and the condition of the patient. One skilled in the art will be aware of factors that may affect the dosage and time required to provide an amount sufficient to provide a therapeutic or prophylactic effect.

[0247] The dose to achieve a therapeutic effect, for example, the dose in vector genomes/per kilogram of body weight (vg/kg), will vary depending on several factors including, but not limited to: the route of administration, the level of expression of the heterologous polynucleotide required for achieving a therapeutic effect, the specific disease being treated, any immune response of the body against the viral vector, the immune response of the body to the heterologous polynucleotide or expression product (protein), and the stability of the expressed protein. One of ordinary skill in the art will be able to determine the dose range of the rAAV/genome vector for treating a patient having a particular disease or disorder based on the above factors as well as other factors.

[0248] Typically, doses range from at least 1×10 ⁸ or more, for example, 1×10 ⁹ , 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 ¹² , 1×10 ¹³ or 1×10 ¹⁴ or more, vector genomes per kilogram (vg/kg) of patient body weight, to achieve a therapeutic effect. The effective dose of AAV was in the range of 1x10 ¹⁰ -1x10 ¹¹ in mice and 1x10 ¹² -1x10 ¹³ in dogs. Doses may be smaller, for example, less than 6 x 10 ¹² vector genomes per kilogram (vg/kg). In particular, the dose is 5×10 ¹¹ vg/kg or 1×10 ¹² vg/kg.

[0249] Using hemophilia B as an example, it is generally believed that in order to achieve a therapeutic effect from a change from a severe disease phenotype to a mild phenotype, a coagulation factor concentration is required that exceeds 1% of the factor concentration found in a healthy individual. The severe phenotype is characterized by joint damage and life-threatening bleeding. It is believed that the transition from a moderate to a mild phenotype requires that the clotting factor concentration exceed 5% of the normal value.

[0250] FVIII levels in healthy individuals are approximately 150-200 ng/mL in plasma, but may be lower (e.g., in the range of approximately 100-150 ng/mL) or higher (e.g., in the range of approximately 200-300 ng/mL ), and are considered normal when determining functional coagulation, for example, using a one-stage coagulation assay with the determination of activated partial thromboplastin time (aPTT). Thus, a therapeutic effect can be achieved when FVIII or hFVIII-BDD is expressed when the total amount of FVIII in the patient/person exceeds 1% of the FVIII level present in healthy patients/humans, e.g. 1% of 100-300 ng/ml .

[0251] Doses of the rAAV vector may be at a level generally at the lower end of the dose range at which there is no significant immune response against FVIII or the AAV vector. In particular, the dose is up to but less than 6×10 ¹² vg/kg, for example, from about 5×10 ¹¹ to about 5×10 ¹² vg/kg, or more specifically, about 5×10 ¹¹ vg/kg or approximately 1×10 ¹² vg/kg.

[0252] Doses in an "effective amount" or "sufficient amount" for treatment (e.g., to alleviate or achieve a therapeutic effect or improvement) are generally effective in producing a response to one, many, or all of the adverse symptoms, effects, or complications of a disease, one or a plurality of undesirable symptoms, disorders, diseases, pathologies or complications, such as those caused by or associated with a disease, to a measurable extent, although a satisfactory result of treatment is the reduction, reduction, inhibition, suppression, limitation or control of the progression or worsening of the disease.

[0253] An effective amount or sufficient amount may, but need not necessarily, be provided in a single administration, multiple administrations may be required, and administration may, but need not be, provided alone or in combination with another composition (e.g., agent) , treatment, protocol or therapeutic regimen. For example, the amount may be proportionally increased according to the needs of the patient, the type, condition and severity of the disease being treated, or the side effects (if any) of the treatment. In addition, an effective amount or sufficient amount need not be effective or sufficient when administered in single or multiple doses without a second composition (e.g., another drug or substance), treatment, protocol, or therapeutic regimen because additional doses, amounts, or durations above and beyond such dosages or additional compositions (eg, drugs or substances), treatments, protocols or therapeutic regimens may be included to be considered effective or sufficient for a given patient. Amounts considered effective also include amounts that result in a reduction in the use of another treatment, treatment regimen or protocol, such as the administration of a recombinant coagulation factor protein (eg, FVIII) for the treatment of a bleeding disorder (eg, hemophilia A).

[0254] Thus, the methods and applications of the invention also include, but are not limited to, methods and applications that result in a reduction in the need or use of another compound, substance, drug, therapeutic regimen, treatment protocol, process or treatment agent. For example, with respect to a blood clotting disorder, the method or use of the invention is therapeutic if the patient requires less frequent administration or dose reduction, or discontinuation of the recombinant coagulation factor to replace the deficient or defective (pathological or mutant) endogenous coagulation factor. Thus, the invention relates to methods and uses for reducing the need for or use of other treatment or therapy.

[0255] An effective amount or sufficient amount need not be effective in any patient being treated, nor in the majority of patients being treated in a given group or population. An effective amount or sufficient amount means effectiveness or sufficiency in a particular patient rather than in a group or general population. As is typical with such modalities, some patients exhibit greater or lesser response, or no response, to a given treatment or application.

[0256] The term “relief” means a detectable or measurable reduction in the severity of a disease or symptom in a patient or an improvement in the baseline cellular response. Detectable or measurable reduction in severity includes patient or objective reduction, reduction, inhibition, suppression, limitation or control of the occurrence, frequency, severity, progression or duration of a disease or complication caused by or associated with a disease, or alleviation of a symptom or underlying cause or consequence of a disease, or elimination of the disease. In the case of HemA, an effective amount will be an amount that reduces the frequency or severity of acute bleeding episodes in a patient, for example, or an amount that reduces clotting time, for example, as measured in a blood clotting assay.

[0257] Thus, the pharmaceutical compositions of the invention include compositions wherein the active substances are contained in an effective amount to achieve the intended therapeutic purpose. Determination of a therapeutically effective dose is well known to the skilled practitioner using the techniques and principles presented in the invention.

[0258] Therapeutic dosages will depend on, among other factors, the age and general condition of the patient, the severity of the aberrant coagulation phenotype, and the effectiveness of regulatory sequences regulating FVIII expression levels. Thus, the therapeutically effective amount in a person falls within a relatively wide range, which can be determined by a health care provider based on the individual patient's response to FVIII vector-based treatment. Such doses may be administered alone or in combination with an immunosuppressant or drug.

[0259] Compositions, such as pharmaceutical compositions, can be delivered to a patient to provide factor VIII (FVIII) production. In a specific embodiment, pharmaceutical compositions comprising sufficient genetic material to cause the recipient to produce a therapeutically effective amount of FVIII polypeptide can affect hemostasis in a patient.

[0260] The compositions may be administered separately. In some embodiments, the recombinant AAV particle provides a therapeutic effect without an immunosuppressive agent. The therapeutic effect of FVIII does not necessarily last for a period of time, for example, 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25, 25-30 or 30-50 days or longer, for example 50-75, 75-100, 100-150, 150-200 days or longer, without administration of an immunosuppressive agent. Thus, in some embodiments, the CpG rAAV viral particle provides a therapeutic effect without administration of an immunosuppressive agent for a period of time.

[0261] The compositions may be administered in combination with at least one other agent. In some embodiments, the rAAV vector is administered in combination with one or more immunosuppressive agents before, substantially at the same time as, or after administration of the rAAV vector. In some embodiments, for example, after 1-12, 12-24, or 24-48 hours, or after 2-4, 4-6, 6-8, 8-10, 10-14, 14-20, 20-25 , 25-30, 30-50 or more than 50 days after administration of the rAAV vector. Such administration of immunosuppressive agents is carried out over a period of time after administration of the rAAV vector if there is a decrease in FVIII after initial expression levels over a period of time, for example, after 20-25, 25-30, 30-50, 50-75, 75-100 , 100-150, 150-200 or more than 200 days after rAAV vector.

[0262] In some embodiments, the immunosuppressant agent is an anti-inflammatory agent. In some embodiments, the immunosuppressive agent is a steroid. In some embodiments, the immunosuppressant agent is cyclosporine (eg, cyclosporine A), mycophenolate, rituximab, or a derivative thereof. Additional specific agents include a stabilizing compound.

[0263] The compositions may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, sodium chloride solution, buffered sodium chloride solution, dextrose solution, and water. The compositions may be administered to a patient alone or in combination with other agents (eg, cofactors) that affect hemostasis.

[0264] Methods for preparing adenoviral vectors and administering them to patients have been described in US Patents 5,998,205; 6,228,646; 6,093,699; 6,100,242; and International Applications WO 94/17810 and WO 94/23744, which are incorporated herein by reference in their entirety. In particular, for example, AAV vectors are used to deliver factor VIII (FVIII), encoded by CpG-reduced nucleic acid variants, to a patient.

[0265] The methods and uses of the invention include delivery and administration systemically, regionally or locally, or by any route, such as injection or infusion. In vivo delivery of pharmaceutical compositions can generally be accomplished by injection using a conventional syringe, although other delivery methods such as convection delivery are possible (see, for example, US Pat. No. 5,720,720). For example, the compositions may be delivered subcutaneously, dermal, intradermal, intrathecal, intraorbital, intramucosal, intraperitoneal, intravenous, intrapleural, intraarterial, oral, intrahepatic, portal vein, or intramuscular. Other routes of administration include oral and pulmonary administration, suppositories and transdermal applications. A clinician specializing in the treatment of patients with bleeding disorders may determine the optimal route of administration of adenovirus-based vectors based on multiple criteria including, but not limited to, the patient's condition and the goal of treatment (eg, improvement or reduction of blood clotting).

[0266] The methods and uses of the invention can be combined with any compound, substance, drug, treatment or other therapeutic regimen or protocol having the desired therapeutic, beneficial, additive, synergistic or complementary activity or effect. Examples of combination compositions and treatments include a second active ingredient such as biologics (proteins), agents (eg, immunosuppressants), and drugs. Such biological products (proteins), agents, drugs, treatments and therapies can be administered or used before, substantially simultaneously with or after any other method or use according to the invention, for example, a therapeutic method for treating a patient with a disease associated with a blood clotting disorder , such as hemophilia A.

[0267] A compound, agent, drug, treatment, or other therapeutic regimen or protocol may be administered as a combination composition or administered separately, for example, in parallel or in order, or sequentially (before or after), relative to the delivery or administration of the nucleic acid, vector , a recombinant vector (eg, rAAV) or a recombinant viral particle. Thus, the invention provides combinations in which the method or use of the invention is carried out in combination with any compound, agent, drug, therapeutic regimen, treatment protocol, process, therapeutic agent or composition provided herein or known to one skilled in the art. The compound, agent, drug, therapeutic regimen, treatment protocol, process, therapeutic agent, or composition may be administered or used before, substantially simultaneously with, or after administration of a nucleic acid, vector, recombinant vector (eg, rAAV), or recombinant viral particle to a patient according to invention.

[0268] The invention can be used in animals, including human and veterinary applications. Therefore, suitable patients include mammals such as humans as well as non-human mammals. The term "patient" refers to an animal, usually a mammal such as a human, non-human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (birds such as chickens) and ducks, horses, cows, goats, sheep, pigs) and experimental animals (mice, rats, rabbits, guinea pigs). Human patients include fetal, neonatal, infant, pediatric and adolescent, and adult patients. Patients include animal models of diseases, such as mice and other animals, models of diseases associated with bleeding disorders, such as hemophilia A, and other diseases known to those skilled in the art.

[0269] Patients suitable for treatment in accordance with the invention include patients having or at risk of insufficient production or deficiency of a functional gene product (eg, FVIII protein), or with production of an aberrant, incompletely functional or nonfunctional gene product (eg, FVIII protein ), which can lead to illness. Patients suitable for treatment in accordance with the invention also include patients who have, or are at risk for, the production of an aberrant or defective (mutant) gene product (protein) that results in a disease, whereby there is a decrease in the amount, expression or function of the aberrant or defective (mutant) gene product. the gene product (protein) may result in the treatment of a disease or the reduction of one or more symptoms or amelioration of the disease. Thus, target patients include patients who have aberrant, deficient, or lacking production of coagulation factor, such as patients with hemophilia (eg, hemophilia A).

[0270] Patients may be tested for immune response, such as the presence of anti-AAV antibodies. Candidate hemophilia patients may be screened before treatment according to the method of the invention. Patients may also be tested for anti-AAV antibodies after treatment and may not necessarily be monitored for a period of time after treatment. Patients with antibodies may receive an immunosuppressant or administration of one or more additional amounts of the AAV vector.

[0271] Patients suitable for treatment in accordance with the invention also include patients who have or are at risk of producing anti-AAV antibodies. rAAV vectors can be administered or delivered to such patients using several techniques. For example, an empty AAV capsid (i.e., an AAV that lacks FVIII nucleic acid) can be delivered to bind to AAV antibodies in a patient, allowing the patient's cells to be transformed with an AAV vector carrying a nucleic acid or nucleic acid variant encoding FVIII and FVIII-BDD .

[0272] The ratio of empty capsids to rAAV vector can be from about 2:1 to about 50:1, or from about 2:1 to about 25:1, or from about 2:1 to about 20:1, or from about 2: 1 to about 15:1, or about 2:1 to about 10:1. Ratios can also be approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0273] Amounts of empty AAV capsid to be administered can be calibrated based on the amount (titer) of AAV antibodies produced in a particular patient. The empty capsid may be of any AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8.

[0274] Alternatively or additionally, the AAV vector can be delivered by direct intramuscular injection (eg, into one or more slow-twitch muscle fibers). In another alternative, a femoral artery catheter can be used to deliver AAV vectors to the liver via the hepatic artery. Non-surgical techniques such as endoscopic retrograde cholangiopancreatography (ERCP) can also be used to deliver AAV vectors directly to the liver, thereby bypassing the bloodstream and avoiding interaction with anti-AAV antibodies. Other ductal systems, such as the submandibular gland ducts, can also be used as a gateway to deliver AAV vectors to a patient who is beginning to develop or pre-existing anti-AAV antibodies.

[0275] Administration or in vivo delivery to a patient may be performed prior to the development of an undesirable symptom, condition, complication, etc., caused by or associated with a disease. For example, screening (eg, genetic) can be used to identify such patients as candidates for the compositions, methods and uses of the invention. Thus, such patients include patients who screen positive for an insufficient amount or deficiency of a functional gene product (eg, FVIII protein), or patients who produce an aberrant, incompletely functional or nonfunctional gene product (eg, FVIII protein).

[0276] Administration or in vivo delivery to a patient in accordance with the methods and uses of the invention as disclosed herein may occur 1-2, 2-4, 4-12, 12-24, or 24-72 hours after the patient has been identified with a disease to be treated, has experienced one or more symptoms of the disease, or has been screened with a positive result as described herein, even if the patient does not have one or more symptoms of the disease. Of course, the methods and applications of the invention can be carried out 1-7, 7-14, 14-21, 21-48 or more days, months or years after the patient has been identified with the disease to be treated, one or more symptoms of the disease, or the patient has been screened with a positive result as described in the present invention.

[0277] "Unit dosage form" as used in the present invention refers to physically discrete units that are suitable as unitary doses for a patient to be treated, each unit containing a predetermined amount, optionally in combination with a pharmaceutical carrier (excipient, diluent , base or excipient) which, when administered in one or more doses, is calculated to produce the desired effect (eg, prophylactic or therapeutic effect). Unit dosage forms may be, for example, in ampoules and vials, which may include a liquid composition or a freeze-dried or lyophilized composition; a sterile liquid carrier which, for example, may be added prior to administration or in vivo delivery. Individual unit dosage forms may be included in multi-dose kits or containers. Recombinant vector sequences (eg, rAAV), recombinant viral particles, and pharmaceutical compositions thereof may be packaged in one or multiple unit dosage forms for ease of administration and uniformity of dosing.

[0278] Patients may be tested for the amount of FVIII and FVIII-BDD or the activity of FVIII and FVIII-BDD to determine whether such patients are suitable for treatment in accordance with the method of the invention. Candidate hemophilia patients may be tested for the amount or activity of FVIII and FVIII-BDD prior to treatment according to the method of the invention. Patients may also be tested for the amount or activity of FVIII and FVIII-BDD after treatment according to the method of the invention. Such treated patients may be periodically monitored after treatment for the amount of FVIII and FVIII-BDD or the activity of FVIII and FVIII-BDD, for example every 1-4 weeks or 1-6 months.

[0279] Patients may be tested for the presence of one or more liver enzymes, for an adverse response, or to determine whether such patients are suitable for treatment in accordance with the method of the invention. Candidate hemophilia patients may thus be screened for levels of one or more liver enzymes before treatment according to the method of the invention. Patients may also be tested for the amount of one or more liver enzymes after treatment in accordance with the method of the invention. Such treated patients may be monitored periodically after treatment for elevated liver enzymes, for example every 1-4 weeks or 1-6 months.

[0280] Examples of liver enzymes include alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), but other enzymes may also be observed indicative of liver damage. The normal level of these enzymes in the circulation is generally defined as a range having an upper limit, above which the enzyme level is considered elevated and therefore indicates liver damage. The normal range depends in part on the standards used by the clinical laboratory performing the study.

[0281] Patients may be monitored for bleeding episodes to determine whether such patients are eligible or responsive to treatment and/or the level or duration of response. Patients may be monitored for bleeding episodes to determine whether such patients require additional treatment, such as subsequent administration of an AAV vector or administration of an immunosuppressive agent, or more frequent monitoring. Thus, patients with hemophilia can be monitored for bleeding episodes before and after treatment according to the method of the invention. Patients may also be tested for the frequency and severity of bleeding episodes during or after treatment according to the method of the invention.

[0282] The invention relates to kits with packaging material and one or more components therein. The kit typically includes a label or package insert that includes a description of the components or instructions for using the components contained therein in vitro , in vivo , or ex vivo . The kit may contain a group of such components, for example, a nucleic acid, a recombinant vector, a viral (eg, AAV) vector or viral particle and, optionally, a second active ingredient such as another compound, agent, drug or composition.

[0283] A kit refers to a physical structure housing one or more kit components. The packaging material can keep the components sterile and can be made from material commonly used for such purposes (eg, paper, corrugated cardboard, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0284] Labels or package inserts may include identifying information about one or more components, dosage levels, clinical pharmacology of the active substance(s), including mechanism of action, pharmacokinetics, and pharmacodynamics. Labels or inserts may include information identifying the manufacturer, lot number, place and date of manufacture, and expiration dates. Labels or inserts may include information about the disease for which a component of the kit may be used. The labels or inserts may include instructions for a healthcare provider or patient for use of one or more components of the kit in a method, application or protocol of treatment or therapeutic regimen. The instructions may include dosage levels, frequencies or durations, as well as instructions for the practice of any of the methods, uses, treatment protocols or prophylactic or therapeutic regimens described in the present invention.

[0285] Labels or inserts may include information about any effect that the component may cause, such as a prophylactic or therapeutic effect. Labels or package inserts may include information about potential side effects, complications, or reactions, such as warnings to the patient or health care provider regarding situations when a particular composition is not appropriate for use. Undesirable side effects or complications may also occur when the patient is, will be, or is currently taking one or more other drugs that may be incompatible with the composition, or the patient will be or is currently using another treatment protocol or therapeutic regimen that will be incompatible with the composition, and thus the instructions may include information about such incompatibilities.

[0286] Labels or inserts include “printed materials,” such as paper or cardboard, placed separately or attached to a component, kit, or packaging material (e.g., a box), or attached to a vial, tube, or vial containing a kit component. The labels or inserts may further include a machine-readable medium such as a bar code label, a disk, an optical disk such as a CD or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage device such as RAM and ROM, or their hybrids, such as magnetic/optical storage devices, flash drives or memory cards.

[0287] Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly known to those skilled in the art to which the present invention relates. Although methods and materials similar or equivalent to those described in the present invention may be used in the practice or testing of the present invention, suitable methods and materials are described in the present invention.

[0288] All patents, patent applications, publications and other sources, GenBank entries and ATCC entries cited in the present invention are incorporated by reference in their entirety. In the event of a conflict, this description, including definitions, will control.

[0289] Various terms relating to the biological molecules of the invention are used above and throughout the specification and claims.

[0290] All features disclosed in the present invention can be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, the disclosed features (e.g., CpG reduced nucleic acid variants encoding FVIII, vector, plasmid, expression/recombinant vector sequence (e.g., rAAV), or recombinant virus particle) are examples of the same or similar kind functions.

[0291] When used in the present invention, singular forms include plural forms unless the context clearly requires otherwise. Thus, for example, a reference to a “nucleic acid” includes a plurality of such nucleic acids, a reference to a “vector” includes a plurality of such vectors, and a reference to a “virus” or “particle” includes a plurality of such viruses/particles.

[0292] As used in the present invention, all numeric values or numeric ranges include integers in such ranges and fractional numbers of values or integers in ranges unless the context clearly requires otherwise. Thus, for illustration purposes, indicating 80% or greater 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., etc.

[0293] A reference to an integer with the phrase greater than (greater than) or less than includes any number greater than or less than the specified numeric value, respectively. So, for example, a reference to less than 100 includes 99, 98, 97, etc., up to one (1); and less than 10 includes 9, 8, 7, etc. to one (1).

[0294] As used in the present invention, all numerical values or ranges include fractions of values and integers in such ranges, as well as fractions of integers in such ranges, unless the context clearly requires otherwise. Thus, for illustration purposes, reference to a numeric 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., etc. The reference to the range 1-50 thus includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 etc., up to 50 inclusive, 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., etc.

[0295] A range series reference includes ranges that combine the boundary values of different ranges in the series. Thus, for illustration purposes, reference is made to a series of ranges such as 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 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.

[0296] The invention is disclosed generally in the present invention using affirmative expressions to describe numerous embodiments and aspects. The invention also, in particular, includes embodiments in which a particular subject, such as substances or materials, method steps and conditions, protocols or procedures, is completely or partially excluded. For example, in some embodiments or aspects of the invention, materials and/or method steps are eliminated. Thus, even though the invention as a whole is not expressed in the present invention, the invention does not include aspects that are not expressly excluded in the present invention, but are nevertheless disclosed in the present invention.

[0297] A number of embodiments of the invention have been described. However, one skilled in the art, without departing from the spirit and scope of the invention, will be able to make various changes and modifications to the invention to adapt it to different applications and conditions. Accordingly, the following examples are intended to illustrate and not to limit the scope of the invention claimed in any way.

EXAMPLE 1

CpG-reduced factor VIII DNA sequences and some vector constructs, plasmid constructs, and AAV vector-producing cell lines .

[0298] 18 different CpG-reduced nucleic acid variants encoding FVIII (SEQ ID NO: 1-18) were prepared and evaluated in expression assays. CpG-reduced human FVIII cDNA constructs were generated with the transthyretin promoter mutant (TTRmut) (SEQ ID NO:22).

[0299] The AAV-SPK-8011 expression cassette has a CpG-reduced FVIII-X07 nucleic acid sequence and an LK03 capsid for packaging. The LK03 capsid has significant homology to AAV3, a non-pathogenic, naturally occurring replication-deficient single-stranded DNA virus.

[0300] Packaging plasmid pLK03 is a 7484 kb plasmid construct that carries the AAV2 Rep and AAV-LK03 Cap genes under the control of the AAV2 p5 promoter, a bacterial origin of replication, and a gene conferring kanamycin resistance to bacterial cells. In this construct, the p5 rep promoter was moved 3′ of the cap gene to reduce the likelihood of generating wild-type or pseudowild-type AAV species and increase vector yield.

[0301] The cloned gene transfer DNA is a gene expression cassette packaged within the AAV-LK03 capsid as a single-stranded genome encoding human coagulation factor VIII (hFVIII) under the control of a liver-specific promoter. The expression plasmid is called pAAV-TTRmut-hFVIII-X07. It was modified by introducing 4 point mutations into the TTR promoter, and the coding region was optimized to increase the expression of human factor VIII. The AAV expression cassette contains the following elements:

- ITR AAV2

- Transthyretin (TTR) promoter: liver-specific transthyretin (TTR) promoter with 4 point mutations increasing gene expression compared to the wild type promoter (Costa et al. 1991)

- Synthetic intron: Derived from the human elongation factor EF-1 alpha gene

- FVIII coding sequence: codon-optimized B-domain deletion sequence coding for human FVIII.

- Rabbit poly-A beta-globin signal sequence (Levitt et al. 1989).

- ITR AAV2

[0302] Three plasmid DNA constructs were used to transfect human embryonic kidney 293 cells to produce the SPK-8011 vector in a process without a helper virus (Matsushita et al. 1998):

- The gene cassette (hFVIII coding sequence and corresponding regulatory elements) was cloned into a plasmid to obtain the vector plasmid pAAV-TTRmut-hFVIII-X07.

- The AAV viral genome (rep and cap) without viral ITR repeats was cloned into a plasmid to produce an AAV packaging plasmid, pLK03, providing the necessary AAV2 rep and AAV-LK03 cap genes in trans configuration for AAV vector packaging. The viral promoter (p5) for the rep gene was moved into a plasmid to prevent the generation of replication-competent AAV by nonhomologous recombination.

- Three genes from adenovirus-2 were cloned into a third plasmid (pCCVC-AD2HP) to obtain the necessary helper virus genes for vector production. Plasmid pCCVC-AD2HPv2 is an 11,832 kb plasmid construct carrying three adenovirus genes, E2A, E4 and VA RNA to provide 'helper' functions for AAV vector replication and encapsidation. Plasmid pCCVC-AD2HPv2 is a derivative of pCCVC-AD2HP in which the 1882 kb DrdI-DrdI restriction fragment containing the AmpR gene and part of the pUC ori sequence was removed and replaced by the DrdI-DrdI fragment from plasmid pAAV2-hRPE65v2 containing the complete Kan ^R gene and part pUC ori sequences.

[0303] The cell substrate used to produce the AAV vector is derived from human embryonic kidney (HEK) 293 cells. The HEK293 cell line is a stable line that is transformed with hydrodynamically fragmented human adenovirus type 5 (Ad5) DNA (Graham et al. 1977). The working cell bank was obtained from the HEK293 Master Cell Bank at the Center for Cellular and Molecular Therapeutics (CCMT) at the Children's Hospital of Philadelphia (CHOP).

EXAMPLE 2

Evaluation of AAV-SPK-8005 and AAV-SPK-8011 vectors (LK03 capsid, FVIII-X07 (SEQ ID NO:7)) in non-human primates (NHPs) .

[0304] FVIII transgene constructs packaged in adeno-associated virus (AAV) vectors were delivered to non-human primates (NHPs). A pilot study and a GLP standard study were conducted.

[0305] Briefly, a dose-finding study was conducted in male cynomolgus monkeys that received a single intravenous infusion of AAV-SPK-8005 or AAV-SPK-8011 (LK03 capsid). hFVIII expression was assessed over 8 weeks. Animal groups and dose levels of each vector (pilot study) are shown in Figure 1.

[0306] The NHPs received intravenous infusion through the saphenous vein of the leg 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 titer of <1:3 before vector administration. This was done to ensure successful hepatic transduction, since even low titers inhibit vector uptake into 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.

[0307] Plasma hFVIII levels were measured using a human protein-specific ELISA in which non-endogenous cynomolgus FVIII was not detected. All animals in the study, with the exception of one macaque in the medium dose cohort, expressed hFVIII after vector delivery. Human factor VIII antigen levels peaked approximately 1 to 2 weeks after vector administration. One week after gene transfer, NNPs transduced with 2-1012 μg/kg AAV-SPK-8005 expressed hFVIII antigen levels of 13.2 ± 3% (mean ± SEM). One week after gene transfer, mean hFVIII levels in two of three animals in the next treatment cohort (5-1012 μg/kg) were 27 ± 0.2%. Human factor VIII was not detected in a third macaque in this cohort at any time point. Upon retesting of the original plasma samples, it was determined that this animal was in fact positive for anti-AAV antibodies and that the initially determined titer of <1:3 was incorrect. Finally, at the highest dose tested, 1,1013 μg/kg, peak hFVIII antigen levels of 54.1 ± 15.6% were observed after AAV infusion.

[0308] Human FVIII expression decreased in approximately one third of the animals at approximately 4 weeks, concomitant with the emergence of hFVIII inhibitory antibodies in these 3 macaques (indicated by ε in Figure 2 ). The development of species-specific antibodies to hFVIII has been previously described in non-human primates and is likely due to differences in several amino acid residues between the human transgene product and endogenous cynomolgus FVIII (McIntosh, J. et al., Blood 121:3335-44 (2013 )).

[0309] To evaluate the potential for thrombus formation due to continuous expression of human FVIII, D-dimer antigen levels were measured in this study. It should be noted that there are few reports of clinical significance or even normal values of D-dimer antigen levels in cynomolgus monkeys; As a reference value, the normal range of D-dimers in humans is less than 500 ng/mL. Because the animals express endogenous cynomolgus FVIII, production of hFVIII by gene transfer in the liver results in FVIII activity exceeding physiological levels.

[0310] An animal dosed at 5x10 ¹² vg/kg but not expressing human FVIII had a peak of 863 ng/ml two weeks after AAV infusion. The remaining animals did not show a significant increase in D-dimer antigen levels compared to baseline values. Taken together, these results suggest that expression of human FVIII at levels targeted in this study is not associated with an increased risk of thrombosis.

[0311] Four weeks after vector administration, no vector-related changes were observed. Liver function tests showed normal values with minor fluctuations, which did not appear to be related to vector dose as they were present before dosing in most cases ( Figure 3 ).

[0312] D-dimer levels up to week 5 are shown in Figure 4 . One animal in the high dose cohort had a small (577 ng/ml) transient increase in D-dimer levels one week after vector administration when circulating human FVIII peaked at around 100%; D-dimer levels quickly returned to normal after this single increase in measurement. In particular, no correlation was observed between D-dimer levels and hFVIII antigen levels ( Figure 4 , bottom panels).

[0313] For the AAV-SPK-8011(LK03 capsid) vector, in a pilot study, three cohorts of cynomolgus monkeys (n=3) received escalating doses of AAV-SPK-8011 (LK03 capsid) ( ^2x1012 , ^6x1012 , and 2 ×10 ¹³ (vg/kg); Figure 1 ). The GLP study used doses of 3×10 ¹² , 6×10 ¹² and 2×10 ¹³ vg/kg vector (AAV-SPK-8011(LK03 capsid)).

[0314] A total of 11 NCPs were used in each study. The pilot study included an observation period of 10 weeks in the absence of immunosuppression. This was followed by a 12-week immunosuppressive phase, which was included to eliminate anti-hFVIII antibodies that had formed during the first 10 weeks of the study. After this, the animals were observed for another 20 weeks.

[0315] The animals were monitored for clinical observations, body weight, and clinical laboratory parameters (biochemical blood test, complete blood count, blood coagulation, urinalysis). In addition, hFVIII antigen levels, FVIII inhibitory antibodies, and D-dimer levels were assessed during the study.

[0316] Data from the hFVIII antigen pilot study are shown in Figure 6 . Mean hFVIII antigen levels peaked around 2–3 weeks, with 22.3±6.2% of hFVIII observed in the low dose cohort and 61.6±15.7% and 153±58.1% observed in the intermediate and high dose cohorts. dose, respectively, using 150 ng/ml as 100% normal hFVIII antigen level ( Figures 6A-6D ).

[0317] In a GLP toxicology study, hepatic gene transfer by peripheral venous infusion of SPK-8011 also resulted in hFVIII expression in all animals. At the low dose of 3 x 10 ¹² vg/kg, hFVIII antigen levels ranged from 5-40% of normal values, with a mean peak level around 2 weeks after AAV administration of 20.3 ± 11% (mean ± SEM). Mean hFVIII antigen levels in the 6×10 ¹² vg/kg cohort were 40.7±4% of normal values.

[0318] Thus, AAV capsid serotype LK03 effectively transduces NHP hepatocytes in vivo , unlike mouse liver. Despite therapeutic levels of hFVIII observed shortly after gene transfer, levels began to decline around 4 weeks in most animals.

[0319] The humoral response to hFVIII in the plasma of cynomolgus monkeys was measured after administration of AAV-SPK-8011(LK03 capsid). Animals were assessed for IgG antibodies against hFVIII by ELISA at baseline and at the indicated time points.

[0320] The majority of vector-treated animals in both the pilot and GLP studies developed neutralizing antibodies against FVIII, which is expected based on preclinical studies in cynomolgus monkeys as well as other reports (McIntosh, J. et al., Blood 121:3335-44 (2013)). Neutralizing antibodies against human FVIII protein, which typically begin to appear three weeks after AAV infusion in macaques, interfere with the detection of circulating hFVIII antigen. As a result, peak hFVIII antigen levels around 2–3 weeks (i.e., before the appearance of inhibitory anti-hFVIII antibodies) can be used to assess the appropriate initial dose of the vector in human patients. The dose response curves of SPK-8011 in the pilot and GLP studies on NHPs are shown in Figure 7 .

[0321] The FVIII expression levels achieved with AAV-SPK-8011 (LK03 capsid) were compared with the reported FVIII levels achieved with the AAV capsid-based FVIII delivery vectors AAV5 and AAV8. The comparison showed that the levels of FVIII achieved with AAV-SPK-8011 (LK03 capsid) exceeded the reported levels of FVIII delivered by AAV vectors with AAV5 and AAV8 capsids ( Figure 8 ).

EXAMPLE 3

Biodistribution of AAV-LK03 capsid in non-human primates (NHPs) .

[0322] The biodistribution of the AAV-LK03 capsid in non-human primates was assessed in a non-GLP study. Intravenous administration of the encapsulated AAV-LK03 vector encoding human coagulation factor IX (AAV-LK03-hFIX) showed that the two main target tissues were the liver and spleen ( Figure 9 ). Spleen tropism is not a unique characteristic of AAV-LK03. For example, the AAV5 capsid, which has been used in several liver-targeted gene therapy studies (eg, NCT02396342, NCT02082860, NCT02576795) and has been shown to be highly safe, targets the spleen with equal, if not greater, efficacy than the liver in non-liver patients. human primates (Paneda et al. 2013). The SPK-8011 expression cassette uses the murine transthyretin or TTR promoter, which is considered liver-specific (Costa, 1991). To further confirm the liver-specific properties of the promoter, a PCR-based expression assay measured vector-mediated FVIII expression in the liver and spleen of mice following administration of a different AAV vector packaging the same expression cassette as SPK-8011 (i.e. AAV-SPK-8005). As shown in Figure 10 , expression of human factor VIII in the spleen was several orders of magnitude lower than in hepatocytes.

[0323] This is the first clinical trial using AAV-LK03, although studies have been conducted with other AAV vectors, including several for the treatment of hemophilia B (NCT02396342, NCT01620801 NCT00076557, NCT02484092, NCT02618915, NCT00979238, NCT01687608) and one for treatment of hemophilia A (NCT02576795). The study, conducted by St. Jude Children's Research Hospital in collaboration with University College London, used an AAV8 vector carrying a self-complementary genome encoding a codon-optimized human factor IX cDNA, scAAV2/8-LP1-hFIXco. Ten patients treated with vector had stable factor IX levels of 1–6% over an average of 3.2 years, with all participants either discontinuing the study early or reducing their use of prophylactic factor replacement therapy (Nathwani et al. 2014). An AAV5 encapsulated vector encoding human FVIII was used in a clinical trial for the treatment of hemophilia A (NCT02576795). Preliminary data presented in 2016 demonstrate an increase in FVIII activity after gene transfer in several patients ranging from 2-60% with follow-up of up to 16 weeks (BioMarin, April 2016).

EXAMPLE 4

Transduction efficiency with the AAV-LK03 capsid analyzed in vitro .

[0324] Primary hepatocytes obtained from cynomolgus monkeys and humans were transduced with the AAV-LK03 luciferase-expressing vector 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.

[0325] The AAV-LK03 capsid clearly demonstrated significantly higher efficiency in transducing human hepatocytes in culture. In a representative example shown in Figure 11 , LK03 demonstrated approximately 5-fold higher efficiency in transducing human hepatocytes compared to non-human primate hepatocytes in vitro . Importantly, these results persisted across different MOIs and across repeated studies.

EXAMPLE 5

Dose calculations for a human clinical trial

[0326] Based on hFVIII levels observed in non-human primates (NHP), expected FVIII levels in humans were estimated at a proposed starting dose of 5×10 ¹¹ vg/kg. Since different batches of vectors may have slightly different hepatic transduction efficiencies, data from the GLP NHP pilot and toxicology study were used to interpolate a range of FVIII concentrations following administration of 5 x 10 ¹¹ vg/kg. For this analysis, a linear regression model was used ( Figure 12 ), i.e. the relationship between AAV dose and resulting hFVIII expression levels was found to show no significant deviation from linearity ( Table 2 ).

table 2

Pilot GLP Best Fit Values Incline 6.099e-012±7.962e-013 5.170e-012±6.421e-013 Y-intercept when X=0.0 0 0 X-intercept when Y=0.0 0 0 1/tilt angle 1.64E+11 1.934E+11 95% confidence intervals Incline 4.346e-012-7.851e-012 3.756e-012-6.583e-012 Compliance criterion Sy, x 28.93 15.29 Is the slope significantly different from zero? t 7.66 8,051 DF eleven eleven P-value <0.0001 <0.0001 Deviation from zero? Meaningful Meaningful Data Number of X values 4 4 Maximum number of Y repetitions 3 3 Total number of values 12 12 Number of missing values 3 3 Series test Points above the line 2 2 Points below the line 1 1 Number of episodes 2 2 P-value (series test) 0.6667 0.6667 Deviation from linearity Insignificant Insignificant The equation Y=6.099e-012*X-0.0 Y=5.170e-012*X-0.0

[0327] Using the linear regression model shown above, it was estimated that mean FVIII levels with an infusion of SPK-8011 at a dose of 5×10 ¹¹ vg/kg would be approximately 2.6% to 3.0% of normal values. However, this linear regression curve appears to underestimate the actual values observed in animals at low and intermediate doses when the equation in Table 2 is used to back-calculate the expected FVIII expression values at doses of ^2x1012 vg/kg, ^3x1012 vg/kg and 6×10 ¹² vg/kg ( Table 3 ).

Table 3

Pilot GLP Dose FVIII
(interpolation) FVIII
(actual) Interpolation vs. Actual (%) Dose FVIII
(interpolation) FVIII
(actual) Interpolation vs. Actual (%) 2E+12 12.2 22.3 54.8 3E+12 15.5 20.3 76.4 6E+12 36.6 61.6 59.4 6E+12 31.0 40.7 76.2 2E+13 122.0 113.5 107.5 1.2E+13 62.0 56.0 110.8

[0328] It is possible that hFVIII expression may follow a linear dose-response relationship at some vector doses, reaching saturation as AAV vector load increases. The high dose cohort was excluded from the previous analysis, the linear regression curve was re-calculated, and the predicted hFVIII expression levels at the specific SPK-8011 dose of 5×10 ¹¹ vg/kg were re-estimated ( Table 4 and Figure 13 ).

Table 4

Pilot GLP Best Fit Values Incline 6.099e-012±7.962e-013 5.170e-012±6.421e-013 Y-intercept when X=0.0 0 0 X-intercept when Y=0.0 0 0 1/tilt angle 1.64E+11 1.934E+11 95% confidence intervals Incline 4.346e-012-7.851e-012 3.756e-012-6.583e-012 Compliance criterion Sy, x 28.93 15.29 Is the slope significantly different from zero? t 7.66 8,051 DF eleven eleven P-value <0.0001 <0.0001 Deviation from zero? Meaningful Meaningful Data Number of X values 4 4 Maximum number of Y repetitions 3 3 Total number of values 12 12 Number of missing values 12 12 Series test Points above the line 2 2 Points below the line 1 1 Number of episodes 2 2 P-value (series test) 0.6667 0.6667 Deviation from linearity Insignificant Insignificant The equation Y=6.099e-012*X-0.0 Y=5.170e-012*X-0.0

[0329] Based on the linear regression curves shown in Figure 13, mean FVIII levels during SPK-8011 infusion at 5 x 10 ¹¹ vg/kg were estimated to be approximately 3.4% to 5.2% of normal values.

EXAMPLE 6

Human clinical trial design

[0330] Suitability

- Age eligible for inclusion in the study: 18 years and older (adults, elderly)

- Gender eligible for inclusion in the study: men

- Inclusion of healthy volunteers: no

[0331] Criteria : Inclusion criteria:

- Men 18 years of age or older

- Confirmed diagnosis of hemophilia A as documented in their medical history, with plasma FVIII activity levels ≤2% of normal values

- Received >150 dosing days (ED) of FVIII concentrates or cryoprecipitate

- Had >10 bleeding events in the previous 12 months only while receiving therapy as needed and having a baseline FVIII level of 1-2% of normal values

- No previously reported cases of allergic reaction to any FVIII-containing product

- Absence of any measurable inhibitor against factor VIII as assessed by the central laboratory and absence of any previously reported positive laboratory results for inhibitors of the FVIII protein

- Consent to use reliable barrier contraception

[0332] Criteria : Exclusion criteria:

- Confirmed active hepatitis B or C

- Current use of antiviral therapy to treat hepatitis B or C

- Presence of significant primary liver disease

- Confirmed presence in the serum* of HIV-1, or HIV-2 with a CD4 count≤200/ ^mm3 (*participants who are HIV+ and have a stable CD4 count>200/ ^mm3 and an undetectable viral load are eligible to participate in the study),

- Presence of detectable antibodies reacting with the AAV-Spark200 capsid

- Participation in a gene transfer study within the last 52 weeks or in a clinical trial with an investigational drug within the last 12 weeks

EXAMPLE 7

Expected FVIII levels at different doses of AAV-SPK-8011 (LK03 capsid)-hFVIII

[0333] Clinical trial NCT03003533 ('Gene Transfer Study for Hemophilia A') is a first-in-human study of the AAV capsid known as LK03 (SEQ ID NO:27). Studies in non-human primates indicate that increasing doses of AAV-SPK-8011(LK03 capsid)-hFVIII results in increased levels of circulating human FVIII in a dose-dependent manner that, at least for some dose ranges, does not appear to deviate significantly from linearity. Mean steady-state FVIII levels (± standard error of the mean) in the first cohort were approximately 11.7 ± 2.3% of normal values. Given that the n of participants in this dose cohort was two, it is difficult to predict whether the relatively low variability in observed FVIII levels would continue when more participants were enrolled in the study.

[0334] Recent experience with rAAV vectors to mediate hepatic coagulation factor expression using the investigational product rAAV-FIX for the treatment of hemophilia B (NCT02484092) may provide a useful reference material for assessing variability in a larger cohort of patients. Steady-state FIX expression was achieved 12 weeks after rAAV-FIX vector infusion, resulting in an average FIX activity (FIX:C) of approximately 33%. It is important to note that the highest levels of FIX:C were about 79% (patient 9), and the lowest levels were about 14% (patient 7). It should be noted that interpretation of vector activity in patient 7 was complicated by the development of an immune response against the rAAV-FIX vector capsid, which resulted in partial loss of FIX expression before administration of a short course of steroids. However, patient 6, who did not exhibit a cellular immune response, had stable levels of approximately 18%. Thus, the difference between the highest and lowest FIX:C levels in study NCT02484092 was approximately 4-fold. Other clinical studies of AAV for the treatment of hemophilia showed significantly higher variability (Pasi et al. (2017) Thromb Haemost. 117(3):508–518). Table 5 shows the expected mean FVIII levels at various doses of AAV-SPK-8011(LK03 capsid)-hFVIII, indicating a linear dose response. The observed variability in the hemophilia B study was used as a conservative approach to estimate variability in the hemophilia A study.

Table 5

Dose (vg/kg) Proposed minimal expressor Estimated average* Estimated maximum expressor 5.00E+11 6 12 24 1.00E+12 12 24 48 2.00E+12 24 48 96 4.00E+12 48 96 192 6.00E+12 96 192 384 *Actual mean observed in the 5x10 ¹¹ vg/kg cohort.

EXAMPLE 8

Results of a human clinical trial

[0335] A dose escalation study was conducted in twelve men with severe (N=11) or moderately severe (N=1) hemophilia A. Patients ranged in age from 18 to 52 years. Before gene therapy, 8 of 12 patients were receiving prophylactic treatment, and 4 of 12 patients were receiving episodic treatment. Patients were enrolled in one of three dosing cohorts and received an infusion of SPK-8011 (AAV-hFVIII, LK03 capsid) at a dose of ^5x1011 vg/kg (N=2, Patients 1 and 2), ^1x1012 vg/kg kg (N=3, Patients 3, 4 and 6) or 2x10 ¹² vg/kg (N=7, Patients 5 and 7-12).

[0336] Figures 14-28 show data obtained from a dose response study of 12 human patients receiving three different doses of AAV-SPK-8011(LK03 capsid)-hFVIII. FVIII activity values determined in patients are relative to 100% FVIII in normal plasma. Typically, plasma was pooled from a large number (say 50 or 100) of healthy volunteers and FVIII activity in this "normal mixed plasma" was determined to be 100%. Dilutions of this plasma were used to construct a calibration curve of FVIII activity against any assay used to determine FIX levels. This calibration curve was then used to determine the amount or percentage (%) of FVIII in the patient sample using the same assay.

[0337] All vector doses resulted in the expression of levels of FVIII sufficient to prevent bleeding and allow prophylaxis to be discontinued. In 12 patients, a 97% reduction in annual bleeding rate (ABR) and a 97% reduction in annual infusion rate were observed with 3 doses. The data indicate that overall kinetics showed a gradual increase to a steady plateau of FVIII.

[0338] In the first dose cohort, FVIII levels were 14% and 15% at weeks 66 and 51, with no bleeding events, no elevated transaminases, and no steroid use. FVIII expression remained stable throughout the observation period. Data from this low-dose cohort indicate that even moderate FVIII levels of 15% can provide bleeding prevention over a follow-up period of up to 66 weeks.

[0339] In the second dose cohort, FVIII levels were 9%, 26%, and 17% at 33, 46, and 31 weeks postinfusion. The first patient in this dose cohort (Patient 3) received a single dose infusion of clotting factor concentrate for spontaneous hemarthrosis on day 159, and the second patient in this dose cohort (Patient 4) received multiple infusions for traumatic bleeding beginning on day 195. These patients received a tapering course of steroids administered at 12 and 7 weeks after vector infusion, which was caused by a decrease in FVIII levels, with eventual stabilization of FVIII levels. The third patient in this dose cohort (Patient 6) did not bleed and did not receive clotting factor infusions or steroids.

[0340] In the third dose cohort (N=7), five of seven patients currently have FVIII levels of 12% with a range of 16-49%; in these patients, the mean FVIII level starting 12 weeks after vector infusion is 30% and the median is 22%. No bleeding was recorded in these patients starting 4 weeks after vector infusion.

[0341] Separately, five of 7 at a dose of 2×10 ¹² vg/kg AAV-LK03 (FVIII) vector received a course of steroids initiated at time points 6 to 11 weeks after vector infusion due to one or more of the following: decreased FVIII levels, an increase in ALT above the patient's baseline value, or an increase in IFN-γ by ELISPOT assays for the AAV capsid. Initiation of steroids was associated with a decrease in ALT to the normal range and decreased ELISPOT signal in all cases; two of seven patients showed limited success in stabilizing FVIII levels, falling <5%, possibly due to immune responses. In one of them, bleeding was not recorded during the 12 weeks of follow-up after treatment; the other had 4 bleeds during the 37-week post-treatment follow-up.

[0342] Overall, a favorable safety profile was observed, with only two patients experiencing ALT elevations above the upper limit of normal. Ninety-one percent (91%) of patients to date have experienced an ABR≤1 after vector infusion. All patients experienced an increase in FVIII levels after vector infusion, but the limited success in preventing a decrease in FVIII levels in two patients indicates that the addition of prophylactic steroids to the regimen may be warranted.

[0343] Based on hFVIII levels observed in non-NPPs, and taking into account that different batches of vectors may have slightly different activities, average human FVIII levels after infusion of SPK-8011 at a dose of 5 x 10 ¹¹ vg/kg were assumed to be may be approximately 3.4-5.8%, subject to linear extrapolation. FVIII activity in the first patient reached a plateau at approximately 9.15 ± 0.53% of normal values and 13.50 ± 0.50% in the second patient. Thus, the average FVIII activity in the low-dose cohort was approximately 11.3%, which is 2-4 times higher than expected based on studies in non-human primates.

[0344] A significant 2- to 4-fold difference (depending on the linear regression curve used) in the low-dose cohort between expected FVIII levels based on preclinical studies using a phylogenetically related species such as macaques and actual results in humans participating in the study , highlights the limitations of current animal models in determining AAV vector doses for humans. Data indicating the existence of much higher FVIII activity in humans than expected based on FVIII activity in NHPs administered AAV-SPK-8011 (LK03 capsid)-hFVIII was not expected.

[0345] Although there is no universal preclinical model for determining AAV dosing in humans, previous experience using AAV2, AAV8, and AAV-Spk vectors in primates to mediate liver-targeted expression of coagulation factor IX has shown that macaques are a good, but not ideal, model for predicting the effectiveness of the AAV vector in humans. Recently, chimeric “humanized” mice, whose livers were partially populated with human hepatocytes, have become a valuable tool for determining the efficiency of hepatic transduction of various viral capsids. Two independent studies reported that measured transduction in human hepatocytes benefits from this mouse model. It was reported that there was an approximately 10-fold difference in the percentage of transduced human hepatocytes between LK03 and AAV8 (43.3±11% and 3.6±1.1% with LK03 and AAV8 vector infusion, respectively (Lisowski L, et al. Nature 506:382-6 (2014)).

[0346] Ultimately, infusion of SPK-8011 in 12 patients with severe or moderately severe hemophilia A resulted in safe, sustained, dose-dependent FVIII activity associated with a 97% reduction in ABR and a 97% reduction in recombinant FVIII use over a period of up to 66 weeks after gene transfer.

EXAMPLE 9

TTR promoter

[0347] The properties of the transthyretin (TTR) promoter were 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.

TTR promoter with 4 nucleotide mutation (TTRmut), SEQ ID NO:22

GTCTGTCTGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCTAGGCAAGGTTCATATT GACT TAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAGCTCCT

EXAMPLE 10

CpG-reduced transgenic constructs encoding FVIII and examples of AAV capsids .

FVIII encoding nucleic acid variant with reduced CpG content X01 (SEQ ID NO:1)

atgcagattg agctgtctac ctgcttcttc ctgtgcctgc tgaggttctg cttctctgct accaggaggt actacctggg ggctgtggag ctgagctggg attacatgca gtctgacctg ggggagctgc ctgtggatgc caggtttccc cccagggtgc ccaagagctt ccccttcaat acctctgtgg tgtataagaa gaccctgttt gtggagttca ctgatcatct gttcaacatt gctaaaccca ggcccccctg gatggggctg ctgggcccta ccatccaggc tgaggtgtat gacactgtgg tgatcactct gaagaacatg gctagccatc ctgtgtctct gcatgctgtg ggggtgagct actggaaggc ttctgagggg gctgagtatg atgatcagac tagccagagg gagaaggagg atgacaaggt gttccctggg ggctctcaca cctatgtctg gcaggtgctg aaggagaatg gccccatggc ctctgatcct ctgtgtctga cctatagcta cctgagccat gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgtagggag gggagcctgg ccaaggagaa gacccagacc ctgcacaagt tcattctgct gtttgctgtg tttgatgagg gcaagagctg gcattctgaa accaagaaca gcctgatgca ggacagggat gctgcctctg ctagggcctg gcccaagatg cacactgtga atgggtatgt caataggtct ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tgggatgggc accacccctg aggtgcacag catctttctg gagggccaca ccttcc tggt gaggaatcac agacaggcca gcctggagat cagccccatc accttcctga ctgcccagac cctgctgatg gacctgggcc agtttctgct gttctgccac atctctagcc accagcatga tggcatggag gcctatgtga aggtggactc ctgccctgag gagccccagc tgaggatgaa gaataatgag gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gagattgat gatgacaatt ctcccagctt cattcagatc aggtctgtgg ccaagaagca tcccaagacc tgggt gcact acattgctgc tgaggaggag gactgggact atgcccccct ggtgctggcc cctgatgaca ggagctataa gagccagtac ctgaataatg gcccccagag gattgggagg aagtataaga aggtgaggtt catggcctat actgatgaaa ccttcaagac cgagaggcc atccagcatg agtctgggat cctggggccc ctg ctgtatg gggaggtggg ggacaccctg ctgatcatct tcaagaacca ggccagcagg ccctacaaca tctaccctca tggcatcact gatgtgaggc ctctgtacag cagaaggctg cccaaggggg tgaagcatct gaaggacttc cccattctgc ctggggagat tttcaagtac aagtggactg tgactgtgga ggatggccca accaagtctg accctaggtg cctgactagg tactacagca gctttgtgaa tatggagagg gacctggcct ctggcct gat tggccccctg ctgatctgct acaaggagtc tgtggatcag aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag aacaggagct ggtacctgac tgagaacatt cagaggtttc tgcccaaccc tgctggggtg cagctggagg accctgaatt ccaggcct ct aacatcatgc acagcattaa tggctatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta cattctgagc attggggccc agactgactt cctgtctgtg ttcttctctg gctacacctt taagcacaag atggtgtatg aggataccct gaccctgttt cctttctctg gggagactgt gttcatgagc atggagaacc ctggcctgtg gatcctgggc tgccacaact ctgacttcag gaacaggggg atgactgctc tgctgaaggt gagcagctg t gataagaaca ctggggacta ctatgaggac agctatgagg acatctctgc ctatctgctg agcaagaata atgctattga gcccaggagc ttctctcaga acccccctgt gctgaagagg caccagaggg agatcaccag aactactctg cagtctgacc aggaggagat tgactatgat gacaccatct ctgtggagat gaagaaggag gattttgata tttatgatga ggatgaaaac cagagcccca ggagctttca gaagaagact aggcactatt tcattgctgc tgtggagagg ctgtgggact atggcatgtc ttctagcccc catgtgctga ggaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttc caggagttca ctgatggcag cttcactcag cccctgtaca ggggggagct gaatgagcac ctggggctgc tgggccctta tatcagggct gaggtggagg ataa catcat ggtgaccttc aggaaccagg ccagcaggcc ctacagcttc tactctagcc tgatcagcta tgaggaggac cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacttat ttctggaagg tgcagcacca tatggccccc accaaggatg agtttgattg caaagcctgg gcc tacttct ctgatgtgga cctggagaag gatgtgcact ctgggctgat tggccccctg ctggtgtgcc acaccaacac tctgaaccct gcccatggca ggcaggtgac tgtgcaggag tttgccctgt tcttcaccat ctttgatgag actaagagct ggtacttcac tgagaacatg gagaggaact gcagggcccc ctgcaatatc cagatggagg accccacctt taaggaaaat tataggtttc atgccattaa tggctacatc atggacaccc tgcctggcct ggtgatggcc caggaccaga ggat caggtg gtacctgctg agcatgggca gcaatgagaa cattcacagc atccacttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg tataatctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc tggagggtgg agtgcctgat tgggg agcac ctgcatgctg gcatgagcac cctgttcctg gtgtattcta acaagtgtca gacccccctg ggcatggcct ctggccatat cagggacttc cagatcactg cctctggcca gtatgggcag tgggccccca agctggccag gctgcattac tctggcagca tcaatgcctg gagcaccaag gagccattca gctggattaa ggtggacctg ctggctccaa tgattatcca tggcatcaag acccagggggg ccaggcagaa gtttagcagc ctgtacatct ctcagtttat catcatgtac tctctggatg gca aaaagtg gcagacctac aggggcaatt ctactggcac tctgatggtg ttctttggca atgtggacag ctctgggatc aagcacaaca tctttaaccc ccctatcatt gccaggtaca ttaggctgca ccccacccat tacagcatca ggagcaccct gaggatggag ctgatgggct gtgatctgaa cagctgcagc at gcccctgg gcatggagag caaggctatc tctgatgccc agattactgc cagcagctac ttcaccaata tgtttgccac ctggagcccc agcaaggcca ggctgcacct gcagggcagg tctaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag aagaccatga aggtgactgg ggtgaccacc cagggggtga agagcctgct gactagcatg tatgtgaagg agttcctgat cagcagcagc caggatggcc atcagtggac cctg ttcttc cagaatggca aggtgaaggt gttccagggc aatcaggaca gcttcacccc tgtggtgaac agcctggacc cccccctgct gaccagatac ctgaggatcc acccccagag ctgggtgcat cagattgccc tgaggatgga ggtgctgggg tgtgaggccc aggacctgta ctga

FVIII encoding nucleic acid variant with reduced CpG content X02 (SEQ ID NO:2)

atgcagattg agctgtctac ctgctttttc ctgtgtctgc tgaggttctg cttctctgcc actaggaggt actacctggg ggctgtggag ctgtcttggg attacatgca gtctgatctg ggggagctgc ctgtggatgc caggtttcct cccagggtgc ccaagtcttt cccctt caat acctctgtgg tgtataagaa gaccctgttt gtggagttta ctgatcacct gttcaacatt gccaagccca ggcccccttg gatgggcctg ctggggccca ccatccaggc tgaggtgtat gacactgtgg tgatcaccct gaagaacatg gcctctcacc ctgtgagcct gcatgctgtg ggggtga gct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg gagaaggagg atgataaggt gttccctggg gggagccaca cttatgtgtg gcaggtgctg aaggagaatg gcccaatggc ctctgatccc ctgtgcctga cctattctta cctgagccat gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag ggctctctgg ctaaggagaa gacccagacc ctgcacaagt tcatcctgct gtttgctgtg tttgatgagg ggaagagctg gcactctgag accaagaaca gcctgatgca ggacagggat gctgcctctg ccagggcctg gcccaaaatg cacactgtga atggctatgt gaataggagc ctgcctggcc tgattggctg ccacaggaag tctgtgtatt ggcatgtgat tggcatgggc accacccctg aggtgcactc tatcttcctg gagggccata ct ttcctggt gaggaatcat aggcaggcca gcctggagat tagccccatt acctttctga ctgcccagac cctgctgatg gacctgggcc agttcctgct gttttgccac atcagctctc accagcatga tggcatggag gcctatgtga aggtggatag ctgccctgag gagccccagc tgaggatgaa gaacaatgag gaggctgagg attatgatga tgatctgact gattctgaaa tggatgtggt gaggtttgat gatgacaata gcccctcttt catccagatc aggtctgtgg ccaagaagca tcctaagacc tgggtgcact acattgctgc tgaggaggag gactgggact atgctcccct ggtgctggcc cctgatgaca ggtcttacaa gagccagtac ctgaacaatg gcccccagag aattgggagg aagtataaga aggtgagatt catggcttac actgatgaga ccttcaagac tagggaggcc atccagcatg agtctggcat tctgggcccc ctgctgtatg gggaggtggg ggacaccctg ctgatcatct tcaagaacca ggcctctagg ccctacaata tttaccccca tgggatcact gatgtgaggc ccctgtacag caggaggctg cctaaggggg tgaagcatct gaaggacttc cccatcctgc ctggggagat cttcaagtat aagtggactg tgactgtgga agatggcccc accaagtctg accctaggtg cctgaccagg tactactctt cttttgtgaa catggagagg gacctggcct ctggcc tgat tggccccctg ctgatctgct acaaggagtc tgtggaccag agggggaacc agattatgtc tgacaagagg aatgtgattc tgttctctgt gtttgatgag aacaggagct ggtatctgac tgagaacatc cagaggttcc tgcccaatcc tgctggggtg cagctggagg accctgagtt ccagg ccagc aacatcatgc acagcatcaa tgggtatgtg tttgattctc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc attggggctc agactgattt cctgtctgtg ttcttttctg gctacacctt taagcataag atggtgtatg aggacactct gaccctgttt cccttctctg gggagactgt gtttatgagc atggagaacc ctggcctgtg gatcctgggc tgccacaact ctgatttcag gaacaggggc atgactgctc tgctgaaggt gtct tcttgt gacaagaaca ctggggacta ttatgaggac agctatgagg acatctctgc ctacctgctg agcaagaaca atgctattga gcccagatct ttcagccaga acccccctgt gctgaagagg caccagaggg agatcactag gaccaccctg cagtctgacc aggaggagat tgactatgat gacactatct ctgtggagat gaagaaggag gactttgata tctatgatga ggatgagaac cagtctccca ggagcttcca gaaaaagacc aggcactact tcattgctgc tgtggagagg ctgtgggact atggcatgtc ttctagcccc catgtgctga ggaacagggc ccagtctggg tctgtgcccc agttcaagaa ggtggtgttc caggagttca ctgatggggag cttcacccag cctctgtaca ggggggagct gaatgagcac ctggggctgc tgggccctta tattagggct gaggtggagg acaacat cat ggtgactttc aggaatcagg cctctaggcc ctatagcttc tacagctctc tgatcagcta tgaggaggat cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacctac ttctggaagg tgcagcacca catggctcct accaaggatg agtttgactg caaggcctgg gcctact ttt ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggccccctg ctggtgtgtc ataccaacac cctgaaccct gcccatggca ggcaggtgac tgtgcaggag tttgccctgt tcttcaccat ctttgatgag accaagagct ggtactttac tgagaacatg gagaggaatt gcagagcccc ttgcaacatc cagatggagg acccaacctt caaagagaac tacaggttcc atgccatcaa tgggtacatc atggacaccc tgcctggcct ggtgatggct caggaccaga ggatcaggt g gtatctgctg agcatgggca gcaatgagaa tatccatagc attcacttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg tataacctgt accctggggt gtttgagact gtggagatgc tgccaagcaa ggctgggatt tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgtctac cctgttcctg gtgtactcca ataagtgcca gacccccctg ggcatggcct ctggccacat cagggacttc cagatcactg cctctggcca gtatgggcag tgggccccaa agctggccag gctgcactat tctgggagca tcaatgcttg gagcaccaag gagcctttca gctggattaa ggtggatctg ctggccccca tgatcattca tggcatcaaa acccagggggg ctagacagaa gttttctagc ctgtacatca gccagttcat catcatgtac agcctggat g gcaagaagtg gcagacttac aggggcaata gcactggcac cctgatggtg ttttttggca atgtggacag ctctggcatc aagcacaaca tctttaaccc ccccattatt gccaggtata tcaggctgca tcccacccac tattctatta ggtctactct gagaatggag ctgatgggct gtgacctgaa cagctgtagc atg cccctgg ggatggagag caaggctatc tctgatgccc agatcactgc cagctcttat ttcaccaata tgtttgccac ctggtctccc tctaaggcca ggctgcacct gcagggcagg agcaatgctt ggaggcccca ggtgaataac cccaaggagt ggctgcaggt ggacttccag aagaccatga aggtgactgg ggtgactacc caggggggtga agtctctgct gactagcatg tatgtgaagg agttcctgat cagcagcagc caggatgggc atcagtggac tctg ttcttc cagaatggca aggtgaaggt cttccagggg aaccaggata gcttcactcc tgtggtgaac tctctggacc cccccctgct gactaggtat ctgaggatcc acccccagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ttga

FVIII encoding nucleic acid variant with reduced CpG content X03 (SEQ ID NO:3)

atgcagattg aactgtctac ttgtttcttc ctgtgcctgc tgaggttttg cttctctgct actaggaggt actatctggg ggctgtggag ctgtcttggg actatatgca gtctgacctg ggggagctgc ctgtggatgc taggtttccc cccagggtgc ccaagagctt ccccttta ac acctctgtgg tgtataagaa gactctgttt gtggagttca ctgaccatct gttcaacatt gccaagccaa ggcccccctg gatgggcctg ctgggcccca ccatccaggc tgaggtgtat gacactgtgg tgattactct gaagaacatg gccagccatc ctgtgagcct gcatgctgtg ggggtgtctt actggaaggc ctctgagggg gctgagtatg atgaccagac ctctcagagg gagaaggagg atgacaaggt gttccctggg ggctctcata cctatgtgtg gcaggtcctg aaggagaatg ggcccatggc ctctgacccc ctgtgcctga cctactctta tctgtctcat gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag ggcagcctgg ctaaggagaa gacccagact ctgcacaagt tcatcctgct gtttgctgtg tttgatgagg gcaagagctg gcactctgag accaagaaca gcctgatgca ggacagggat gctgcctctg ctagggcctg gcccaagatg cacactgtga atgggtatgt gaacaggac ctgccaggcc tgattggctg ccataggaag tctgtgtatt ggcatgtgat tgggatgggg actacccctg aggtccacag cattttcctg gaggggcata cctttct ggt gaggaaccac aggcaggcct ctctggagat ctctcccatt actttcctga ctgcccagac cctgctgatg gacctgggcc agttcctgct gttctgccac atcagcagcc accagcatga tggcatggag gcctatgtga aggtggatag ctgccctgag gagccccagc tgaggatgaa aaacaatgag gaggctgagg attatgatga tgacctgact gattctgaga tggatgtggt gaggtttgat gatgataaca gccccagctt catccagatt aggtctgtgg ccaagaagca tcccaagacc tgg gtgcact acattgctgc tgaggaggag gattgggact atgctcctct ggtgctggcc cctgatgaca ggagctacaa gagccagtac ctgaataatg gcccccagag gattggcagg aagtataaga aggtgaggtt catggcctac actgatgaga cctttaagac cagggaggcc atccagcatg aatctgggat cctgggcccc ct gctgtatg gggaggtggg ggacaccctg ctgattatct ttaagaacca ggctagcagg ccctacaaca tttaccccca tggcattact gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcacct gaaggatttc cccattctgc ctggggagat ctttaagtac aaatggactg tgactgtgga ggatggccct actaagtctg atcccaggtg tctgaccaga tactacagca gctttgtgaa tatggagagg gacctggctt ctggcct gat tggccccctg ctgatctgct acaaggagtc tgtggaccag aggggcaatc agattatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag aacagaagct ggtacctgac tgagaacatc cagaggttcc tgcccaaccc tgctggggtg cagctggagg accctgagtt ccaggc tagc aatatcatgc acagcattaa tggctatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctattggta cattctgagc attggggccc agactgattt cctgtctgtg ttcttttctg gctacacctt caagcacaag atggtgtatg aggatactct gaccctgttt cccttctctg gggagactgt gttcatgagc atggagaacc ctggcctgtg gatcctgggc tgtcacaact ctgacttcag gaacaggggc atgactgccc tgctgaaggt gagctct tgt gataagaaca ctggggacta ctatgaggac tcttatgagg acatctctgc ctacctgctg agcaagaaca atgctattga gcccaggagc ttctctcaga atccccctgt gctgaagagg catcagaggg agatcactag gactaccctg cagtctgacc aggaagagat tgactatgat gacaccatct ctgtggaaat gaaga aggag gactttgata tctatgatga ggatgaaaac cagagcccca ggagcttcca gaagaagacc aggcattact tcattgctgc tgtggagagg ctgtgggact atgggatgag ctcttctccc catgtgctga ggaatagggc tcagtctggc tctgtcccac agttcaagaa ggtggtgttt caggagttca ctgatggcag cttcactcag cccctgtaca ggggggagct gaatgagcat ctgggcctgc tggggcccta catcagggct gaggtggagg ataac attat ggtgactttc aggaaccagg cctctaggcc ctacagcttc tacagcagcc tgatcagcta tgaggaggac cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac taagacctat ttctggaagg tgcagcatca catggctccc actaaagatg agtttgactg caaggcctgg g cctacttct ctgatgtgga tctggagaag gatgtgcatt ctgggctgat tggccctctg ctggtctgcc atactaacac cctgaatcct gcccatggca ggcaggtgac tgtgcaggag tttgccctgt tctttaccat ctttgatgag accaagtctt ggtacttcac tgagaacatg gagaggaact gcagggcccc ctgtaacatc cagatggagg accccacctt taaggagaac tacaggttcc atgccatcaa tggctacatc atggacactc tgcctggcct ggtgatggcc caggaccaga ggat caggtg gtacctgctg tctatgggct ctaatgagaa cattcattct atccacttct ctggccatgt gtttactgtg aggaagaagg aggagtacaa gatggccctg tacaatctgt accctggggt gtttgaaact gtggagatgc tgccctctaa ggctggcatc tggagggtgg agtgcctgat tgggg aacac ctgcatgctg gcatgagcac cctgttcctg gtctatagca ataagtgcca gacccccctg gggatggcct ctgggcatat cagagacttc cagatcactg cctctggcca gtatggccag tgggccccca agctggccag gctgcactac tctggcagca ttaatgcctg gagcaccaag gagcccttct cttggatcaa ggtggacctg ctggctccca tgatcatcca tgggatcaag acccagggggg ccaggcagaa gttcagcagc ctgtacatct ctcagttcat catcatgtac tctctggatg gcaagaagtg gcagacctac aggggcaata gcactgggac cctgatggtg ttctttggga atgtggacag ctctggcatc aagcacaata tcttcaaccc ccccatcatt gccaggtaca tcagactgca ccccactcat tacagcatca ggagcactct gaggatggag ctgatgggct gtgacctgaa tagctgctct atgcccctgg gcatggagag caaggccatt tctgatgccc agattactgc ctcttcttac ttcactaata tgtttgccac ctggagcccc agcaaggcca ggctgcatct gcaggggagg agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag aagactatga aggtgactgg ggtgaccact caggggggtga agagcctgct gaccagcatg tatgtgaagg agttcctgat ctcttctagc caggatgggc accagtggac cctg tttttc cagaatggga aggtgaaggt gtttcagggc aatcaggaca gctttactcc tgtggtgaac agcctggacc cccccctgct gactaggtac ctgaggattc acccccagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga

FVIII encoding nucleic acid variant with reduced CpG content X04 (SEQ ID NO:4)

atgcagattg agctgtctac ctgcttcttt ctgtgcctgc tgaggttctg tttctctgcc actaggaggt attatctggg ggctgtggag ctgtcctggg actacatgca gtctgatctg ggggagctgc ctgtggatgc caggttccct cccagggtgc ccaagtcttt cccttt caat acctctgtgg tgtacaagaa gactctgttt gtggagttta ctgatcacct gtttaacatt gccaagccca ggcccccctg gatggggctg ctgggcccca ccatccaggc tgaggtgtat gacactgtgg tgattactct gaagaatatg gcttctcacc ctgtgagcct gcatgctgtg ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg gagaaggagg atgacaaggt gttccctggg ggcagccaca cttatgtgtg gcaggtgctg aaggagaatg gcccaatggc ctctgacccc ctgtgcctga cctacagcta tctgagccat gtggatctgg tgaaggatct gaactctggc ctgattgggg ccctgctggt gtgcagggag ggctctctgg ccaaggagaa gactcagact ctgcacaagt tcatcctgct gtttgctgtg tttgatgagg gcaagagctg gcactctgag accaagaact ctctgatgca ggataggat gctgcttctg ccagggcctg gcccaagatg cacactgtga atgggtatgt gaataggagc ctgcctgggc tgattgggtg tcacaggaag tctgtgtact ggcatgtgat tggcatgggc accactcctg aggtgcacag catctttctg gagggccaca ctt ttctggt gaggaatcac aggcaggcca gcctggagat cagccccatc accttcctga ctgcccagac cctgctgatg gatctgggcc agttcctgct gttttgccat atcagcagcc atcagcatga tgggatggag gcttatgtga aggtggactc ttgccctgag gagcctcagc tgaggatgaa gaataatgaa gaggctgagg actatgatga tgatctgact gactctgaga tggatgtggt gaggtttgat gatgacaaca gccccagctt tatccagatt aggtctgtgg ccaagaagca ccccaagacc t gggtgcatt acattgctgc tgaggaagag gattgggact atgcccccct ggtgctggcc cctgatgaca ggagctacaa gtctcagtac ctgaacaatg gccctcagag gattggcagg aagtacaaga aggtgaggtt catggcttac actgatgaga ccttcaagac cagggaggcc attcagcatg aatctgggat cctggg cccc ctgctgtatg gggaggtggg ggacaccctg ctgattattt tcaagaacca ggccagcagg ccctacaaca tttatcctca tggcattact gatgtgagac ccctgtacag caggaggctg cctaaggggg tgaagcacct gaaggacttc cccatcctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggcccc actaagtctg accccaggtg cctgactagg tactactcca gctttgtgaa catggagagg gacctggcct ctggcc tgat tggccccctg ctgatctgct acaaggagtc tgtggatcag aggggcaacc agatcatgtc tgacaagaga aatgtgatcc tgttctctgt gtttgatgag aataggtctt ggtacctgac tgagaacatc cagaggtttc tgcctaatcc tgctggggtg cagctggagg atcctgagtt ccaggcctct aacattatgc acagcatcaa tgggtatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc attggggccc agactgactt tctgtctgtg ttcttctctg gctacacctt taagcataag atggtgtatg aggacaccct gactctgttc cccttctctg gggagactgt gttcatgagc atggagaacc caggcctgtg gatcctgggc tgccacaact ctgatttcag gaataggggc atgactgccc tgctgaaggt gagcagctgt gataagaaca ctggggacta ttatgaggat agctatgagg acatctctgc ctacctgctg agcaagaaca atgccattga gcccaggagc ttcagccaga atcctcctgt gctgaagagg caccagaggg agatcaccag gaccaccctg cagtctgatc aggaggat tgactatgat gacactatct ctgtggagat gaagaagg ag gactttgaca tctatgatga ggatgagaat cagagcccca ggagcttcca gaagaagact agacactact ttattgctgc tgtggagagg ctgtgggact atggcatgag ctcttctccc catgtgctga gaaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtcttc caggagttca ctgatggctc tttcacccag cctctgtata gaggggagct gaatgagcac ctgggcctgc tgggccctta catcagggct gaggtggagg acaata tcat ggtgaccttc aggaaccagg ctagcaggcc ctactctttc tacagcagcc tgatcagcta tgaggaggac cagaggcagg gggctgagcc taggaagaat tttgtgaagc ccaatgagac caagacctac ttctggaagg tgcagcacca catggctccc actaaggatg agtttgactg caaggcctgg gcctact ttt ctgatgtgga cctggagaag gatgtgcatt ctggcctgat tggccccctg ctggtctgcc acaccaatac tctgaaccct gctcatggga gacaggtgac tgtgcaggag tttgccctgt tcttcaccat ctttgatgag accaagtcct ggtactttac tgagaacatg gagaggaatt gcagggcccc ttgcaacatc cagatggagg accccacctt caaggaaaat tataggttcc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggcc caggaccaga ggatca ggtg gtatctgctg tctatgggct ctaatgagaa catccacagc atccatttct ctggccatgt gttcactgtg aggaagaagg aggagtataa gatggctctg tacaacctgt accctggggt ctttgagact gtggagatgc tgcccagcaa ggctggcatt tggagggtgg agtgcctgat tgggga acac ctgcatgctg ggatgagcac cctgttcctg gtgtactcta acaagtgcca gaccccactg ggcatggctt ctggccacat cagggatttc cagattactg cctctggcca gtatggccag tgggctccca agctggctag gctgcactac tctgggagca tcaatgcctg gtctactaag gagcctttct cttggatcaa agtggacctg ctggccccta tgatcatcca tgggatcaag actcagggg ccaggcagaa gttcagcagc ctgtacatct ctcagttcat cattatgtac agcc tggatg gcaagaagtg gcagacctac aggggcaaca gcactggcac cctgatggtg ttctttggga atgtggacag ctctgggatt aagcacaaca tctttaaccc ccccatcatt gccaggtata tcaggctgca ccctacccac tacagcatta ggagcaccct gaggatggag ctgatgggct gtgacctgaa cagctg cagc atgcccctgg ggatggagag caaggccatt tctgatgctc agatcactgc ttctagctac ttcactaaca tgtttgccac ctggtctccc agcaaggcta gactgcacct gcaggggagg agcaatgcct ggaggcccca ggtgaataat cccaaggagt ggctgcaggt ggatttccag aaaaccatga aggtgactgg ggtgactacc caggggggtga agtctctgct gaccagcatg tatgtgaagg agttcctgat cagcagcagc caggatgggc atcagtggac cctg ttcttt cagaatggga aggtgaaggt gtttcagggc aatcaggaca gcttcacccc tgtggtgaac agcctggacc cccccctgct gaccaggtac ctgaggatcc acccccagag ctgggtgcat cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ctga

FVIII encoding nucleic acid variant with reduced CpG content X05 (SEQ ID NO:5)

atgcagattg agctgtctac ttgcttcttc ctgtgcctgc tgaggttctg cttctctgcc actaggaggt attacctggg ggctgtggag ctgagctggg actatatgca gtctgacctg ggggagctgc ctgtggatgc caggtttcct cccagggtgc ctaagagctt ccccttcaac acctctgtgg tgtacaagaa gactctgttt gtggagttta ctgatcatct gttcaacatt gccaagccca ggcctccttg gatggggctg ctgggcccca ccatccaggc tgaggtgtat gacactgtgg tgattaccct gaagaatatg gccagccatc ctgtgagcct gcatgctgtg ggggtgagct att ggaaggc ctctgagggg gctgagtatg atgatcagac tagccagagg gagaaggagg atgacaaggt gttccctggg gggagccata cctatgtgtg gcaggtgctg aaggagaatg gccccatggc ctctgaccct ctgtgcctga cttatagcta cctgagccat gtggatctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag ggcagcctgg ccaaggagaa gactcagacc ctgcacaagt tcatcctgct gtttgctgtg tttgatgagg ggaagtcctg gcactct gag actaagaaca gcctgatgca ggataggat gctgcttctg ccaggggcctg gcctaagatg cacactgtga atggctatgt gaataggagc ctgcctggcc tgattggctg ccataggaag tctgtgtact ggcatgtgat tgggatgggc accacccctg aggtgcactc tattttcctg gagggccata ct ttcctggt gaggaaccat aggcaggcca gcctggagat cagccccatc actttcctga ctgcccagac tctgctgatg gacctgggcc agttcctgct gttctgccac atcagcagcc atcagcatga tggcatggag gcttatgtga aggtggacag ctgccctgag gagcctcagc tgaggatgaa gaataatgag gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat gatgacaact ctccctcttt catccagatc aggtctgtgg ccaagaagca ccctaagacc tgg gtgcact acattgctgc tgaggaggag gattgggact atgcccccct ggtgctggcc ccagatgaca ggagctacaa gtcccagtac ctgaacaatg gcccccagag gattggcagg aagtacaaga aggtgaggtt catggcttat actgatgaga ctttcaagac cagggaggcc atccagcatg agtctggcat cctggggcc ct ctgctgtatg gggaggtggg ggacaccctg ctgattatct tcaagaacca ggcttctagg ccctacaata tctaccctca tggcatcact gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcatct gaaggatttc cccatcctgc ctggggagat ctttaagtat aagtggactg tgactgtgga ggatggcccc actaagtctg accccaggtg cctgaccagg tattacagca gctttgtgaa catggagagg gatctggctt ctgggctga t tggccccctg ctgatctgct acaaggagtc tgtggaccag aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag aataggagct ggtacctgac tgagaacatc cagaggtttc tgcccaatcc tgctggggtg cagctggagg atcctgagtt tcaggcct ct aatatcatgc acagcatcaa tggctatgtg tttgactctc tgcagctgtc tgtgtgcctg catgaggtgg cctattggta catcctgagc attggggccc agactgactt tctgtctgtg tttttttctg gctacacctt caagcacaag atggtgtatg aggatactct gactctgttc cctttttctg gggagactgt gttcatgtct atggagaacc ctgggctgtg gattctgggc tgccacaatt ctgacttcag gaacagaggc atgactgctc tgctgaaggt gagcagct gt gacaagaaca ctggggacta ctatgaggac tcttatgagg acatttctgc ctacctgctg agcaagaaca atgccattga gcccagaagc ttttctcaga acccccctgt gctgaagagg caccagaggg agatcaccag gaccaccctg cagtctgacc aggaggat tgactatgat gatactattt ctgtggagat ga agaaggag gactttgaca tctatgatga ggatgagaac cagagcccca ggtctttcca gaagaagact aggcactact ttattgctgc tgtggagagg ctgtgggact atgggatgtc tagctctcct catgtgctga ggaacagggc ccagtctggc tctgtgcccc agtttaaaaa ggtggtgttc caggaattca ctgatggcag ctttacccag cctctgtaca ggggggagct gaatgagcac ctggggctgc tggggcctta cattagggct gaggtggagg acaacat cat ggtgaccttc aggaatcagg ccagcaggcc ctactctttc tacagcagcc tgatctctta tgaggaggac cagaggcagg gggctgaacc caggaagaac tttgtgaagc ccaatgagac caagacctac ttctggaagg tgcagcacca catggctccc accaaggatg agtttgattg caaggcctgg gcttactt ct ctgatgtgga tctggagaag gatgtgcact ctgggctgat tggccccctg ctggtgtgcc acaccaacac tctgaaccct gcccatggca gacaggtgac tgtgcaggag tttgccctgt tcttcactat ctttgatgag actaagagct ggtacttcac tgagaacatg gagaggaatt gcagggcccc ttgcaacatc cagatggagg accccacctt taaggagaac tacaggtttc atgccattaa tggctacatc atggacaccc tgcctggcct ggtgatggcc caggaccaga ggat caggtg gtacctgctg tctatgggga gcaatgagaa catccacagc attcacttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg tacaacctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctgggatc tggagggtgg agtgcctgat t ggggagcac ctgcatgctg ggatgagcac cctgttcctg gtgtatagca acaagtgcca gacccccctg ggcatggcct ctggccacat cagagacttt cagattactg cctctggcca gtatgggcag tgggccccca agctggccag gctgcactat tctggctcta ttaatgcctg gagcactaag gagcccttca gctggattaa ggtggacctg ctggctccca tgatcatcca tggcatcaag actcagggggg ccaggcagaa gttctcttct ctgtacatca gccagttcat tatcatgtac tccctggatg g caagaagtg gcagacctat aggggcaaca gcactggcac cctgatggtg ttctttggga atgtggacag ctctggcatc aagcataata tcttcaatcc ccccatcatt gctaggtaca tcaggctgca ccccacccac tactctatta ggtctaccct gaggatggag ctgatgggct gtgacctgaa cagctgcagc atg cctctgg gcatggagag caaagccatc tctgatgccc agatcactgc cagcagctac tttaccaaca tgtttgctac ttggagcccc agcaaggcca ggctgcacct gcaggggagg tctaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag aagactatga aggtgactgg ggtgaccacc caggggggtga agagcctgct gacctctatg tatgtgaagg agttcctgat tagcagcagc caggatggcc accagtggac cctg tttttc cagaatggga aggtgaaggt gtttcagggg aaccaggaca gcttcactcc tgtggtgaac tctctggacc cccccctgct gaccaggtat ctgaggatcc accctcagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ctga

FVIII encoding nucleic acid variant with reduced CpG content X06 (SEQ ID NO:6)

atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttttg cttctctgcc accaggaggt actacctggg ggctgtggag ctgagctggg attacatgca gtctgacctg ggggagctgc ctgtggatgc caggttccct cccagggtgc ccaagtcttt ccccttca ac acttctgtgg tgtacaagaa gaccctgttt gtggagttta ctgaccacct gttcaacatt gccaagccca ggcctccctg gatgggcctg ctgggcccca ccattcaggc tgaggtgtat gacactgtgg tcatcaccct gaaaaatatg gctagccacc ctgtgtctct gcatgctgtg ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac tagccagagg gagaaggagg atgacaaggt gttccctggg ggcagccaca cttatgtgtg gcaggtgctg aaagagaatg gccccatggc ttctgatccc ctgtgtctga cctatagcta cctgagccat gtggatctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag ggcagcctgg ctaaggagaa gacccagacc ctgcataagt tcatcctgct gtttgctgtg tttgatgagg gcaagagctg gcactct gag actaagaaca gcctgatgca ggataggat gctgcttctg ccaggggcctg gcccaagatg cacactgtga atgggtatgt gaacaggagc ctgcctggcc tgattggctg ccataggaag tctgtctatt ggcatgtgat tggcatgggc actactcctg aggtgcacag catctttctg gagggccaca ccttcc tggt gaggaaccac aggcaggcca gcctggagat ctctcccatc actttcctga ctgctcagac cctgctgatg gacctgggcc agttcctgct gttctgtcac atctctagcc accagcatga tggcatggag gcctatgtga aggtggatag ctgccctgag gaaccccagc tgaggatgaa gaacaatgag gaggctgagg attatgatga tgatctgact gattctgaga tggatgtggt gaggtttgat gatgacaatt ctcctagctt cattcagatc agatctgtgg ccaaaaagca tcctaag act tgggtgcatt atattgctgc tgaggaggag gattgggatt atgcccccct ggtgctggct cctgatgata ggagctacaa gtctcagtac ctgaataatg ggccccagag gattggcagg aagtacaaga aggtgaggtt catggcctac actgatgaga ccttcaagac cagggaggcc attcagcatg agtctgggat tct ggggccc ctgctgtatg gggaggtggg ggataccctg ctgatcattt tcaagaacca ggccagcagg ccctacaaca tctaccccca tgggattact gatgtgaggc ccctgtactc taggaggctg cctaaggggg tgaagcacct gaaggatttt cctatcctgc ctggggaaat cttcaagtac aagtggactg tgactgtgga ggatggcccc actaagtctg atcccaggtg tctgaccagg tattatagct cttttgtgaa catggagagg gatctggcct ctgg gctgat tggccctctg ctgatctgct acaaggagtc tgtggaccag aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag aacaggagct ggtatctgac tgagaacatc cagaggtttc tgcccaatcc tgctggggtg cagctggagg atcctgagtt ccaggctagc aacatcatgc acagcatcaa tgggtatgtg tttgacagcc tgcagctgtc tgtgtgtctg catgaggtgg cctactggta tatcctgtct attggggccc agactgactt cctgtctgtg tttttttctg ggtatacttt taagcacaag atggtgtatg aggacaccct gactctgttc cccttctctg gggagactgt gtttatgagc atggagaacc ctggcctgtg gatcctgggc tgccacaatt ctgacttcag gaataggggg atgactgccc tgctgaaggt gag cagctgt gataagaata ctggggacta ctatgaggac tcttatgagg acatttctgc ctatctgctg tctaagaaca atgccattga acccaggagc ttctctcaga acccccctgt gctgaagagg caccagaggg aaatcaccag aactactctg cagtctgatc aggaggaaat tgactatgat gacactattt ctgtggagat ga agaaggag gactttgaca tctatgatga ggatgagaac cagagcccaa ggagcttcca gaagaagact aggcactact tcattgctgc tgtggagagg ctgtgggact atggcatgag cagcagcccc catgtgctga gaaacagggc ccagtctggg tctgtgcccc agttcaagaa ggtggtgttc caggagttca ctgatgggag cttcacccag cccctgtata ggggggagct gaatgagcac ctgggcctgc tgggccccta tattagggct gaggtggagg acaa catcat ggtgaccttc aggaatcagg cctctaggcc ctacagcttc tacagcagcc tgattagcta tgaggaggat cagaggcagg gggctgaacc caggaagaac tttgtgaagc ccaatgagac caagacctat ttctggaagg tgcagcatca catggccccc accaaggatg agtttgactg caaggcctgg gcctact tct ctgatgtgga tctggagaag gatgtgcact ctggcctgat tggccccctg ctggtgtgcc acaccaacac cctgaaccct gctcatggca ggcaggtgac tgtgcaggag tttgccctgt tcttcaccat ctttgatgag actaagtctt ggtacttcac tgagaatatg gagaggaatt gcagggcccc ctgcaatatt cagatggaag accccacctt caaggagaat tacaggttcc atgccattaa tggctacatc atggataccc tgcctggcct ggtgatggcc caggatcaga ggatca ggtg gtacctgctg agcatgggca gcaatgagaa catccactct atccacttct ctggccatgt gtttactgtg aggaagaagg aggagtataa gatggccctg tacaacctgt accctggggt ctttgagact gtggagatgc tgccttctaa ggctggcatt tggagggtgg agtgcctgat tgggga acac ctgcatgctg gcatgtctac cctgttcctg gtgtacagca ataagtgcca gacccccctg ggcatggcct ctgggcatat cagggatttc cagatcactg cctctggcca gtatggccag tgggccccaa agctggctag gctgcactac tctgggagca tcaatgcctg gagcactaag gagcccttca gctggatcaa ggtggacctg ctggccccca tgattatcca tgggattaag actcagggg ccaggcagaa gttcagcagc ctgtacatca gccagttcat tatcatgtac agcctggat g gcaagaagtg gcagacctat aggggcaact ctactgggac cctgatggtg ttctttggga atgtggatag ctctgggatc aagcacaata tcttcaaccc ccccatcatt gccaggtata tcaggctgca ccccacccac tacagcatta ggtctaccct gaggatggag ctgatgggct gtgatctgaa cagctgtagc atg cctctgg gcatggagtc taaggccatt tctgatgccc agattactgc tagcagctac ttcaccaaca tgtttgccac ctggtctccc agcaaggcca ggctgcatct gcagggcagg tctaatgctt ggaggcccca ggtgaacaac ccaaaggagt ggctgcaggt ggatttccag aagactatga aggtgactgg ggtgaccact caggggggtga agtctctgct gacctctatg tatgtgaagg agttcctgat ctctagcagc caggatggcc atcagtggac cct gttcttc cagaatggca aggtgaaagt gttccagggc aatcaggata gcttcactcc agtggtgaac agcctggatc cccctctgct gactaggtac ctgaggatcc acccccagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ctga

FVIII encoding nucleic acid variant with reduced CpG content X07 (SEQ ID NO:7)

atgcagattg agctgagcac ctgcttcttc ctgtgtctgc tgaggttctg cttctctgcc accaggaggt attacctggg ggctgtggag ctgagctggg actatatgca gtctgacctg ggggagctgc ctgtggatgc taggttcccc cccagggtgc ccaagagctt cccctttaac acttctgtgg tgtacaagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt gccaagccca ggcccccctg gatggggctg ctggggccca ccatccaggc tgaggtgtat gacactgtgg tgatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg ggggtgagct actggaa ggc ttctgagggg gctgagtatg atgaccagac tagccagagg gagaaggagg atgacaaggt gtttcctggg ggcagccata cctatgtgtg gcaggtgctg aaggagaatg gccccatggc ctctgacccc ctgtgcctga cctacagcta cctgtctcat gtggacctgg tgaaggacct gaactctggc ctgattgggg ctctgctggt gtgtagggag ggcagcctgg ctaaggaaaa gacccagacc ctgcataagt ttatcctgct gtttgctgtg tttgatgagg gcaagagctg gcactct gag accaagaaca gcctgatgca ggatagggat gctgcctctg ccaggcttg gcctaagatg cacactgtga atgggtatgt gaataggagc ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tgggatgggc accacccctg aggtccatag catcttcctg gagggccaca ctt tcctggt gaggaaccac agacaggcct ctctggagat ctctcccatc accttcctga ctgctcagac tctgctgatg gacctgggcc agttcctgct gttttgccat attagcagcc accagcatga tgggatggag gcctatgtga aggtggatag ctgccctgag gagcctcagc tgaggatgaa gaacaatgag gaggctgaag actatgatga tgacctgact gattctgaga tggatgtggt gaggtttgat gatgacaata gccccagctt cattcagatc aggtctgtgg ccaagaaaca ccag acc tgggtgcact acattgctgc tgaggaagag gactgggact atgctcccct ggtgctggcc cctgatgata ggtcttataa gagccagtac ctgaacaatg ggccccagag gattggcagg aagtacaaga aggtgaggtt catggcctac actgatgaaa ccttcaaaac cagggaggcc attcagcatg agtctggcat c ctgggccct ctgctgtatg gggaggtggg ggacaccctg ctgatcatct tcaagaacca ggccagcagg ccctacaaca tctatcctca tggcatcact gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcacct gaaagacttc cccatcctgc ctggggagat ctttaagtat aagtggactg tgactgtgga ggatggccct accaagtctg accccaggtg tctgaccagg tactattcta gctttgtgaa catggagagg gacctggcct ctggcct gat tgggcccctg ctgatctgct acaaggagtc tgtggaccag aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttttctgt gtttgatgag aataggagct ggtacctgac tgagaacatc cagaggtttc tgcccaatcc tgctggggtg cagctggagg atcctgagtt cca ggccagc aatatcatgc atagcatcaa tggctatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc attggggccc agactgactt tctgtctgtg ttcttttctg gctatacctt caagcacaag atggtgtatg aggataccct gaccctgttc cccttctctg gggagactgt gttcatgagc atggagaatc ctgggctgtg gatcctgggg tgccacaact ctgattttag gaacaggggg atgactgccc tgctgaaggt gtctagctgt gataagaaca ctggggacta ctatgaggac agctatgagg acatttctgc ttatctgctg tctaagaata atgccattga gcccagaagc ttcagccaga atccccctgt gctgaagaga catcagagg agatcaccag aactaccctg cagtctgatc aggaggagat tgactatgat gacactatct ctgtggagat gaagaaggag g actttgaca tctatgatga ggatgagaat cagtctccca ggagctttca gaagaagacc agacattact tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagccct catgtgctga ggaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttc caggaattca ctgatggcag cttcacccag cccctgtaca ggggggagct gaatgagcac ctgggcctgc tggggcctta tatcagggct gaggtggagg ataatatt at ggtgactttc aggaaccagg ccagcaggcc ctactctttc tatagcagcc tgatctctta tgaggaggat cagaggcagg gggctgagcc taggaagaac tttgtgaagc ccaatgagac taagacctac ttctggaagg tccagcacca catggcccct accaaggatg agtttgactg caaggcctgg gcctattt ct ctgatgtgga tctggagaag gatgtccatt ctgggctgat tggccccctg ctggtgtgcc acactaacac tctgaatcct gcccatggca ggcaggtgac tgtccaggag tttgccctgt tcttcactat ctttgatgag accaagagct ggtactttac tgagaacatg gagaggaact gcagagctcc ttgcaatatt cagatggagg accccacctt caaggagaat tacaggttcc atgccattaa tgggtacatc atggacaccc tgcctggcct ggtgatggct caggaccaga ggatcaggt g gtacctgctg agcatgggct ctaatgagaa tatccacagc atccacttct ctgggcatgt gttcactgtg aggaagaagg aggagtacaa gatggctctg tataatctgt accctggggt gtttgaaact gtggagatgc tgccctctaa ggctggcatc tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgagcac cctgttcctg gtgtacagca acaagtgcca gacccccctg ggcatggcct ctggccacat cagggacttc cagatcactg cctctggcca gtatggccag tgggccccca agctggccag gctgcactat tctggcagca tcaatgcctg gagcaccaag gagcccttca gctggatcaa ggtggacctg ctggccccca tgatcattca tggcatcaag acccagggggg ccaggcagaa gttcagctct ctgtacatct ctcagttcat catcatgtac tctctggatg gga agaagtg gcagacctac aggggcaaca gcactggcac cctgatggtg ttctttggga atgtggactc ttctggcatc aagcacaaca tcttcaatcc ccccatcatt gctaggtata ttaggctgca tcccacccac tacagcatca ggtctaccct gaggatggag ctgatgggct gtgacctgaa ctcttgcagc at gcccctgg gcatggagtc taaggccatc tctgatgccc agattactgc cagcagctac ttcaccaaca tgtttgccac ctggagcccc tctaaggcca ggctgcatct gcaggggagg agcaatgcct ggaggcctca ggtgaacaac cccaaggagt ggctgcaggt ggatttccag aagaccatga aggtgactgg ggtgaccacc caggggggtca agagcctgct gaccagcatg tatgtgaagg agttcctgat cagcagcagc caggatggcc accagtggac tctg ttcttt cagaatggga aggtgaaggt gtttcagggc aatcaggact ctttcacccc tgtggtgaac agcctggacc cccccctgct gaccagatac ctgaggatcc acccccagtc ttgggtgcat cagattgccc tgaggatgga ggtgctgggc tgtgaggctc aggatctgta ctga

FVIII encoding nucleic acid variant with reduced CpG content X08 (SEQ ID NO:8)

atgcagattg agctgagcac ttgctttttt ctgtgcctgc tgaggttttg tttttctgcc accaggaggt actacctggg ggctgtggag ctgagctggg actatatgca gtctgatctg ggggagctgc ctgtggatgc caggttcccc cccagggtgc ccaagtcttt tccct tcaac acctctgtgg tgtataagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt gctaagccta ggcccccctg gatgggcctg ctgggcccta ccattcaggc tgaggtgtat gacactgtgg tgatcaccct gaagaacatg gccagccatc ctgtgagcct gcatgctgtg gg ggtctctt actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagaga gagaaggagg atgacaaggt cttccctggg ggctctcaca cctatgtgtg gcaggtgctg aaggaaaatg gccccatggc ctctgacccc ctgtgcctga cctacagcta tctgagccat gtggatctgg tgaaggacct gaattctggc ctgattgggg ccctgctggt gtgcagggag ggcagcctgg ccaaggagaa gacccagacc ctgcacaagt ttatcctgct gtttgctgtg tttgatgagg gcaagtcttg gcact ctgag actaagaaca gcctgatgca ggacagggat gctgcctctg ccaggggcctg gcccaagatg cacactgtga atggctatgt gaacagggc ctgcctgggc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc accacccctg aggtgcacag catcttcctg gaagg ccaca ctttcctggt gaggaaccat aggcaggcca gcctggagat cagccctatc accttcctga ctgcccagac cctgctgatg gatctggggc agttcctgct gttctgccac atctctagcc accagcatga tgggatggag gcctatgtga aggtggacag ctgcccagag gagcctcagc tgaggatgaa aaacaatgaa gaggctgagg attatgatga tgatctgact gactctgaga tggatgtggt gagatttgat gatgacaata gccctagctt tattcagatc aggtctgtgg ctaagaagca ccccaagacc t gggtgcatt acattgctgc tgaggaggag gactgggatt atgctcctct ggtgctggcc cctgatgata ggagctacaa gagccagtac ctgaataatg gccctcagag gattggcagg aagtacaaga aggtgaggtt catggcttac actgatgaga ccttcaagac tagggaggcc atccagcatg agtctgggat cctggggccc ctgctgtatg gggaggtggg ggacaccctg ctgatcatct tcaagaacca ggctagcagg ccttacaaca tctatcccca tgggatcact gatgtgagac ctctgtacag caggaggctg cccaaggggg tcaagcatct gaaagacttc cccatcctgc ctggggagat ctttaagtat aagtggactg tgactgtgga ggatgggccc accaagtctg accccaggtg cctgaccagg tattacagca gctttgtgaa catggagagg gatctggcct ctgggct gat tggccccctg ctgatctgtt acaaggaatc tgtggatcag aggggcaatc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag aataggtctt ggtacctgac tgaaaacatc cagaggttcc tgcccaaccc tgctggggtc cagctggagg atcctgagtt ccaggctagc aacatcatgc acagcatcaa tgggtatgtg tttgatagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgtct attggggccc agactgactt cctgtctgtg ttcttttctg gctacacctt caagcacaag atggtgtatg aggacaccct gaccctgttc cccttctctg gggagactgt ctttatgagc atggagaacc ctgggctgtg gatcctgggc tgccacaact ctgatttcag gaataggggc atgactgctc tgctgaaggt gagctctt gt gacaagaaca ctggggatta ctatgaggac agctatgagg acatttctgc ctacctgctg agcaagaaca atgccattga gcctaggagc tttagccaga atcctcctgt cctgaagagg caccagaggg agatcaccag gaccaccctg cagtctgacc aggaggat tgactatgat gataccatct ctgtggagat gaagaa ggag gactttgaca tctatgatga ggatgagaat cagtctccca ggagcttcca gaagaagacc aggcactatt tcattgctgc tgtggagagg ctgtgggact atggcatgag cagctctcct catgtgctga ggaatagggc tcagtctggc tctgtgcccc agttcaagaa agtggtgttt caggagttca ctgatggctc tttcacccag cctctgtata ggggggagct gaatgagcac ctggggctgc tgggccccta tatcaggct gaggtggagg ataacat cat ggtgaccttc aggaaccagg cctctaggcc ctacagcttc tatagcagcc tgatcagcta tgaggaggac cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacttac ttctggaagg tgcagcatca catggccccc accaaggatg agtttgactg taaggcctgg gcc tacttct ctgatgtgga tctggagaag gatgtgcact ctggcctgat tggccccctg ctggtgtgcc ataccaatac tctgaaccct gctcatggca ggcaggtgac tgtgcaggag tttgctctgt tcttcactat ctttgatgag accaagtctt ggtatttcac tgagaatatg gagaggaact gcagggcccc ctgcaacatc cagatggagg accccacctt taaggagaac tataggtttc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggcc caggatcaga gg atcaggtg gtacctgctg agcatggggt ctaatgagaa catccacagc atccacttct ctggccatgt gtttactgtg agaaagaagg aggagtacaa gatggctctg tacaatctgt accctggggt ctttgagact gtggagatgc tgcctagcaa ggctgggatc tggagggtgg agtgcctgat tgg ggaacat ctgcatgctg ggatgtctac tctgttcctg gtgtacagca acaagtgcca gacccccctg ggcatggctt ctggccatat cagggacttt cagattactg cctctgggca gtatggccag tgggccccca agctggctag gctgcattat tctggcagca tcaatgcctg gtctactaag gagcccttca gctggatcaa ggtggatctg ctggccccca tgatcatcca tggcatcaag acccagggg ccaggcagaa gtttagctct ctgtacatta gccagttcat catcatgtac agcctggatg ggaaga agtg gcagacctac aggggcaatt ctactggcac cctgatggtg ttctttggca atgtggacag ctctggcatc aagcacaaca tctttaaccc ccctatcatt gctaggtaca tcaggctgca tcccacccat tacagcatca ggagcaccct gaggatggag ctgatgggct gtgacctgaa ctcttgcagc atgcc cctgg gcatggagag caaggccatt tctgatgccc agattactgc cagcagctac ttcactaaca tgtttgccac ctggtctccc agcaaggcca ggctgcacct gcagggcagg agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggatttccag aagaccatga aggtgactgg ggtgaccacc caggggggtga agagcctgct gactagcatg tatgtgaagg agttcctgat cagctctagc caggatggcc accagtggac tctgt ttttc cagaatggca aggtgaaggt gttccagggc aaccaggact ctttcactcc tgtggtgaac agcctggacc cccccctgct gaccaggtat ctgaggattc acccccagtc ttgggtgcat cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga

FVIII encoding nucleic acid variant with reduced CpG content X09 (SEQ ID NO:9)

atgcagattg agctgagcac ctgcttcttc ctgtgtctgc tgagattttg cttttctgcc actaggaggt attacctggg ggctgtggag ctgtcttggg actacatgca gtctgatctg ggggagctgc ctgtggatgc caggttccca cctagggtgc ctaagagctt tcccttca at acctctgtgg tgtacaagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt gccaagccta ggcccccctg gatgggcctg ctgggcccta ccatccaggc tgaagtgtat gacactgtgg tgatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg ggggtgtct t actggaaggc ctctgagggg gctgagtatg atgatcagac cagccagagg gagaaggaag atgacaaggt gttccctggg ggcagccaca cctatgtctg gcaggtgctg aaggagaatg gccccatggc ctctgatccc ctgtgcctga cctactctta cctgagccat gtggacctgg tgaaggatct gaattctggc ctgattgggg ccctgctggt gtgcagggag ggcagcctgg ccaaggagaa gacccagacc ctgcataagt tcatcctgct gtttgctgtg tttgatgaag ggaagagctg gcactctga g actaagaaca gcctgatgca ggacagggat gctgcttctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaatagaagc ctgcctggcc tgattgggtg ccacaggaag tctgtgtact ggcatgtgat tgggatgggc actacccctg aggtgcatag catcttcctg gaaggccata cct tcctggt gaggaatcat aggcaggctt ctctggaaat ttctcccatc actttcctga ctgctcagac cctgctgatg gacctgggcc agttcctgct gttctgccac atcagctctc accagcatga tgggatggag gcctatgtga aggtggacag ctgtcctgag gagccccagc tgaggatgaa gaacaatgag gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt caggtttgat gatgacaata gcccctcttt catccagatc aggtctgtgg ccaagaagca ccccaagact t gggtgcact acattgctgc tgaggaggag gattgggatt atgcccctct ggtgctggcc cctgatgaca ggagctataa gtctcagtac ctgaataatg gcccccagag gattgggagg aagtataaga aggtgaggtt tatggcctac actgatgaga ccttcaagac cagggaggcc atccagcatg agtctggcat cctgggcccc ctgctgtatg gggaggtggg ggataccctg ctgatcatct tcaagaacca ggcctctagg ccctacaata tctaccctca tggcatcact gatgtgagac ccctgtatag caggaggctg cctaaggggg tgaagcacct gaaggacttc cccatcctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatggcccc accaagtctg accccaggtg cctgaccagg tattacagct cttttgtgaa catggagagg gatctggcct ctggg ctgat tggcccactg ctgatctgct acaaggagtc tgtggatcag aggggcaatc agatcatgtc tgacaagagg aatgtgatcc tgttttctgt gtttgatgaa aataggtctt ggtatctgac tgagaacatc cagaggtttc tgcccaatcc tgctggggtg cagctggagg atcctgagt t tcaggcctct aatatcatgc attctatcaa tggctatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc attggggctc agactgactt cctgtctgtg ttcttttctg gctatacttt caagcacaag atggtgtatg aggacactct gaccctgttc cccttctctg gggagactgt gttcatgtct atggaaaatc ctgggctgtg gattctgggc tgccacaatt ctgacttcag gaataggggg atgactgccc tgctgaaggt gtctag ctgt gataagaaca ctggggatta ctatgaggac tcttatgaag atatctctgc ctatctgctg agcaagaaca atgccattga gcccaggagc ttcagccaga acccccctgt gctgaagagg caccagaggg agatcaccag gaccactctg cagtctgatc aggaggagat tgactatgat gacactatct ctgtggagat ga agaaggag gattttgaca tttatgatga ggatgagaac cagtctccca ggagcttcca gaagaagacc aggcattact ttattgctgc tgtggagagg ctgtgggact atgggatgag cagctctcct catgtgctga ggaacagggc ccagtctggg tctgtgcccc agttcaagaa ggtggtgttc caggagttca ctgatgggag cttcacccag cccctgtata ggggggagct gaatgagcac ctgggcctgc tgggccccta catcagggct gaggtggagg ataata tcat ggtgaccttc aggaaccagg ctagcaggcc ttacagcttt tacagcagcc tgatctctta tgaagaagac cagaggcagg gggctgagcc caggaagaat tttgtgaagc ctaatgagac caagacttat ttttggaagg tgcagcatca catggctcct accaaggatg agtttgactg caaggcctgg gcctact ttt ctgatgtgga tctggagaag gatgtgcact ctggcctgat tggccctctg ctggtgtgcc atactaacac tctgaaccct gcccatggga ggcaggtgac tgtgcaggag tttgccctgt tcttcactat ttttgatgag accaagtctt ggtatttcac tgagaacatg gagaggaact gcagggctcc ctgcaacatc cagatggaag accccacctt caaggagaac tataggttcc atgccatcaa tgggtacatc atggataccc tgcctggcct ggtgatggcc caggatcaga ggatta ggtg gtatctgctg agcatgggct ctaatgagaa catccacagc atccatttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggctctg tacaacctgt atcctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc tggagggtgg aatgcctgat tggggagca c ctgcatgctg gcatgagcac tctgttcctg gtgtatagca acaagtgcca gacccccctg ggcatggcct ctggccatat cagggatttc cagatcactg cttctggcca gtatggccag tgggccccca agctggccag gctgcactat tctggcagca tcaatgcctg gagcactaag gagccttttt cttggatcaa ggtggacctg ctggccccta tgattattca tggcatcaag acccagggggg ccaggcagaa gttctctagc ctgtacatct ctcagttcat cattatgtat agcct ggatg gcaagaagtg gcagacctac aggggcaata gcactggcac cctgatggtg ttttttggga atgtggactc ttctgggatc aagcacaaca tctttaaccc ccccatcatt gccaggtata ttaggctgca ccccacccac tacagcatca ggagcaccct gaggatggag ctgatgggct gtgatctgaa ttctt gctct atgcccctgg gcatggagag caaggccatc tctgatgccc agatcactgc cagctcttac ttcaccaaca tgtttgccac ctggtctcct agcaaggcca ggctgcatct gcagggcagg agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag aagaccatga aggtgactgg ggtgaccact caggggggtga agagcctgct gacctctatg tatgtgaagg agttcctgat cagcagcagc caggatggcc accagtggac tctg ttcttc cagaatggga aggtgaaggt gttccagggc aaccaggata gctttacccc tgtggtgaac agcctggacc ctcctctgct gaccagatac ctgaggatcc atcctcagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga

FVIII encoding nucleic acid variant with reduced CpG content X10 (SEQ ID NO:10)

atgcagattg agctgagcac ttgcttcttc ctgtgcctgc tgaggttctg cttttctgct actaggaggt actacctggg ggctgtggag ctgagctggg attacatgca gtctgacctg ggggagctgc cagtggatgc caggttcccc cccagggtgc ccaagtcttt tcctttcaac acctctgtgg tgtacaagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt gccaagccca ggcccccctg gatggggctg ctggggccca ccatccaggc tgaggtgtat gacactgtgg tgattaccct gaagaacatg gctagccacc ctgtgagcct gcatgctgtg ggggtgagct attgga aggc ctctgagggg gctgagtatg atgatcagac cagccagagg gaaaaggagg atgacaaggt gttccctggg ggcagccata cttatgtgtg gcaggtgctg aaggagaatg ggcccatggc ctctgacccc ctgtgcctga cttacagcta tctgagccat gtggacctgg tgaaggatct gaactctggc ctgattgggg ctctgctggt gtgcagggag ggcagcctgg ctaaggagaa gactcagact ctgcataagt tcatcctgct gtttgctgtg tttgatgaag gcaagagctg gcactct gag accaagaact ctctgatgca ggataggat gctgcctctg ccaggcttg gcccaagatg cacactgtga atggctatgt gaacaggagc ctgcctggcc tgattgggtg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc accacccctg aggtgcacag cattttcctg gagggccaca c cttcctggt gaggaatcac aggcaggcca gcctggagat cagccccatc accttcctga ctgcccagac cctgctgatg gacctggggc agtttctgct gttctgccac atcagcagcc atcagcatga tggcatggag gcctatgtga aggtggactc ttgccctgag gagccccagc tgaggatgaa gaacaatgag gaggctgagg attatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat gatgacaata gccccagctt catccagatt aggtctgtgg ccaagaagca ccctaagacc tggg tgcact acattgctgc tgaggaggag gattgggatt atgcccccct ggtgctggct cctgatgaca ggtcttataa gagccagtac ctgaacaatg ggccccagag gattggcagg aagtacaaga aggtgaggtt catggcttac actgatgaga ccttcaagac tagggaggcc atccagcatg agtctggcat cctgggcccc ct gctgtatg gggaggtggg ggataccctg ctgatcatct tcaagaacca ggccagcagg ccctacaaca tttaccctca tggcatcact gatgtgaggc ccctgtacag caggagactg cccaaggggg tgaagcacct gaaggatttt cccattctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggcccc accaagtctg atcccaggtg cctgactagg tactactctt ctttttgtgaa tatggagagg gatctggcct ctggcc tgat tggccccctg ctgatctgct acaaggagtc tgtggaccag aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag aataggagct ggtacctgac tgagaatatc cagaggttcc tgcctaatcc tgctggggtc cagctggagg atcctgagtt ccaggc tagc aacattatgc acagcatcaa tggctatgtg tttgattctc tgcagctgtc tgtgtgcctg catgaggtgg cttactggta catcctgtct attggggccc agactgattt cctgtctgtg ttcttctctg gctacacttt caagcataag atggtgtatg aggataccct gaccctgttc cccttctctg gggagactgt gttcatgtct atggagaacc ctggcctgtg gatcctgggc tgtcataact ctgacttcag aaacaggggc atgactgccc tgctgaaggt gagcagct gt gacaagaaca ctggggacta ctatgaggac agctatgagg atatctctgc ttatctgctg agcaagaata atgccattga gcccaggagc ttcagccaga acccccctgt gctgaagagg caccagaggg agatcactag gactaccctg cagtctgatc aggaggat tgactatgat gacaccatct ctgtggagat gaaga aggag gactttgaca tctatgatga ggatgagaac cagtccccca ggtctttcca gaagaagacc aggcactact tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagcccc catgtgctga ggaacagggc tcagtctggc tctgtgcccc agttcaagaa ggtggtcttc caggagttca ctgatggctc ttttacccag cctctgtaca gaggggagct gaatgagcac ctgggcctgc tgggccccta catcagggct gaggtggagg ataatatcat ggtgaccttc agaaaccagg cctctaggcc ctacagcttc tacagcagcc tgatctctta tgaggaggat cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacctac ttctggaagg tgcagcacca tatggcccct actaaggatg agtttgactg caaggcctgg gcttatt ttt ctgatgtgga cctggagaag gatgtgcact ctgggctgat tggccccctg ctggtgtgcc acaccaacac cctgaaccct gcccatggca ggcaggtgac tgtgcaggag tttgccctgt tcttcactat ctttgatgag accaagagct ggtacttcac tgagaacatg gagagaaatt gtagggctcc ctgcaatatc cagatggagg accccacctt caaagaaaat tacagattcc atgccatcaa tgggtacatc atggataccc tgcctgggct ggtgatggct caggaccaga ggatcaggtg gtacctgctg agcatggggt ctaatgagaa catccactct atccatttct ctggccatgt gttcactgtg agaaagaagg aggagtataa gatggctctg tacaacctgt acccaggggt gtttgagact gtggaaatgc tgcccagcaa agctgggatc tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgtctac cctgttcctg gtgtacagca acaagtgcca gactcccctg ggcatggcct ctgggcacat cagggatttt cagatcactg cctctggcca gtatggccag tgggccccca agctggccag gctgcactac tctggcagca ttaatgcttg gagcactaag gagcccttca gctggatcaa ggtggatctg ctggccccca tgatcatcca tggcatcaag acccagggg ccaggcagaa gttctctagc ctgtacattt ctcagttcat catcatgtac agcctggatg ggaagaagtg gcagacctac agggggaaca gcactgggac cctgatggtg ttctttggca atgtggatag ctctggcatc aagcacaata tcttcaatcc ccccattatt gccaggtaca ttaggctgca tcctactcac tactctatta ggagcaccct gaggatggag ctgatggggt gtgacctgaa cagctgttct atg cccctgg gcatggagtc taaggctatc tctgatgccc agatcactgc cagcagctac ttcactaata tgtttgccac ctggagccct agcaaggcca gactgcacct gcagggcagg agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag aagaccatga aggtgactgg ggtgaccact caggggggtga agagcctgct gaccagcatg tatgtgaagg agttcctgat cagcagcagc caggatggcc accagtggac cctgt tcttc cagaatggga aggtgaaggt gttccagggc aaccaggact ctttcacccc tgtggtgaac agcctggatc ctcccctgct gaccaggtac ctgaggatcc acccccagag ctgggtgcac cagattgctc tgaggatgga agtgctgggc tgtgaggccc aggatctgta ctga

FVIII encoding nucleic acid variant with reduced CpG content X11 (SEQ ID NO:11)

atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttttg cttctctgct accaggaggt actacctggg ggctgtggag ctgagctggg actatatgca gtctgacctg ggggagctgc ctgtggatgc taggttccct cccagggtgc ccaagagctt cccctttaat acctctgtgg tgtacaagaa aaccctgttt gtggagttca ctgaccatct gttcaacatt gccaagccca ggcccccttg gatgggcctg ctgggcccca ccattcaggc tgaggtgtat gacactgtgg tcattaccct gaagaacatg gcttctcacc ctgtgagcct gcatgctgtg ggggtgagct act ggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg gagaaggagg atgataaggt gttccctggg ggcagccaca cctatgtgtg gcaggtgctg aaggagaatg gccccatggc ctctgatccc ctgtgcctga cctactctta tctgtctcat gtggacctgg tgaaggacct gaactctggc ctgattgggg ctctgctggt gtgcagggag ggctctctgg ccaaggagaa gacccagacc ctgcacaagt ttattctgct gtttgctgtc tttgatgagg gcaagagctg gcattctgag accaagaaca gcctgatgca ggacagggat gctgcctctg ccagggcctg gcccaaaatg cacactgtga atggctatgt gaacaggagc ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc accacccctg aggtgcacag catcttcctg gagggccaca cct ttctggt gaggaatcac aggcaggcca gcctggagat tagccccatc accttcctga ctgcccagac cctgctgatg gacctgggcc agttcctgct gttctgccac atcagcagcc accagcatga tggcatggag gcctatgtga aggtggatag ctgccctgag gagccccagc tgaggatgaa aaacaatgag gaggctgagg attatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat gatgacaata gccccagctt tattcagatt aggtctgtgg ctaagaagca ccccaagact tgg gtgcact acattgctgc tgaggaggag gattgggact atgcccctct ggtcctggcc cctgatgata ggtcttacaa gagccagtat ctgaacaatg gcccccagag gattggcagg aagtacaaga aggtgaggtt catggcctac actgatgaga cctttaagac cagggaggcc attcagcatg agtctgggat cctggggcc cc ctgctgtatg gggaggtggg ggacactctg ctgatcatct tcaagaacca ggccagcagg ccttataaca tctaccctca tgggatcact gatgtgaggc ccctgtactc tagaaggctg cccaaggggg tcaagcacct gaaggatttt cccatcctgc ctggggagat tttcaagtac aagtggactg tgactgtgga ggatggcccc accaagtctg accctaggtg cctgaccagg tactacagct cttttgtgaa catggagagg gacctggcct ctggcc tgat tggccctctg ctgatttgct acaaggagtc tgtggaccag aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttttctgt gtttgatgag aacaggtctt ggtacctgac tgagaacatc cagaggttcc tgcctaaccc agctggggtg cagctggagg atcctgagtt ccaggccagc aatattatgc atagcattaa tggctatgtg tttgatagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc attggggccc agactgactt tctgtctgtg ttcttctctg gctacacctt caagcataag atggtgtatg aggacaccct gactctgttc cctttttctg gggagactgt gtttatgagc atggagaatc ctggcctgtg gatcctgggc tgccataatt ctgacttcag gaacaggggc atgactgccc tgctgaaagt gagca gctgt gacaagaata ctggggacta ctatgaagac agctatgagg acatctctgc ctacctgctg agcaagaaca atgccattga gcccaggagc ttcagccaga accccccagt gctgaagagg caccagagag agatcaccag gactaccctg cagtctgacc aggagagat tgactatgat gacaccattt ctgtggagat gaaga aggag gactttgaca tttatgatga ggatgagaat cagagcccca ggagcttcca gaagaagact aggcactatt ttattgctgc tgtggagagg ctgtgggact atggcatgag cagctctccc catgtgctga ggaatagggc ccagtctggc tctgtgcctc agttcaagaa ggtggtgttc caggagttca ctgatggcag ctttacccag cccctgtata ggggggagct gaatgagcac ctgggcctgc tgggccccta tatcagggct gaggtggagg aca atattat ggtgaccttt aggaaccagg ccagcaggcc ctactctttc tatagcagcc tgatcagcta tgaggaggac cagaggcagg gggctgagcc caggaagaat tttgtgaagc ctaatgagac caagacctac ttctggaagg tgcagcatca catggccccc accaaggatg agtttgactg caaggcttgg g cctatttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggccccctg ctggtgtgcc acactaacac tctgaatcct gcccatggca ggcaggtgac tgtgcaggag tttgccctgt tcttcaccat ctttgatgag accaagagct ggtacttcac tgagaacatg gagaggaact gcagggcccc ctgcaacatc cagatggagg atcccacctt caaggagaac tacaggtttc atgccatcaa tggctacatc atggacactc tgcctggcct ggtgatggcc caggatcaga ggat caggtg gtacctgctg agcatgggct ctaatgagaa tatccatagc atccacttct ctggccatgt gttcactgtc aggaagaagg aggagtacaa gatggctctg tataatctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc tggagggtgg agtgcctgat tggggag cac ctgcatgctg ggatgagcac cctgtttctg gtgtactcta acaagtgcca gacccccctg ggcatggcct ctgggcacat cagggatttc cagatcactg cttctggcca gtatggccag tgggccccca agctggccag gctgcactac tctggcagca tcaatgcctg gtctaccaag gagccctttt cttggattaa ggtggacctg ctggccccca tgatcatcca tggcatcaag acccagggggg ccaggcagaa gttcagcagc ctgtacatca gccagttcat catcatgtac agcctggat g gcaaaaagtg gcagacctac aggggcaata gcactgggac tctgatggtg ttctttggca atgtggacag ctctgggatc aagcacaata tcttcaaccc tcccatcatt gctaggtaca tcaggctgca ccccacccac tatagcatca ggtctaccct gaggatggag ctgatgggct gtgacctgaa ctcttg cagc atgcccctgg gcatggagtc caaagctatc tctgatgccc agattactgc cagcagctac ttcaccaaca tgtttgccac ctggtctccc tctaaggcca ggctgcacct gcagggcagg agcaatgcct ggaggcccca ggtgaacaat cccaaggagt ggctgcaggt ggatttccag aaaactatga aggtgactgg ggtgaccacc caggggggtga agtctctgct gaccagcatg tatgtgaagg agttcctgat ctcttctagc caggatggcc accagtggac tct gttcttc cagaatggca aggtgaaggt gttccagggc aaccaggaca gcttcacccc tgtggtgaac tctctggatc cccccctgct gaccaggtac ctgaggattc atccccagag ctgggtgcac cagattgctc tgagaatgga ggtgctgggg tgtgaggctc aggacctgta ttga

FVIII encoding nucleic acid variant with reduced CpG content X12 (SEQ ID NO:12)

atgcagattg agctgtctac ttgttttttt ctgtgcctgc tgaggttctg cttctctgcc accaggaggt attacctggg ggctgtggag ctgagctggg attacatgca gtctgatctg ggggagctgc ctgtggatgc caggttcccc cccagggtgc ccaagagctt cccctt caac acctctgtgg tgtataagaa gaccctgttt gtggagttca ctgatcatct gtttaacatt gccaagccca ggcccccctg gatgggcctg ctgggcccaa ctatccaggc tgaggtgtat gacactgtgg tcatcaccct gaagaatatg gccagccatc ctgtgagcct gcatgctgtg ggggtga gct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg gagaaggagg atgacaaggt gttccctggg ggcagccaca cctatgtgtg gcaggtgctg aaggagaatg gccccatggc ctctgacccc ctgtgcctga cttatagcta cctgtctcat gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt ctgtagggaa ggcagcctgg ccaaggagaa gacccagacc ctgcacaagt ttattctgct gtttgctgtg tttgatgaag gcaagagctg gcactctga g accaagaatt ctctgatgca ggataggat gctgcctctg ccaggcctg gcccaagatg catactgtga atggctatgt gaacagaagc ctgcctggcc tgattggctg ccataggaag tctgtgtatt ggcatgtgat tgggatgggc actacccctg aagtgcacag cattttcctg gagggccaca ctttcct ggt gaggaaccac aggcaggcct ctctggagat cagccccatt actttcctga ctgcccagac cctgctgatg gatctgggcc agttcctgct gttctgccac atctctagcc accagcatga tggcatggag gcctatgtga aggtggacag ctgccctgag gagccccagc tgaggatgaa gaataatgag gaggctgagg attatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat gatgataata gccccagctt catccagatc aggtctgtgg ccaagaagca tcccaagacc tggg tgcact atattgctgc tgaagaggag gactgggact atgcccctct ggtgctggct cctgatgaca ggagctataa gagccagtat ctgaacaatg ggccccagag gattgggagg aagtacaaga aggtgaggtt catggcctac actgatgaga cctttaagac cagggaggcc atccagcatg agtctggcat tctggggccc ctgct gtatg gggaggtggg ggacactctg ctgatcattt tcaagaacca ggccagcagg ccctacaata tttaccccca tggcatcact gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcacct gaaggacttc cccatcctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggccct accaagtctg accctaggtg tctgactagg tactacagca gctttgtgaa catggagaga gacctggctt ctggcc tgat tggccccctg ctgatctgct acaaggagtc tgtggatcag aggggcaacc agattatgtc tgataagagg aatgtcatcc tgttctctgt gtttgatgag aacaggagct ggtatctgac tgagaacatt cagaggttcc tgcccaaccc tgctggggtg cagctggagg accctgagtt ccaggcca gc aacatcatgc attctattaa tggctatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc attggggccc agactgactt tctgtctgtg tttttctctg ggtacacctt caagcacaag atggtctatg aggacaccct gaccctgttc cccttttctg gggaaactgt gtttatgagc atggagaacc ctgggctgtg gatcctgggc tgccacaact ctgactttag gaataggggc atgactgccc tgctgaaggt gagcag ctgt gacaagaata ctggggatta ctatgaggac agctatgagg atatctctgc ctacctgctg agcaagaaca atgccattga gcctaggagc ttcagccaga acccccctgt gctgaagagg caccagaggg agatcaccag gaccaccctg cagtctgatc aggaggagat tgactatgat gacaccatct ctgtggagat ga agaaggag gactttgata tttatgatga ggatgagaac cagagcccca ggagcttcca gaagaagacc aggcactatt tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagcccc catgtgctga ggaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttc caggaattta ctgatggcag ctttacccag cccctgtaca gaggggagct gaatgagcac ctgggcctgc tgggccccta catcagggct gaggtggagg ataatatcat ggtgaccttt aggaaccagg cctctaggcc ctattctttt tacagcagcc tgatcagcta tgaggaggac cagaggcagg gggctgagcc taggaagaac tttgtgaagc ccaatgagac caagacctac ttttggaaag tgcagcacca catggccccc actaaggatg agtttgattg caaggcctgg gcctatt tct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggccccctg ctggtgtgcc acaccaacac tctgaaccct gcccatggca ggcaggtgac tgtgcaggag tttgccctgt tctttaccat ctttgatgag actaagagct ggtatttcac tgagaacatg gagaggaact gcagagcccc ttgcaacatc cagatggagg accctacctt caaggagaac tataggttcc atgccatcaa tgggtacatc atggataccc tgcctggcct ggtgatggct caggaccaga ggatcaggt g gtacctgctg agcatgggga gcaatgagaa cattcatagc atccacttct ctgggcatgt gttcactgtg aggaagaagg aggagtataa gatggccctg tacaacctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgagcac tctgttcctg gtgtacagca acaagtgcca gacccccctg ggcatggcct ctggccacat cagggacttc cagattactg cctctgggca gtatgggcag tgggccccca agctggccag gctgcactac tctgggtcta tcaatgcttg gagcaccaag gagcctttca gctggatcaa ggtggatctg ctggccccca tgatcattca tgggatcaag acccagggggg ccaggcagaa gttcagcagc ctgtatattt ctcagttcat catcatgtat tct ctggatg gcaaaaagtg gcagacctat agagggaaca gcactgggac cctgatggtg ttttttggca atgtggatag ctctggcatc aagcacaata tcttcaaccc ccccattatt gccaggtaca tcaggctgca ccccacccac tactctatca ggagcaccct gaggatggag ctgatgggct gtgatctgaa cag ctgctct atgcctctgg ggatggaaag caaggccatc tctgatgccc agatcactgc cagcagctat ttcaccaata tgtttgccac ttggagccct agcaaggcta ggctgcatct gcagggcagg tctaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag aagactatga aagtgactgg ggtgaccacc caggggggtga aaagcctgct gaccagcatg tatgtgaagg agttcctgat tagcagcagc caggatggcc accagtggac cctgtt cttc cagaatggga aggtgaaggt gtttcagggc aatcaggata gcttcacccc agtggtgaac agcctggacc cccccctgct gaccaggtac ctgaggatcc acccccagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga

FVIII encoding nucleic acid variant with reduced CpG content X13 (SEQ ID NO:13)

atgcagattg agctgagcac ctgctttttc ctgtgcctgc tgaggttctg cttctctgct accaggaggt actacctggg ggctgtggag ctgtcttggg attacatgca gtctgacctg ggggagctgc ctgtggatgc caggtttccc cccagggtgc ccaagtcttt cccct ttaac acctctgtgg tgtataagaa gactctgttt gtggagttca ctgatcacct gttcaatatt gccaagccca ggcccccttg gatgggcctg ctgggcccca ctatccaggc tgaggtgtat gacactgtgg tcatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg ggggtga gct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg gagaaggagg atgacaaggt gttcccaggg gggtctcaca cttatgtgtg gcaggtgctg aaggagaatg ggcccatggc ctctgaccct ctgtgcctga cttatagcta cctgtctcat gtggatctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag gggagcctgg ccaaggagaa gacccagacc ctgcacaagt tcatcctgct gtttgctgtg tttgatgagg ggaagagctg gcactctgag accaagaata gcctgatgca ggacagggat gctgcttctg ctagggcctg gcctaagatg cacactgtga atggctatgt gaacaggagc ctgcctggcc tgattgggtg tcacaggaag tctgtgtact ggcatgtgat tggcatgggg actactccag aagtgcacag catcttcctg gaggggcaca ccttcctgg t gaggaatcac aggcaggcca gcctggagat ttctcccatc actttcctga ctgcccagac cctgctgatg gatctggggc agttcctgct gttctgccac atcagcagcc atcagcatga tgggatggag gcctatgtga aggtggacag ctgccctgag gagcctcagc tgaggatgaa gaacaatgag gaggctgagg actatgatga tgatctgact gactctgaga tggatgtggt gaggtttgat gatgacaact ctcccagctt catccagatc aggtctgtgg ccaagaagca ccccaagacc tgggtgcact acattgctgc tgaggaggag gattgggatt atgctcccct ggtgctggct cctgatgata ggagctacaa gagccagtat ctgaataatg ggccccagag gattggcagg aagtataaga aggtgaggtt catggcctac actgatgaga cctttaagac cagggaggct attcagcatg agtctggcat cctggggcc cc ctgctgtatg gggaggtggg ggacaccctg ctgatcattt tcaagaacca ggccagcagg ccctataaca tctatcccca tgggatcact gatgtgaggc ccctgtactc taggaggctg cccaaggggg tcaagcacct gaaggacttc cccatcctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggcccc actaagtctg accccaggtg cctgactagg tactacagca gctttgtgaa catggagaga gatctggcct ctggcct gat tggccccctg ctgatctgct acaaagagtc tgtggatcag aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag aacagaagct ggtacctgac tgagaacatt cagaggtttc tgcccaaccc tgctggggtc cagctggagg accctgagtt tcaggcca gc aacatcatgc acagcatcaa tgggtatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta tatcctgagc attggggccc agactgattt cctgtctgtg ttcttctctg gctacacttt caagcacaag atggtgtatg aggataccct gaccctgttc cctttctctg gggaaactgt gttcatgagc atggagaacc ctgggctgtg gatcctgggg tgccacaatt ctgatttcag gaacagaggc atgactgctc tgctgaaggt gtctagctgt gacaagaaca ctggggacta ctatgaggac agctatgagg acatctctgc ctacctgctg agcaagaaca atgctattga acccaggtct ttcagccaga acccccctgt gctgaagagg caccagaggg agatcactag gaccaccctg cagtctgatc aggaggat tgactatgat gacaccatct ctgtggagat gaagaagg ag gactttgaca tctatgatga ggatgagaat cagtctccca ggagcttcca gaagaagact aggcattact tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagccct catgtgctga ggaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttt caggagttca ctgatggcag cttcacccag cccctgtaca ggggggagct gaatgagcat ctgggcctgc tgggccccta catcagggct gaggtggagg acaacatcat ggtgaccttc agaaatcagg ctagcaggcc ctacagcttc tacagcagcc tgatctctta tgaggaggac cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacctat ttctggaagg tgcagcacca catggccccc accaaggatg agtttgattg caaggcctgg gcctacttct ctgatgtgga cctggagaag gatgtgcatt ctgggctgat tggccctctg ctggtgtgcc acaccaacac cctgaatcct gcccatggca ggcaggtgac tgtgcaggag tttgccctgt tctttactat ctttgatgag accaagtctt ggtattttac tgagaacatg gagaggaact gcagggcccc ctgcaacatc cagatggagg accccacctt caaggagaac tacagattcc atgccatcaa tggctacatt atggacactc tgcctggcct ggtgatggcc caggaccaga ggatcaggtg gtacctgctg tctatgggca gcaatgagaa cattcactct atccacttct ctgggcatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg tacaacctgt accctggggt gtttgagact gtggagatgc tgcctagcaa ggctgggatc tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgtctac cctgttcctg gtgtacagca acaagtgcca gacccccctg ggcatggcct ctggccacat cagagatttt cagatcactg cctctggcca gtatggccag tgggctccta agctggccag gctgcactac tctggcagca tcaatgcctg gagcaccaag gagcccttta gctggatcaa ggtggacctg ctggccccca tgatcatcca tggcatcaag actcagggggg ccaggcagaa gttctctagc ctgtacatta gccagttcat catcatgtat agcctggatg gcaaga agtg gcagacctac aggggcaaca gcactgggac cctgatggtg ttctttggga atgtggacag ctctgggatc aagcacaata tcttcaaccc ccccattatt gccaggtata ttaggctgca ccccactcac tacagcatta ggagcaccct gaggatggag ctgatgggct gtgatctgaa cagctgcagc atgcccc tgg gcatggagtc taaggccatc tctgatgccc agatcactgc cagctcttac ttcaccaaca tgtttgccac ttggagcccc agcaaggcca ggctgcacct gcagggcagg agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggatttccag aagactatga aggtgactgg ggtgaccact caggggggtga agagcctgct gactagcatg tatgtgaagg agttcctgat cagctctagc caggatggcc accagtggac cctgt tcttt cagaatggca aggtgaaggt gttccagggc aaccaggact ctttcacccc tgtggtgaat tctctggacc ctcccctgct gactaggtat ctgaggattc atccccagag ctgggtgcat cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ttga

FVIII encoding nucleic acid variant with reduced CpG content X14 (SEQ ID NO:14)

atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttttg cttttctgcc actaggaggt actacctggg ggctgtggag ctgtcttggg attacatgca gtctgacctg ggggagctgc cagtggatgc caggttcccc ccaagggtgc ccaagtcttt tcccttca at acctctgtgg tgtacaagaa gaccctgttt gtggagttta ctgatcatct gtttaacatt gccaagccca ggcccccctg gatggggctg ctgggcccca ccatccaggc tgaggtgtat gatactgtgg tgattaccct gaagaatatg gccagccatc ctgtgtctct gcatgctgtg ggggtgtctt at tggaaggc ctctgagggg gctgagtatg atgatcagac cagccagagg gagaaggagg atgataaggt gttccctggg ggctctcaca cctatgtgtg gcaggtgctg aaggagaatg ggcctatggc ctctgaccca ctgtgcctga cttacagcta tctgagccat gtggacctgg tgaaggacct gaactctggg ctgattgggg ccctgctggt gtgcagggag ggcagcctgg ccaaggagaa gactcagacc ctgcacaagt tcatcctgct gtttgctgtg tttgatgagg gcaagtcttg gcact ctgag accaagaaca gcctgatgca ggatagggat gctgcctctg ccaggggcctg gcccaagatg cacactgtga atggctatgt gaacaggtct ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc accacccctg aggtgcatag cattttcctg gagggccaca c cttcctggt gaggaaccac aggcaggcta gcctggagat cagccccatc actttcctga ctgcccagac cctgctgatg gacctgggcc agttcctgct gttctgccac atctctagcc accagcatga tggcatggag gcctatgtga aggtggactc ttgtcctgag gagccccagc tgaggatgaa gaacaatgag gaggctgagg attatgatga tgatctgact gattctgaga tggatgtggt gaggtttgat gatgacaaca gcccctcttt catccagatc aggtctgtgg ccaagaagca ccccaag acc tgggtgcact acattgctgc tgaggaggag gattgggatt atgcccccct ggtgctggcc cctgatgaca ggagctataa gtctcagtac ctgaacaatg gcccccagag aattggcagg aagtacaaga aggtgaggtt catggcctat actgatgaga ccttcaaaac cagggaggcc attcagcatg agtctggcat cct ggggccc ctgctgtatg gggaggtggg ggacaccctg ctgatcatct tcaagaacca ggctagcagg ccttacaaca tctaccccca tgggatcact gatgtgaggc ccctgtacag caggaggctg cctaaggggg tgaagcacct gaaggacttt cccattctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatgggccc accaagtctg accccaggtg cctgactagg tactactcta gctttgtgaa catggagagg gacctggcct ctgg gctgat tggccccctg ctgatctgtt acaaggagtc tgtggaccag aggggcaacc agatcatgtc tgataagagg aatgtgatcc tgttctctgt gtttgatgag aacaggagct ggtacctgac tgagaacatc cagagattcc tgcccaaccc tgctggggtg cagctggagg atcctgagtt cca ggccagc aacatcatgc attctatcaa tgggtatgtg tttgatagcc tgcagctgtc tgtgtgtctg catgaggtgg cctactggta cattctgagc attggggccc agactgactt cctgtctgtg ttcttctctg gctacacttt caaacacaag atggtgtatg aggacaccct gaccctgttc cccttctctg gggagactgt gtttatgagc atggagaacc ctgggctgtg gattctgggc tgccacaact ctgacttcag aacaggggc atgactgccc tgctgaaggt gtcttct tgt gataagaaca ctggggacta ttatgaagac agctatgagg acatctctgc ctacctgctg agcaagaata atgctattga gcccaggtct ttctctcaga acccccctgt gctgaagagg caccagaggg agatcaccag gaccaccctg cagtctgatc aggaggat tgactatgat gacactattt ctgtggagat gaaga aggaa gactttgata tctatgatga ggatgagaac cagagcccta ggagcttcca gaagaagact aggcattact tcattgctgc tgtggagagg ctgtgggact atggcatgag cagcagcccc catgtgctga ggaatagggc tcagtctggc tctgtgcctc agttcaagaa ggtggtgttc caggaattca ctgatggcag cttcactcag cccctgtaca ggggggagct gaatgagcac ctggggctgc tgggccctta catcaggct gaggtggagg acaatatcat ggtgaccttt aggaaccagg cctctaggcc ttacagcttc tactctagcc tgatctctta tgaagaggac cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac taagacttac ttctggaagg tgcagcacca catggctccc accaaggatg agtttgactg caaggcttgg gcctacttct ctgatgtgga cctggagaag gatgtgcact ctgggctgat tgggcccctg ctggtgtgcc acactaacac tctgaatcct gcccatggca gacaggtgac tgtgcaggag tttgccctgt tttttaccat ctttgatgag actaagtctt ggtacttcac tgagaacatg gagaggaact gcagggcccc ctgcaacatc cagatggagg atcccacctt caaggagaac tacaggtttc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggct caggaccaga gg attaggtg gtatctgctg agcatgggca gcaatgagaa tatccactct atccacttct ctgggcatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg tataacctgt atcctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc tggagagtgg agtgcctgat tgggg agcac ctgcatgctg gcatgagcac tctgtttctg gtgtatagca acaagtgtca gacccctctg ggcatggcct ctgggcacat tagggacttt cagatcactg cttctggcca gtatgggcag tgggctccca agctggccag gctgcactat tctggcagca ttaatgcctg gagcaccaag gagcctttca gctggatcaa ggtggacctg ctggccccca tgatcatcca tgggatcaag acccagggggg ctaggcagaa gttcagcagc ctgtacatca gccagtttat catcatgtat tctctggat g gcaagaagtg gcagacctac aggggcaatt ctactggcac tctgatggtg ttctttggga atgtggatag ctctgggatc aagcataata tcttcaatcc ccccattatt gctaggtata tcaggctgca ccccacccac tatagcatca ggagcaccct gaggatggag ctgatggggt gtgacctgaa cagctgcagc atgcccctgg gcatggagag caaggctatt tctgatgccc agatcactgc cagcagctac tttactaata tgtttgccac ctggagcccc agcaaggcca gactgcacct gcagggcagg tctaatgcct ggaggcctca ggtgaataac cccaaggagt ggctgcaggt ggacttccag aaaaccatga aggtgactgg ggtgactacc caggggggtga agtctctgct gaccagcatg tatgtgaagg agttcctgat ctcttctagc caggatggcc accagtggac cctg ttcttt cagaatggga aggtgaaggt cttccagggc aaccaggata gcttcacccc tgtggtgaat agcctggatc ctcctctgct gaccaggtat ctgaggatcc acccccagag ctgggtgcat cagattgccc tgaggatgga ggtgctgggc tgtgaggctc aggacctgta ctga

FVIII encoding nucleic acid variant with reduced CpG content X15 (SEQ ID NO:15)

atgcagattg agctgagcac ctgtttcttc ctgtgcctgc tgaggttctg tttctctgcc actaggaggt actacctggg ggctgtggag ctgagctggg actatatgca gtctgacctg ggggagctgc ctgtggatgc caggttcccc cccagggtgc ctaagagctt ccccttcaat act tctgtgg tgtacaagaa gactctgttt gtggagttta ctgaccacct gttcaacatt gctaagccca ggcctccctg gatggggctg ctgggcccca ccatccaggc tgaggtgtat gatactgtgg tgattaccct gaagaacatg gcctctcatc cagtgagcct gcatgctgtg ggggtgagct actggaagg c ctctgaaggg gctgagtatg atgaccagac cagccagagg gagaaggagg atgacaaggt gttccctggg ggcagccaca cctatgtgtg gcaggtgctg aaggagaatg gcccaatggc ctctgacccc ctgtgcctga cttatagcta cctgagccat gtggatctgg tgaaggacct gaattctggc ctgattgggg ccctgctggt gtgcagagag ggctctctgg ctaaggagaa gacccagact ctgcacaagt tcatcctgct gtttgctgtg tttgatgagg gcaagagctg gcactctgag actaagaata gcctgatgca ggacagggat gctgcttctg ccagggcctg gcccaagatg catactgtga atggctatgt gaacaggagc ctgcctggcc tgattggctg tcacaggaaa tctgtctact ggcatgtgat tgggatgggc actacccctg aggtgcactc tatcttcctg gagggccata ccttcc tggt gaggaaccac aggcaggcca gcctggagat ctctcccatt accttcctga ctgcccagac cctgctgatg gatctgggcc agttcctgct gttctgccac atcagcagcc accagcatga tgggatggag gcttatgtga aggtggatag ctgccctgag gagccccagc tgaggatgaa gaacaatgag gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat gatgacaact ctcccagctt tattcagatc aggtctgtgg ctaagaagca ccccaagact tgggtgcact acattgctgc tgaggaggag gactgggact atgcccctct ggtgctggct cctgatgaca ggtcttacaa gtctcagtac ctgaataatg gccctcagag gattggcagg aagtacaaga aggtgaggtt catggcctac actgatgaga ccttcaagac cagggaggcc atccagcatg agtctggcat cctggggcc cc ctgctgtatg gggaggtggg ggataccctg ctgatcatct tcaagaatca ggccagcagg ccctacaaca tctaccccca tggcatcact gatgtgaggc cactgtacag caggaggctg cccaaggggg tgaagcatct gaaggacttc cccattctgc ctggggagat cttcaagtac aaatggactg tgactgtgga ggatggccct accaagtctg accccaggtg tctgaccagg tactacagca gcttttgtgaa tatggagagg gacctggcct ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggaccag aggggcaatc agatcatgtc tgataagagg aatgtgattc tgttctctgt gtttgatgag aacaggagct ggtacctgac tgagaacatc cagaggttcc tgcccaatcc tgctggggtg cagctggagg accctgagtt ccaggccag c aatatcatgc acagcatcaa tggctatgtc tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cttactggta tattctgagc attggggccc agactgattt cctgtctgtg ttcttttctg gctatacctt taagcacaag atggtgtatg aggacaccct gaccctgttc cccttctctg gggagactgt gttcatgtct atggagaacc ctgggctgtg gatcctgggc tgccacaact ctgacttcag gaacaggggg atgactgccc tgctgaaggt gtctagct gt gataagaaca ctggggacta ttatgaggac agctatgagg acatctctgc ttacctgctg agcaagaaca atgccattga gcccaggtct ttcagccaga atccccctgt gctgaagagg catcagaggg agatcaccag gaccaccctg cagtctgatc aggaggat tgattatgat gacactatct ctgtggaaat gaaga aggag gactttgaca tctatgatga ggatgagaac cagagcccca ggagcttcca gaagaagacc aggcactact tcattgctgc tgtggagagg ctgtgggatt atggcatgag cagctctccc catgtgctga ggaacagagc ccagtctggc tctgtgcctc agttcaagaa ggtggtcttc caggagttca ctgatggctc tttcacccag cccctgtaca ggggggagct gaatgagcac ctgggcctgc tggggcccta cattagggct gaggtggagg ataacatcat ggtgactttc agaaaccagg ccagcaggcc ttacagcttt tactcttctc tgattagcta tgaggaggat cagaggcagg gggctgagcc taggaagaac tttgtgaagc ccaatgagac caagacctat ttctggaagg tgcagcacca catggctccc actaaggatg agtttgactg caaggcttgg gcctacttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tgggcccctg ctggtgtgcc acaccaacac cctgaaccct gcccatggca ggcaggtgac tgtgcaggag tttgccctgt tcttcaccat ctttgatgag actaagagct ggtacttcac tgagaacatg gagaggaact gcagggcccc ctgcaacatc cagatggagg accccacctt caaggagaat tacaggttcc atgccatcaa tggctacatt atggacaccc tgcctggcct ggtgatggcc caggatcaga ggatcagg tg gtatctgctg agcatgggct ctaatgagaa catccacagc atccacttct ctggccatgt gtttactgtg aggaagaagg aggaatacaa gatggctctg tataacctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctgggatc tggagggtgg agtgcctgat tggggagca c ctgcatgctg ggatgagcac cctgttcctg gtgtatagca ataagtgcca gacccccctg ggcatggctt ctggccacat cagggatttc cagatcactg cttctggcca gtatggccag tgggctccca agctggctag gctgcattac tctgggtcta tcaatgcctg gagcactaag gagcccttca gctggatcaa ggtggacctg ctggccccca tgatcattca tggcatcaag acccagggggg ctaggcagaa gttcagcagc ctgtacatca gccagttcat cattatgtac agcctgg atg gcaagaagtg gcagacttac aggggcaata gcactgggac tctgatggtg ttctttggca atgtggactc ttctggcatc aagcacaaca tcttcaaccc tcccatcatt gccaggtaca ttaggctgca ccctacccac tactctatca ggagcaccct gaggatggag ctgatggggt gtgatctgaa ctcttgcag c atgcctctgg gcatggaaag caaagccatc tctgatgccc agatcactgc ctctagctat ttcaccaata tgtttgccac ctggagccct agcaaggcca ggctgcacct gcagggcaga tctaatgcct ggaggcccca ggtgaacaat cccaaggagt ggctgcaggt ggacttccag aagaccatga aggtgactgg ggtgaccact caggggggtga agagcctgct gactagcatg tatgtgaagg agttcctgat ctcttctagc caggatggcc accagtggac cctg ttcttc cagaatggca aggtgaaagt gttccagggc aaccaggata gcttcactcc tgtggtgaac tctctggacc ctcccctgct gactaggtac ctgaggattc atccccagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga

FVIII encoding nucleic acid variant with reduced CpG content X16 (SEQ ID NO:16)

atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttctg cttctctgcc accaggaggt actacctggg ggctgtggag ctgtcttggg actatatgca gtctgacctg ggggagctgc cagtggatgc caggttcccc cccagggtgc ccaagagctt tcctttcaac action ggaaggc ctctgagggg gctgagtatg atgatcagac ttctcagagg gagaaggagg atgataaggt gttccctggg ggctctcaca cttatgtgtg gcaggtgctg aaggagaatg gccccatggc ttctgatcca ctgtgcctga cctactctta cctgagccat gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag ggcagcctgg ccaaggagaa gacccagacc ctgcataagt tcatcctgct gtttgctgtg tttgatgagg ggaagagctg gcactctgag accaagaatt ctctgatgca ggacagggat gctgcctctg ccagggcctg gcctaagatg cacactgtga atggctatgt gaacaggtct ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc actacccctg aggtgcacag cattttcctg gagggccaca ccttcc tggt caggaaccat aggcaggcct ctctggagat cagccccatc actttcctga ctgcccagac cctgctgatg gacctgggcc agttcctgct gttctgccac attagcagcc accagcatga tggcatggag gcctatgtga aggtggactc ttgccctgag gagccccagc tgaggatgaa gaacaatgag gaagctgagg attatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat gatgacaaca gccccagctt catccagatc aggtctgtgg ccaagaagca ccccaagacc t gggtgcact acattgctgc tgaggaggag gattgggact atgctcccct ggtgctggct cctgatgata ggagctacaa gtctcagtac ctgaataatg gcccccagag gattggcagg aagtacaaga aggtgaggtt catggcctac actgatgaga ccttcaagac cagagaggct atccagcatg agtctgggat cctggggcc c ctgctgtatg gggaggtggg ggacaccctg ctgatcatct tcaagaacca ggccagcaga ccctacaaca tctaccccca tgggatcact gatgtgaggc ccctgtacag caggaggctg cctaaggggg tgaagcacct gaaggacttc cccatcctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatgggccc accaagtctg accctaggtg cctgactagg tactactcta gctttgtgaa catggagagg gacctggcct ct ggcctgat tggccccctg ctgatttgct acaaggagtc tgtggatcag aggggcaatc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag aataggtctt ggtacctgac tgagaacatc cagaggttcc tgcctaatcc tgctggggtg cagctggagg accctgagt t tcaggccagc aacatcatgc acagcatcaa tggctatgtg tttgactctc tgcagctgtc tgtgtgcctg catgaggtgg cttactggta tatcctgagc attggggctc agactgactt cctgtctgtg ttcttttctg gctacacttt taagcacaag atggtgtatg aggacaccct gaccctgttc cccttttctg gggagactgt gttcatgtct atggagaacc ctgggctgtg gattctgggc tgtcacaact ctgacttcag aaacaggggc atgactgccc tgctgaaggt gtc tagctgt gacaagaata ctggggacta ctatgaggac agctatgagg acatttctgc ctatctgctg agcaagaaca atgccattga gcccaggagc ttttctcaga atccccctgt gctgaagagg caccagagag agatcaccag gaccactctg cagtctgatc aggaggagat tgattatgat gacactatct ctgtggagat gaagaaagag gactttgata tctatgatga ggatgagaat cagtctccca ggagcttcca gaagaagact agacactact tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagccct catgtgctga ggaacagggc ccagtctggg tctgtgcccc agttcaagaa ggtggtgttc caggagttca ctgatggcag ctttacccag cccctgtata ggggggagct gaatgagcat ctgggcctgc tgggccccta tattagggct gaagtggagg acaacatcat ggtgaccttt aggaaccagg ccagcaggcc ctacagcttt tacagcagcc tgattagcta tgaggaggat cagagacagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacctac ttctggaagg tgcagcacca catggcccct accaaggatg agtttgactg caaggcctgg gcttactt ct ctgatgtgga cctggagaaa gatgtgcact ctggcctgat tgggcccctg ctggtgtgcc acaccaacac cctgaaccct gcccatggga ggcaggtgac tgtgcaggag tttgccctgt ttttcaccat ctttgatgag accaagagct ggtacttcac tgagaacatg gagaggaact gcagggcccc ctgtaacatc cagatggagg atcctacttt caaggagaac tacaggttcc atgccattaa tgggtacatc atggacaccc tgcctgggct ggtgatggcc caggatcaga ggat taggtg gtatctgctg tctatgggct ctaatgagaa catccactct atccacttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg tacaacctgt accctggggt gtttgaaact gtggagatgc tgccctctaa agctgggatc tggagggtgg agtgcctgat tgggg agcac ctgcatgctg gcatgagcac cctgttcctg gtgtacagca ataagtgcca gactcccctg ggcatggctt ctgggcacat cagggatttc cagatcactg cctctggcca gtatggccag tgggccccca agctggctag gctgcactac tctggcagca tcaatgcctg gagcaccaag gagcccttct cttggattaa ggtggacctg ctggctccca tgatcattca tggcatcaag acccagggggg ccaggcagaa gttttctagc ctgtatatta gccagttcat catcatgtat agcctggat g ggaagaagtg gcagacctac agggggaata gcactggcac cctgatggtg ttttttggca atgtggattc ttctggcatc aagcataaca tcttcaatcc ccctatcatt gccaggtaca ttaggctgca tcccacccat tactctatca ggagcaccct gaggatggag ctgatggggt gtgatctgaa cagctgtagc at gcccctgg gcatggagtc caaggctatc tctgatgccc agatcactgc cagcagctac ttcaccaaca tgtttgccac ctggagcccc agcaaggcca ggctgcacct gcagggcagg tctaatgcct ggaggcccca ggtgaacaat cccaaggagt ggctgcaggt ggacttccag aagactatga aggtgactgg ggtgaccact caggggggtga agagcctgct gaccagcatg tatgtgaagg agttcctgat ctcttctagc caggatgggc atcagtggac cct gtttttt cagaatggca aagtgaaggt gtttcagggg aatcaggaca gctttacccc tgtggtgaac agcctggatc ctcctctgct gactagatac ctgaggatcc acccccagag ctgggtccac cagattgctc tgaggatgga ggtgctgggg tgtgaggctc aggacctgta ctga

FVIII encoding nucleic acid variant with reduced CpG content X17 (SEQ ID NO:17)

atgcagattg agctgagcac ctgcttcttt ctgtgcctgc tgaggttctg cttctctgcc accaggaggt actacctggg ggctgtggaa ctgagctggg actatatgca gtctgacctg ggggagctgc ctgtggatgc caggttcccc cccagggtgc ccaagtcttt ccccttta ac acttctgtgg tgtacaagaa gaccctgttt gtggagttta ctgaccacct gttcaatatt gccaagccca ggcccccctg gatgggcctg ctgggcccaa ccatccaggc tgaggtgtat gatactgtgg tgatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg ggggtgagct at tggaaggc ttctgagggg gctgagtatg atgaccagac tagccagagg gagaaggagg atgacaaggt gttccctggg gggtctcata cctatgtgtg gcaggtgctg aaggagaatg gccccatggc ctctgacccc ctgtgcctga cctattctta cctgagccat gtggacctgg tcaaggacct gaactctggc ctgattgggg ctctgctggt gtgcagggag ggcagcctgg ccaaggagaa gactcagact ctgcataagt tcatcctgct gtttgctgtg tttgatgagg gcaagagctg gcactctgag accaagaact ctctgatgca ggataggat gctgcctctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaataggtct ctgcctggcc tgattggctg ccataggaag tctgtgtact ggcatgtgat tggcatgggc actacccctg aggtgcactc tatcttcctg gaggggcaca ccttcctgg t gaggaaccac aggcaggcca gcctggagat ctctcccatc accttcctga ctgcccagac tctgctgatg gacctgggcc agttcctgct gttctgccat atcagcagcc accagcatga tggcatggag gcctatgtga aggtggacag ctgcccagag gaaccccagc tgaggatgaa gaacaatgag gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat gatgacaaca gccccagctt tattcagatc aggtctgtgg ccaagaagca ccccaagacc t gggtgcact acattgctgc tgaggaggag gactgggatt atgcccccct ggtgctggcc cctgatgaca ggtcttacaa gtctcagtac ctgaacaatg gcccccagag gattgggagg aagtacaaga aggtgaggtt catggcctac actgatgaga ccttcaagac cagggaggcc atccagcatg agtctggcat cctggggcc c ctgctgtatg gggaggtggg ggataccctg ctgattatct tcaagaacca ggctagcagg ccctataaca tctaccccca tggcattact gatgtgaggc ccctgtactc taggagactg cccaaggggg tgaagcacct gaaagacttc cccatcctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatggcccc actaagtctg accccaggtg cctgaccagg tattacagca gctttgtgaa tatggagagg gatctggctt ctggcct gat tgggcctctg ctgatttgct acaaggagtc tgtggatcag agggggaacc agattatgtc tgacaagagg aatgtgattc tgttctctgt gtttgatgag aacaggagct ggtacctgac tgagaatatc cagaggttcc tgcctaatcc tgctggggtg cagctggagg accctgagtt cca ggctagc aacattatgc acagcatcaa tggctatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cttactggta cattctgtct attggggccc agactgactt cctgtctgtg ttcttctctg gctacacctt caagcacaag atggtgtatg aggacactct gaccctgttc cccttctctg gggagactgt gttcatgagc atggagaatc ctgggctgtg gattctgggg tgccacaact ctgatttcag gaacaggggc atgactgccc tgctgaaggt gagcagctgt gacaagaaca ctggggatta ttatgaggac agctatgagg acatttctgc ctacctgctg agcaagaaca atgccattga gcctaggagc ttcagccaga atccccctgt gctgaagaga caccagaggg agatcactag gaccactctg cagtctgatc aggaggat tgactatgat gacaccattt ctgtggagat gaagaagg ag gactttgata tttatgatga ggatgagaac cagagcccca gaagcttcca gaagaagacc aggcactact tcattgctgc tgtggagagg ctgtgggatt atggcatgtc ttctagcccc catgtgctga ggaacagggc tcagtctggc tctgtgcctc agttcaagaa ggtggtgttc caggagttca ctgatggggag cttcacccag cctctgtaca ggggggagct gaatgaacat ctgggcctgc tggggcccta catcagggct gaggtggagg ataata tcat ggtgactttc aggaatcagg cctctaggcc ctacagcttc tactctagcc tgatcagcta tgaggaggac cagaggcagg gggctgagcc taggaagaat tttgtgaaac ccaatgagac caagacctac ttttggaagg tgcagcacca catggcccct accaaggatg agtttgactg taaggcctgg gcc tacttct ctgatgtgga cctggagaag gatgtgcatt ctgggctgat tggccccctg ctggtgtgcc acaccaacac cctgaaccct gcccatggca ggcaggtgac tgtgcaggag tttgccctgt tcttcaccat ctttgatgag actaagagct ggtatttcac tgagaacatg gagaggaact gtagggctcc ctgcaacatc cagatggagg atccaacttt caaggagaac tacaggttcc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggcc caggaccaga ggattaggtg gtacctgctg agcatgggct ctaatgagaa catccactct atccacttct ctggccatgt gtttactgtg aggaagaagg aggagtacaa gatggctctg tacaacctgt accctggggt gtttgagact gtggagatgc tgcctagcaa ggctggcatt tggagagtgg agtgtctgat tggggagcac ctg catgctg ggatgtctac cctgttcctg gtgtactcta acaagtgcca gacccccctg gggatggctt ctgggcacat cagagatttt cagattactg cttctgggca gtatggccag tgggctccca agctggccag actgcattac tctggctcta ttaatgcttg gagcaccaag gagcctttca gctggatcaa ggtggacctg ctggctccca tgatcatcca tggcattaag actcaggggg ctaggcagaa gttcagcagc ctgtatattt ctcagtttat tatcatgtat tctctggat g gcaagaagtg gcagacttac aggggcaaca gcactggcac cctgatggtg ttctttggca atgtggacag ctctgggatc aagcataaca tcttcaaccc ccccattatt gccaggtaca tcaggctgca ccccacccac tattctatca ggagcactct gaggatggag ctgatggggt gtgacctgaa cagctgctct atgcccctgg gcatggagag caaggccatc tctgatgccc agatcactgc cagctcttat ttcaccaaca tgtttgccac ctggagcccc agcaaggcca ggctgcacct gcagggcaga agcaatgcct ggaggcccca ggtgaacaat cctaaggagt ggctgcaggt ggacttccag aagactatga aggtgactgg ggtgactacc cagggggtga agagcctgct gaccagcatg tatgtgaagg agttcctgat tagcagcagc caggatgggc atcagtggac cct gttcttc cagaatggga aggtgaaggt gttccagggc aatcaggaca gcttcacccc tgtggtgaac agcctggacc cccccctgct gaccaggtac ctgaggatcc atccccagag ctgggtgcac cagattgctc tgagaatgga ggtgctgggc tgtgaggccc aggacctgta ttga

FVIII encoding nucleic acid variant with reduced CpG content X18 (SEQ ID NO:18)

atgcagattg agctgtctac ctgttttttt ctgtgcctgc tgaggttctg cttctctgct accaggaggt attatctggg ggctgtggag ctgagctggg actacatgca gtctgacctg ggggagctgc ctgtggatgc caggtttcct cccagggtgc ctaagagctt cccct tcaac acctctgtgg tgtacaagaa gactctgttt gtggagttca ctgaccacct gttcaacatt gccaagccca ggcccccctg gatggggctg ctgggcccca ctatccaggc tgaggtgtat gatactgtgg tgattaccct gaagaacatg gcctctcacc ctgtgtctct gcatgctgtg ggggtgag ct actggaaggc ttctgagggg gctgaatatg atgatcagac ctctcagagg gagaaggagg atgacaaggt gtttcctggg ggcagccaca cctatgtgtg gcaggtgctg aaggagaatg ggcccatggc ctctgatccc ctgtgcctga cctacagcta cctgagccat gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag ggcagcctgg ccaaggaaaa gacccagacc ctgcataagt tcatcctgct gtttgctgtg tttgatgagg gcaagtcttg gc actctgag accaagaaca gcctgatgca ggacagggat gctgcctctg ctagggcctg gcccaagatg cacactgtga atgggtatgt gaacagatct ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggg accacccctg aggtgcatag catcttcctg gaggggcaca c cttcctggt gagaaatcat aggcaggcca gcctggagat tagccccatc accttcctga ctgcccagac cctgctgatg gacctgggcc agttcctgct gttctgccac atttctagcc accagcatga tggcatggag gcctatgtga aggtggatag ctgccctgaa gagccccagc tgaggatgaa gaacaatgag gaggctgagg attatgatga tgatctgact gactctgaga tggatgtggt gaggtttgat gatgacaaca gccccagctt catccagatc aggtctgtgg ccaagaagca ccctaagacc t gggtgcact acattgctgc tgaagaggag gactgggact atgcccccct ggtgctggcc ccagatgaca ggtcttacaa gagccagtac ctgaataatg gcccccagag gattgggagg aagtataaga aagtgaggtt catggcttac actgatgaga cctttaagac tagggaggcc attcagcatg agtctgggat tctgggccct ctgctgtatg gggaggtggg ggacaccctg ctgatcattt tcaagaacca ggccagcagg ccctataata tttatcccca tgggattact gatgtcaggc ccctgtacag caggaggctg cctaaggggg tgaagcacct gaaggacttc cccattctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatggcccc accaagtctg atcctaggtg cctgaccagg tactatagca gctttgtgaa catggagagg gacctggctt ctggcct gat tggccccctg ctgatctgct acaaggaatc tgtggaccag aggggcaacc agattatgtc tgacaagagg aatgtgatcc tgttttctgt gtttgatgag aataggagct ggtatctgac tgagaacatc cagaggttcc tgcccaatcc tgctggggtg cagctggagg accctgagtt ccaggctt ct aacatcatgc atagcatcaa tgggtatgtg tttgactctc tgcagctgtc tgtgtgcctg catgaggtgg cctattggta catcctgagc attggggccc agactgactt cctgtctgtg ttcttctctg gctacacctt caagcacaag atggtgtatg aggacaccct gaccctgttc cctttctctg gggagactgt gttcatgagc atggagaacc ctggcctgtg gattctgggc tgccataatt ctgacttcag aaacaggggc atgactgctc tgctgaaggt gagcagctgt gacaagaata ctggggacta ctatgaggac tcttatgagg atatttctgc ctacctgctg agcaagaaca atgctattga gcccaggagc ttcagccaga acccccctgt cctgaagagg catcagaggg agatcactag gaccaccctg cagtctgatc aggaggat tgactatgat gacactatct ctgtggaaat gaagaa ggag gactttgata tctatgatga ggatgagaac cagagcccca ggtctttcca gaagaagacc aggcactact tcattgctgc tgtggagagg ctgtgggact atggcatgtc tagcagcccc catgtgctga ggaacagagc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttt caggagttca ctgatgggag cttcactcag cccctgtata ggggggagct gaatgagcat ctgggcctgc tggggcccta catcagggct gaggtggagg ataacatcat ggtgaccttc aggaaccagg ccagcaggcc ctactctttc tactcttctc tgatcagcta tgaggaggat cagaggcagg gggctgagcc taggaagaac tttgtcaagc ctaatgagac taagacctac ttttggaagg tgcagcacca catggctccc actaaggatg agtttgattg caaggcctgg gcct acttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggccccctg ctggtgtgtc acaccaatac cctgaaccct gcccatggca ggcaggtcac tgtgcaggag tttgccctgt ttttcactat ctttgatgag actaagtctt ggtacttcac tgagaacatg gaaaggaatt gcagggctcc ctgcaacatc cagatggagg accccacctt caaggagaac tacaggtttc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggct caggatcaga ggattaggtg gtatctgctg agcatgggca gcaatgagaa catccacagc atccactttt ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggctctg tacaatctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctgggatc tggagggtgg agtgcct gat tggggaacac ctgcatgctg gcatgtctac cctgttcctg gtgtactcta acaagtgcca gactcccctg ggcatggcct ctgggcacat cagggacttc cagatcactg cctctgggca gtatggccag tgggccccta agctggctag gctgcattac tctggcagca tcaatgcctg gagcaccaag gagcccttca gctggatcaa ggtggacctg ctggccccta tgatcatcca tggcatcaag acccagggggg ccagacagaa gttctcttct ctgtacatct ctcagttcat catcatgtac tctctggatg gcaaga agtg gcagacctac agggggaatt ctactggcac tctgatggtg ttctttggga atgtggatag ctctgggatc aagcataata ttttcaaccc ccccattatt gctaggtaca tcaggctgca cccaacccac tactctatta ggtctaccct gaggatggag ctgatgggct gtgacctgaa ctcttgtagc atgcccc tgg gcatggagag caaggctatc tctgatgccc agatcactgc cagcagctac tttaccaaca tgtttgctac ttggagcccc agcaaggcca ggctgcacct gcagggcagg agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggattttcag aagaccatga aggtgactgg ggtgaccact caggggggtga aaagcctgct gactagcatg tatgtgaagg agtttctgat cagcagctct caggatggcc atcagtggac cctg ttcttc cagaatggca aggtgaaggt gttccagggc aaccaggata gcttcacccc tgtggtgaat agcctggacc cccccctgct gaccaggtac ctgaggatcc atccccagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaagccc aggacctgta ctga

Factor VIII-BDD wild type cDNA (SEQ ID NO:19)

ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG CTTTAGTGCC ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG ACTATATGCA AAGTGATCTC GGTGAGCTGC CTGTGGACGC AAGATTTCCT CCTAGAGTGC CAAAATCTTT TCCATTCAAC ACCTCAGTCG TGTACAAAAA GACTCTGTTT GTAG AATTCA CGGATCACCT TTTCAACATC GCTAAGCCAA GGCCACCCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC TGAGGTTTAT GATACAGTGG TCATTACACT TAAGAACATG GCTTCCCATC CTGTCAGTCT TCATGCTGTT GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATG ATGATCAGAC CAGTCAAAGG GAGAAAGAAG ATG ATAAAGT CTTCCCTGGT GGAAGCCATA CATATGTCTG GCAGGTCCTG AAAGAGAATG GTCCAATGGC CTCTGACCCA CTGTGCCTTA CCTACTCATA TCTTTCTCAT GTGGACCTGG TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTAGT ATGTAGAGAA GGGAGTCTGG CCAAGGAAAA GACACAGACC TTGCACAAAT TTATACTACT TTTTGCTGTA TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACT CCTTGATGCA GGATAGGGAT GCTGCATCTG CTCGGGCCTG GCCTAAAATG CACACAGTCA ATGGTTATGT AAACAGGTCT CTGCCAGGTC TGATTGGATG CCACAGGAAA TCAGTCTATT GGCATGTGAT TGGAATGGGC ACCACTCCTG AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT GAGGAACCAT CGCCAGGCGT CCTTGGAAAT CTCGCCAATA ACTTTCCTTA CTGCTCAAAC ACTCTTGATG GACCTTGGAC AGTTTCTACT GTTTTGTCAT ATCTCTTCCC ACCAACATGA TGGCATGGAA GCTTATGTCA AAGTAGACAG CTGTCCAGAG GAACCCCAAC TACGAATGAA AAATAATGAA GAAGCGGAAG ACTATGATGA TGATCTTACT GATTCTGAAA TGGATGTGGT CAGGTTTGAT GATGACAACT CTCCTTCCTT TATCCAAATT CGCTCAGTTG CCAAGAAGCA TCCTAAAACT TGGGTACATT ACATTGCTGC T GAAGAGGAG GACTGGGACT ATGCTCCCTT AGTCCTCGCC CCCGATGACA GAAGTTATAA AAGTCAATAT TTGAACAATG GCCCTCAGCG GATTGGTAGG AAGTACAAAA AAGTCCGATT TATGGCATAC ACAGATGAAA CCTTTAAGAC TCGTGAAGCT ATTCAGCATG AATCAGGAAT CTTGGGACCT TTACTTTATG GGGAAGTTGG AGACACACTG TTGATTATAT TTAAGAATCA AGCAAGCAGA CCATATAACA TCTACCCTCA CGGAATCACT GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAAGGTG TAAAACATTT GAAGGATTTT CCAATTCTGC CAGGAGAAAT ATTCAAATAT AAATGGACAG TGACTGTAGA AGATGGGCCA ACTAAATCAG ATCCTCGGTG CCTGACCCGC TATTACTCTA GTTTCGTTAA TATGGAGAGA GATCTAGCTT CAGGACTCAT TGGCCCTCTC CTCATCTGCT AC AAAGAATC TGTAGATCAA AGAGGAAACC AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT ATTTGATGAG AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTC TCCCCAATCC AGCTGGAGTG CAGCTTGAGG ATCCAGAGTT CCAAGCCTCC AACATCATGC ACAGCATCAA TGGCTATGTT TTTGATAGTT TGCAGTTGTC AGTTTGTTTG CATGAGGTGG CATACTGGTA CATTCTAAGC ATTGGAGCAC AGACTGACTT CCTTTCTGTC TTCTTCTCTG GATATACCTT CAAACACAAA ATGGTCTATG AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CTTCATGTCG ATGGAAAACC CAGGTCTATG GATTCTGGGG TGCCACAACT CAGACTTTCG GAACAGAGGC ATGACCGCCT TACTGAAGGT TTCTAGTTGT GACAAGAACA CTGGTGATTA TTACGAGGAC AGTTATGAAG ATATTTCAGC ATACTTGCTG AGTAAAACA ATGCCATTGA ACCAAGAAGC TTCTCCCAAA ACCCACCAGT CTTGAAACGC CATCAACGGG AAATAACTCG TACTACTCTT CAGTCAGATC AAGAGGAAAT TGACTATGAT GATACCATAT CAGTTGAAAT GAAGAAGGAAA GATTTTGACA TTTATGATGA GGATGAAAAT CAGAGCCCCC GCAGCT TTCA AAAGAAAACA CGACACTATT TTATTGCTGC AGTGGAGAGG CTCTGGGATT ATGGGATGAG TAGCTCCCCA CATGTTCTAA GAAACAGGGC TCAGAGTGGC AGTGTCCCTC AGTTCAAGAA AGTTGTTTTC CAGGAATTTA CTGATGGCTC CTTTACTCAG CCCTTATACC GTGGAGAACT AAATGAACAT TTGGGACTCC TGGGGCCATA TATAAGAGCA GAAGTTGAAG ATAATATCAT GGTAACTTTC AGAAATCAGG CCTCTCGTCC CTATTCCTTC TATTCTAGCC TTATTTCTTA TGAGGAAGAT CAGAGGCAAG GAGCAGAACC TAGAAAAAAC TTTGTCAAGC CTAATGAAAC CAAAACTTAC TTTTGGAAAG TGCAACATCA TATGGCACCC ACTAAAGATG AGTTTGACTG CAAAGCCTGG GCTTATTTCT CTGATGTTGA CCTGGAAAAA GATGTGCACT CAGGCCTGAT TGGACCCC TT CTGGTCTGCC ACACTAACAC ACTGAACCCT GCTCATGGGA GACAAGTGAC AGTACAGGAA TTTGCTCTGT TTTTCACCAT CTTTGATGAG ACCAAAAGCT GGTACTTCAC TGAAAATATG GAAAGAAACT GCAGGGCTCC CTGCAATATC CAGATGGAAG ATCCCACTTT TAAAGAGAAT TATCGCTTCC ATGCAATCAA TGGCTACATA ATGGATACAC TACCTGGCTT AGTAATGGCT CAGGATCAAA GGATTCGATG GTATCTGCTC AGCATGGGCA GCAATGA AAA CATCCATTCT ATTCATTTCA GTGGACATGT GTTCACCGTA CGAAAAAAAG AGGAGTATAA AATGGCACTG TACAATCTCT ATCCAGGTGT TTTTGAGACA GTGGAAATGT TACCATCCAA AGCTGGAATT TGGCGGGTGG AATGCCTTAT TGGCGAGCAT CTACATGCTG GGATGAGCAC ACTTTTTCTG GTGTACAGCA ATAAGTGTCA GACTCCCCTG GGAATGGCTT CTGGACACAT TAGAGATTTT CAGATTACAG CTTCAGGACA ATATGGACAG TGGGCCCCAA AGCTGGCCAG ACTTCATTAT TCCGGATCAA TCAATGCCTG GAGCACCAAG GAGCCCTTTT CTTGGATCAA GGTGGATCTG TTGGCACCAA TGATTATTCA CGGCATCAAG ACCCAGGGTG CCCGTCAGAA GTTCTCCAGC CTCTACATCT CTCAGTTTAT CATCATGTAT AGTCTTGATG GGAAGAAGTG GCAG A GATTACTGC TTCATCCTAC TTTACCAATA TGTTTGCCAC CTGGTCTCCT TCAAAAGCTC GACTTCACCT CCAAGGGAGG AGTAATGCCT GGAGACCTCA GGTGAATAAT CCAAAAGAGT GGCTGCAAGT GGACTTCCAG AAGACAATGA AAGTCACAGG AGTAACTACT CAGGGAGTAA AATCTCTGCT TACCAGCATG TATGTGAAGG AGTTCCTCAT CTCCAGCAGT CAAGATGGCC ATCAGTGGAC TCTCTTTTTT CAGAATGGCA AAG TAAAGGT TTTTCAGGGA AATCAAGACT CCTTCACACC TGTGGTGAAC TCTCTAGACC CACCGTTACT GACTCGCTAC CTTCGAATTC ACCCCCAGAG TTGGGTGCAC CAGATTGCCC TGAGGATGGA GGTTCTGGGC TGCGAGGCAC AGGACCTCTA CTGA

Factor VIII V3 cDNA (SEQ ID NO:20)

ATGCAGATTGAGCTGAGCACCTGCTTCTTCCTGTGCCTGCTGAGGTTCTGCTTCTCTGCCACCAGGAGATACCTGGGGGCTGTGGAGCTGAGCTGGGACTACATGCAGTCTGACCTGGGGGAGCTGCCTGTGGATGCCAGGTTCCCCCCCAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCTGTGGTGTACAAGAAGACCCTGTTTGTGGAGTTCACTGACCACCTGTTCAA CATTGCCAAGCCCAGGCCCCCCTGGATGGGCCTGCTGGGCCCCACCATCCAGGCTGAGGTGTATGACACTGTGGTGATCACCCTGAAGAACATGGCCAGCCACCCTGTGAGCCTGCATGCTGTGGGGGTGAGCTACTGGAAGGCCTCTGAGGGGGCTGAGTATGATGACCAGACCAGCCAGAGGGAAGGAGGATGACAAGGTGTTCCCTGGGGGCAGCCACACCTATGTGTGGCAGGTGCTGA AGGAGAATGGCCCCATGGCCTCTGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCATGTGGACCTGGTGAAGGACCTGAACTCTGGCCTGATTGGGGCCCTGCTGGTGTGCAGGGAGGGCAGCCTGGCCAAGGAGAAGACCCAGACCCTGCACAAGTTCATCCTGCTGTTTGCTGTGTTTGATGAGGGCAAGAGCTGGCACTCTGAAACCAAGAACAGCCTGATGCAGGACAGGGATGCTGCCT CTGCCAGGGCCTGGCCCAAGATGCACACTGTGAATGGCTATGTGAACAGGAGCCTGCCTGGCCTGATTGGCTGCCACAGGAAGTCTGTGTACTGGCATGTGATTGGCATGGGCACCACCCCTGAGGTGCACAGCATCTTCCTGGAGGGCCACACCTTCCTGGTCAGGAACCACAGGCAGGCCAGCCTGGAGATCAGCCCATCACCTTCCTGACTGCCCAGACCCTGCTGATGGACCTGGGCCAG TTCCTGCTGTTCTGCCACATCAGCAGCCACCAGCATGATGGCATGGAGGCCTATGTGAAGGTGGACAGCTGCCCTGAGGAGCCCCAGCTGAGGATGAAGAACAATGAGGAGGCTGAGGACTATGATGATGACCTGACTGACTCTGAGATGGATGTGGTGAGGTTTGATGATGACAACAGCCCCAGCTTCATCCAGATCAGGTCTGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTACATTG CTGCTGAGGAGGAGGACTGGGACTATGCCCCCCTGGTGCTGGCCCCTGATGACAGGAGCTACAAGAGCCAGTACCTGAACAATGGCCCCCAGAGGATTGGCAGGAAGTACAAGAAGGTCAGGTTCATGGCCTACACTGATGAAACCTTCAAGACCAGGGAGGCCATCCAGCATGAGTCTGGCATCCTGGGCCCCCTGCTGTATGGGGAGGTGGGGGACACCCTGCTGATCATCTTCAAGAACCAGG CCAGCAGGCCCTACAACATCTACCCCCATGGCATCACTGATGTGAGGCCCCTGTACAGCAGGAGGCTGCCCAAGGGGGTGAAGCACCTGAAGGACTTCCCCATCCTGCCTGGGGAGATCTTCAAGTACAAGTGGACTGTGACTGTGGAGGATGGCCCCACCAAGTCTGACCCCAGGTGCCTGACCAGATACTACAGCAGCTTTGTGAACATGGAGAGGGACCTGGCCTCTGGCCTGATTGGCCCCCT GCTGATCTGCTACAAGGAGTCTGTGGACCAGAGGGGCAACCAGATCATGTCTGACAAGAGGAATGTGATCCTGTTCTCTGTGTTTGATGAGAACAGGAGCTGGTACCTGACTGAGAACATCCAGAGGTTCCTGCCCAACCCTGCTGGGGTGCAGCTGGAGGACCCTGAGTTCCAGGCCAGCAACATCATGCACAGCATCAATGGCTATGTGTTTGACAGCCTGCAGCTGTCTGTGTGCCTGCATGA GGTGGCCTACTGGTACATCCTGAGCATTGGGGCCCAGACTGACTTCCTGTCTGTGTTCTTCTCTGGCTACACCTTCAAGCACAAGATGGTGTATGAGGACACCCTGACCCTGTTCCCCTTCTCTGGGGAGACTGTGTTCATGAGCATGGAGAACCCTGGCCTGTGGATTCTGGGCTGCCACAACTCTGACTTCAGGAACAGGGCATGACTGCCCTGCTGAAAGTCTCCAGCTGTGACAAGAACACTGG GGACTACTATGAGGACAGCTATGAGGACATCTCTGCCTACCTGCTGAGCAAGAACAATGCCATTGAGCCCAGGAGCTTCAGCCAGAACAGCAGGCACCCCAGCACCAGGCAGAAGCAGTTCAATGCCACCACCATCCCTGAGAATGACATAGAGAAGACAGACCCATGGTTTGCCCACCGGACCCCCATGCCCAAGATCCAGAATGTGAGCAGCTCTGACCTGCTGATGCTGCTGAGGCAGAGCCCCAC CCCCCATGGCCTGAGCCTGTCTGACCTGCAGGAGGCCAAGTATGAAACCTTCTCTGATGACCCCAGCCCTGGGGCCATTGACAGCAACAACAGCCTGTCTGAGATGACCCACTTCAGGCCCCAGCTGCACCACTCTGGGGACATGGTGTTCACCCCTGAGTCTGGCCTGCAGCTGAGGCTGAATGAAGCTGGGCACCACTGCTGCCACTGAGCTGAAGAAGCTGGACTTCAAAGTCTCCAGCACCAGC AACAACCTGATCAGCACCATCCCCCTCTGACAACCTGGCTGCTGGCACTGACAACACCAGCAGCCTGGGCCCCCCCAGCATGCCTGTGCACTATGACAGCCAGCTGGACACCACCCTGTTTGGCAAGAAGAGCAGCCCCCTGACTGAGTCTGGGGGCCCCCTGAGCCTGTCTGAGGAGAACAATGACAGCAAGCTGCTGGAGTCTGGCCTGATGAACAGCCAGGAGAGCAGCTGGGGCAAGAA TGTGAGCACCAGGAGCTTCCAGAAGAAGACCAGGCACTACTTCATTGCTGCTGTGGAGAGGCTGTGGGACTATGGCATGAGCAGCAGCCCCCATGTGCTGAGGAACAGGGCCCAGTCTGGCTCTGTGCCCCAGTTCAAGAAGGTGGTGTTCCAGGAGTTCACTGATGGCAGCTTCACCCAGCCCCTGTACAGAGGGGAGCTGAATGAGCACCTGGGCCTGCTGGGCCCCTACATCAGGCTGAGG TGGAGGACAACATCATGGTGACCTTCAGGAACCAGGCCAGCAGGCCCTACAGCTTCTACAGCAGCCTGATCAGCTATGAGGAGGACCAGAGGCAGGGGGCTGAGCCCAGGAAACTTTGTGAAGCCCAATGAAACCAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACTTCTCTGATGTGGACCTGGAGAAGGATGTGCACTCT GGCCTGATTGGCCCCCTGCTGGTGTGCCACACCAACACCCTGAACCCTGCCCATGGCAGGCAGGTGACTGTGCAGGAGTTTGCCCTGTTCTTCACCATCTTTGATGAAACCAAGAGCTGGTACTTCACTGAGAACATGGAGAGGAACTGCAGGGCCCCCTGCAACATCCAGATGGAGGACCCCACCTTCAAGGAGAACTACAGGTTCCATGCCATCAATGGCTACATCATGGACACCCTGCCTGGCCTGG TGATGGCCCAGGACCAGAGGATCAGGTGGTACCTGCTGAGCATGGGCAGCAATGAGAACATCCACAGCATCCACTTCTCTGGCCATGTGTTCACTGTGAGGAAGAAGGAGGAGTACAAGATGGCCCTGTACAACCTGTACCCTGGGGTGTTTGAGACTGTGGAGATGCTGCCCAGCAAGGCTGGCATCTGGAGGGTGGAGTGCCTGATTGGGGAGCACCTGCATGCTGGCATGAGCACCCTG TTCCTGGTGTACAGCAACAAGTGCCAGACCCCCCTGGGCATGGCCTCTGGCCACATCAGGGACTTCCAGATCACTGCCTCTGGCCAGTATGGCCAGTGGGCCCCCAAGCTGGCCAGGCTGCACTACTCTGGCAGCATCAATGCCTGGAGCACCAAGGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCCATGATCATCCATGGCATCAAGACCCAGGGGGCCAGGCAGAAGTTCAGCAGCC TGTACATCAGCCAGTTCATCATCATGTACAGCCTGGATGGCAAGAAGTGGCAGACCTACAGGGGCAACAGCACTGGCACCCTGATGGTGTTCTTTGGCAATGTGGACAGCTCTGGCATCAAGCACAACATCTTCAACCCCCCCATCATTGCCAGATACATCAGGCTGCACCCCACCCACTACAGCATCAGGAGCACCCTGAGGATGGAGCTGATGGGCTGTGACCTGAACAGCTGCAGCATGCCCCTGGGG CATGGAGAGCAAGGCCATCTCTGATGCCCAGATCACTGCCAGCAGCTACTTCACCAACATGTTTGCCACCTGGAGCCCCAGCAAGGCCAGGCTGCACCTGCAGGGCAGGAGCAATGCCTGGAGGCCCCAGGTCAACAACCCCAAGGAGTGGCTGCAGGTGGACTTCCAGAAGACCATGAAGGTGACTGGGGTGACCACCCAGGGGGTGAAGAGCCTGCTGACCAGCATGTATGTGAAGGAGTTCCT GATCAGCAGCAGCCAGGATGGCCACCAGTGGACCCTGTTCTTCCAGAATGGCAAGGTGAAGGTGTTCCAGGGCAACCAGGACAGCTTCACCCCTGTGGTGAACAGCCTGGACCCCCCCCTGCTGACCAGATACCTGAGGATTCACCCCCAGAGCTGGGTGCACCAGATTGCCCTGAGGATGGAGGTGCTGGGCTGTGAGGCCCAGGACCTGTACTGA

Factor VIII CO3 cDNA (SEQ ID NO:21)

atgcagattg agctgtcaac ttgctttttc ctgtgcctgc tgagattttg tttttccgct actagaagat actacctggg ggctgtggaa ctgtcttggg attacatgca gagtgacctg ggagagctgc cagtggacgc acgatttcca cctagagtcc ctaaatcatt ccccttcaac acca gcgtgg tctataagaa aacactgttc gtggagttta ctgatcacct gttcaacatc gctaagcctc ggccaccctg gatgggactg ctgggaccaa caatccaggc agaggtgtac gacaccgtgg tcattacact gaaaaacatg gcctcacacc ccgtgagcct gcatgctgtg ggcgtcagct actggaaggc ttccgaaggg gcagagtatg acgatcagac ttcccagaga gaaaaagagg acgataaggt gtttcctggc gggtctcata cctatgtgtg gcaggtcctg aaagagaatg gccccatggc ttccgaccct ctgtgcctga cctactctta tctgagtcac gtggacctgg tcaaggatct gaacagcgga ctgatcggag cactgctggt gtgtagggaa gggagcctgg ctaaggagaa aacccagaca ctgcataagt tcattctgct gttcgccgtg tttgacgaag gaaaatcatg gcacag cgag acaaagaata gtctgatgca ggaccgggat gccgcttcag ccagagcttg gcccaaaatg cacactgtga acggctacgt caatcgctca ctgcctggac tgatcggctg ccaccgaaag agcgtgtatt ggcatgtcat cggaatgggc accacacctg aagtgcactc cattttcctg ga ggggcata cctttctggt ccgcaaccac cgacaggcct ccctggagat ctctccaatt accttcctga cagctcagac tctgctgatg gatctgggac agttcctgct gttttgccac atcagctccc accagcatga tggcatggag gcctacgtga aagtggacag ctgtcccgag gaacctcagc tgaggatgaa gaacaatgag gaagctgaag actatgacga tgacctgacc gactccgaga tggatgtggt ccgattcgat gacgataaca gcccctcctt tatccagatt agatctgtgg ccaagaaaca ccctaaga ca tgggtccatt acatcgcagc cgaggaagag gactgggatt atgcaccact ggtgctggca ccagacgatc gatcctacaa atctcagtat ctgaacaatg gaccacagcg gattggcaga aagtacaaga aagtgaggtt catggcttat accgatgaaa ccttcaagac tcgcgaagca atccagcacg agagcggga t tctgggacca ctgctgtacg gagaagtggg ggacaccctg ctgatcattt ttaagaacca ggccagcagg ccttacaata tctatccaca tggaattaca gatgtgcgcc ctctgtacag ccggagactg ccaaagggcg tcaaacacct gaaggacttc ccaatcctgc ccggggaaat ttttaagtat aaatggactg tcaccgtcga ggatggcccc actaagagcg accctaggtg cctgacccgc tactattcta gtttcgtgaa tatggaaagg g atctggcca gcggactgat cggcccactg ctgatttgtt acaaagagag cgtggatcag agaggcaacc agatcatgtc cgacaagagg aatgtgattc tgttcagtgt ctttgacgaa aaccggtcat ggtatctgac cgagaacatc cagagattcc tgcctaatcc agccggagtg cagctggaag atcct gagtt tcaggcttct aacatcatgc atagtattaa tggctacgtg ttcgacagtc tgcagctgtc agtgtgtctg cacgaggtcg cttactggta tatcctgagc attggagcac agacagattt cctgagcgtg ttcttttccg gctacacttt taagcataaa atggtgtatg aggacacact gactctgttc cccttcagcg gcgaaaccgt gtttatgtcc atggagaatc ccgggctgtg gatcctggga tgccacaaca gcgatttcag gaatcgcggg atgactgccc tgctgaa agt gtcaagctgt gacaagaaca ccggagacta ctatgaagat tcatacgagg acatcagcgc atatctgctg tccaaaaaca atgccattga acccaggtct tttagtcaga atcctccagt gctgaagagg caccagcgcg agatcacccg cactaccctg cagagtgatc aggaagagat cgactacgac gatacaattt ctgtggaaat gaagaaagag gacttcgata tctatgacga agatgagaac cagagtcctc gatcattcca gaagaaaacc cggcattact ttattgctgc agtggagcgc ctgtgggatt atggcatgtc ctctagtcct cacgtgctgc gaaatcgggc ccagtcaggg agcgtcccac agttcaagaa agtggtcttc caggagttta cagacggatc ctttactcag ccactgtacc ggggcgaact gaacgagcac ctggggctgc tgggacccta tatcagagct gaagtgga gg ataacattat ggtcaccttc agaaatcagg catctaggcc ttacagtttt tattcaagcc tgatctctta cgaagaggac cagaggcagg gagcagaacc acgaaaaaac ttcgtgaagc ctaatgagac caaaacatac ttttggaagg tgcagcacca tatggcccca acaaaagacg aattcgattg caaggcatgg gcctattttt ctgacgtgga tctggagaag gacgtccaca gtggcctgat cgggccactg ctggtgtgtc atactaacac cctgaatccc gcacacggca ggcaggtcac tgtccaggaa ttcgccctgt tctttaccat ctttgatgag acaaaaagct ggtacttcac cgaaaacatg gagcgaaatt gccgggctcc atgtaatatt cagatggaag accccacatt caaggagaac taccgctttc atgccatcaa tgggtatatt atggatactc tgcccggact ggtcatggct caggaccaga gaatcaggt g gtacctgctg agcatggggt ccaacgagaa tatccactca attcatttca gcggacacgt gtttactgtc cggaagaaag aagagtataa aatggccctg tacaacctgt atcccggcgt gttcgaaacc gtcgagatgc tgcctagcaa ggcagggatc tggagagtgg aatgcctgat tggggag cac ctgcatgccg gaatgtctac cctgtttctg gtgtacagta ataagtgtca gacacccctg gggatggctt ccggacatat ccgggatttc cagattaccg catctggaca gtacggccag tgggccccta agctggctag actgcactat tccgggtcta tcaacgcttg gtccacaaaa gagcctttct cttggattaa ggtggacctg ctggcaccaa tgatcattca tggcatcaaa actcagggggg ccaggcagaa gttctcctct ctgtacatct cacagtttat catcatgtac agcc tggatg gcaagaaatg gcagacatac cgcggcaata gcacagggac tctgatggtg ttctttggca acgtggacag ttcagggatc aagcacaaca ttttcaatcc ccctatcatt gctagataca tcaggctgca cccaacccat tattctattc gaagtacact gcggatggaa ctgatggggt gcgatctgaa cagt tgttca atgcccctgg gaatggagtc caaggcaatc tctgacgccc agattaccgc tagctcctac ttcactaata tgtttgctac ctggagcccc tccaaagcac gactgcatct gcagggacga agcaacgcat ggcgaccaca ggtgaacaat cccaaggagt ggctgcaggt cgattttcag aaaactatga aggtgaccgg agtcacaact cagggcgtga aaagtctgct gacctcaatg tacgtcaagg agttcctgat ctctagttca caggacggcc accagtggac act gttcttt cagaacggaa aggtgaaagt cttccagggc aatcaggatt cctttacacc tgtggtcaac tctctggacc cacccctgct gactcgctac ctgcgaatcc acccacagtc ctgggtgcat cagattgcac tgagaatgga agtcctgggc tgcgaggccc aggacctgta ttga

Full-length cassette including TTR promoter mutant (TTRmut), synthetic intron, CpG-reduced Factor VIII cDNA, poly-A and ITR repeats (SEQ ID NO:23)

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc tacgcgtgtc tgtctgcaca tttcgtagag cga gtgttcc gatactctaa tctccctagg caaggttcat attgacttag gttacttatt ctccttttgt tgactaagtc aataatcaga atcagcaggt ttggagtcag cttggcaggg atcagcagcc tgggttggaa ggagggggta taaaagcccc ttcaccagga gaagccgtca cacagatcca caag ctcctg ctagcaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactg acactgacat ccactttttc tttttctcca caggtttaaa cgccaccatg cagattgagc tgagcacctg cttcttcctg tgtctgctga ggttctgctt ctctgccacc aggaggtatt acctgggggc tgtggagctg agctgggact atatgcagtc tgacctgggg gagctgcctg tggatgctag gttccccccc agggtgccca agagcttccc ctttaacact t ctgtggtgt acaagaagac cctgtttgtg gagttcactg accacctgtt caacattgcc aagcccaggc ccccctggat ggggctgctg gggcccacca tccaggctga ggtgtatgac actgtggtga tcaccctgaa gaacatggcc agccaccctg tgagcctgca tgctgtgggg gtgagctact gga aggcttc tgaggggggct gagtatgatg accagactag ccagagggag aaggaggatg acaaggtgtt tcctggggggc agccatacct atgtgtggca ggtgctgaag gagaatggcc ccatggcctc tgaccccctg tgcctgacct acagctacct gtctcatgtg gacctggtga aggacctgaa ctctggcctg attggggctc tgctggtgtg tagggagggc agcctggcta aggaaaagac ccagaccctg cataagttta tcctgctgtt tgctgtgttt gatgagggca agagctggca ctctga gacc aagaacagcc tgatgcagga tagggatgct gcctctgcca gggcttggcc taagatgcac actgtgaatg ggtatgtgaa taggagcctg cctggcctga ttggctgcca caggaagtct gtgtactggc atgtgattgg gatgggcacc acccctgagg tccatagcat cttcctggag ggccacactt tcctggt gag gaaccacaga caggcctctc tggagatctc tcccatcacc ttcctgactg ctcagactct gctgatggac ctgggccagt tcctgctgtt ttgccatatt agcagccacc agcatgatgg gatggaggcc tatgtgaagg tggatagctg ccctgaggag cctcagctga ggatgaagaa caatgaggag gctgaagact atgatgatga cctgactgat tctgagatgg atgtggtgag gtttgatgat gacaatagcc ccagcttcat tcagatcagg tctgtggcca agaaacaccc caagacctgg g tgcactaca ttgctgctga ggaagaggac tgggactatg ctcccctggt gctggcccct gatgataggt cttataagag ccagtacctg aacaatgggc cccagaggat tggcaggaag tacaagaagg tgaggttcat ggcctacact gatgaaacct tcaaaaccag ggaggccatt cagcatgagt ctggcatcct ggg ccctctg ctgtatgggg aggtggggga caccctgctg atcatcttca agaaccaggc cagcaggccc tacaacatct atcctcatgg catcactgat gtgaggcccc tgtacagcag gaggctgccc aagggggtga agcacctgaa agacttcccc atcctgcctg gggagatctt taagtataag tggactgtga ctgtggagga tggccctacc aagtctgacc ccaggtgtct gaccaggtac tattctagct ttgtgaacat ggagagggac ctggcctct gcc tgattgg gcccctgctg atctgctaca aggagtctgt ggaccagagg ggcaaccaga tcatgtctga caagaggaat gtgatcctgt tttctgtgtt tgatgagaat aggagctggt acctgactga gaacatccag aggtttctgc ccaatcctgc tggggtgcag ctggaggatc ctgagttcca ggccagcaat atcatgcata gcatcaatgg ctatgtgttt gacagcctgc agctgtctgt gtgcctgcat gaggtggcct actggtacat cctgagcatt ggggcccaga ctgactttct gtctgtgttc ttttctggct ataccttcaa gcacaagatg gtgtatgagg ataccctgac cctgttcccc ttctctgggg agactgtgtt catgagcatg gagaatcctg ggctgtggat cctggggtgc cacaactctg attttaggaa cagggggatg actgccctgc tgaaggtgtc tagctgtga t aagaacactg gggactacta tgaggacagc tatgaggaca tttctgctta tctgctgtct aagaataatg ccattgagcc cagaagcttc agccagaatc cccctgtgct gaagagacat cagagggaga tcaccagaac taccctgcag tctgatcagg aggagattga ctatgatgac actatctctg tggagatgaa gaaggaggac tttgacatct atgatgagga tgagaatcag tctcccagga gctttcagaa gaagaccaga cattacttca ttgctgctgt ggagaggctg tgggactatg gcatgagctc tagccctcat gtgctgagga acagggccca gtctggctct gtgccccagt tcaagaaggt ggtgttccag gaattcactg atggcagctt cacccagccc ctgtacaggg gggagctgaa tgagcacctg ggcctgctgg ggccttatat cagggctgag gtggaggata atattatggt gacttt cagg aaccaggcca gcaggcccta ctctttctat agcagcctga tctcttatga ggaggatcag aggcaggggg ctgagcctag gaagaacttt gtgaagccca atgagactaa gacctacttc tggaaggtcc agcaccacat ggcccctacc aaggatgagt ttgactgcaa ggcctgggcc tatttctctg atgt ggatct ggagaaggat gtccattctg ggctgattgg ccccctgctg gtgtgccaca ctaacactct gaatcctgcc catggcaggc aggtgactgt ccaggagttt gccctgttct tcactatctt tgatgagacc aagagctggt actttactga gaacatggag aggaactgca gagctccttg caatattcag atggaggacc ccaccttcaa ggagaattac aggttccatg ccattaatgg gtacatcatg gacaccctgc ctggcctggt gatggctcag gaccagagga tcaggtggta cctgct gagc atgggctcta atgagaatat ccacagcatc cacttctctg ggcatgtgtt cactgtgagg aagaaggagg agtacaagat ggctctgtat aatctgtacc ctggggtgtt tgaaactgtg gagatgctgc cctctaaggc tggcatctgg agggtggagt gcctgattgg ggagcacctg catgctggca tgagcaccct gttcctggtg tacagcaaca agtgccagac ccccctgggc atggcctctg gccacatcag ggacttccag atcactgcct ctggccagta tggccagtgg gcccccaagc tggccaggct gcactattct ggcagcatca atgcctggag caccaaggag cccttcagct ggatcaaggt ggacctgctg gcccccatga tcattcatgg catcaagacc caggggggcca ggcagaagtt cagctctctg tacatctctc agttcatcat catgtactct ctggatggga agaagtggca g acctacagg ggcaacagca ctggcaccct gatggtgttc tttgggaatg tggactcttc tggcatcaag cacaacatct tcaatccccc catcattgct aggtatatta ggctgcatcc cacccactac agcatcaggt ctaccctgag gatggagctg atgggctgtg acctgaactc ttgcagcatg cccctgggca tggagt ctaa ggccatctct gatgcccaga ttactgccag cagctacttc accaacatgt ttgccacctg gagcccctct aaggccaggc tgcatctgca ggggagaggagc aatgcctgga ggcctcaggt gaacaacccc aaggagtggc tgcaggtgga tttccagaag accatgaagg tgactggggt gaccacccag ggggtcaaga gcctgctgac cagcatgtat gtgaaggagt tcctgatcag cagcagccag gatggccacc agtggactct gttct ttcag aatgggaagg tgaaggtgtt tcagggcaat caggactctt tcacccctgt ggtgaacagc ctggaccccc ccctgctgac cagatacctg aggatccacc cccagtcttg ggtgcatcag attgccctga ggatggaggt gctgggctgt gaggctcagg atctgtactg agcggccgca ataa aagatc agagctctag agatctgtgt gttggttttt tgtgtaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcagg

Full-length plasmid including TTR promoter mutant (TTRmut), synthetic intron, CpG-reduced Factor VIII cDNA, poly-A and ITR repeats (SEQ ID NO:24)

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc tacgcgtgtc tgtctgcaca tttcgtagag cga gtgttcc gatactctaa tctccctagg caaggttcat attgacttag gttacttatt ctccttttgt tgactaagtc aataatcaga atcagcaggt ttggagtcag cttggcaggg atcagcagcc tgggttggaa ggagggggta taaaagcccc ttcaccagga gaagccgtca cacagatcca caag ctcctg ctagcaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactg acactgacat ccactttttc tttttctcca caggtttaaa cgccaccatg cagattgagc tgagcacctg cttcttcctg tgtctgctga ggttctgctt ctctgccacc aggaggtatt acctgggggc tgtggagctg agctgggact atatgcagtc tgacctgggg gagctgcctg tggatgctag gttccccccc agggtgccca agagcttccc ctttaacact t ctgtggtgt acaagaagac cctgtttgtg gagttcactg accacctgtt caacattgcc aagcccaggc ccccctggat ggggctgctg gggcccacca tccaggctga ggtgtatgac actgtggtga tcaccctgaa gaacatggcc agccaccctg tgagcctgca tgctgtgggg gtgagctact gga aggcttc tgaggggggct gagtatgatg accagactag ccagagggag aaggaggatg acaaggtgtt tcctggggggc agccatacct atgtgtggca ggtgctgaag gagaatggcc ccatggcctc tgaccccctg tgcctgacct acagctacct gtctcatgtg gacctggtga aggacctgaa ctctggcctg attggggctc tgctggtgtg tagggagggc agcctggcta aggaaaagac ccagaccctg cataagttta tcctgctgtt tgctgtgttt gatgagggca agagctggca ctctga gacc aagaacagcc tgatgcagga tagggatgct gcctctgcca gggcttggcc taagatgcac actgtgaatg ggtatgtgaa taggagcctg cctggcctga ttggctgcca caggaagtct gtgtactggc atgtgattgg gatgggcacc acccctgagg tccatagcat cttcctggag ggccacactt tcctggt gag gaaccacaga caggcctctc tggagatctc tcccatcacc ttcctgactg ctcagactct gctgatggac ctgggccagt tcctgctgtt ttgccatatt agcagccacc agcatgatgg gatggaggcc tatgtgaagg tggatagctg ccctgaggag cctcagctga ggatgaagaa caatgaggag gctgaagact atgatgatga cctgactgat tctgagatgg atgtggtgag gtttgatgat gacaatagcc ccagcttcat tcagatcagg tctgtggcca agaaacaccc caagacctgg g tgcactaca ttgctgctga ggaagaggac tgggactatg ctcccctggt gctggcccct gatgataggt cttataagag ccagtacctg aacaatgggc cccagaggat tggcaggaag tacaagaagg tgaggttcat ggcctacact gatgaaacct tcaaaaccag ggaggccatt cagcatgagt ctggcatcct ggg ccctctg ctgtatgggg aggtggggga caccctgctg atcatcttca agaaccaggc cagcaggccc tacaacatct atcctcatgg catcactgat gtgaggcccc tgtacagcag gaggctgccc aagggggtga agcacctgaa agacttcccc atcctgcctg gggagatctt taagtataag tggactgtga ctgtggagga tggccctacc aagtctgacc ccaggtgtct gaccaggtac tattctagct ttgtgaacat ggagagggac ctggcctct gcc tgattgg gcccctgctg atctgctaca aggagtctgt ggaccagagg ggcaaccaga tcatgtctga caagaggaat gtgatcctgt tttctgtgtt tgatgagaat aggagctggt acctgactga gaacatccag aggtttctgc ccaatcctgc tggggtgcag ctggaggatc ctgagttcca ggccagcaat atcatgcata gcatcaatgg ctatgtgttt gacagcctgc agctgtctgt gtgcctgcat gaggtggcct actggtacat cctgagcatt ggggcccaga ctgactttct gtctgtgttc ttttctggct ataccttcaa gcacaagatg gtgtatgagg ataccctgac cctgttcccc ttctctgggg agactgtgtt catgagcatg gagaatcctg ggctgtggat cctggggtgc cacaactctg attttaggaa cagggggatg actgccctgc tgaaggtgtc tagctgtga t aagaacactg gggactacta tgaggacagc tatgaggaca tttctgctta tctgctgtct aagaataatg ccattgagcc cagaagcttc agccagaatc cccctgtgct gaagagacat cagagggaga tcaccagaac taccctgcag tctgatcagg aggagattga ctatgatgac actatctctg tggagatgaa gaaggaggac tttgacatct atgatgagga tgagaatcag tctcccagga gctttcagaa gaagaccaga cattacttca ttgctgctgt ggagaggctg tgggactatg gcatgagctc tagccctcat gtgctgagga acagggccca gtctggctct gtgccccagt tcaagaaggt ggtgttccag gaattcactg atggcagctt cacccagccc ctgtacaggg gggagctgaa tgagcacctg ggcctgctgg ggccttatat cagggctgag gtggaggata atattatggt gacttt cagg aaccaggcca gcaggcccta ctctttctat agcagcctga tctcttatga ggaggatcag aggcaggggg ctgagcctag gaagaacttt gtgaagccca atgagactaa gacctacttc tggaaggtcc agcaccacat ggcccctacc aaggatgagt ttgactgcaa ggcctgggcc tatttctctg atgt ggatct ggagaaggat gtccattctg ggctgattgg ccccctgctg gtgtgccaca ctaacactct gaatcctgcc catggcaggc aggtgactgt ccaggagttt gccctgttct tcactatctt tgatgagacc aagagctggt actttactga gaacatggag aggaactgca gagctccttg caatattcag atggaggacc ccaccttcaa ggagaattac aggttccatg ccattaatgg gtacatcatg gacaccctgc ctggcctggt gatggctcag gaccagagga tcaggtggta cctgct gagc atgggctcta atgagaatat ccacagcatc cacttctctg ggcatgtgtt cactgtgagg aagaaggagg agtacaagat ggctctgtat aatctgtacc ctggggtgtt tgaaactgtg gagatgctgc cctctaaggc tggcatctgg agggtggagt gcctgattgg ggagcacctg catgctggca tgagcaccct gttcctggtg tacagcaaca agtgccagac ccccctgggc atggcctctg gccacatcag ggacttccag atcactgcct ctggccagta tggccagtgg gcccccaagc tggccaggct gcactattct ggcagcatca atgcctggag caccaaggag cccttcagct ggatcaaggt ggacctgctg gcccccatga tcattcatgg catcaagacc caggggggcca ggcagaagtt cagctctctg tacatctctc agttcatcat catgtactct ctggatggga agaagtggca g acctacagg ggcaacagca ctggcaccct gatggtgttc tttgggaatg tggactcttc tggcatcaag cacaacatct tcaatccccc catcattgct aggtatatta ggctgcatcc cacccactac agcatcaggt ctaccctgag gatggagctg atgggctgtg acctgaactc ttgcagcatg cccctgggca tggagt ctaa ggccatctct gatgcccaga ttactgccag cagctacttc accaacatgt ttgccacctg gagcccctct aaggccaggc tgcatctgca ggggagaggagc aatgcctgga ggcctcaggt gaacaacccc aaggagtggc tgcaggtgga tttccagaag accatgaagg tgactggggt gaccacccag ggggtcaaga gcctgctgac cagcatgtat gtgaaggagt tcctgatcag cagcagccag gatggccacc agtggactct gttct ttcag aatgggaagg tgaaggtgtt tcagggcaat caggactctt tcacccctgt ggtgaacagc ctggaccccc ccctgctgac cagatacctg aggatccacc cccagtcttg ggtgcatcag attgccctga ggatggaggt gctgggctgt gaggctcagg atctgtactg agcggccgca ataa aagatc agagctctag agatctgtgt gttggttttt tgtgtaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccggggcttt gcccggggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggca gcttgaagga aatactaagg caaaggtact gcaagtgctc gcaacattcg cttatgcgga ttattgccgt agtgccgcga cgccgggggc aagatgcaga gattgccat g gtacaggccg tgcggttgat attgccaaaa cagagctgtg ggggagagtt gtcgagaaag agtgcggaag atgcaaaggc gtcggctatt caaggatgcc agcaagcgca gcatatcgcg ctgtgacgat gctaatccca aaccttaccc aacccacctg gtcacgcact gttaagccg c tgtatgacgc tctggtggtg caatgccaca aagaagagtc aatcgcagac aacattttga atgcggtcac acgttagcag catgattgcc acggatggca acatattaac ggcatgatat tgacttattg aataaaattg ggtaaatttg actcaacgat gggttaattc gctcgttgtg gtagtgagat gaaaagaggc ggcgcttact accgattccg cctagttggt cacttcgacg tatcgtctgg aactccaacc atcgcaggca gagaggtctg caaaatgcaa tcccgaaaca gttcgcaggt aatagttaga gcctg cataa cggtttcggg attttttata tctgcacaac aggtaagagc attgagtcga taatcgtgaa gagtcggcga gcctggttag ccagtgctct ttccgttgtg ctgaattaag cgaataccgg aagcagaacc ggatcaccaa atgcgtacag gcgtcatcgc cgcccagcaa cagcacaacc caaactgagc cgtagccact gtctgtcctg aattcattag taatagttac gctgcggcct tttacacatg accttcgtga aagcgggtgg caggaggtcg cgctaacaac ctcctgccgt tttgcccgtg catalog tgcaatg gcgtaccttc gcggcagata taatggcggt gcgtttacaa aaacagtaat cgacgcaacg atgtgcgcca ttatcgccta gttcattcgt gaccttctcg acttcgccgg actaagtagc aatctcgctt atataacgag cgtgtttatc ggctacatcg gtactgactc gatt ggttcg cttatcaaac gcttcgctgc taaaaaagcc ggagtagaag atggtagaaa tcaataatca acgtaaggcg ttcctcgata tgctggcgtg gtcggaggga actgataacg gacgtcagaa aaccagaaat catggttatg acgtcattgt aggcggagag ctatttactg attactccga tcaccctcgc aaacttgtca cgctaaaccc aaaactcaaa tcaacaggcg ccggacgcta ccagcttctt tcccgttggt gggatgccta ccgcaagcag cttggcctga aagacttctc tccgaa aagt caggacgctg tggcattgca gcagattaag gagcgtggcg ctttacctat gattgatcgt ggtgatatcc gtcaggcaat cgaccgttgc agcaatatct gggcttcact gccgggcgct ggttatggtc agttcgagca taaggctgac agcctgattg caaaattcaa agaagc gggc ggaacggtca gagagattga tgtatgagca gagtcaccgc gattatctcc gctctggtta tctgcatcat cgtctgcctg tcatgggctg ttaatcatta ccgtgataac gccattacct acaaagccca gcgcgacaaa aatgccagag aactgaagct ggcgaacgcg gcaattactg acatgcagat gcgtcagcgt gatgttgctg cgctcgatgc aaaatacacg aaggagttag ctgatgctaa agctgaaaat gatgctctgc gtgatgatgt tgccgctggt cgtcgtcggt tgcacatcaa agcagtctgt cagtcagtgc gtgaagccac caccgcctcc ggcgtggata atgcagcctc cccccgactg gcagacaccg ctgaacggga ttatttcacc ctcagagaga ggctgatcac tatgcaaaaa caactggaag gaacccagaa gtatattaat gagcagtgca gatagagtt g cccatatcga tgggcaactc atgcaattat tgtgagcaat acacacgcgc ttccagcgga gtataaatgc ctaaagtaat aaaaccgagc aatccatta cgaatgtttg ctgggtttct gttttaacaa cattttctgc gccgccacaa attttggctg catcgacagt tttcttctgc ccaattccag aaacgaagaa atgatgggtg atggtttcct ttggtgctac tgctgccggt ttgttttgaa cagtaaacgt ctgttgagca catcctgtaa taagcagggc cagcgcagta gcgagtagca tttttt tcat ggtgttattc ccgatgcttt ttgaagttcg cagaatcgta tgtgtagaaa attaaacaaa ccctaaacaa tgagttgaaa tttcatattg ttaatattta ttaatgtatg tcaggtgcga tgaatcgtca ttgtattccc ggattaacta tgtccacagc cctgacgggg aactt ctctg cgggagtgtc cgggaataat taaaacgatg cacacaggt ttagcgcgta cacgtattgc attatgccaa cgccccggtg ctgacacgga agaaaccgga cgttatgatt tagcgtggaa agatttgtgt agtgttctga atgctctcag taaatagtaa tgaattatca aaggtatagt aatatctttt atgttcatgg atatttgtaa cccatcggaa aactcctgct ttagcaagat tttccctgta ttgctgaaat gtgatttctc ttgatttcaa cctatcatag gacgtttcta taagatgcgt gtttcttgag aatttaa cat ttacaacctt tttaagtcct tttattaaca cggtgttatc gttttctaac acgatgtgaa tattatctgt ggctagatag taaatataat gtgagacgtt gtgacgtttt agttcagaat aaaacaattc acagtctaaa tcttttcgca cttgatcgaa tatttcttta aa aatggcaa cctgagccat tggtaaaacc ttccatgtga tacgagggcg cgtagtttgc attatcgttt ttatcgtttc aatctggtct gacctccttg tgttttgttg atgatttatg tcaaatatta ggaatgtttt cacttaatag tattggttgc gtaacaaagt gcggtcctgc tggcattctg gagggaaata caaccgacag atgtatgtaa ggccaacgtg ctcaaatctt catacagaaa gatttgaagt aatattttaa ccgctagatg aagagcaagc gcatggagcg acaaaatgaa taaagaacaa tctgctgatg atccctccgt ggatctgatt cgtgtaaaaa atatgcttaa tagcaccatt tctatgagtt accctgatgt tgtaattgca tgtatagaac ataaggtgtc tctggaagca ttcagagcaa ttgaggcagc gttggtgaag cacgataata atatgaagga ttattccctg gtggttgact gatcaccata actgc taatc attcaaacta tttagtctgt gacagagcca acacgcagtc tgtcactgtc aggaaagtgg taaaactgca actcaattac tgcaatgccc tcgtaattaa gtgaatttac aatatcgtcc tgttcggagg gaagaacgcg ggatgttcat tcttcatcac ttttaattga tgtatatgct ctcttttctg acgttagtct ccgacggcag gcttcaatga cccaggctga gaaattcccg gacccttttt gctcaagagc gatgttaatt tgttcaatca tttggttagg aa agcggatg ttgcgggttg ttgttctgcg ggttctgttc ttcgttgaca tgaggttgcc ccgtattcag tgtcgctgat ttgtattgtc tgaagttgtt tttacgttaa gttgatgcag atcaattaat acgatacctg cgtcataatt gattatttga cgtggttt ga tggcctccac gcacgttgtg atatgtagat gataatcatt atcactttac gggtcctttc cggtgatccg acaggttacg gggcggcgac ctgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatc gggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ct cgggctat tcttttgatt tagacctgca ggcatgcaag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgcgatttat tcaacaaagc cgccgtcccg tcaagtcagc gtaatgctct gccagtgtta caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatca a taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg t gagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaa a tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc cat gttggaa tttaatcgcg gcttcgagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg tggctttgtt gaataaatcg aacttttgct gagttgaagg atcagatcac gcatcttccc gacaacgcag accgttccgt ggcaaagcaa aagttcaaaa tcaccaactg gtccacctac aacaaagctc t catcaaccg tggctccctc actttctggc tggatgatgg ggcgattcag gcctggtatg agtcagcaac accttcttca cgaggcagac ctctcgacgg agttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc t tgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacg acctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgt cgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt

FVIII-BDD, encoded by nucleic acid sequences X01-X18. Sequence SQ is bold/underlined (SEQ ID NO:25)

SFSQNPPVLKRHQR

Wild type FVIII with BDD (SEQ ID NO:26)

MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCRE GSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHY IAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSW YLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLL MLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFK VSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQ EKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIP QANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYG TQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRA EVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKK EEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMEL MGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

AAV-LK03 VP1 capsid (SEQ ID NO:27)

MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQ PLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNN GSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYG TVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL

AAV-SPK VP1 capsid (SEQ ID NO:28) used in AAV-SPK-8005 and AAV-SPK-hFIX

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQ PIGEPPA A PSG V G PN

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

Some definitions/abbreviations used

BDD: All or at least part of the B-domain (BD) has been removed

FVIII-BDD: FVIII with B-domain deletion

SQ: SFSQNPPVLKRHQR (SEQ ID NO:29)

FVIII/SQ: FVIII with SQ

FVIIIX01-X18: FVIII encoding CpG reduced nucleic acid variants shown as SEQ ID NO:1-18, respectively.

TTRmut: TTR promoter with 4 mutations, changing TAmGTGTAG to TATTGACTTAG

CO3: codon optimized FVIII nucleic acid variant shown as SEQ ID NO:21

NHP: non-human primate

ALT: alanine aminotransferase

D-dimer: a protein fragment formed as a result of the breakdown of a blood clot

SPK-8005: AAV capsid (SEQ ID NO:28) + TTRmut-hFVIII-X07; also called AAV-SPK-8005

SPK-8011: AAV capsid LK03 (SEQ ID NO:27) + TTRmut-hFVIII-X07; also called AAV-SPK-8011

[0348] Although some of the embodiments of the invention have been described and specifically exemplified above, the invention should not 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.

--->

LIST OF SEQUENCES

<110> SPARK THERAPEUTICS, INC.

ANGUELA, Xavier

<120> METHODS OF GENOTHERAPY USING THE FACTOR VIII (FVIII) GENE

<130> 023637-0460703

<140> PCT/US2018/044892

<141> 2018-08-01

<150> 62/540,053

<151> 2017-08-01

<150> 62/583,890

<151> 2017-11-09

<150> 62/596,535

<151> 2017-12-08

<150> 62/596,670

<151> 2017-12-08

<160> 29

<170> Patent In version 3.5

<210> 1

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 1

atgcagattg agctgtctac ctgcttcttc ctgtgcctgc tgaggttctg cttctctgct 60

accaggaggt actacctggg ggctgtggag ctgagctggg attacatgca gtctgacctg 120

ggggagctgc ctgtggatgc caggtttccc cccagggtgc ccaagagctt ccccttcaat 180

acctctgtgg tgtataagaa gaccctgttt gtggagttca ctgatcatct gttcaacatt 240

gctaaaccca ggcccccctg gatggggctg ctgggcccta ccatccaggc tgaggtgtat 300

gacactgtgg tgatcactct gaagaacatg gctagccatc ctgtgtctct gcatgctgtg 360

ggggtgagct actggaaggc ttctgagggg gctgagtatg atgatcagac tagccagagg 420

gagaaggagg atgacaaggt gttccctggg ggctctcaca cctatgtctg gcaggtgctg 480

aaggagaatg gccccatggc ctctgatcct ctgtgtctga cctatagcta cctgagccat 540

gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgtagggag 600

gggagcctgg ccaaggagaa gacccagacc ctgcacaagt tcattctgct gtttgctgtg 660

tttgatgagg gcaagagctg gcattctgaa accaagaaca gcctgatgca ggacagggat 720

gctgcctctg ctagggcctg gcccaagatg cacactgtga atgggtatgt caataggtct 780

ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tgggatgggc 840

accacccctg aggtgcacag catctttctg gagggccaca ccttcctggt gaggaatcac 900

agacaggcca gcctggagat cagccccatc accttcctga ctgcccagac cctgctgatg 960

gacctgggcc agtttctgct gttctgccac atctctagcc accagcatga tggcatggag 1020

gcctatgtga aggtggactc ctgccctgag gagccccagc tgaggatgaa gaataatgag 1080

gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gagattgat 1140

gatgacaatt ctcccagctt cattcagatc aggtctgtgg ccaagaagca tcccaagacc 1200

tgggtgcact acattgctgc tgaggaggag gactgggact atgcccccct ggtgctggcc 1260

cctgatgaca ggagctataa gagccagtac ctgaataatg gcccccagag gattgggagg 1320

aagtataaga aggtgaggtt catggcctat actgatgaaa ccttcaagac cagagaggcc 1380

atccagcatg agtctgggat cctggggccc ctgctgtatg gggaggtggg ggacaccctg 1440

ctgatcatct tcaagaacca ggccagcagg ccctacaaca tctaccctca tggcatcact 1500

gatgtgaggc ctctgtacag cagaaggctg cccaaggggg tgaagcatct gaaggacttc 1560

cccattctgc ctggggagat tttcaagtac aagtggactg tgactgtgga ggatggccca 1620

accaagtctg accctaggtg cctgactagg tactacagca gctttgtgaa tatggagagg 1680

gacctggcct ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggatcag 1740

aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag 1800

aacaggact ggtacctgac tgagaacatt cagaggtttc tgcccaaccc tgctggggtg 1860

cagctggagg accctgaatt ccaggcctct aacatcatgc acagcattaa tggctatgtg 1920

tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta cattctgagc 1980

attggggccc agactgactt cctgtctgtg ttcttctctg gctacacctt taagcacaag 2040

atggtgtatg aggataccct gaccctgttt cctttctctg gggagactgt gttcatgagc 2100

atggagaacc ctggcctgtg gatcctgggc tgccacaact ctgacttcag gaacaggggg 2160

atgactgctc tgctgaaggt gagcagctgt gataagaaca ctggggacta ctatgaggac 2220

agctatgagg acatctctgc ctatctgctg agcaagaata atgctattga gcccaggagc 2280

ttctctcaga acccccctgt gctgaagagg caccagaggg agatcaccag aactactctg 2340

cagtctgacc aggagagat tgactatgat gacaccatct ctgtggagat gaagaaggag 2400

gattttgata tttatgatga ggatgaaaac cagagcccca ggagctttca gaagaagact 2460

aggcactatt tcattgctgc tgtggagagg ctgtgggact atggcatgtc ttctagcccc 2520

catgtgctga ggaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttc 2580

caggagttca ctgatggcag cttcactcag cccctgtaca ggggggagct gaatgagcac 2640

ctggggctgc tgggccctta tatcagggct gaggtggagg ataacatcat ggtgaccttc 2700

aggaaccagg ccagcaggcc ctacagcttc tactctagcc tgatcagcta tgaggaggac 2760

cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacttat 2820

ttctggaagg tgcagcacca tatggccccc accaaggatg agtttgattg caaagcctgg 2880

gcctacttct ctgatgtgga cctggagaag gatgtgcact ctgggctgat tggccccctg 2940

ctggtgtgcc acaccaacac tctgaaccct gcccatggca ggcaggtgac tgtgcaggag 3000

tttgccctgt tcttcaccat ctttgatgag actaagagct ggtacttcac tgagaacatg 3060

gagaggaact gcagggcccc ctgcaatatc cagatggagg accccacctt taaggaaaat 3120

tataggtttc atgccattaa tggctacatc atggacaccc tgcctggcct ggtgatggcc 3180

caggaccaga ggatcaggtg gtacctgctg agcatgggca gcaatgagaa cattcacagc 3240

atccacttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg 3300

tataatctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc 3360

tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgagcac cctgttcctg 3420

gtgtattcta acaagtgtca gacccccctg ggcatggcct ctggccatat cagggacttc 3480

cagatcactg cctctggcca gtatgggcag tgggccccca agctggccag gctgcattac 3540

tctggcagca tcaatgcctg gagcaccaag gagccattca gctggattaa ggtggacctg 3600

ctggctccaa tgattatcca tggcatcaag acccagggg ccaggcagaa gtttagcagc 3660

ctgtacatct ctcagtttat catcatgtac tctctggatg gcaaaaagtg gcagacctac 3720

aggggcaatt ctactggcac tctgatggtg ttctttggca atgtggacag ctctgggatc 3780

aagcacaaca tctttaaccc ccctatcatt gccaggtaca ttaggctgca ccccacccat 3840

tacagcatca ggagcaccct gaggatggag ctgatgggct gtgatctgaa cagctgcagc 3900

atgcccctgg gcatggagag caaggctatc tctgatgccc agattactgc cagcagctac 3960

ttcaccaata tgtttgccac ctggagcccc agcaaggcca ggctgcacct gcagggcagg 4020

tctaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag 4080

aagaccatga aggtgactgg ggtgaccacc cagggggtga agagcctgct gactagcatg 4140

tatgtgaagg agttcctgat cagcagcagc caggatggcc atcagtggac cctgttcttc 4200

cagaatggca aggtgaaggt gttccagggc aatcaggaca gcttcacccc tgtggtgaac 4260

agcctggacc cccccctgct gaccagatac ctgaggatcc acccccagag ctgggtgcat 4320

cagattgccc tgaggatgga ggtgctgggg tgtgaggccc aggacctgta ctga 4374

<210> 2

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 2

atgcagattg agctgtctac ctgctttttc ctgtgtctgc tgaggttctg cttctctgcc 60

actaggaggt actacctggg ggctgtggag ctgtcttggg attacatgca gtctgatctg 120

ggggagctgc ctgtggatgc caggtttcct cccagggtgc ccaagtcttt ccccttcaat 180

acctctgtgg tgtataagaa gaccctgttt gtggagttta ctgatcacct gttcaacatt 240

gccaagccca ggcccccttg gatgggcctg ctggggccca ccatccaggc tgaggtgtat 300

gacactgtgg tgatcaccct gaagaacatg gcctctcacc ctgtgagcct gcatgctgtg 360

ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg 420

gagaaggagg atgataaggt gttccctggg gggagccaca cttatgtgtg gcaggtgctg 480

aaggagaatg gcccaatggc ctctgatccc ctgtgcctga cctattctta cctgagccat 540

gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag 600

ggctctctgg ctaaggagaa gacccagacc ctgcacaagt tcatcctgct gtttgctgtg 660

tttgatgagg ggaagagctg gcactctgag accaagaaca gcctgatgca ggacagggat 720

gctgcctctg ccagggcctg gcccaaaatg cacactgtga atggctatgt gaataggagc 780

ctgcctggcc tgattggctg ccacaggaag tctgtgtatt ggcatgtgat tggcatgggc 840

accacccctg aggtgcactc tatcttcctg gagggccata ctttcctggt gaggaatcat 900

aggcaggcca gcctggagat tagccccatt acctttctga ctgccgac cctgctgatg 960

gacctgggcc agttcctgct gttttgccac atcagctctc accagcatga tggcatggag 1020

gcctatgtga aggtggatag ctgccctgag gagccccagc tgaggatgaa gaacaatgag 1080

gaggctgagg attatgatga tgatctgact gattctgaaa tggatgtggt gaggtttgat 1140

gatgacaata gcccctcttt catccagatc aggtctgtgg ccaagaagca tcctaagacc 1200

tgggtgcact acattgctgc tgaggaggag gactgggact atgctcccct ggtgctggcc 1260

cctgatgaca ggtcttacaa gagccagtac ctgaacaatg gcccccagag aattgggagg 1320

aagtataaga aggtgagatt catggcttac actgatgaga ccttcaagac tagggaggcc 1380

atccagcatg agtctggcat tctgggcccc ctgctgtatg gggaggtggg ggacaccctg 1440

ctgatcatct tcaagaacca ggcctctagg ccctacaata tttaccccca tgggatcact 1500

gatgtgaggc ccctgtacag caggaggctg cctaaggggg tgaagcatct gaaggacttc 1560

cccatcctgc ctggggagat cttcaagtat aagtggactg tgactgtgga agatggcccc 1620

accaagtctg accctaggtg cctgaccagg tactactctt cttttgtgaa catggagagg 1680

gacctggcct ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggaccag 1740

agggggaacc agattatgtc tgacaagagg aatgtgattc tgttctctgt gtttgatgag 1800

aacaggagct ggtatctgac tgagaacatc cagaggttcc tgcccaatcc tgctggggtg 1860

cagctggagg accctgagtt ccaggccagc aacatcatgc acagcatcaa tgggtatgtg 1920

tttgattctc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc 1980

attggggctc agactgattt cctgtctgtg ttcttttctg gctacacctt taagcataag 2040

atggtgtatg aggacactct gaccctgttt cccttctctg gggagactgt gtttatgagc 2100

atggagaacc ctggcctgtg gatcctgggc tgccacaact ctgatttcag gaacaggggc 2160

atgactgctc tgctgaaggt gtcttcttgt gacaagaaca ctggggacta ttatgaggac 2220

agctatgagg acatctctgc ctacctgctg agcaagaaca atgctattga gccgatct 2280

ttcagccaga acccccctgt gctgaagagg caccagaggg agatcactag gaccaccctg 2340

cagtctgacc aggaggagat tgactatgat gacactatct ctgtggagat gaagaaggag 2400

gactttgata tctatgatga ggatgagaac cagtctccca ggagcttcca gaaaaagacc 2460

aggcactact tcattgctgc tgtggagagg ctgtgggact atggcatgtc ttctagcccc 2520

catgtgctga ggaacagggc ccagtctggg tctgtgcccc agttcaagaa ggtggtgttc 2580

caggagttca ctgatgggag cttcacccag cctctgtaca ggggggagct gaatgagcac 2640

ctggggctgc tgggccctta tattagggct gaggtggagg acaacatcat ggtgactttc 2700

aggaatcagg cctctaggcc ctatagcttc tacagctctc tgatcagcta tgaggaggat 2760

cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacctac 2820

ttctggaagg tgcagcacca catggctcct accaaggatg agtttgactg caaggcctgg 2880

gcctactttt ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggccccctg 2940

ctggtgtgtc ataccaacac cctgaaccct gcccatggca ggcaggtgac tgtgcaggag 3000

tttgccctgt tcttcaccat ctttgatgag accaagagct ggtactttac tgagaacatg 3060

gagaggaatt gcagagcccc ttgcaacatc cagatggagg acccaacctt caaagagaac 3120

tacaggttcc atgccatcaa tgggtacatc atggacaccc tgcctggcct ggtgatggct 3180

caggaccaga ggatcaggtg gtatctgctg agcatgggca gcaatgagaa tatccatagc 3240

attcacttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg 3300

tataacctgt accctggggt gtttgagact gtggagatgc tgccaagcaa ggctgggatt 3360

tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgtctac cctgttcctg 3420

gtgtactcca ataagtgcca gacccccctg ggcatggcct ctggccacat cagggacttc 3480

cagatcactg cctctggcca gtatgggcag tgggccccaa agctggccag gctgcactat 3540

tctgggagca tcaatgcttg gagcaccaag gagcctttca gctggattaa ggtggatctg 3600

ctggccccca tgatcattca tggcatcaaa acccaggggg ctagacagaa gttttctagc 3660

ctgtacatca gccagttcat catcatgtac agcctggatg gcaagaagtg gcagacttac 3720

aggggcaata gcactggcac cctgatggtg ttttttggca atgtggacag ctctggcatc 3780

aagcacaaca tctttaaccc ccccattatt gccaggtata tcaggctgca tccaccaccac 3840

tattctatta ggtctactct gagaatggag ctgatgggct gtgacctgaa cagctgtagc 3900

atgcccctgg ggatggagag caaggctatc tctgatgccc agatcactgc cagctcttat 3960

ttcaccaata tgtttgccac ctggtctccc tctaaggcca ggctgcacct gcagggcagg 4020

agcaatgctt ggaggcccca ggtgaataac cccaaggagt ggctgcaggt ggacttccag 4080

aagaccatga aggtgactgg ggtgactacc cagggggtga agtctctgct gactagcatg 4140

tatgtgaagg agttcctgat cagcagcagc caggatgggc atcagtggac tctgttcttc 4200

cagaatggca aggtgaaggt cttccagggg aaccaggata gcttcactcc tgtggtgaac 4260

tctctggacc cccccctgct gactaggtat ctgaggatcc acccccagag ctgggtgcac 4320

cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ttga 4374

<210> 3

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 3

atgcagattg aactgtctac ttgtttcttc ctgtgcctgc tgaggttttg cttctctgct 60

actaggaggt actatctggg ggctgtggag ctgtcttggg actatatgca gtctgacctg 120

ggggagctgc ctgtggatgc taggtttccc cccagggtgc ccaagagctt cccctttaac 180

acctctgtgg tgtataagaa gactctgttt gtggagttca ctgaccatct gttcaacatt 240

gccaagccaa ggcccccctg gatgggcctg ctgggcccca ccatccaggc tgaggtgtat 300

gacactgtgg tgattactct gaagaacatg gccagccatc ctgtgagcct gcatgctgtg 360

ggggtgtctt actggaaggc ctctgagggg gctgagtatg atgaccagac ctctcagagg 420

gagaaggagg atgacaaggt gttccctggg ggctctcata cctatgtgtg gcaggtcctg 480

aaggagaatg ggcccatggc ctctgacccc ctgtgcctga cctactctta tctgtctcat 540

gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag 600

ggcagcctgg ctaaggagaa gacccagact ctgcacaagt tcatcctgct gtttgctgtg 660

tttgatgagg gcaagagctg gcactctgag accaagaaca gcctgatgca ggacagggat 720

gctgcctctg ctagggcctg gcccaagatg cacactgtga atgggtatgt gaacaggagc 780

ctgccaggcc tgattggctg ccataggaag tctgtgtatt ggcatgtgat tgggatgggg 840

actacccctg aggtccacag cattttcctg gaggggcata cctttctggt gaggaaccac 900

aggcaggcct ctctggagat ctctcccatt actttcctga ctgcccagac cctgctgatg 960

gacctgggcc agttcctgct gttctgccac atcagcagcc accagcatga tggcatggag 1020

gcctatgtga aggtggatag ctgccctgag gagccccagc tgaggatgaa aaacaatgag 1080

gaggctgagg attatgatga tgacctgact gattctgaga tggatgtggt gaggtttgat 1140

gatgataaca gccccagctt catccagatt aggtctgtgg ccaagaagca tcccaagacc 1200

tgggtgcact acattgctgc tgaggaggag gattgggact atgctcctct ggtgctggcc 1260

cctgatgaca ggagctacaa gagccagtac ctgaataatg gcccccagag gattggcagg 1320

aagtataaga aggtgaggtt catggcctac actgatgaga cctttaagac cagggaggcc 1380

atccagcatg aatctgggat cctgggcccc ctgctgtatg gggaggtggg ggacaccctg 1440

ctgattatct ttaagaacca ggctagcagg ccctacaaca tttaccccca tggcattact 1500

gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcacct gaaggatttc 1560

cccattctgc ctggggagat ctttaagtac aaatggactg tgactgtgga ggatggccct 1620

actaagtctg atcccaggtg tctgaccaga tactacagca gctttgtgaa tatggagagg 1680

gacctggctt ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggaccag 1740

aggggcaatc agattatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag 1800

aacagaagct ggtacctgac tgagaacatc cagaggttcc tgcccaaccc tgctggggtg 1860

cagctggagg accctgagtt ccaggctagc aatatcatgc acagcattaa tggctatgtg 1920

tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctattggta cattctgagc 1980

attggggccc agactgattt cctgtctgtg ttcttttctg gctacacctt caagcacaag 2040

atggtgtatg aggatactct gaccctgttt cccttctctg gggagactgt gttcatgagc 2100

atggagaacc ctggcctgtg gatcctgggc tgtcacaact ctgacttcag gaacaggggc 2160

atgactgccc tgctgaaggt gagctcttgt gataagaaca ctggggacta ctatgaggac 2220

tcttatgagg acatctctgc ctacctgctg agcaagaaca atgctattga gcccaggagc 2280

ttctctcaga atccccctgt gctgaagagg catcagaggg agatcactag gactaccctg 2340

cagtctgacc aggaagagat tgactatgat gacaccatct ctgtggaaat gaagaaggag 2400

gactttgata tctatgatga ggatgaaaac cagagcccca ggagcttcca gaagaagacc 2460

aggcattact tcattgctgc tgtggagagg ctgtgggact atgggatgag ctcttctccc 2520

catgtgctga ggaatagggc tcagtctggc tctgtcccac agttcaagaa ggtggtgttt 2580

caggagttca ctgatggcag cttcactcag cccctgtaca ggggggagct gaatgagcat 2640

ctgggcctgc tggggcccta catcagggct gaggtggagg ataacattat ggtgactttc 2700

aggaaccagg cctctaggcc ctacagcttc tacagcagcc tgatcagcta tgaggaggac 2760

cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac taagacctat 2820

ttctggaagg tgcagcatca catggctccc actaaagatg agtttgactg caaggcctgg 2880

gcctacttct ctgatgtgga tctggagaag gatgtgcatt ctgggctgat tggccctctg 2940

ctggtctgcc atactaacac cctgaatcct gcccatggca ggcaggtgac tgtgcaggag 3000

tttgccctgt tctttaccat ctttgatgag accaagtctt ggtacttcac tgagaacatg 3060

gagaggaact gcagggcccc ctgtaacatc cagatggagg accccacctt taaggagaac 3120

tacaggttcc atgccatcaa tggctacatc atggacactc tgcctggcct ggtgatggcc 3180

caggaccaga ggatcaggtg gtacctgctg tctatgggct ctaatgagaa cattcattct 3240

atccacttct ctggccatgt gtttactgtg aggaagaagg aggagtacaa gatggccctg 3300

tacaatctgt accctggggt gtttgaaact gtggagatgc tgccctctaa ggctggcatc 3360

tggagggtgg agtgcctgat tggggaacac ctgcatgctg gcatgagcac cctgttcctg 3420

gtctatagca ataagtgcca gacccccctg gggatggcct ctgggcatat cagagacttc 3480

cagatcactg cctctggcca gtatggccag tgggccccca agctggccag gctgcactac 3540

tctggcagca ttaatgcctg gagcaccaag gagcccttct cttggatcaa ggtggacctg 3600

ctggctccca tgatcatcca tgggatcaag acccaggggg ccaggcagaa gttcagcagc 3660

ctgtacatct ctcagttcat catcatgtac tctctggatg gcaagaagtg gcagacctac 3720

aggggcaata gcactgggac cctgatggtg ttctttggga atgtggacag ctctggcatc 3780

aagcacaata tcttcaaccc ccccatcatt gccaggtaca tcagactgca ccccactcat 3840

tacagcatca ggagcactct gaggatggag ctgatgggct gtgacctgaa tagctgctct 3900

atgcccctgg gcatggagag caaggccatt tctgatgccc agattactgc ctcttcttac 3960

ttcactaata tgtttgccac ctggagcccc agcaaggcca ggctgcatct gcaggggagg 4020

agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag 4080

aagactatga aggtgactgg ggtgaccact cagggggtga agagcctgct gaccagcatg 4140

tatgtgaagg agttcctgat ctcttctagc caggatgggc accagtggac cctgtttttc 4200

cagaatggga aggtgaaggt gtttcagggc aatcaggaca gctttactcc tgtggtgaac 4260

agcctggacc cccccctgct gactaggtac ctgaggattc acccccagag ctgggtgcac 4320

cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga 4374

<210> 4

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 4

atgcagattg agctgtctac ctgcttcttt ctgtgcctgc tgaggttctg tttctctgcc 60

actaggaggt attatctggg ggctgtggag ctgtcctggg actacatgca gtctgatctg 120

ggggagctgc ctgtggatgc caggttccct cccagggtgc ccaagtcttt ccctttcaat 180

acctctgtgg tgtacaagaa gactctgttt gtggagttta ctgatcacct gtttaacatt 240

gccaagccca ggcccccctg gatggggctg ctgggcccca ccatccaggc tgaggtgtat 300

gacactgtgg tgattactct gaagaatatg gcttctcacc ctgtgagcct gcatgctgtg 360

ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg 420

gagaaggagg atgacaaggt gttccctggg ggcagccaca cttatgtgtg gcaggtgctg 480

aaggagaatg gcccaatggc ctctgacccc ctgtgcctga cctacagcta tctgagccat 540

gtggatctgg tgaaggatct gaactctggc ctgattgggg ccctgctggt gtgcagggag 600

ggctctctgg ccaaggagaa gactcagact ctgcacaagt tcatcctgct gtttgctgtg 660

tttgatgagg gcaagagctg gcactctgag accaagaact ctctgatgca ggatagggat 720

gctgcttctg ccagggcctg gcccaagatg cacactgtga atgggtatgt gaataggagc 780

ctgcctgggc tgattgggtg tcacaggaag tctgtgtact ggcatgtgat tggcatgggc 840

accactcctg aggtgcacag catctttctg gagggccaca cttttctggt gaggaatcac 900

aggcaggcca gcctggagat cagccccatc accttcctga ctgcccagac cctgctgatg 960

gatctgggcc agttcctgct gttttgccat atcagcagcc atcagcatga tgggatggag 1020

gcttatgtga aggtggactc ttgccctgag gagcctcagc tgaggatgaa gaataatgaa 1080

gaggctgagg actatgatga tgatctgact gactctgaga tggatgtggt gaggtttgat 1140

gatgacaaca gccccagctt tatccagatt aggtctgtgg ccaagaagca ccccaagacc 1200

tgggtgcatt acattgctgc tgaggaagag gattgggact atgcccccct ggtgctggcc 1260

cctgatgaca ggagctacaa gtctcagtac ctgaacaatg gccctcagag gattggcagg 1320

aagtacaaga aggtgaggtt catggcttac actgatgaga ccttcaagac cagggaggcc 1380

attcagcatg aatctgggat cctgggcccc ctgctgtatg gggaggtggg ggacaccctg 1440

ctgattattt tcaagaacca ggccagcagg ccctacaaca tttatcctca tggcattact 1500

gatgtgagac ccctgtacag caggaggctg cctaaggggg tgaagcacct gaaggacttc 1560

cccatcctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggcccc 1620

actaagtctg accccaggtg cctgactagg tactactcca gctttgtgaa catggagagg 1680

gacctggcct ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggatcag 1740

aggggcaacc agatcatgtc tgacaagaga aatgtgatcc tgttctctgt gtttgatgag 1800

aataggtctt ggtacctgac tgagaacatc cagaggtttc tgcctaatcc tgctggggtg 1860

cagctggagg atcctgagtt ccaggcctct aacattatgc acagcatcaa tgggtatgtg 1920

tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc 1980

attggggccc agactgactt tctgtctgtg ttcttctctg gctacacctt taagcataag 2040

atggtgtatg aggacaccct gactctgttc cccttctctg gggagactgt gttcatgagc 2100

atggagaacc caggcctgtg gatcctgggc tgccacaact ctgatttcag gaataggggc 2160

atgactgccc tgctgaaggt gagcagctgt gataagaaca ctggggacta ttatgaggat 2220

agctatgagg acatctctgc ctacctgctg agcaagaaca atgccattga gcccaggagc 2280

ttcagccaga atcctcctgt gctgaagagg caccagaggg agatcaccag gaccaccctg 2340

cagtctgatc aggaggagat tgactatgat gacactatct ctgtggagat gaagaaggag 2400

gactttgaca tctatgatga ggatgagaat cagagcccca ggagcttcca gaagaagact 2460

agacactact ttattgctgc tgtggagagg ctgtgggact atggcatgag ctcttctccc 2520

catgtgctga gaaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtcttc 2580

caggagttca ctgatggctc tttcacccag cctctgtata gaggggagct gaatgagcac 2640

ctgggcctgc tgggccctta catcagggct gaggtggagg acaatatcat ggtgaccttc 2700

aggaaccagg ctagcaggcc ctactctttc tacagcagcc tgatcagcta tgaggaggac 2760

cagaggcagg gggctgagcc taggaagaat tttgtgaagc ccaatgagac caagacctac 2820

ttctggaagg tgcagcacca catggctccc actaaggatg agtttgactg caaggcctgg 2880

gcctactttt ctgatgtgga cctggagaag gatgtgcatt ctggcctgat tggccccctg 2940

ctggtctgcc acaccaatac tctgaaccct gctcatggga gacaggtgac tgtgcaggag 3000

tttgccctgt tcttcaccat ctttgatgag accaagtcct ggtactttac tgagaacatg 3060

gagaggaatt gcagggcccc ttgcaacatc cagatggagg accccacctt caaggaaaat 3120

tataggttcc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggcc 3180

caggaccaga ggatcaggtg gtatctgctg tctatgggct ctaatgagaa catccacagc 3240

atccatttct ctggccatgt gttcactgtg aggaagaagg aggagtataa gatggctctg 3300

tacaacctgt accctggggt ctttgagact gtggagatgc tgcccagcaa ggctggcatt 3360

tggagggtgg agtgcctgat tggggaacac ctgcatgctg ggatgagcac cctgttcctg 3420

gtgtactcta acaagtgcca gaccccactg ggcatggctt ctggccacat cagggatttc 3480

cagattactg cctctggcca gtatggccag tgggctccca agctggctag gctgcactac 3540

tctgggagca tcaatgcctg gtctactaag gagcctttct cttggatcaa agtggacctg 3600

ctggccccta tgatcatcca tgggatcaag actcaggggg ccaggcagaa gttcagcagc 3660

ctgtacatct ctcagttcat cattatgtac agcctggatg gcaagaagtg gcagacctac 3720

aggggcaaca gcactggcac cctgatggtg ttctttggga atgtggacag ctctgggatt 3780

aagcacaaca tctttaaccc ccccatcatt gccaggtata tcaggctgca ccctacccac 3840

tacagcatta ggagcaccct gaggatggag ctgatgggct gtgacctgaa cagctgcagc 3900

atgcccctgg ggatggagag caaggccatt tctgatgctc agatcactgc ttctagctac 3960

ttcactaaca tgtttgccac ctggtctccc agcaaggcta gactgcacct gcaggggagg 4020

agcaatgcct ggaggcccca ggtgaataat cccaaggagt ggctgcaggt ggatttccag 4080

aaaaccatga aggtgactgg ggtgactacc cagggggtga agtctctgct gaccagcatg 4140

tatgtgaagg agttcctgat cagcagcagc caggatgggc atcagtggac cctgttcttt 4200

cagaatggga aggtgaaggt gtttcagggc aatcaggaca gcttcacccc tgtggtgaac 4260

agcctggacc cccccctgct gaccaggtac ctgaggatcc acccccagag ctgggtgcat 4320

cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ctga 4374

<210> 5

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 5

atgcagattg agctgtctac ttgcttcttc ctgtgcctgc tgaggttctg cttctctgcc 60

actaggaggt attacctggg ggctgtggag ctgagctggg actatatgca gtctgacctg 120

ggggagctgc ctgtggatgc caggtttcct cccagggtgc ctaagagctt ccccttcaac 180

acctctgtgg tgtacaagaa gactctgttt gtggagttta ctgatcatct gttcaacatt 240

gccaagccca ggcctccttg gatggggctg ctgggcccca ccatccaggc tgaggtgtat 300

gacactgtgg tgattaccct gaagaatatg gccagccatc ctgtgagcct gcatgctgtg 360

ggggtgagct attggaaggc ctctgagggg gctgagtatg atgatcagac tagccagagg 420

gagaaggagg atgacaaggt gttccctggg gggagccata cctatgtgtg gcaggtgctg 480

aaggagaatg gccccatggc ctctgaccct ctgtgcctga cttatagcta cctgagccat 540

gtggatctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag 600

ggcagcctgg ccaaggagaa gactcagacc ctgcacaagt tcatcctgct gtttgctgtg 660

tttgatgagg ggaagtcctg gcactctgag actaagaaca gcctgatgca ggatagggat 720

gctgcttctg ccagggcctg gcctaagatg cacactgtga atggctatgt gaataggagc 780

ctgcctggcc tgattggctg ccataggaag tctgtgtact ggcatgtgat tgggatgggc 840

accacccctg aggtgcactc tattttcctg gagggccata ctttcctggt gaggaaccat 900

aggcaggcca gcctggagat cagccccatc actttcctga ctgcccagac tctgctgatg 960

gacctgggcc agttcctgct gttctgccac atcagcagcc atcagcatga tggcatggag 1020

gcttatgtga aggtggacag ctgccctgag gagcctcagc tgaggatgaa gaataatgag 1080

gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat 1140

gatgacaact ctccctcttt catccagatc aggtctgtgg ccaagaagca ccctaagacc 1200

tgggtgcact acattgctgc tgaggaggag gattgggact atgcccccct ggtgctggcc 1260

ccagatgaca ggagctacaa gtcccagtac ctgaacaatg gcccccagag gattggcagg 1320

aagtacaaga aggtgaggtt catggcttat actgatgaga ctttcaagac cagggaggcc 1380

atccagcatg agtctggcat cctgggccct ctgctgtatg gggaggtggg ggacaccctg 1440

ctgattatct tcaagaacca ggcttctagg ccctacaata tctaccctca tggcatcact 1500

gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcatct gaaggatttc 1560

cccatcctgc ctggggagat ctttaagtat aagtggactg tgactgtgga ggatggcccc 1620

actaagtctg accccaggtg cctgaccagg tattacagca gctttgtgaa catggagagg 1680

gatctggctt ctgggctgat tggccccctg ctgatctgct acaaggagtc tgtggaccag 1740

aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag 1800

aataggagct ggtacctgac tgagaacatc cagaggtttc tgcccaatcc tgctggggtg 1860

cagctggagg atcctgagtt tcaggcctct aatatcatgc acagcatcaa tggctatgtg 1920

tttgactctc tgcagctgtc tgtgtgcctg catgaggtgg cctattggta catcctgagc 1980

attggggccc agactgactt tctgtctgtg tttttttctg gctacacctt caagcacaag 2040

atggtgtatg aggatactct gactctgttc cctttttctg gggagactgt gttcatgtct 2100

atggagaacc ctgggctgtg gattctgggc tgccacaatt ctgacttcag gaacagaggc 2160

atgactgctc tgctgaaggt gagcagctgt gacaagaaca ctggggacta ctatgaggac 2220

tcttatgagg acatttctgc ctacctgctg agcaagaaca atgccattga gcccagaagc 2280

ttttctcaga acccccctgt gctgaagagg caccagaggg agatcaccag gaccaccctg 2340

cagtctgacc aggaggat tgactatgat gatactattt ctgtggagat gaagaaggag 2400

gactttgaca tctatgatga ggatgagaac cagagcccca ggtctttcca gaagaagact 2460

aggcactact ttattgctgc tgtggagagg ctgtgggact atgggatgtc tagctctcct 2520

catgtgctga ggaacagggc ccagtctggc tctgtgcccc agtttaaaaa ggtggtgttc 2580

caggaattca ctgatggcag ctttacccag cctctgtaca ggggggagct gaatgagcac 2640

ctggggctgc tggggcctta cattagggct gaggtggagg acaacatcat ggtgaccttc 2700

aggaatcagg ccagcaggcc ctactctttc tacagcagcc tgatctctta tgaggaggac 2760

cagaggcagg gggctgaacc caggaagaac tttgtgaagc ccaatgagac caagacctac 2820

ttctggaagg tgcagcacca catggctccc accaaggatg agtttgattg caaggcctgg 2880

gcttacttct ctgatgtgga tctggagaag gatgtgcact ctgggctgat tggccccctg 2940

ctggtgtgcc acaccaacac tctgaaccct gcccatggca gacaggtgac tgtgcaggag 3000

tttgccctgt tcttcactat ctttgatgag actaagagct ggtacttcac tgagaacatg 3060

gagaggaatt gcagggcccc ttgcaacatc cagatggagg accccacctt taaggagaac 3120

tacaggtttc atgccattaa tggctacatc atggacaccc tgcctggcct ggtgatggcc 3180

caggaccaga ggatcaggtg gtacctgctg tctatgggga gcaatgagaa catccacagc 3240

attcacttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg 3300

tacaacctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctgggatc 3360

tggagggtgg agtgcctgat tggggagcac ctgcatgctg ggatgagcac cctgttcctg 3420

gtgtatagca acaagtgcca gacccccctg ggcatggcct ctggccacat cagagacttt 3480

cagattactg cctctggcca gtatgggcag tgggccccca agctggccag gctgcactat 3540

tctggctcta ttaatgcctg gagcactaag gagcccttca gctggattaa ggtggacctg 3600

ctggctccca tgatcatcca tggcatcaag actcaggggg ccaggcagaa gttctcttct 3660

ctgtacatca gccagttcat tatcatgtac tccctggatg gcaagaagtg gcagacctat 3720

aggggcaaca gcactggcac cctgatggtg ttctttggga atgtggacag ctctggcatc 3780

aagcataata tcttcaatcc ccccatcatt gctaggtaca tcaggctgca cccccaccac 3840

tactctatta ggtctaccct gaggatggag ctgatgggct gtgacctgaa cagctgcagc 3900

atgcctctgg gcatggagag caaagccatc tctgatgccc agatcactgc cagcagctac 3960

tttaccaaca tgtttgctac ttggagcccc agcaaggcca ggctgcacct gcaggggagg 4020

tctaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag 4080

aagactatga aggtgactgg ggtgaccacc cagggggtga agagcctgct gacctctatg 4140

tatgtgaagg agttcctgat tagcagcagc caggatggcc accagtggac cctgtttttc 4200

cagaatggga aggtgaaggt gtttcagggg aaccaggaca gcttcactcc tgtggtgaac 4260

tctctggacc cccccctgct gaccaggtat ctgaggatcc accctcagag ctgggtgcac 4320

cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ctga 4374

<210> 6

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 6

atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttttg cttctctgcc 60

accaggaggt actacctggg ggctgtggag ctgagctggg attacatgca gtctgacctg 120

ggggagctgc ctgtggatgc caggttccct cccagggtgc ccaagtcttt ccccttcaac 180

acttctgtgg tgtacaagaa gaccctgttt gtggagttta ctgaccacct gttcaacatt 240

gccaagccca ggcctccctg gatgggcctg ctgggcccca ccattcaggc tgaggtgtat 300

gacactgtgg tcatcaccct gaaaaatatg gctagccacc ctgtgtctct gcatgctgtg 360

ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac tagccagagg 420

gagaaggagg atgacaaggt gttccctggg ggcagccaca cttatgtgtg gcaggtgctg 480

aaagagaatg gccccatggc ttctgatccc ctgtgtctga cctatagcta cctgagccat 540

gtggatctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag 600

ggcagcctgg ctaaggagaa gacccagacc ctgcataagt tcatcctgct gtttgctgtg 660

tttgatgagg gcaagagctg gcactctgag actaagaaca gcctgatgca ggatagggat 720

gctgcttctg ccagggcctg gcccaagatg cacactgtga atgggtatgt gaacaggagc 780

ctgcctggcc tgattggctg ccataggaag tctgtctatt ggcatgtgat tggcatgggc 840

actactcctg aggtgcacag catctttctg gagggccaca ccttcctggt gaggaaccac 900

aggcaggcca gcctggagat ctctcccatc actttcctga ctgctcagac cctgctgatg 960

gacctgggcc agttcctgct gttctgtcac atctctagcc accagcatga tggcatggag 1020

gcctatgtga aggtggatag ctgccctgag gaaccccagc tgaggatgaa gaacaatgag 1080

gaggctgagg attatgatga tgatctgact gattctgaga tggatgtggt gaggtttgat 1140

gatgacaatt ctcctagctt cattcagatc agatctgtgg ccaaaaagca tcctaagact 1200

tgggtgcatt atattgctgc tgaggaggag gattgggatt atgcccccct ggtgctggct 1260

cctgatgata ggagctacaa gtctcagtac ctgaataatg ggccccagag gattggcagg 1320

aagtacaaga aggtgaggtt catggcctac actgatgaga ccttcaagac cagggaggcc 1380

attcagcatg agtctgggat tctggggccc ctgctgtatg gggaggtggg ggataccctg 1440

ctgatcattt tcaagaacca ggccagcagg ccctacaaca tctaccccca tgggattact 1500

gatgtgaggc ccctgtactc taggaggctg cctaaggggg tgaagcacct gaaggatttt 1560

cctatcctgc ctggggaaat cttcaagtac aagtggactg tgactgtgga ggatggcccc 1620

actaagtctg atcccaggtg tctgaccagg tattatagct cttttgtgaa catggagagg 1680

gatctggcct ctgggctgat tggccctctg ctgatctgct acaaggagtc tgtggaccag 1740

aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag 1800

aacaggagct ggtatctgac tgagaacatc cagaggtttc tgcccaatcc tgctggggtg 1860

cagctggagg atcctgagtt ccaggctagc aacatcatgc acagcatcaa tgggtatgtg 1920

tttgacagcc tgcagctgtc tgtgtgtctg catgaggtgg cctactggta tatcctgtct 1980

attggggccc agactgactt cctgtctgtg tttttttctg ggtatacttt taagcacaag 2040

atggtgtatg aggacaccct gactctgttc cccttctctg gggagactgt gtttatgagc 2100

atggagaacc ctggcctgtg gatcctgggc tgccacaatt ctgacttcag gaatagggg 2160

atgactgccc tgctgaaggt gagcagctgt gataagaata ctggggacta ctatgaggac 2220

tcttatgagg acatttctgc ctatctgctg tctaagaaca atgccattga acccaggagc 2280

ttctctcaga acccccctgt gctgaagagg caccagaggg aaatcaccag aactactctg 2340

cagtctgatc aggaggaaat tgactatgat gacactattt ctgtggagat gaagaaggag 2400

gactttgaca tctatgatga ggatgagaac cagagcccaa ggagcttcca gaagaagact 2460

aggcactact tcattgctgc tgtggagagg ctgtgggact atggcatgag cagcagcccc 2520

catgtgctga gaaacagggc ccagtctggg tctgtgcccc agttcaagaa ggtggtgttc 2580

caggagttca ctgatgggag cttcacccag cccctgtata ggggggagct gaatgagcac 2640

ctgggcctgc tgggccccta tattagggct gaggtggagg acaacatcat ggtgaccttc 2700

aggaatcagg cctctaggcc ctacagcttc tacagcagcc tgattagcta tgaggaggat 2760

cagaggcagg gggctgaacc caggaagaac tttgtgaagc ccaatgagac caagacctat 2820

ttctggaagg tgcagcatca catggccccc accaaggatg agtttgactg caaggcctgg 2880

gcctacttct ctgatgtgga tctggagaag gatgtgcact ctggcctgat tggccccctg 2940

ctggtgtgcc acaccaacac cctgaaccct gctcatggca ggcaggtgac tgtgcaggag 3000

tttgccctgt tcttcaccat ctttgatgag actaagtctt ggtacttcac tgagaatatg 3060

gagaggaatt gcagggcccc ctgcaatatt cagatggaag accccacctt caaggagaat 3120

tacaggttcc atgccattaa tggctacatc atggataccc tgcctggcct ggtgatggcc 3180

caggatcaga ggatcaggtg gtacctgctg agcatgggca gcaatgagaa catccactct 3240

atccacttct ctggccatgt gtttactgtg aggaagaagg aggagtataa gatggccctg 3300

tacaacctgt accctggggt ctttgagact gtggagatgc tgccttctaa ggctggcatt 3360

tggagggtgg agtgcctgat tggggaacac ctgcatgctg gcatgtctac cctgttcctg 3420

gtgtacagca ataagtgcca gacccccctg ggcatggcct ctgggcatat cagggatttc 3480

cagatcactg cctctggcca gtatggccag tgggccccaa agctggctag gctgcactac 3540

tctgggagca tcaatgcctg gagcactaag gagcccttca gctggatcaa ggtggacctg 3600

ctggccccca tgattatcca tgggattaag actcaggggg ccaggcagaa gttcagcagc 3660

ctgtacatca gccagttcat tatcatgtac agcctggatg gcaagaagtg gcagacctat 3720

aggggcaact ctactgggac cctgatggtg ttctttggga atgtggatag ctctgggatc 3780

aagcacaata tcttcaaccc ccccatcatt gccaggtata tcaggctgca cccacccac 3840

tacagcatta ggtctaccct gaggatggag ctgatgggct gtgatctgaa cagctgtagc 3900

atgcctctgg gcatggagtc taaggccatt tctgatgccc agattactgc tagcagctac 3960

ttcaccaaca tgtttgccac ctggtctccc agcaaggcca ggctgcatct gcagggcagg 4020

tctaatgctt ggaggcccca ggtgaacaac ccaaaggagt ggctgcaggt ggatttccag 4080

aagactatga aggtgactgg ggtgaccact cagggggtga agtctctgct gacctctatg 4140

tatgtgaagg agttcctgat ctctagcagc caggatggcc atcagtggac cctgttcttc 4200

cagaatggca aggtgaaagt gttccagggc aatcaggata gcttcactcc agtggtgaac 4260

agcctggatc cccctctgct gactaggtac ctgaggatcc acccccagag ctgggtgcac 4320

cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ctga 4374

<210> 7

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 7

atgcagattg agctgagcac ctgcttcttc ctgtgtctgc tgaggttctg cttctctgcc 60

accaggaggt attacctggg ggctgtggag ctgagctggg actatatgca gtctgacctg 120

ggggagctgc ctgtggatgc taggttcccc cccagggtgc ccaagagctt cccctttaac 180

acttctgtgg tgtacaagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt 240

gccaagccca ggcccccctg gatggggctg ctggggccca ccatccaggc tgaggtgtat 300

gacactgtgg tgatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg 360

ggggtgagct actggaaggc ttctgagggg gctgagtatg atgaccagac tagccagagg 420

gagaaggagg atgacaaggt gtttcctggg ggcagccata cctatgtgtg gcaggtgctg 480

aaggagaatg gccccatggc ctctgacccc ctgtgcctga cctacagcta cctgtctcat 540

gtggacctgg tgaaggacct gaactctggc ctgattgggg ctctgctggt gtgtagggag 600

ggcagcctgg ctaaggaaaa gacccagacc ctgcataagt ttatcctgct gtttgctgtg 660

tttgatgagg gcaagagctg gcactctgag accaagaaca gcctgatgca ggatagggat 720

gctgcctctg ccagggcttg gcctaagatg cacactgtga atgggtatgt gaataggagc 780

ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tgggatgggc 840

accacccctg aggtccatag catcttcctg gagggccaca ctttcctggt gaggaaccac 900

agacaggcct ctctggagat ctctcccatc accttcctga ctgctcagac tctgctgatg 960

gacctgggcc agttcctgct gttttgccat attagcagcc accagcatga tgggatggag 1020

gcctatgtga aggtggatag ctgccctgag gagcctcagc tgaggatgaa gaacaatgag 1080

gaggctgaag actatgatga tgacctgact gattctgaga tggatgtggt gaggtttgat 1140

gatgacaata gccccagctt cattcagatc aggtctgtgg ccaagaaaca ccccaagacc 1200

tgggtgcact acattgctgc tgaggaagag gactgggact atgctcccct ggtgctggcc 1260

cctgatgata ggtcttataa gagccagtac ctgaacaatg ggccccagag gattggcagg 1320

aagtacaaga aggtgaggtt catggcctac actgatgaaa ccttcaaaac cagggaggcc 1380

attcagcatg agtctggcat cctgggccct ctgctgtatg gggaggtggg ggacaccctg 1440

ctgatcatct tcaagaacca ggccagcagg ccctacaaca tctatcctca tggcatcact 1500

gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcacct gaaagacttc 1560

cccatcctgc ctggggagat ctttaagtat aagtggactg tgactgtgga ggatggccct 1620

accaagtctg accccaggtg tctgaccagg tactattcta gctttgtgaa catggagagg 1680

gacctggcct ctggcctgat tgggcccctg ctgatctgct acaaggagtc tgtggaccag 1740

aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttttctgt gtttgatgag 1800

aataggagct ggtacctgac tgagaacatc cagaggtttc tgcccaatcc tgctggggtg 1860

cagctggagg atcctgagtt ccaggccagc aatatcatgc atagcatcaa tggctatgtg 1920

tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc 1980

attggggccc agactgactt tctgtctgtg ttcttttctg gctatacctt caagcacaag 2040

atggtgtatg aggataccct gaccctgttc cccttctctg gggagactgt gttcatgagc 2100

atggagaatc ctgggctgtg gatcctgggg tgccacaact ctgattttag gaacaggggg 2160

atgactgccc tgctgaaggt gtctagctgt gataagaaca ctggggacta ctatgaggac 2220

agctatgagg acatttctgc ttatctgctg tctaagaata atgccattga gcccagaagc 2280

ttcagccaga atccccctgt gctgaagaga catcagaggg agatcaccag aactaccctg 2340

cagtctgatc aggaggagat tgactatgat gacactatct ctgtggagat gaagaaggag 2400

gactttgaca tctatgatga ggatgagaat cagtctccca ggagctttca gaagaagacc 2460

agacattact tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagccct 2520

catgtgctga ggaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttc 2580

caggaattca ctgatggcag cttcacccag cccctgtaca ggggggagct gaatgagcac 2640

ctgggcctgc tggggcctta tatcagggct gaggtggagg ataatattat ggtgactttc 2700

aggaaccagg ccagcaggcc ctactctttc tatagcagcc tgatctctta tgaggaggat 2760

cagaggcagg gggctgagcc taggaagaac tttgtgaagc ccaatgagac taagacctac 2820

ttctggaagg tccagcacca catggcccct accaaggatg agtttgactg caaggcctgg 2880

gcctatttct ctgatgtgga tctggagaag gatgtccatt ctgggctgat tggccccctg 2940

ctggtgtgcc acactaacac tctgaatcct gcccatggca ggcaggtgac tgtccaggag 3000

tttgccctgt tcttcactat ctttgatgag accaagagct ggtactttac tgagaacatg 3060

gagaggaact gcagagctcc ttgcaatatt cagatggagg accccacctt caaggagaat 3120

tacaggttcc atgccattaa tgggtacatc atggacaccc tgcctggcct ggtgatggct 3180

caggaccaga ggatcaggtg gtacctgctg agcatgggct ctaatgagaa tatccacagc 3240

atccacttct ctgggcatgt gttcactgtg aggaagaagg aggagtacaa gatggctctg 3300

tataatctgt accctggggt gtttgaaact gtggagatgc tgccctctaa ggctggcatc 3360

tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgagcac cctgttcctg 3420

gtgtacagca acaagtgcca gacccccctg ggcatggcct ctggccacat cagggacttc 3480

cagatcactg cctctggcca gtatggccag tgggccccca agctggccag gctgcactat 3540

tctggcagca tcaatgcctg gagcaccaag gagcccttca gctggatcaa ggtggacctg 3600

ctggccccca tgatcattca tggcatcaag acccagggg ccaggcagaa gttcagctct 3660

ctgtacatct ctcagttcat catcatgtac tctctggatg ggaagaagtg gcagacctac 3720

aggggcaaca gcactggcac cctgatggtg ttctttggga atgtggactc ttctggcatc 3780

aagcacaaca tcttcaatcc ccccatcatt gctaggtata ttaggctgca tccaccaccac 3840

tacagcatca ggtctaccct gaggatggag ctgatgggct gtgacctgaa ctcttgcagc 3900

atgcccctgg gcatggagtc taaggccatc tctgatgccc agattactgc cagcagctac 3960

ttcaccaaca tgtttgccac ctggagcccc tctaaggcca ggctgcatct gcaggggagg 4020

agcaatgcct ggaggcctca ggtgaacaac cccaaggagt ggctgcaggt ggatttccag 4080

aagaccatga aggtgactgg ggtgaccacc cagggggtca agagcctgct gaccagcatg 4140

tatgtgaagg agttcctgat cagcagcagc caggatggcc accagtggac tctgttcttt 4200

cagaatggga aggtgaaggt gtttcagggc aatcaggact ctttcacccc tgtggtgaac 4260

agcctggacc cccccctgct gaccagatac ctgaggatcc acccccagtc ttgggtgcat 4320

cagattgccc tgaggatgga ggtgctgggc tgtgaggctc aggatctgta ctga 4374

<210> 8

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 8

atgcagattg agctgagcac ttgctttttt ctgtgcctgc tgaggttttg tttttctgcc 60

accaggaggt actacctggg ggctgtggag ctgagctggg actatatgca gtctgatctg 120

ggggagctgc ctgtggatgc caggttcccc cccagggtgc ccaagtcttt tcccttcaac 180

acctctgtgg tgtataagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt 240

gctaagccta ggcccccctg gatgggcctg ctgggcccta ccattcaggc tgaggtgtat 300

gacactgtgg tgatcaccct gaagaacatg gccagccatc ctgtgagcct gcatgctgtg 360

ggggtctctt actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagaga 420

gagaaggagg atgacaaggt cttccctggg ggctctcaca cctatgtgtg gcaggtgctg 480

aaggaaaatg gccccatggc ctctgacccc ctgtgcctga cctacagcta tctgagccat 540

gtggatctgg tgaaggacct gaattctggc ctgattgggg ccctgctggt gtgcagggag 600

ggcagcctgg ccaaggagaa gacccagacc ctgcacaagt ttatcctgct gtttgctgtg 660

tttgatgagg gcaagtcttg gcactctgag actaagaaca gcctgatgca ggacagggat 720

gctgcctctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaacaggagc 780

ctgcctgggc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc 840

accacccctg aggtgcacag catcttcctg gaaggccaca ctttcctggt gaggaaccat 900

aggcaggcca gcctggagat cagccctatc accttcctga ctgcccagac cctgctgatg 960

gatctggggc agttcctgct gttctgccac atctctagcc accagcatga tgggatggag 1020

gcctatgtga aggtggacag ctgcccagag gagcctcagc tgaggatgaa aaacaatgaa 1080

gaggctgagg attatgatga tgatctgact gactctgaga tggatgtggt gagattgat 1140

gatgacaata gccctagctt tattcagatc aggtctgtgg ctaagaagca ccccaagacc 1200

tgggtgcatt acattgctgc tgaggaggag gactgggatt atgctcctct ggtgctggcc 1260

cctgatgata ggagctacaa gagccagtac ctgaataatg gccctcagag gattggcagg 1320

aagtacaaga aggtgaggtt catggcttac actgatgaga ccttcaagac tagggaggcc 1380

atccagcatg agtctgggat cctggggccc ctgctgtatg gggaggtggg ggacaccctg 1440

ctgatcatct tcaagaacca ggctagcagg ccttacaaca tctatcccca tgggatcact 1500

gatgtgagac ctctgtacag caggaggctg cccaaggggg tcaagcatct gaaagacttc 1560

cccatcctgc ctggggagat ctttaagtat aagtggactg tgactgtgga ggatgggccc 1620

accaagtctg accccaggtg cctgaccagg tattacagca gctttgtgaa catggagagg 1680

gatctggcct ctgggctgat tggccccctg ctgatctgtt acaaggaatc tgtggatcag 1740

aggggcaatc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag 1800

aataggtctt ggtacctgac tgaaaacatc cagaggttcc tgcccaaccc tgctggggtc 1860

cagctggagg atcctgagtt ccaggctagc aacatcatgc acagcatcaa tgggtatgtg 1920

tttgatagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgtct 1980

attggggccc agactgactt cctgtctgtg ttcttttctg gctacacctt caagcacaag 2040

atggtgtatg aggacaccct gaccctgttc cccttctctg gggagactgt ctttatgagc 2100

atggagaacc ctgggctgtg gatcctgggc tgccacaact ctgatttcag gaataggggc 2160

atgactgctc tgctgaaggt gagctcttgt gacaagaaca ctggggatta ctatgaggac 2220

agctatgagg acatttctgc ctacctgctg agcaagaaca atgccattga gcctaggagc 2280

tttagccaga atcctcctgt cctgaagagg caccagaggg agatcaccag gaccaccctg 2340

cagtctgacc aggagagat tgactatgat gataccatct ctgtggagat gaagaaggag 2400

gactttgaca tctatgatga ggatgagaat cagtctccca ggagcttcca gaagaagacc 2460

aggcactatt tcattgctgc tgtggagagg ctgtgggact atggcatgag cagctctcct 2520

catgtgctga ggaatagggc tcagtctggc tctgtgcccc agttcaagaa agtggtgttt 2580

caggagttca ctgatggctc tttcacccag cctctgtata ggggggagct gaatgagcac 2640

ctggggctgc tgggccccta tatcagggct gaggtggagg ataacatcat ggtgaccttc 2700

aggaaccagg cctctaggcc ctacagcttc tatagcagcc tgatcagcta tgaggaggac 2760

cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacttac 2820

ttctggaagg tgcagcatca catggccccc accaaggatg agtttgactg taaggcctgg 2880

gcctacttct ctgatgtgga tctggagaag gatgtgcact ctggcctgat tggccccctg 2940

ctggtgtgcc ataccaatac tctgaaccct gctcatggca ggcaggtgac tgtgcaggag 3000

tttgctctgt tcttcactat ctttgatgag accaagtctt ggtatttcac tgagaatatg 3060

gagaggaact gcagggcccc ctgcaacatc cagatggagg accccacctt taaggagaac 3120

tataggtttc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggcc 3180

caggatcaga ggatcaggtg gtacctgctg agcatggggt ctaatgagaa catccacagc 3240

atccacttct ctggccatgt gtttactgtg agaaagaagg aggagtacaa gatggctctg 3300

tacaatctgt accctggggt ctttgagact gtggagatgc tgcctagcaa ggctgggatc 3360

tggagggtgg agtgcctgat tggggaacat ctgcatgctg ggatgtctac tctgttcctg 3420

gtgtacagca acaagtgcca gacccccctg ggcatggctt ctggccatat cagggacttt 3480

cagattactg cctctgggca gtatggccag tgggccccca agctggctag gctgcattat 3540

tctggcagca tcaatgcctg gtctactaag gagcccttca gctggatcaa ggtggatctg 3600

ctggccccca tgatcatcca tggcatcaag acccagggg ccaggcagaa gtttagctct 3660

ctgtacatta gccagttcat catcatgtac agcctggatg ggaagaagtg gcagacctac 3720

aggggcaatt ctactggcac cctgatggtg ttctttggca atgtggacag ctctggcatc 3780

aagcacaaca tctttaaccc ccctatcatt gctaggtaca tcaggctgca tcccacccat 3840

tacagcatca ggagcaccct gaggatggag ctgatgggct gtgacctgaa ctcttgcagc 3900

atgcccctgg gcatggagag caaggccatt tctgatgccc agattactgc cagcagctac 3960

ttcactaaca tgtttgccac ctggtctccc agcaaggcca ggctgcacct gcagggcagg 4020

agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggatttccag 4080

aagaccatga aggtgactgg ggtgaccacc cagggggtga agagcctgct gactagcatg 4140

tatgtgaagg agttcctgat cagctctagc caggatggcc accagtggac tctgtttttc 4200

cagaatggca aggtgaaggt gttccagggc aaccaggact ctttcactcc tgtggtgaac 4260

agcctggacc cccccctgct gaccaggtat ctgaggattc acccccagtc ttgggtgcat 4320

cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga 4374

<210> 9

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 9

atgcagattg agctgagcac ctgcttcttc ctgtgtctgc tgagattttg cttttctgcc 60

actaggaggt attacctggg ggctgtggag ctgtcttggg actacatgca gtctgatctg 120

ggggagctgc ctgtggatgc caggttccca cctagggtgc ctaagagctt tcccttcaat 180

acctctgtgg tgtacaagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt 240

gccaagccta ggcccccctg gatgggcctg ctgggcccta ccatccaggc tgaagtgtat 300

gacactgtgg tgatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg 360

ggggtgtctt actggaaggc ctctgagggg gctgagtatg atgatcagac cagccagagg 420

gagaaggaag atgacaaggt gttccctggg ggcagccaca cctatgtctg gcaggtgctg 480

aaggagaatg gccccatggc ctctgatccc ctgtgcctga cctactctta cctgagccat 540

gtggacctgg tgaaggatct gaattctggc ctgattgggg ccctgctggt gtgcagggag 600

ggcagcctgg ccaaggagaa gacccagacc ctgcataagt tcatcctgct gtttgctgtg 660

tttgatgaag ggaagagctg gcactctgag actaagaaca gcctgatgca ggacagggat 720

gctgcttctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaatagaagc 780

ctgcctggcc tgattgggtg ccacaggaag tctgtgtact ggcatgtgat tgggatgggc 840

actacccctg aggtgcatag catcttcctg gaaggccata ccttcctggt gaggaatcat 900

aggcaggctt ctctggaaat ttctcccatc actttcctga ctgctcagac cctgctgatg 960

gacctgggcc agttcctgct gttctgccac atcagctctc accagcatga tgggatggag 1020

gcctatgtga aggtggacag ctgtcctgag gagccccagc tgaggatgaa gaacaatgag 1080

gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt caggtttgat 1140

gatgacaata gcccctcttt catccagatc aggtctgtgg ccaagaagca ccccaagact 1200

tgggtgcact acattgctgc tgaggaggag gattgggatt atgcccctct ggtgctggcc 1260

cctgatgaca ggagctataa gtctcagtac ctgaataatg gcccccagag gattgggagg 1320

aagtataaga aggtgaggtt tatggcctac actgatgaga ccttcaagac cagggaggcc 1380

atccagcatg agtctggcat cctgggcccc ctgctgtatg gggaggtggg ggataccctg 1440

ctgatcatct tcaagaacca ggcctctagg ccctacaata tctaccctca tggcatcact 1500

gatgtgagac ccctgtatag caggaggctg cctaaggggg tgaagcacct gaaggacttc 1560

cccatcctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatggcccc 1620

accaagtctg accccaggtg cctgaccagg tattacagct cttttgtgaa catggagagg 1680

gatctggcct ctgggctgat tggcccactg ctgatctgct acaaggagtc tgtggatcag 1740

aggggcaatc agatcatgtc tgacaagagg aatgtgatcc tgttttctgt gtttgatgaa 1800

aataggtctt ggtatctgac tgagaacatc cagaggtttc tgcccaatcc tgctggggtg 1860

cagctggagg atcctgagtt tcaggcctct aatatcatgc attctatcaa tggctatgtg 1920

tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc 1980

attggggctc agactgactt cctgtctgtg ttcttttctg gctatacttt caagcacaag 2040

atggtgtatg aggacactct gaccctgttc cccttctctg gggagactgt gttcatgtct 2100

atggaaaatc ctgggctgtg gattctggggc tgccacaatt ctgacttcag gaatagggg 2160

atgactgccc tgctgaaggt gtctagctgt gataagaaca ctggggatta ctatgaggac 2220

tcttatgaag atatctctgc ctatctgctg agcaagaaca atgccattga gcccaggagc 2280

ttcagccaga acccccctgt gctgaagagg caccagaggg agatcaccag gaccactctg 2340

cagtctgatc aggaggagat tgactatgat gacactatct ctgtggagat gaagaaggag 2400

gattttgaca tttatgatga ggatgagaac cagtctccca ggagcttcca gaagaagacc 2460

aggcattact ttattgctgc tgtggagagg ctgtgggact atgggatgag cagctctcct 2520

catgtgctga ggaacagggc ccagtctggg tctgtgcccc agttcaagaa ggtggtgttc 2580

caggagttca ctgatgggag cttcacccag cccctgtata ggggggagct gaatgagcac 2640

ctgggcctgc tgggccccta catcagggct gaggtggagg ataatatcat ggtgaccttc 2700

aggaaccagg ctagcaggcc ttacagcttt tacagcagcc tgatctctta tgaagaagac 2760

cagaggcagg gggctgagcc caggaagaat tttgtgaagc ctaatgagac caagacttat 2820

ttttggaagg tgcagcatca catggctcct accaaggatg agtttgactg caaggcctgg 2880

gcctactttt ctgatgtgga tctggagaag gatgtgcact ctggcctgat tggccctctg 2940

ctggtgtgcc atactaacac tctgaaccct gcccatggga ggcaggtgac tgtgcaggag 3000

tttgccctgt tcttcactat ttttgatgag accaagtctt ggtatttcac tgagaacatg 3060

gagaggaact gcagggctcc ctgcaacatc cagatggaag accccacctt caaggagaac 3120

tataggttcc atgccatcaa tgggtacatc atggataccc tgcctggcct ggtgatggcc 3180

caggatcaga ggattaggtg gtatctgctg agcatgggct ctaatgagaa catccacagc 3240

atccatttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggctctg 3300

tacaacctgt atcctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc 3360

tggagggtgg aatgcctgat tggggagcac ctgcatgctg gcatgagcac tctgttcctg 3420

gtgtatagca acaagtgcca gacccccctg ggcatggcct ctggccatat cagggatttc 3480

cagatcactg cttctggcca gtatggccag tgggccccca agctggccag gctgcactat 3540

tctggcagca tcaatgcctg gagcactaag gagccttttt cttggatcaa ggtggacctg 3600

ctggccccta tgattattca tggcatcaag acccagggg ccaggcagaa gttctctagc 3660

ctgtacatct ctcagttcat cattatgtat agcctggatg gcaagaagtg gcagacctac 3720

aggggcaata gcactggcac cctgatggtg ttttttggga atgtggactc ttctgggatc 3780

aagcacaaca tctttaaccc ccccatcatt gccaggtata ttaggctgca cccccaccac 3840

tacagcatca ggagcaccct gaggatggag ctgatgggct gtgatctgaa ttcttgctct 3900

atgcccctgg gcatggagag caaggccatc tctgatgccc agatcactgc cagctcttac 3960

ttcaccaaca tgtttgccac ctggtctcct agcaaggcca ggctgcatct gcagggcagg 4020

agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag 4080

aagaccatga aggtgactgg ggtgaccact cagggggtga agagcctgct gacctctatg 4140

tatgtgaagg agttcctgat cagcagcagc caggatggcc accagtggac tctgttcttc 4200

cagaatggga aggtgaaggt gttccagggc aaccaggata gctttacccc tgtggtgaac 4260

agcctggacc ctcctctgct gaccagatac ctgaggatcc atcctcagag ctgggtgcac 4320

cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga 4374

<210> 10

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 10

atgcagattg agctgagcac ttgcttcttc ctgtgcctgc tgaggttctg cttttctgct 60

actaggaggt actacctggg ggctgtggag ctgagctggg attacatgca gtctgacctg 120

ggggagctgc cagtggatgc caggttcccc cccagggtgc ccaagtcttt tcctttcaac 180

acctctgtgg tgtacaagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt 240

gccaagccca ggcccccctg gatggggctg ctggggccca ccatccaggc tgaggtgtat 300

gacactgtgg tgattaccct gaagaacatg gctagccacc ctgtgagcct gcatgctgtg 360

ggggtgagct attggaaggc ctctgagggg gctgagtatg atgatcagac cagccagagg 420

gaaaaggagg atgacaaggt gttccctggg ggcagccata cttatgtgtg gcaggtgctg 480

aaggagaatg ggcccatggc ctctgacccc ctgtgcctga cttacagcta tctgagccat 540

gtggacctgg tgaaggatct gaactctggc ctgattgggg ctctgctggt gtgcagggag 600

ggcagcctgg ctaaggagaa gactcagact ctgcataagt tcatcctgct gtttgctgtg 660

tttgatgaag gcaagagctg gcactctgag accaagaact ctctgatgca ggatagggat 720

gctgcctctg ccagggcttg gcccaagatg cacactgtga atggctatgt gaacaggagc 780

ctgcctggcc tgattgggtg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc 840

accacccctg aggtgcacag cattttcctg gagggccaca ccttcctggt gaggaatcac 900

aggcaggcca gcctggagat cagccccatc accttcctga ctgcccagac cctgctgatg 960

gacctggggc agtttctgct gttctgccac atcagcagcc atcagcatga tggcatggag 1020

gcctatgtga aggtggactc ttgccctgag gagccccagc tgaggatgaa gaacaatgag 1080

gaggctgagg attatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat 1140

gatgacaata gccccagctt catccagatt aggtctgtgg ccaagaagca ccctaagacc 1200

tgggtgcact acattgctgc tgaggaggag gattgggatt atgcccccct ggtgctggct 1260

cctgatgaca ggtcttataa gagccagtac ctgaacaatg ggccccagag gattggcagg 1320

aagtacaaga aggtgaggtt catggcttac actgatgaga ccttcaagac tagggaggcc 1380

atccagcatg agtctggcat cctgggcccc ctgctgtatg gggaggtggg ggataccctg 1440

ctgatcatct tcaagaacca ggccagcagg ccctacaaca tttaccctca tggcatcact 1500

gatgtgaggc ccctgtacag caggagactg cccaaggggg tgaagcacct gaaggatttt 1560

cccattctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggcccc 1620

accaagtctg atcccaggtg cctgactagg tactactctt cttttgtgaa tatggagagg 1680

gatctggcct ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggaccag 1740

aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag 1800

aataggagct ggtacctgac tgagaatatc cagaggttcc tgcctaatcc tgctggggtc 1860

cagctggagg atcctgagtt ccaggctagc aacattatgc acagcatcaa tggctatgtg 1920

tttgattctc tgcagctgtc tgtgtgcctg catgaggtgg cttactggta catcctgtct 1980

attggggccc agactgattt cctgtctgtg ttcttctctg gctacacttt caagcataag 2040

atggtgtatg aggataccct gaccctgttc cccttctctg gggagactgt gttcatgtct 2100

atggagaacc ctggcctgtg gatcctgggc tgtcataact ctgacttcag aaacaggggc 2160

atgactgccc tgctgaaggt gagcagctgt gacaagaaca ctggggacta ctatgaggac 2220

agctatgagg atatctctgc ttatctgctg agcaagaata atgccattga gcccaggagc 2280

ttcagccaga acccccctgt gctgaagagg caccagaggg agatcactag gactaccctg 2340

cagtctgatc aggaggagat tgactatgat gacaccatct ctgtggagat gaagaaggag 2400

gactttgaca tctatgatga ggatgagaac cagtccccca ggtctttcca gaagaagacc 2460

aggcactact tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagcccc 2520

catgtgctga ggaacagggc tcagtctggc tctgtgcccc agttcaagaa ggtggtcttc 2580

caggagttca ctgatggctc ttttacccag cctctgtaca gaggggagct gaatgagcac 2640

ctgggcctgc tgggccccta catcagggct gaggtggagg ataatatcat ggtgaccttc 2700

agaaaccagg cctctaggcc ctacagcttc tacagcagcc tgatctctta tgaggaggat 2760

cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacctac 2820

ttctggaagg tgcagcacca tatggcccct actaaggatg agtttgactg caaggcctgg 2880

gcttattttt ctgatgtgga cctggagaag gatgtgcact ctgggctgat tggccccctg 2940

ctggtgtgcc acaccaacac cctgaaccct gcccatggca ggcaggtgac tgtgcaggag 3000

tttgccctgt tcttcactat ctttgatgag accaagagct ggtacttcac tgagaacatg 3060

gagagaaatt gtagggctcc ctgcaatatc cagatggagg accccacctt caaagaaaat 3120

tacagattcc atgccatcaa tgggtacatc atggataccc tgcctgggct ggtgatggct 3180

caggaccaga ggatcaggtg gtacctgctg agcatggggt ctaatgagaa catccactct 3240

atccatttct ctggccatgt gttcactgtg agaaagaagg aggagtataa gatggctctg 3300

tacaacctgt acccaggggt gtttgagact gtggaaatgc tgcccagcaa agctgggatc 3360

tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgtctac cctgttcctg 3420

gtgtacagca acaagtgcca gactcccctg ggcatggcct ctgggcacat cagggatttt 3480

cagatcactg cctctggcca gtatggccag tgggccccca agctggccag gctgcactac 3540

tctggcagca ttaatgcttg gagcactaag gagcccttca gctggatcaa ggtggatctg 3600

ctggccccca tgatcatcca tggcatcaag acccagggg ccaggcagaa gttctctagc 3660

ctgtacattt ctcagttcat catcatgtac agcctggatg ggaagaagtg gcagacctac 3720

agggggaaca gcactgggac cctgatggtg ttctttggca atgtggatag ctctggcatc 3780

aagcacaata tcttcaatcc ccccattatt gccaggtaca ttaggctgca tcctactcac 3840

tactctatta ggagcaccct gaggatggag ctgatggggt gtgacctgaa cagctgttct 3900

atgcccctgg gcatggagtc taaggctatc tctgatgccc agatcactgc cagcagctac 3960

ttcactaata tgtttgccac ctggagccct agcaaggcca gactgcacct gcagggcagg 4020

agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag 4080

aagaccatga aggtgactgg ggtgaccact cagggggtga agagcctgct gaccagcatg 4140

tatgtgaagg agttcctgat cagcagcagc caggatggcc accagtggac cctgttcttc 4200

cagaatggga aggtgaaggt gttccagggc aaccaggact ctttcacccc tgtggtgaac 4260

agcctggatc ctcccctgct gaccaggtac ctgaggatcc acccccagag ctgggtgcac 4320

cagattgctc tgaggatgga agtgctgggc tgtgaggccc aggatctgta ctga 4374

<210> 11

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 11

atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttttg cttctctgct 60

accaggaggt actacctggg ggctgtggag ctgagctggg actatatgca gtctgacctg 120

ggggagctgc ctgtggatgc taggttccct cccagggtgc ccaagagctt cccctttaat 180

acctctgtgg tgtacaagaa aaccctgttt gtggagttca ctgaccatct gttcaacatt 240

gccaagccca ggcccccttg gatgggcctg ctgggcccca ccattcaggc tgaggtgtat 300

gacactgtgg tcattaccct gaagaacatg gcttctcacc ctgtgagcct gcatgctgtg 360

ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg 420

gagaaggagg atgataaggt gttccctggg ggcagccaca cctatgtgtg gcaggtgctg 480

aaggagaatg gccccatggc ctctgatccc ctgtgcctga cctactctta tctgtctcat 540

gtggacctgg tgaaggacct gaactctggc ctgattgggg ctctgctggt gtgcagggag 600

ggctctctgg ccaaggagaa gacccagacc ctgcacaagt ttattctgct gtttgctgtc 660

tttgatgagg gcaagagctg gcattctgag accaagaaca gcctgatgca ggacagggat 720

gctgcctctg ccagggcctg gcccaaaatg cacactgtga atggctatgt gaacaggagc 780

ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc 840

accacccctg aggtgcacag catcttcctg gagggccaca cctttctggt gaggaatcac 900

aggcaggcca gcctggagat tagccccatc accttcctga ctgcccagac cctgctgatg 960

gacctgggcc agttcctgct gttctgccac atcagcagcc accagcatga tggcatggag 1020

gcctatgtga aggtggatag ctgccctgag gagccccagc tgaggatgaa aaacaatgag 1080

gaggctgagg attatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat 1140

gatgacaata gccccagctt tattcagatt aggtctgtgg ctaagaagca ccccaagact 1200

tgggtgcact acattgctgc tgaggaggag gattgggact atgcccctct ggtcctggcc 1260

cctgatgata ggtcttacaa gagccagtat ctgaacaatg gcccccagag gattggcagg 1320

aagtacaaga aggtgaggtt catggcctac actgatgaga cctttaagac cagggaggcc 1380

attcagcatg agtctgggat cctgggcccc ctgctgtatg gggaggtggg ggacactctg 1440

ctgatcatct tcaagaacca ggccagcagg ccttataaca tctaccctca tgggatcact 1500

gatgtgaggc ccctgtactc tagaaggctg cccaaggggg tcaagcacct gaaggatttt 1560

cccatcctgc ctggggagat tttcaagtac aagtggactg tgactgtgga ggatggcccc 1620

accaagtctg accctaggtg cctgaccagg tactacagct cttttgtgaa catggagagg 1680

gacctggcct ctggcctgat tggccctctg ctgatttgct acaaggagtc tgtggaccag 1740

aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttttctgt gtttgatgag 1800

aacaggtctt ggtacctgac tgagaacatc cagaggttcc tgcctaaccc agctggggtg 1860

cagctggagg atcctgagtt ccaggccagc aatattatgc atagcattaa tggctatgtg 1920

tttgatagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc 1980

attggggccc agactgactt tctgtctgtg ttcttctctg gctacacctt caagcataag 2040

atggtgtatg aggacaccct gactctgttc cctttttctg gggagactgt gtttatgagc 2100

atggagaatc ctggcctgtg gatcctgggc tgccataatt ctgacttcag gaacaggggc 2160

atgactgccc tgctgaaagt gagcagctgt gacaagaata ctggggacta ctatgaagac 2220

agctatgagg acatctctgc ctacctgctg agcaagaaca atgccattga gcccaggagc 2280

ttcagccaga accccccagt gctgaagagg caccagagag agatcaccag gactaccctg 2340

cagtctgacc aggaggat tgactatgat gacaccattt ctgtggagat gaagaaggag 2400

gactttgaca tttatgatga ggatgagaat cagagcccca ggagcttcca gaagaagact 2460

aggcactatt ttattgctgc tgtggagagg ctgtgggact atggcatgag cagctctccc 2520

catgtgctga ggaatagggc ccagtctggc tctgtgcctc agttcaagaa ggtggtgttc 2580

caggagttca ctgatggcag ctttacccag cccctgtata ggggggagct gaatgagcac 2640

ctgggcctgc tgggccccta tatcagggct gaggtggagg acaatattat ggtgaccttt 2700

aggaaccagg ccagcaggcc ctactctttc tatagcagcc tgatcagcta tgaggaggac 2760

cagaggcagg gggctgagcc caggaagaat tttgtgaagc ctaatgagac caagacctac 2820

ttctggaagg tgcagcatca catggccccc accaaggatg agtttgactg caaggcttgg 2880

gcctatttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggccccctg 2940

ctggtgtgcc acactaacac tctgaatcct gcccatggca ggcaggtgac tgtgcaggag 3000

tttgccctgt tcttcaccat ctttgatgag accaagagct ggtacttcac tgagaacatg 3060

gagaggaact gcagggcccc ctgcaacatc cagatggagg atcccacctt caaggagaac 3120

tacaggtttc atgccatcaa tggctacatc atggacactc tgcctggcct ggtgatggcc 3180

caggatcaga ggatcaggtg gtacctgctg agcatgggct ctaatgagaa tatccatagc 3240

atccacttct ctggccatgt gttcactgtc aggaagaagg aggagtacaa gatggctctg 3300

tataatctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc 3360

tggagggtgg agtgcctgat tggggagcac ctgcatgctg ggatgagcac cctgtttctg 3420

gtgtactcta acaagtgcca gacccccctg ggcatggcct ctgggcacat cagggatttc 3480

cagatcactg cttctggcca gtatggccag tgggccccca agctggccag gctgcactac 3540

tctggcagca tcaatgcctg gtctaccaag gagccctttt cttggattaa ggtggacctg 3600

ctggccccca tgatcatcca tggcatcaag acccagggg ccaggcagaa gttcagcagc 3660

ctgtacatca gccagttcat catcatgtac agcctggatg gcaaaaagtg gcagacctac 3720

aggggcaata gcactgggac tctgatggtg ttctttggca atgtggacag ctctgggatc 3780

aagcacaata tcttcaaccc tcccatcatt gctaggtaca tcaggctgca ccccacccac 3840

tatagcatca ggtctaccct gaggatggag ctgatgggct gtgacctgaa ctcttgcagc 3900

atgcccctgg gcatggagtc caaagctatc tctgatgccc agattactgc cagcagctac 3960

ttcaccaaca tgtttgccac ctggtctccc tctaaggcca ggctgcacct gcagggcagg 4020

agcaatgcct ggaggcccca ggtgaacaat cccaaggagt ggctgcaggt ggatttccag 4080

aaaactatga aggtgactgg ggtgaccacc cagggggtga agtctctgct gaccagcatg 4140

tatgtgaagg agttcctgat ctcttctagc caggatggcc accagtggac tctgttcttc 4200

cagaatggca aggtgaaggt gttccagggc aaccaggaca gcttcacccc tgtggtgaac 4260

tctctggatc cccccctgct gaccaggtac ctgaggattc atccccagag ctgggtgcac 4320

cagattgctc tgagaatgga ggtgctgggg tgtgaggctc aggacctgta ttga 4374

<210> 12

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 12

atgcagattg agctgtctac ttgttttttt ctgtgcctgc tgaggttctg cttctctgcc 60

accaggaggt attacctggg ggctgtggag ctgagctggg attacatgca gtctgatctg 120

ggggagctgc ctgtggatgc caggttcccc cccagggtgc ccaagagctt ccccttcaac 180

acctctgtgg tgtataagaa gaccctgttt gtggagttca ctgatcatct gtttaacatt 240

gccaagccca ggcccccctg gatgggcctg ctgggcccaa ctatccaggc tgaggtgtat 300

gacactgtgg tcatcaccct gaagaatatg gccagccatc ctgtgagcct gcatgctgtg 360

ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg 420

gagaaggagg atgacaaggt gttccctggg ggcagccaca cctatgtgtg gcaggtgctg 480

aaggagaatg gccccatggc ctctgacccc ctgtgcctga cttatagcta cctgtctcat 540

gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt ctgtagggaa 600

ggcagcctgg ccaaggagaa gacccagacc ctgcacaagt ttattctgct gtttgctgtg 660

tttgatgaag gcaagagctg gcactctgag accaagaatt ctctgatgca ggatagggat 720

gctgcctctg cccaggcctg gcccaagatg catactgtga atggctatgt gaacagaagc 780

ctgcctggcc tgattggctg ccataggaag tctgtgtatt ggcatgtgat tgggatgggc 840

actacccctg aagtgcacag cattttcctg gagggccaca ctttcctggt gaggaaccac 900

aggcaggcct ctctggagat cagccccatt actttcctga ctgcccagac cctgctgatg 960

gatctgggcc agttcctgct gttctgccac atctctagcc accagcatga tggcatggag 1020

gcctatgtga aggtggacag ctgccctgag gagccccagc tgaggatgaa gaataatgag 1080

gaggctgagg attatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat 1140

gatgataata gccccagctt catccagatc aggtctgtgg ccaagaagca tcccaagacc 1200

tgggtgcact atattgctgc tgaagaggag gactgggact atgcccctct ggtgctggct 1260

cctgatgaca ggagctataa gagccagtat ctgaacaatg ggccccagag gattgggagg 1320

aagtacaaga aggtgaggtt catggcctac actgatgaga cctttaagac cagggaggcc 1380

atccagcatg agtctggcat tctggggccc ctgctgtatg gggaggtggg ggacactctg 1440

ctgatcattt tcaagaacca ggccagcagg ccctacaata tttaccccca tggcatcact 1500

gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcacct gaaggacttc 1560

cccatcctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggccct 1620

accaagtctg accctaggtg tctgactagg tactacagca gctttgtgaa catggagaga 1680

gacctggctt ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggatcag 1740

aggggcaacc agattatgtc tgataagagg aatgtcatcc tgttctctgt gtttgatgag 1800

aacaggact ggtatctgac tgagaacatt cagaggttcc tgcccaaccc tgctggggtg 1860

cagctggagg accctgagtt ccaggccagc aacatcatgc attctattaa tggctatgtg 1920

tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc 1980

attggggccc agactgactt tctgtctgtg tttttctctg ggtacacctt caagcacaag 2040

atggtctatg aggacaccct gaccctgttc cccttttctg gggaaactgt gtttatgagc 2100

atggagaacc ctgggctgtg gatcctgggc tgccacaact ctgactttag gaataggggc 2160

atgactgccc tgctgaaggt gagcagctgt gacaagaata ctggggatta ctatgaggac 2220

agctatgagg atatctctgc ctacctgctg agcaagaaca atgccattga gcctaggagc 2280

ttcagccaga acccccctgt gctgaagagg caccagaggg agatcaccag gaccaccctg 2340

cagtctgatc aggaggagat tgactatgat gacaccatct ctgtggagat gaagaaggag 2400

gactttgata tttatgatga ggatgagaac cagagcccca ggagcttcca gaagaagacc 2460

aggcactatt tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagcccc 2520

catgtgctga ggaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttc 2580

caggaattta ctgatggcag ctttacccag cccctgtaca gaggggagct gaatgagcac 2640

ctgggcctgc tgggccccta catcagggct gaggtggagg ataatatcat ggtgaccttt 2700

aggaaccagg cctctaggcc ctattctttt tacagcagcc tgatcagcta tgaggaggac 2760

cagaggcagg gggctgagcc taggaagaac tttgtgaagc ccaatgagac caagacctac 2820

ttttggaaag tgcagcacca catggccccc actaaggatg agtttgattg caaggcctgg 2880

gcctatttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggccccctg 2940

ctggtgtgcc acaccaacac tctgaaccct gcccatggca ggcaggtgac tgtgcaggag 3000

tttgccctgt tctttaccat ctttgatgag actaagagct ggtatttcac tgagaacatg 3060

gagaggaact gcagagcccc ttgcaacatc cagatggagg accctacctt caaggagaac 3120

tataggttcc atgccatcaa tgggtacatc atggataccc tgcctggcct ggtgatggct 3180

caggaccaga ggatcaggtg gtacctgctg agcatgggga gcaatgagaa cattcatagc 3240

atccacttct ctgggcatgt gttcactgtg aggaagaagg aggagtataa gatggccctg 3300

tacaacctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc 3360

tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgagcac tctgttcctg 3420

gtgtacagca acaagtgcca gacccccctg ggcatggcct ctggccacat cagggacttc 3480

cagattactg cctctgggca gtatgggcag tgggccccca agctggccag gctgcactac 3540

tctgggtcta tcaatgcttg gagcaccaag gagcctttca gctggatcaa ggtggatctg 3600

ctggccccca tgatcattca tgggatcaag acccaggggg ccaggcagaa gttcagcagc 3660

ctgtatattt ctcagttcat catcatgtat tctctggatg gcaaaaagtg gcagacctat 3720

agagggaaca gcactgggac cctgatggtg ttttttggca atgtggatag ctctggcatc 3780

aagcacaata tcttcaaccc ccccattatt gccaggtaca tcaggctgca ccccacccac 3840

tactctatca ggagcaccct gaggatggag ctgatgggct gtgatctgaa cagctgctct 3900

atgcctctgg ggatggaaag caaggccatc tctgatgccc agatcactgc cagcagctat 3960

ttcaccaata tgtttgccac ttggagccct agcaaggcta ggctgcatct gcagggcagg 4020

tctaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag 4080

aagactatga aagtgactgg ggtgaccacc cagggggtga aaagcctgct gaccagcatg 4140

tatgtgaagg agttcctgat tagcagcagc caggatggcc accagtggac cctgttcttc 4200

cagaatggga aggtgaaggt gtttcagggc aatcaggata gcttcacccc agtggtgaac 4260

agcctggacc cccccctgct gaccaggtac ctgaggatcc acccccagag ctgggtgcac 4320

cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga 4374

<210> 13

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 13

atgcagattg agctgagcac ctgctttttc ctgtgcctgc tgaggttctg cttctctgct 60

accaggaggt actacctggg ggctgtggag ctgtcttggg attacatgca gtctgacctg 120

ggggagctgc ctgtggatgc caggtttccc cccagggtgc ccaagtcttt cccctttaac 180

acctctgtgg tgtataagaa gactctgttt gtggagttca ctgatcacct gttcaatatt 240

gccaagccca ggcccccttg gatgggcctg ctgggcccca ctatccaggc tgaggtgtat 300

gacactgtgg tcatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg 360

ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg 420

gagaaggagg atgacaaggt gttcccaggg gggtctcaca cttatgtgtg gcaggtgctg 480

aaggagaatg ggcccatggc ctctgaccct ctgtgcctga cttatagcta cctgtctcat 540

gtggatctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag 600

gggagcctgg ccaaggagaa gacccagacc ctgcacaagt tcatcctgct gtttgctgtg 660

tttgatgagg ggaagagctg gcactctgag accaagaata gcctgatgca ggacagggat 720

gctgcttctg ctagggcctg gcctaagatg cacactgtga atggctatgt gaacaggagc 780

ctgcctggcc tgattgggtg tcacaggaag tctgtgtact ggcatgtgat tggcatgggg 840

actactccag aagtgcacag catcttcctg gaggggcaca ccttcctggt gaggaatcac 900

aggcaggcca gcctggagat ttctcccatc actttcctga ctgcccagac cctgctgatg 960

gatctggggc agttcctgct gttctgccac atcagcagcc atcagcatga tgggatggag 1020

gcctatgtga aggtggacag ctgccctgag gagcctcagc tgaggatgaa gaacaatgag 1080

gaggctgagg actatgatga tgatctgact gactctgaga tggatgtggt gaggtttgat 1140

gatgacaact ctcccagctt catccagatc aggtctgtgg ccaagaagca ccccaagacc 1200

tgggtgcact acattgctgc tgaggaggag gattgggatt atgctcccct ggtgctggct 1260

cctgatgata ggagctacaa gagccagtat ctgaataatg ggccccagag gattggcagg 1320

aagtataaga aggtgaggtt catggcctac actgatgaga cctttaagac cagggaggct 1380

attcagcatg agtctggcat cctgggcccc ctgctgtatg gggaggtggg ggacaccctg 1440

ctgatcattt tcaagaacca ggccagcagg ccctataaca tctatcccca tgggatcact 1500

gatgtgaggc ccctgtactc taggaggctg cccaaggggg tcaagcacct gaaggacttc 1560

cccatcctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggcccc 1620

actaagtctg accccaggtg cctgactagg tactacagca gctttgtgaa catggagaga 1680

gatctggcct ctggcctgat tggccccctg ctgatctgct acaaagagtc tgtggatcag 1740

aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag 1800

aacagaagct ggtacctgac tgagaacatt cagaggtttc tgcccaaccc tgctggggtc 1860

cagctggagg accctgagtt tcaggccagc aacatcatgc acagcatcaa tgggtatgtg 1920

tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta tatcctgagc 1980

attggggccc agactgattt cctgtctgtg ttcttctctg gctacacttt caagcacaag 2040

atggtgtatg aggataccct gaccctgttc cctttctctg gggaaactgt gttcatgagc 2100

atggagaacc ctgggctgtg gatcctgggg tgccacaatt ctgatttcag gaacagaggc 2160

atgactgctc tgctgaaggt gtctagctgt gacaagaaca ctggggacta ctatgaggac 2220

agctatgagg acatctctgc ctacctgctg agcaagaaca atgctattga acccaggtct 2280

ttcagccaga acccccctgt gctgaagagg caccagaggg agatcactag gaccaccctg 2340

cagtctgatc aggaggagat tgactatgat gacaccatct ctgtggagat gaagaaggag 2400

gactttgaca tctatgatga ggatgagaat cagtctccca ggagcttcca gaagaagact 2460

aggcattact tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagccct 2520

catgtgctga ggaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttt 2580

caggagttca ctgatggcag cttcacccag cccctgtaca ggggggagct gaatgagcat 2640

ctgggcctgc tgggccccta catcagggct gaggtggagg acaacatcat ggtgaccttc 2700

agaaatcagg ctagcaggcc ctacagcttc tacagcagcc tgatctctta tgaggaggac 2760

cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacctat 2820

ttctggaagg tgcagcacca catggccccc accaaggatg agtttgattg caaggcctgg 2880

gcctacttct ctgatgtgga cctggagaag gatgtgcatt ctgggctgat tggccctctg 2940

ctggtgtgcc acaccaacac cctgaatcct gcccatggca ggcaggtgac tgtgcaggag 3000

tttgccctgt tctttactat ctttgatgag accaagtctt ggtattttac tgagaacatg 3060

gagaggaact gcagggcccc ctgcaacatc cagatggagg accccacctt caaggagaac 3120

tacagattcc atgccatcaa tggctacatt atggacactc tgcctggcct ggtgatggcc 3180

caggaccaga ggatcaggtg gtacctgctg tctatgggca gcaatgagaa cattcactct 3240

atccacttct ctgggcatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg 3300

tacaacctgt accctggggt gtttgagact gtggagatgc tgcctagcaa ggctgggatc 3360

tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgtctac cctgttcctg 3420

gtgtacagca acaagtgcca gacccccctg ggcatggcct ctggccacat cagagatttt 3480

cagatcactg cctctggcca gtatggccag tgggctccta agctggccag gctgcactac 3540

tctggcagca tcaatgcctg gagcaccaag gagcccttta gctggatcaa ggtggacctg 3600

ctggccccca tgatcatcca tggcatcaag actcaggggg ccaggcagaa gttctctagc 3660

ctgtacatta gccagttcat catcatgtat agcctggatg gcaagaagtg gcagacctac 3720

aggggcaaca gcactgggac cctgatggtg ttctttggga atgtggacag ctctgggatc 3780

aagcacaata tcttcaaccc ccccattatt gccaggtata ttaggctgca ccccactcac 3840

tacagcatta ggagcaccct gaggatggag ctgatgggct gtgatctgaa cagctgcagc 3900

atgcccctgg gcatggagtc taaggccatc tctgatgccc agatcactgc cagctcttac 3960

ttcaccaaca tgtttgccac ttggagcccc agcaaggcca ggctgcacct gcagggcagg 4020

agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggatttccag 4080

aagactatga aggtgactgg ggtgaccact cagggggtga agagcctgct gactagcatg 4140

tatgtgaagg agttcctgat cagctctagc caggatggcc accagtggac cctgttcttt 4200

cagaatggca aggtgaaggt gttccagggc aaccaggact ctttcacccc tgtggtgaat 4260

tctctggacc ctcccctgct gactaggtat ctgaggattc atccccagag ctgggtgcat 4320

cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ttga 4374

<210> 14

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 14

atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttttg cttttctgcc 60

actaggaggt actacctggg ggctgtggag ctgtcttggg attacatgca gtctgacctg 120

ggggagctgc cagtggatgc caggttcccc ccaagggtgc ccaagtcttt tcccttcaat 180

acctctgtgg tgtacaagaa gaccctgttt gtggagttta ctgatcatct gtttaacatt 240

gccaagccca ggcccccctg gatggggctg ctgggcccca ccatccaggc tgaggtgtat 300

gatactgtgg tgattaccct gaagaatatg gccagccatc ctgtgtctct gcatgctgtg 360

ggggtgtctt attggaaggc ctctgagggg gctgagtatg atgatcagac cagccagagg 420

gagaaggagg atgataaggt gttccctggg ggctctcaca cctatgtgtg gcaggtgctg 480

aaggagaatg ggcctatggc ctctgaccca ctgtgcctga cttacagcta tctgagccat 540

gtggacctgg tgaaggacct gaactctggg ctgattgggg ccctgctggt gtgcagggag 600

ggcagcctgg ccaaggagaa gactcagacc ctgcacaagt tcatcctgct gtttgctgtg 660

tttgatgagg gcaagtcttg gcactctgag accaagaaca gcctgatgca ggatagggat 720

gctgcctctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaacaggtct 780

ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc 840

accacccctg aggtgcatag cattttcctg gagggccaca ccttcctggt gaggaaccac 900

aggcaggcta gcctggagat cagccccatc actttcctga ctgcccagac cctgctgatg 960

gacctgggcc agttcctgct gttctgccac atctctagcc accagcatga tggcatggag 1020

gcctatgtga aggtggactc ttgtcctgag gagccccagc tgaggatgaa gaacaatgag 1080

gaggctgagg attatgatga tgatctgact gattctgaga tggatgtggt gaggtttgat 1140

gatgacaaca gcccctcttt catccagatc aggtctgtgg ccaagaagca ccccaagacc 1200

tgggtgcact acattgctgc tgaggaggag gattgggatt atgcccccct ggtgctggcc 1260

cctgatgaca ggagctataa gtctcagtac ctgaacaatg gcccccagag aattggcagg 1320

aagtacaaga aggtgaggtt catggcctat actgatgaga ccttcaaaac cagggaggcc 1380

attcagcatg agtctggcat cctggggccc ctgctgtatg gggaggtggg ggacaccctg 1440

ctgatcatct tcaagaacca ggctagcagg ccttacaaca tctaccccca tgggatcact 1500

gatgtgaggc ccctgtacag caggaggctg cctaaggggg tgaagcacct gaaggacttt 1560

cccattctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatgggccc 1620

accaagtctg accccaggtg cctgactagg tactactcta gctttgtgaa catggagagg 1680

gacctggcct ctgggctgat tggccccctg ctgatctgtt acaaggagtc tgtggaccag 1740

aggggcaacc agatcatgtc tgataagagg aatgtgatcc tgttctctgt gtttgatgag 1800

aacaggagct ggtacctgac tgagaacatc cagagattcc tgcccaaccc tgctggggtg 1860

cagctggagg atcctgagtt ccaggccagc aacatcatgc attctatcaa tgggtatgtg 1920

tttgatagcc tgcagctgtc tgtgtgtctg catgaggtgg cctactggta cattctgagc 1980

attggggccc agactgactt cctgtctgtg ttcttctctg gctacacttt caaacacaag 2040

atggtgtatg aggacaccct gaccctgttc cccttctctg gggagactgt gtttatgagc 2100

atggagaacc ctgggctgtg gattctgggc tgccacaact ctgacttcag aaacaggggc 2160

atgactgccc tgctgaaggt gtcttcttgt gataagaaca ctggggacta ttatgaagac 2220

agctatgagg acatctctgc ctacctgctg agcaagaata atgctattga gcccaggtct 2280

ttctctcaga acccccctgt gctgaagagg caccagaggg agatcaccag gaccaccctg 2340

cagtctgatc aggaggat tgactatgat gacactattt ctgtggagat gaagaaggaa 2400

gactttgata tctatgatga ggatgagaac cagagcccta ggagcttcca gaagaagact 2460

aggcattact tcattgctgc tgtggagagg ctgtgggact atggcatgag cagcagcccc 2520

catgtgctga ggaatagggc tcagtctggc tctgtgcctc agttcaagaa ggtggtgttc 2580

caggaattca ctgatggcag cttcactcag cccctgtaca ggggggagct gaatgagcac 2640

ctggggctgc tgggccctta catcagggct gaggtggagg acaatatcat ggtgaccttt 2700

aggaaccagg cctctaggcc ttacagcttc tactctagcc tgatctctta tgaagaggac 2760

cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac taagacttac 2820

ttctggaagg tgcagcacca catggctccc accaaggatg agtttgactg caaggcttgg 2880

gcctacttct ctgatgtgga cctggagaag gatgtgcact ctgggctgat tgggcccctg 2940

ctggtgtgcc acactaacac tctgaatcct gcccatggca gacaggtgac tgtgcaggag 3000

tttgccctgt tttttaccat ctttgatgag actaagtctt ggtacttcac tgagaacatg 3060

gagaggaact gcagggcccc ctgcaacatc cagatggagg atcccacctt caaggagaac 3120

tacaggtttc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggct 3180

caggaccaga ggattaggtg gtatctgctg agcatgggca gcaatgagaa tatccactct 3240

atccacttct ctgggcatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg 3300

tataacctgt atcctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc 3360

tggagagtgg agtgcctgat tggggagcac ctgcatgctg gcatgagcac tctgtttctg 3420

gtgtatagca acaagtgtca gacccctctg ggcatggcct ctgggcacat tagggacttt 3480

cagatcactg cttctggcca gtatgggcag tgggctccca agctggccag gctgcactat 3540

tctggcagca ttaatgcctg gagcaccaag gagcctttca gctggatcaa ggtggacctg 3600

ctggccccca tgatcatcca tgggatcaag acccagggg ctaggcagaa gttcagcagc 3660

ctgtacatca gccagtttat catcatgtat tctctggatg gcaagaagtg gcagacctac 3720

aggggcaatt ctactggcac tctgatggtg ttctttggga atgtggatag ctctgggatc 3780

aagcataata tcttcaatcc ccccattatt gctaggtata tcaggctgca ccccacccac 3840

tatagcatca ggagcaccct gaggatggag ctgatggggt gtgacctgaa cagctgcagc 3900

atgcccctgg gcatggagag caaggctatt tctgatgccc agatcactgc cagcagctac 3960

tttactaata tgtttgccac ctggagcccc agcaaggcca gactgcacct gcagggcagg 4020

tctaatgcct ggaggcctca ggtgaataac cccaaggagt ggctgcaggt ggacttccag 4080

aaaaccatga aggtgactgg ggtgactacc cagggggtga agtctctgct gaccagcatg 4140

tatgtgaagg agttcctgat ctcttctagc caggatggcc accagtggac cctgttcttt 4200

cagaatggga aggtgaaggt cttccagggc aaccaggata gcttcacccc tgtggtgaat 4260

agcctggatc ctcctctgct gaccaggtat ctgaggatcc acccccagag ctgggtgcat 4320

cagattgccc tgaggatgga ggtgctgggc tgtgaggctc aggacctgta ctga 4374

<210> 15

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 15

atgcagattg agctgagcac ctgtttcttc ctgtgcctgc tgaggttctg tttctctgcc 60

actaggaggt actacctggg ggctgtggag ctgagctggg actatatgca gtctgacctg 120

ggggagctgc ctgtggatgc caggttcccc cccagggtgc ctaagagctt ccccttcaat 180

acttctgtgg tgtacaagaa gactctgttt gtggagttta ctgaccacct gttcaacatt 240

gctaagccca ggcctccctg gatggggctg ctgggcccca ccatccaggc tgaggtgtat 300

gatactgtgg tgattaccct gaagaacatg gcctctcatc cagtgagcct gcatgctgtg 360

ggggtgagct actggaaggc ctctgaaggg gctgagtatg atgaccagac cagccagagg 420

gagaaggagg atgacaaggt gttccctggg ggcagccaca cctatgtgtg gcaggtgctg 480

aaggagaatg gcccaatggc ctctgacccc ctgtgcctga cttatagcta cctgagccat 540

gtggatctgg tgaaggacct gaattctggc ctgattgggg ccctgctggt gtgcagagag 600

ggctctctgg ctaaggagaa gacccagact ctgcacaagt tcatcctgct gtttgctgtg 660

tttgatgagg gcaagagctg gcactctgag actaagaata gcctgatgca ggacagggat 720

gctgcttctg ccagggcctg gcccaagatg catactgtga atggctatgt gaacaggagc 780

ctgcctggcc tgattggctg tcacaggaaa tctgtctact ggcatgtgat tgggatgggc 840

actacccctg aggtgcactc tatcttcctg gagggccata ccttcctggt gaggaaccac 900

aggcaggcca gcctggagat ctctcccatt accttcctga ctgcccagac cctgctgatg 960

gatctgggcc agttcctgct gttctgccac atcagcagcc accagcatga tgggatggag 1020

gcttatgtga aggtggatag ctgccctgag gagccccagc tgaggatgaa gaacaatgag 1080

gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat 1140

gatgacaact ctcccagctt tattcagatc aggtctgtgg ctaagaagca ccccaagact 1200

tgggtgcact acattgctgc tgaggaggag gactgggact atgcccctct ggtgctggct 1260

cctgatgaca ggtcttacaa gtctcagtac ctgaataatg gccctcagag gattggcagg 1320

aagtacaaga aggtgaggtt catggcctac actgatgaga ccttcaagac cagggaggcc 1380

atccagcatg agtctggcat cctgggcccc ctgctgtatg gggaggtggg ggataccctg 1440

ctgatcatct tcaagaatca ggccagcagg ccctacaaca tctaccccca tggcatcact 1500

gatgtgaggc cactgtacag caggaggctg cccaaggggg tgaagcatct gaaggacttc 1560

cccattctgc ctggggagat cttcaagtac aaatggactg tgactgtgga ggatggccct 1620

accaagtctg accccaggtg tctgaccagg tactacagca gctttgtgaa tatggagagg 1680

gacctggcct ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggaccag 1740

aggggcaatc agatcatgtc tgataagagg aatgtgattc tgttctctgt gtttgatgag 1800

aacaggagct ggtacctgac tgagaacatc cagaggttcc tgcccaatcc tgctggggtg 1860

cagctggagg accctgagtt ccaggccagc aatatcatgc acagcatcaa tggctatgtc 1920

tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cttactggta tattctgagc 1980

attggggccc agactgattt cctgtctgtg ttcttttctg gctatacctt taagcacaag 2040

atggtgtatg aggacaccct gaccctgttc cccttctctg gggagactgt gttcatgtct 2100

atggagaacc ctgggctgtg gatcctgggc tgccacaact ctgacttcag gaacaggggg 2160

atgactgccc tgctgaaggt gtctagctgt gataagaaca ctggggacta ttatgaggac 2220

agctatgagg acatctctgc ttacctgctg agcaagaaca atgccattga gcccaggtct 2280

ttcagccaga atccccctgt gctgaagagg catcagaggg agatcaccag gaccaccctg 2340

cagtctgatc aggaggagat tgattatgat gacactatct ctgtggaaat gaagaaggag 2400

gactttgaca tctatgatga ggatgagaac cagagcccca ggagcttcca gaagaagacc 2460

aggcactact tcattgctgc tgtggagagg ctgtgggatt atggcatgag cagctctccc 2520

catgtgctga ggaacagagc ccagtctggc tctgtgcctc agttcaagaa ggtggtcttc 2580

caggagttca ctgatggctc tttcacccag cccctgtaca ggggggagct gaatgagcac 2640

ctgggcctgc tggggcccta cattagggct gaggtggagg ataacatcat ggtgactttc 2700

agaaaccagg ccagcaggcc ttacagcttt tactcttctc tgattagcta tgaggaggat 2760

cagaggcagg gggctgagcc taggaagaac tttgtgaagc ccaatgagac caagacctat 2820

ttctggaagg tgcagcacca catggctccc actaaggatg agtttgactg caaggcttgg 2880

gcctacttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tgggcccctg 2940

ctggtgtgcc acaccaacac cctgaaccct gcccatggca ggcaggtgac tgtgcaggag 3000

tttgccctgt tcttcaccat ctttgatgag actaagagct ggtacttcac tgagaacatg 3060

gagaggaact gcagggcccc ctgcaacatc cagatggagg accccacctt caaggagaat 3120

tacaggttcc atgccatcaa tggctacatt atggacaccc tgcctggcct ggtgatggcc 3180

caggatcaga ggatcaggtg gtatctgctg agcatgggct ctaatgagaa catccacagc 3240

atccacttct ctggccatgt gtttactgtg aggaagaagg aggaatacaa gatggctctg 3300

tataacctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctgggatc 3360

tggagggtgg agtgcctgat tggggagcac ctgcatgctg ggatgagcac cctgttcctg 3420

gtgtatagca ataagtgcca gacccccctg ggcatggctt ctggccacat cagggatttc 3480

cagatcactg cttctggcca gtatggccag tgggctccca agctggctag gctgcattac 3540

tctgggtcta tcaatgcctg gagcactaag gagcccttca gctggatcaa ggtggacctg 3600

ctggccccca tgatcattca tggcatcaag acccagggg ctaggcagaa gttcagcagc 3660

ctgtacatca gccagttcat cattatgtac agcctggatg gcaagaagtg gcagacttac 3720

aggggcaata gcactgggac tctgatggtg ttctttggca atgtggactc ttctggcatc 3780

aagcacaaca tcttcaaccc tcccatcatt gccaggtaca ttaggctgca ccctacccac 3840

tactctatca ggagcaccct gaggatggag ctgatggggt gtgatctgaa ctcttgcagc 3900

atgcctctgg gcatggaaag caaagccatc tctgatgccc agatcactgc ctctagctat 3960

ttcaccaata tgtttgccac ctggagccct agcaaggcca ggctgcacct gcagggcaga 4020

tctaatgcct ggaggcccca ggtgaacaat cccaaggagt ggctgcaggt ggacttccag 4080

aagaccatga aggtgactgg ggtgaccact cagggggtga agagcctgct gactagcatg 4140

tatgtgaagg agttcctgat ctcttctagc caggatggcc accagtggac cctgttcttc 4200

cagaatggca aggtgaaagt gttccagggc aaccaggata gcttcactcc tgtggtgaac 4260

tctctggacc ctcccctgct gactaggtac ctgaggattc atccccagag ctgggtgcac 4320

cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggatctgta ctga 4374

<210> 16

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 16

atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttctg cttctctgcc 60

accaggaggt actacctggg ggctgtggag ctgtcttggg actatatgca gtctgacctg 120

ggggagctgc cagtggatgc caggttcccc cccagggtgc ccaagagctt tcctttcaac 180

acttctgtgg tgtacaagaa gaccctgttt gtggagttca ctgaccacct gttcaatatt 240

gctaagccca ggccaccctg gatgggcctg ctgggcccta ccattcaggc tgaggtgtat 300

gacactgtgg tgattactct gaagaatatg gccagccacc ctgtgagcct gcatgctgtg 360

ggggtgtctt actggaaggc ctctgagggg gctgagtatg atgatcagac ttctcagagg 420

gagaaggagg atgataaggt gttccctggg ggctctcaca cttatgtgtg gcaggtgctg 480

aaggagaatg gccccatggc ttctgatcca ctgtgcctga cctactctta cctgagccat 540

gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag 600

ggcagcctgg ccaaggagaa gacccagacc ctgcataagt tcatcctgct gtttgctgtg 660

tttgatgagg ggaagagctg gcactctgag accaagaatt ctctgatgca ggacagggat 720

gctgcctctg ccagggcctg gcctaagatg cacactgtga atggctatgt gaacaggtct 780

ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc 840

actacccctg aggtgcacag cattttcctg gagggccaca ccttcctggt caggaaccat 900

aggcaggcct ctctggagat cagccccatc actttcctga ctgcccagac cctgctgatg 960

gacctgggcc agttcctgct gttctgccac attagcagcc accagcatga tggcatggag 1020

gcctatgtga aggtggactc ttgccctgag gagccccagc tgaggatgaa gaacaatgag 1080

gaagctgagg attatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat 1140

gatgacaaca gccccagctt catccagatc aggtctgtgg ccaagaagca ccccaagacc 1200

tgggtgcact acattgctgc tgaggaggag gattgggact atgctcccct ggtgctggct 1260

cctgatgata ggagctacaa gtctcagtac ctgaataatg gcccccagag gattggcagg 1320

aagtacaaga aggtgaggtt catggcctac actgatgaga ccttcaagac cagagaggct 1380

atccagcatg agtctgggat cctggggccc ctgctgtatg gggaggtggg ggacaccctg 1440

ctgatcatct tcaagaacca ggccagcaga ccctacaaca tctaccccca tgggatcact 1500

gatgtgaggc ccctgtacag caggaggctg cctaaggggg tgaagcacct gaaggacttc 1560

cccatcctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatgggccc 1620

accaagtctg accctaggtg cctgactagg tactactcta gctttgtgaa catggagagg 1680

gacctggcct ctggcctgat tggccccctg ctgatttgct acaaggagtc tgtggatcag 1740

aggggcaatc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag 1800

aataggtctt ggtacctgac tgagaacatc cagaggttcc tgcctaatcc tgctggggtg 1860

cagctggagg accctgagtt tcaggccagc aacatcatgc acagcatcaa tggctatgtg 1920

tttgactctc tgcagctgtc tgtgtgcctg catgaggtgg cttactggta tatcctgagc 1980

attggggctc agactgactt cctgtctgtg ttcttttctg gctacacttt taagcacaag 2040

atggtgtatg aggacaccct gaccctgttc cccttttctg gggagactgt gttcatgtct 2100

atggagaacc ctgggctgtg gattctgggc tgtcacaact ctgacttcag aaacaggggc 2160

atgactgccc tgctgaaggt gtctagctgt gacaagaata ctggggacta ctatgaggac 2220

agctatgagg acatttctgc ctatctgctg agcaagaaca atgccattga gcccaggagc 2280

ttttctcaga atccccctgt gctgaagagg caccagagag agatcaccag gaccactctg 2340

cagtctgatc aggaggat tgattatgat gacactatct ctgtggagat gaagaaagag 2400

gactttgata tctatgatga ggatgagaat cagtctccca ggagcttcca gaagaagact 2460

agacactact tcattgctgc tgtggagagg ctgtgggact atggcatgag ctctagccct 2520

catgtgctga ggaacagggc ccagtctggg tctgtgcccc agttcaagaa ggtggtgttc 2580

caggagttca ctgatggcag ctttacccag cccctgtata ggggggagct gaatgagcat 2640

ctgggcctgc tgggccccta tattagggct gaagtggagg acaacatcat ggtgaccttt 2700

aggaaccagg ccagcaggcc ctacagcttt tacagcagcc tgattagcta tgaggaggat 2760

cagagacagg gggctgagcc caggaagaac tttgtgaagc ccaatgagac caagacctac 2820

ttctggaagg tgcagcacca catggcccct accaaggatg agtttgactg caaggcctgg 2880

gcttacttct ctgatgtgga cctggagaaa gatgtgcact ctggcctgat tgggcccctg 2940

ctggtgtgcc acaccaacac cctgaaccct gcccatggga ggcaggtgac tgtgcaggag 3000

tttgccctgt ttttcaccat ctttgatgag accaagagct ggtacttcac tgagaacatg 3060

gagaggaact gcagggcccc ctgtaacatc cagatggagg atcctacttt caaggagaac 3120

tacaggttcc atgccattaa tgggtacatc atggacaccc tgcctgggct ggtgatggcc 3180

caggatcaga ggattaggtg gtatctgctg tctatgggct ctaatgagaa catccactct 3240

atccacttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg 3300

tacaacctgt accctggggt gtttgaaact gtggagatgc tgccctctaa agctgggatc 3360

tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgagcac cctgttcctg 3420

gtgtacagca ataagtgcca gactcccctg ggcatggctt ctgggcacat cagggatttc 3480

cagatcactg cctctggcca gtatggccag tgggccccca agctggctag gctgcactac 3540

tctggcagca tcaatgcctg gagcaccaag gagcccttct cttggattaa ggtggacctg 3600

ctggctccca tgatcattca tggcatcaag acccaggggg ccaggcagaa gttttctagc 3660

ctgtatatta gccagttcat catcatgtat agcctggatg ggaagaagtg gcagacctac 3720

agggggaata gcactggcac cctgatggtg ttttttggca atgtggattc ttctggcatc 3780

aagcataaca tcttcaatcc ccctatcatt gccaggtaca ttaggctgca tcccacccat 3840

tactctatca ggagcaccct gaggatggag ctgatggggt gtgatctgaa cagctgtagc 3900

atgcccctgg gcatggagtc caaggctatc tctgatgccc agatcactgc cagcagctac 3960

ttcaccaaca tgtttgccac ctggagcccc agcaaggcca ggctgcacct gcagggcagg 4020

tctaatgcct ggaggcccca ggtgaacaat cccaaggagt ggctgcaggt ggacttccag 4080

aagactatga aggtgactgg ggtgaccact cagggggtga agagcctgct gaccagcatg 4140

tatgtgaagg agttcctgat ctcttctagc caggatgggc atcagtggac cctgttttttt 4200

cagaatggca aagtgaaggt gtttcagggg aatcaggaca gctttacccc tgtggtgaac 4260

agcctggatc ctcctctgct gactagatac ctgaggatcc acccccagag ctgggtccac 4320

cagattgctc tgaggatgga ggtgctgggg tgtgaggctc aggacctgta ctga 4374

<210> 17

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 17

atgcagattg agctgagcac ctgcttcttt ctgtgcctgc tgaggttctg cttctctgcc 60

accaggaggt actacctggg ggctgtggaa ctgagctggg actatatgca gtctgacctg 120

ggggagctgc ctgtggatgc caggttcccc cccagggtgc ccaagtcttt cccctttaac 180

acttctgtgg tgtacaagaa gaccctgttt gtggagttta ctgaccacct gttcaatatt 240

gccaagccca ggcccccctg gatgggcctg ctgggcccaa ccatccaggc tgaggtgtat 300

gatactgtgg tgatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg 360

ggggtgagct attggaaggc ttctgagggg gctgagtatg atgaccagac tagccagagg 420

gagaaggagg atgacaaggt gttccctggg gggtctcata cctatgtgtg gcaggtgctg 480

aaggagaatg gccccatggc ctctgacccc ctgtgcctga cctattctta cctgagccat 540

gtggacctgg tcaaggacct gaactctggc ctgattgggg ctctgctggt gtgcagggag 600

ggcagcctgg ccaaggagaa gactcagact ctgcataagt tcatcctgct gtttgctgtg 660

tttgatgagg gcaagagctg gcactctgag accaagaact ctctgatgca ggatagggat 720

gctgcctctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaataggtct 780

ctgcctggcc tgattggctg ccataggaag tctgtgtact ggcatgtgat tggcatgggc 840

actacccctg aggtgcactc tatcttcctg gaggggcaca ccttcctggt gaggaaccac 900

aggcaggcca gcctggagat ctctcccatc accttcctga ctgcccagac tctgctgatg 960

gacctgggcc agttcctgct gttctgccat atcagcagcc accagcatga tggcatggag 1020

gcctatgtga aggtggacag ctgcccagag gaaccccagc tgaggatgaa gaacaatgag 1080

gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat 1140

gatgacaaca gccccagctt tattcagatc aggtctgtgg ccaagaagca ccccaagacc 1200

tgggtgcact acattgctgc tgaggaggag gactgggatt atgcccccct ggtgctggcc 1260

cctgatgaca ggtcttacaa gtctcagtac ctgaacaatg gcccccagag gattgggagg 1320

aagtacaaga aggtgaggtt catggcctac actgatgaga ccttcaagac cagggaggcc 1380

atccagcatg agtctggcat cctggggccc ctgctgtatg gggaggtggg ggataccctg 1440

ctgattatct tcaagaacca ggctagcagg ccctataaca tctaccccca tggcattact 1500

gatgtgaggc ccctgtactc taggagactg cccaaggggg tgaagcacct gaaagacttc 1560

cccatcctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatggcccc 1620

actaagtctg accccaggtg cctgaccagg tattacagca gctttgtgaa tatggagagg 1680

gatctggctt ctggcctgat tgggcctctg ctgatttgct acaaggagtc tgtggatcag 1740

agggggaacc agattatgtc tgacaagagg aatgtgattc tgttctctgt gtttgatgag 1800

aacaggagct ggtacctgac tgagaatatc cagaggttcc tgcctaatcc tgctggggtg 1860

cagctggagg accctgagtt ccaggctagc aacattatgc acagcatcaa tggctatgtg 1920

tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cttactggta cattctgtct 1980

attggggccc agactgactt cctgtctgtg ttcttctctg gctacacctt caagcacaag 2040

atggtgtatg aggacactct gaccctgttc cccttctctg gggagactgt gttcatgagc 2100

atggagaatc ctgggctgtg gattctgggg tgccacaact ctgatttcag gaacaggggc 2160

atgactgccc tgctgaaggt gagcagctgt gacaagaaca ctggggatta ttatgaggac 2220

agctatgagg acatttctgc ctacctgctg agcaagaaca atgccattga gcctaggagc 2280

ttcagccaga atccccctgt gctgaagaga caccagaggg agatcactag gaccactctg 2340

cagtctgatc aggaggat tgactatgat gacaccattt ctgtggagat gaagaaggag 2400

gactttgata tttatgatga ggatgagaac cagagcccca gaagcttcca gaagaagacc 2460

aggcactact tcattgctgc tgtggagagg ctgtgggatt atggcatgtc ttctagcccc 2520

catgtgctga ggaacagggc tcagtctggc tctgtgcctc agttcaagaa ggtggtgttc 2580

caggagttca ctgatgggag cttcacccag cctctgtaca ggggggagct gaatgaacat 2640

ctgggcctgc tggggcccta catcagggct gaggtggagg ataatatcat ggtgactttc 2700

aggaatcagg cctctaggcc ctacagcttc tactctagcc tgatcagcta tgaggaggac 2760

cagaggcagg gggctgagcc taggaagaat tttgtgaaac ccaatgagac caagacctac 2820

ttttggaagg tgcagcacca catggcccct accaaggatg agtttgactg taaggcctgg 2880

gcctacttct ctgatgtgga cctggagaag gatgtgcatt ctgggctgat tggccccctg 2940

ctggtgtgcc acaccaacac cctgaaccct gcccatggca ggcaggtgac tgtgcaggag 3000

tttgccctgt tcttcaccat ctttgatgag actaagagct ggtatttcac tgagaacatg 3060

gagaggaact gtagggctcc ctgcaacatc cagatggagg atccaacttt caaggagaac 3120

tacaggttcc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggcc 3180

caggaccaga ggattaggtg gtacctgctg agcatgggct ctaatgagaa catccactct 3240

atccacttct ctggccatgt gtttactgtg aggaagaagg aggagtacaa gatggctctg 3300

tacaacctgt accctggggt gtttgagact gtggagatgc tgcctagcaa ggctggcatt 3360

tggagagtgg agtgtctgat tggggagcac ctgcatgctg ggatgtctac cctgttcctg 3420

gtgtactcta acaagtgcca gacccccctg gggatggctt ctgggcacat cagagatttt 3480

cagattactg cttctgggca gtatggccag tgggctccca agctggccag actngcattac 3540

tctggctcta ttaatgcttg gagcaccaag gagcctttca gctggatcaa ggtggacctg 3600

ctggctccca tgatcatcca tggcattaag actcaggggg ctaggcagaa gttcagcagc 3660

ctgtatattt ctcagtttat tatcatgtat tctctggatg gcaagaagtg gcagacttac 3720

aggggcaaca gcactggcac cctgatggtg ttctttggca atgtggacag ctctgggatc 3780

aagcataaca tcttcaaccc ccccattatt gccaggtaca tcaggctgca ccccacccac 3840

tattctatca ggagcactct gaggatggag ctgatggggt gtgacctgaa cagctgctct 3900

atgcccctgg gcatggagag caaggccatc tctgatgccc agatcactgc cagctcttat 3960

ttcaccaaca tgtttgccac ctggagcccc agcaaggcca ggctgcacct gcagggcaga 4020

agcaatgcct ggaggcccca ggtgaacaat cctaaggagt ggctgcaggt ggacttccag 4080

aagactatga aggtgactgg ggtgactacc cagggggtga agagcctgct gaccagcatg 4140

tatgtgaagg agttcctgat tagcagcagc caggatgggc atcagtggac cctgttcttc 4200

cagaatggga aggtgaaggt gttccagggc aatcaggaca gcttcacccc tgtggtgaac 4260

agcctggacc cccccctgct gaccaggtac ctgaggatcc atccccagag ctgggtgcac 4320

cagattgctc tgagaatgga ggtgctgggc tgtgaggccc aggacctgta ttga 4374

<210> 18

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Option FVIII

<400> 18

atgcagattg agctgtctac ctgttttttt ctgtgcctgc tgaggttctg cttctctgct 60

accaggaggt attatctggg ggctgtggag ctgagctggg actacatgca gtctgacctg 120

ggggagctgc ctgtggatgc caggtttcct cccagggtgc ctaagagctt ccccttcaac 180

acctctgtgg tgtacaagaa gactctgttt gtggagttca ctgaccacct gttcaacatt 240

gccaagccca ggcccccctg gatggggctg ctgggcccca ctatccaggc tgaggtgtat 300

gatactgtgg tgattaccct gaagaacatg gcctctcacc ctgtgtctct gcatgctgtg 360

ggggtgagct actggaaggc ttctgagggg gctgaatatg atgatcagac ctctcagagg 420

gagaaggagg atgacaaggt gtttcctggg ggcagccaca cctatgtgtg gcaggtgctg 480

aaggagaatg ggcccatggc ctctgatccc ctgtgcctga cctacagcta cctgagccat 540

gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag 600

ggcagcctgg ccaaggaaaa gacccagacc ctgcataagt tcatcctgct gtttgctgtg 660

tttgatgagg gcaagtcttg gcactctgag accaagaaca gcctgatgca ggacagggat 720

gctgcctctg ctagggcctg gcccaagatg cacactgtga atgggtatgt gaacagatct 780

ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggg 840

accacccctg aggtgcatag catcttcctg gaggggcaca ccttcctggt gagaaatcat 900

aggcaggcca gcctggagat tagccccatc accttcctga ctgcccagac cctgctgatg 960

gacctgggcc agttcctgct gttctgccac atttctagcc accagcatga tggcatggag 1020

gcctatgtga aggtggatag ctgccctgaa gagccccagc tgaggatgaa gaacaatgag 1080

gaggctgagg attatgatga tgatctgact gactctgaga tggatgtggt gaggtttgat 1140

gatgacaaca gccccagctt catccagatc aggtctgtgg ccaagaagca ccctaagacc 1200

tgggtgcact acattgctgc tgaagaggag gactgggact atgcccccct ggtgctggcc 1260

ccagatgaca ggtcttacaa gagccagtac ctgaataatg gcccccagag gattgggagg 1320

aagtataaga aagtgaggtt catggcttac actgatgaga cctttaagac tagggaggcc 1380

attcagcatg agtctgggat tctgggccct ctgctgtatg gggaggtggg ggacaccctg 1440

ctgatcattt tcaagaacca ggccagcagg ccctataata tttatcccca tgggattact 1500

gatgtcaggc ccctgtacag caggaggctg cctaaggggg tgaagcacct gaaggacttc 1560

cccattctgc ctggggagat cttcaagtat aagtggactg tgactgtgga ggatggcccc 1620

accaagtctg atcctaggtg cctgaccagg tactatagca gctttgtgaa catggagagg 1680

gacctggctt ctggcctgat tggccccctg ctgatctgct acaaggaatc tgtggaccag 1740

aggggcaacc agattatgtc tgacaagagg aatgtgatcc tgttttctgt gtttgatgag 1800

aataggagct ggtatctgac tgagaacatc cagaggttcc tgcccaatcc tgctggggtg 1860

cagctggagg accctgagtt ccaggcttct aacatcatgc atagcatcaa tgggtatgtg 1920

tttgactctc tgcagctgtc tgtgtgcctg catgaggtgg cctattggta catcctgagc 1980

attggggccc agactgactt cctgtctgtg ttcttctctg gctacacctt caagcacaag 2040

atggtgtatg aggacaccct gaccctgttc cctttctctg gggagactgt gttcatgagc 2100

atggagaacc ctggcctgtg gattctgggc tgccataatt ctgacttcag aaacaggggc 2160

atgactgctc tgctgaaggt gagcagctgt gacaagaata ctggggacta ctatgaggac 2220

tcttatgagg atatttctgc ctacctgctg agcaagaaca atgctattga gcccaggagc 2280

ttcagccaga acccccctgt cctgaagagg catcagaggg agatcactag gaccaccctg 2340

cagtctgatc aggaggagat tgactatgat gacactatct ctgtggaaat gaagaaggag 2400

gactttgata tctatgatga ggatgagaac cagagcccca ggtctttcca gaagaagacc 2460

aggcactact tcattgctgc tgtggagagg ctgtgggact atggcatgtc tagcagcccc 2520

catgtgctga ggaacagagc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttt 2580

caggagttca ctgatgggag cttcactcag cccctgtata ggggggagct gaatgagcat 2640

ctgggcctgc tggggcccta catcagggct gaggtggagg ataacatcat ggtgaccttc 2700

aggaaccagg ccagcaggcc ctactctttc tactcttctc tgatcagcta tgaggaggat 2760

cagaggcagg gggctgagcc taggaagaac tttgtcaagc ctaatgagac taagacctac 2820

ttttggaagg tgcagcacca catggctccc actaaggatg agtttgattg caaggcctgg 2880

gcctacttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggccccctg 2940

ctggtgtgtc acaccaatac cctgaaccct gcccatggca ggcaggtcac tgtgcaggag 3000

tttgccctgt ttttcactat ctttgatgag actaagtctt ggtacttcac tgagaacatg 3060

gaaaggaatt gcagggctcc ctgcaacatc cagatggagg accccacctt caaggagaac 3120

tacaggtttc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggct 3180

caggatcaga ggattaggtg gtatctgctg agcatgggca gcaatgagaa catccacagc 3240

atccactttt ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggctctg 3300

tacaatctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctgggatc 3360

tggagggtgg agtgcctgat tggggaacac ctgcatgctg gcatgtctac cctgttcctg 3420

gtgtactcta acaagtgcca gactcccctg ggcatggcct ctgggcacat cagggacttc 3480

cagatcactg cctctgggca gtatggccag tgggccccta agctggctag gctgcattac 3540

tctggcagca tcaatgcctg gagcaccaag gagcccttca gctggatcaa ggtggacctg 3600

ctggccccta tgatcatcca tggcatcaag acccaggggg cgacagaa gttctcttct 3660

ctgtacatct ctcagttcat catcatgtac tctctggatg gcaagaagtg gcagacctac 3720

agggggaatt ctactggcac tctgatggtg ttctttggga atgtggatag ctctgggatc 3780

aagcataata ttttcaaccc ccccattatt gctaggtaca tcaggctgca cccaacccac 3840

tactctatta ggtctaccct gaggatggag ctgatgggct gtgacctgaa ctcttgtagc 3900

atgcccctgg gcatggagag caaggctatc tctgatgccc agatcactgc cagcagctac 3960

tttaccaaca tgtttgctac ttggagcccc agcaaggcca ggctgcacct gcagggcagg 4020

agcaatgcct ggaggcccca ggtgaacaac cccaaggagt ggctgcaggt ggattttcag 4080

aagaccatga aggtgactgg ggtgaccact cagggggtga aaagcctgct gactagcatg 4140

tatgtgaagg agtttctgat cagcagctct caggatggcc atcagtggac cctgttcttc 4200

cagaatggca aggtgaaggt gttccagggc aaccaggata gcttcacccc tgtggtgaat 4260

agcctggacc cccccctgct gaccaggtac ctgaggatcc atccccagag ctgggtgcac 4320

cagattgccc tgaggatgga ggtgctgggc tgtgaagccc aggacctgta ctga 4374

<210> 19

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Factor VIII-BDD wild type

<400> 19

atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60

accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120

ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180

acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240

gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300

gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360

ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420

gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480

aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540

gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600

gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660

tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720

gctgcatctg ctcggggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780

ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840

accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900

cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960

gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020

gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080

gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140

gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200

tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260

cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320

aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380

attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440

ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500

gatgtccgtc ctttgtattc aaggatta ccaaaaggtg taaaacattt gaaggatttt 1560

ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620

actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680

gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740

agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800

aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860

cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920

tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980

attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040

atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100

atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160

atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220

agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280

ttctcccaaa acccaccagt cttgaaacgc catcaacggg aaataactcg tactactctt 2340

cagtcagatc aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa 2400

gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca aaagaaaaca 2460

cgacactatt ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 2520

catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc 2580

caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact aaatgaacat 2640

ttgggactcc tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc 2700

agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta tgaggaagat 2760

cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 2820

ttttggaaag tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880

gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940

ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa 3000

tttgctctgt ttttcaccat ctttgatgag accaaaagct ggtacttcac tgaaaatatg 3060

gaaagaaact gcagggctcc ctgcaatatc cagatggaag atcccacttt taaagagaat 3120

tatcgcttcc atgcaatcaa tggctacata atggatacac tacctggctt agtaatggct 3180

caggatcaaa ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3240

attcatttca gtggacatgt gttcaccgta cgaaaaaaag aggagtataa aatggcactg 3300

tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa agctggaatt 3360

tggcgggtgg aatgccttat tggcgagcat ctacatgctg ggatgagcac actttttctg 3420

gtgtacagca ataagtgtca gactcccctg ggaatggctt ctggacacat tagagatttt 3480

cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540

tccggatcaa tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 3600

ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc 3660

ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg gcagacttat 3720

cgaggaaatt ccactggaac cttaatggtc ttctttggca atgtggattc atctgggata 3780

aaacacaata tttttaaccc tccaattatt gctcgataca tccgtttgca cccaactcat 3840

tatagcattc gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 3900

atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 3960

tttaccaata tgtttgccac ctggtctcct tcaaaagctc gacttcacct ccaagggagg 4020

agtaatgcct ggagaacctca ggtgaataat ccaaaagagt ggctgcaagt ggacttccag 4080

aagacaatga aagtcacagg agtaactact cagggagtaa aatctctgct taccagcatg 4140

tatgtgaagg agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt 4200

cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4260

tctctagacc caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 4320

cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctcta ctga 4374

<210> 20

<211> 4890

<212> DNA

<213> Artificial sequence

<220>

<223> V3 factor VIII

<400> 20

atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttctg cttctctgcc 60

accaggagat actacctggg ggctgtggag ctgagctggg actacatgca gtctgacctg 120

ggggagctgc ctgtggatgc caggttcccc cccagagtgc ccaagagctt ccccttcaac 180

acctctgtgg tgtacaagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt 240

gccaagccca ggcccccctg gatgggcctg ctgggcccca ccatccaggc tgaggtgtat 300

gacactgtgg tgatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg 360

ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg 420

gagaaggagg atgacaaggt gttccctggg ggcagccaca cctatgtgtg gcaggtgctg 480

aaggagaatg gccccatggc ctctgacccc ctgtgcctga cctacagcta cctgagccat 540

gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag 600

ggcagcctgg ccaaggagaa gacccagacc ctgcacaagt tcatcctgct gtttgctgtg 660

tttgatgagg gcaagagctg gcactctgaa accaagaaca gcctgatgca ggacagggat 720

gctgcctctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaacaggagc 780

ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc 840

accacccctg aggtgcacag catcttcctg gagggccaca ccttcctggt caggaaccac 900

aggcaggcca gcctggagat cagccccatc accttcctga ctgcccagac cctgctgatg 960

gacctgggcc agttcctgct gttctgccac atcagcagcc accagcatga tggcatggag 1020

gcctatgtga aggtggacag ctgccctgag gagccccagc tgaggatgaa gaacaatgag 1080

gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat 1140

gatgacaaca gccccagctt catccagatc aggtctgtgg ccaagaagca ccccaagacc 1200

tgggtgcact acattgctgc tgaggaggag gactgggact atgcccccct ggtgctggcc 1260

cctgatgaca ggagctacaa gagccagtac ctgaacaatg gcccccagag gattggcagg 1320

aagtacaaga aggtcaggtt catggcctac actgatgaaa ccttcaagac cagggaggcc 1380

atccagcatg agtctggcat cctgggcccc ctgctgtatg gggaggtggg ggacaccctg 1440

ctgatcatct tcaagaacca ggccagcagg ccctacaaca tctaccccca tggcatcact 1500

gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcacct gaaggacttc 1560

cccatcctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggcccc 1620

accaagtctg accccaggtg cctgaccaga tactacagca gctttgtgaa catggagagg 1680

gacctggcct ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggaccag 1740

aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag 1800

aacaggagct ggtacctgac tgagaacatc cagaggttcc tgcccaaccc tgctggggtg 1860

cagctggagg accctgagtt ccaggccagc aacatcatgc acagcatcaa tggctatgtg 1920

tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc 1980

attggggccc agactgactt cctgtctgtg ttcttctctg gctacacctt caagcacaag 2040

atggtgtatg aggacaccct gaccctgttc cccttctctg gggagactgt gttcatgagc 2100

atggagaacc ctggcctgtg gattctgggc tgccacaact ctgacttcag gaacaggggc 2160

atgactgccc tgctgaaagt ctccagctgt gacaagaaca ctggggacta ctatgaggac 2220

agctatgagg acatctctgc ctacctgctg agcaagaaca atgccattga gcccaggagc 2280

ttcagccaga acagcaggca ccccagcacc aggcagaagc agttcaatgc caccaccatc 2340

cctgagaatg acatagagaa gacagacca tggtttgccc accggacccc catgcccaag 2400

atccagaatg tgagcagctc tgacctgctg atgctgctga ggcagagccc caccccccat 2460

ggcctgagcc tgtctgacct gcaggaggcc aagtatgaaa ccttctctga tgaccccagc 2520

cctggggcca ttgacagcaa caacagcctg tctgagatga cccacttcag gccccagctg 2580

caccactctg gggacatggt gttcacccct gagtctggcc tgcagctgag gctgaatgag 2640

aagctgggca ccactgctgc cactgagctg aagaagctgg acttcaaagt ctccagcacc 2700

agcaacaacc tgatcagcac catcccctct gacaacctgg ctgctggcac tgacaacacc 2760

agcagcctgg gcccccccag catgcctgtg cactatgaca gccagctgga caccaccctg 2820

tttggcaaga agagcagccc cctgactgag tctggggggcc ccctgagcct gtctgaggag 2880

aacaatgaca gcaagctgct ggagtctggc ctgatgaaca gccaggagag cagctggggc 2940

aagaatgtga gcaccaggag cttccagaag aagaccaggc actacttcat tgctgctgtg 3000

gagaggctgt gggactatgg catgagcagc agcccccatg tgctgaggaa cagggcccag 3060

tctggctctg tgccccagtt caagaaggtg gtgttccagg agttcactga tggcagcttc 3120

acccagcccc tgtacagagg ggagctgaat gagcacctgg gcctgctggg cccctacatc 3180

agggctgagg tggaggacaa catcatggtg accttcagga accaggccag caggccctac 3240

agcttctaca gcagcctgat cagctatgag gaggaccaga ggcaggggggc tgagcccagg 3300

aagaactttg tgaagcccaa tgaaaccaag acctacttct ggaaggtgca gcaccacatg 3360

gcccccacca aggatgagtt tgactgcaag gcctgggcct acttctctga tgtggacctg 3420

gagaaggatg tgcactctgg cctgattggc cccctgctgg tgtgccacac caacaccctg 3480

aaccctgccc atggcaggca ggtgactgtg caggagtttg ccctgttctt caccatcttt 3540

gatgaaacca agagctggta cttcactgag aacatggaga ggaactgcag ggccccctgc 3600

aacatccaga tggaggaccc caccttcaag gagaactaca ggttccatgc catcaatggc 3660

tacatcatgg acaccctgcc tggcctggtg atggcccagg accagaggat caggtggtac 3720

ctgctgagca tgggcagcaa tgagaacatc cacagcatcc acttctctgg ccatgtgttc 3780

actgtgagga agaagggagga gtacaagatg gccctgtaca acctgtaccc tggggtgttt 3840

gagactgtgg agatgctgcc cagcaaggct ggcatctgga gggtggagtg cctgattggg 3900

gagcacctgc atgctggcat gagcaccctg ttcctggtgt acagcaacaa gtgccagacc 3960

cccctgggca tggcctctgg ccacatcagg gacttccaga tcactgcctc tggccagtat 4020

ggccagtggg cccccaagct ggccaggctg cactactctg gcagcatcaa tgcctggagc 4080

accaaggagc ccttcagctg gatcaaggtg gacctgctgg cccccatgat catccatggc 4140

atcaagaccc aggggggccag gcagaagttc agcagcctgt acatcagcca gttcatcatc 4200

atgtacagcc tggatggcaa gaagtggcag acctacaggg gcaacagcac tggcaccctg 4260

atggtgttct ttggcaatgt ggacagctct ggcatcaagc acaacatctt caacccccccc 4320

atcattgcca gatacatcag gctgcacccc acccactaca gcatcaggag caccctgagg 4380

atggagctga tgggctgtga cctgaacagc tgcagcatgc ccctgggcat gggagagcaag 4440

gccatctctg atgcccagat cactgccagc agctacttca ccaacatgtt tgccacctgg 4500

agccccagca aggccaggct gcacctgcag ggcaggagca atgcctggag gccccaggtc 4560

aacaacccca aggagtggct gcaggtggac ttccagaaga ccatgaaggt gactggggtg 4620

accacccagg gggtgaagag cctgctgacc agcatgtatg tgaaggagtt cctgatcagc 4680

agcagccagg atggccacca gtggaccctg ttcttccaga atggcaaggt gaaggtgttc 4740

cagggcaacc aggacagctt cacccctgtg gtgaacagcc tggacccccc cctgctgacc 4800

agatacctga ggattcaccc cgagctgg gtgcaccaga ttgccctgag gatggaggtg 4860

ctgggctgtg aggcccagga cctgtactga 4890

<210> 21

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> CO3 factor VIII

<400> 21

atgcagattg agctgtcaac ttgctttttc ctgtgcctgc tgagattttg tttttccgct 60

actagaagat actacctggg ggctgtggaa ctgtcttggg attacatgca gagtgacctg 120

ggagagctgc cagtggacgc acgatttcca cctagagtcc ctaaatcatt ccccttcaac 180

accagcgtgg tctataagaa aacactgttc gtggagttta ctgatcacct gttcaacatc 240

gctaagcctc ggccaccctg gatgggactg ctgggaccaa caatccaggc agaggtgtac 300

gacaccgtgg tcattacact gaaaaacatg gcctcacacc ccgtgagcct gcatgctgtg 360

ggcgtcagct actggaaggc ttccgaaggg gcagagtatg acgatcagac ttcccagaga 420

gaaaaagagg acgataaggt gtttcctggc gggtctcata cctatgtgtg gcaggtcctg 480

aaagagaatg gccccatggc ttccgaccct ctgtgcctga cctactctta tctgagtcac 540

gtggacctgg tcaaggatct gaacagcgga ctgatcggag cactgctggt gtgtagggaa 600

gggagcctgg ctaaggagaa aacccagaca ctgcataagt tcattctgct gttcgccgtg 660

tttgacgaag gaaaatcatg gcacagcgag acaaagaata gtctgatgca ggaccgggat 720

gccgcttcag ccagagcttg gcccaaaatg cacactgtga acggctacgt caatcgctca 780

ctgcctggac tgatcggctg ccaccgaaag agcgtgtatt ggcatgtcat cggaatgggc 840

accacacctg aagtgcactc cattttcctg gaggggcata cctttctggt ccgcaaccac 900

cgacaggcct ccctggagat ctctccaatt accttcctga cagctcagac tctgctgatg 960

gatctgggac agttcctgct gttttgccac atcagctccc accagcatga tggcatggag 1020

gcctacgtga aagtggacag ctgtcccgag gaacctcagc tgaggatgaa gaacaatgag 1080

gaagctgaag actatgacga tgacctgacc gactccgaga tggatgtggt ccgattcgat 1140

gacgataaca gcccctcctt tatccagatt agatctgtgg ccaagaaaca ccctaagaca 1200

tgggtccatt acatcgcagc cgaggaagag gactgggatt atgcaccact ggtgctggca 1260

cgacgatc gatcctacaa atctcagtat ctgaacaatg gaccacagcg gattggcaga 1320

aagtacaaga aagtgaggtt catggcttat accgatgaaa ccttcaagac tcgcgaagca 1380

atccagcacg agagcgggat tctgggacca ctgctgtacg gagaagtggg ggacaccctg 1440

ctgatcattt ttaagaacca ggccagcagg ccttacaata tctatccaca tggaattaca 1500

gatgtgcgcc ctctgtacag ccggagactg ccaaagggcg tcaaacacct gaaggacttc 1560

ccaatcctgc ccggggaaat ttttaagtat aaatggactg tcaccgtcga ggatggcccc 1620

actaagagcg accctaggtg cctgacccgc tactattcta gtttcgtgaa tatggaaagg 1680

gatctggcca gcggactgat cggcccactg ctgatttgtt acaaagagag cgtggatcag 1740

agaggcaacc agatcatgtc cgacaagagg aatgtgattc tgttcagtgt ctttgacgaa 1800

aaccggtcat ggtatctgac cgagaacatc cagagattcc tgcctaatcc agccggagtg 1860

cagctggaag atcctgagtt tcaggcttct aacatcatgc atagtattaa tggctacgtg 1920

ttcgacagtc tgcagctgtc agtgtgtctg cacgaggtcg cttactggta tatcctgagc 1980

attggagcac agacagattt cctgagcgtg ttcttttccg gctacacttt taagcataaa 2040

atggtgtatg aggacacact gactctgttc cccttcagcg gcgaaaccgt gtttatgtcc 2100

atggagaatc ccgggctgtg gatcctggga tgccacaaca gcgatttcag gaatcgcggg 2160

atgactgccc tgctgaaagt gtcaagctgt gacaagaaca ccggagacta ctatgaagat 2220

tcatacgagg acatcagcgc atatctgctg tccaaaaaca atgccattga acccaggtct 2280

tttagtcaga atcctccagt gctgaagagg caccagcgcg agatcacccg cactaccctg 2340

cagagtgatc aggaagagat cgactacgac gatacaattt ctgtggaaat gaagaaagag 2400

gacttcgata tctatgacga agatgagaac cagagtcctc gatcattcca gaagaaaacc 2460

cggcattact ttattgctgc agtggagcgc ctgtgggatt atggcatgtc ctctagtcct 2520

cacgtgctgc gaaatcgggc ccagtcaggg agcgtcccac agttcaagaa agtggtcttc 2580

caggagttta cagacggatc ctttactcag ccactgtacc ggggcgaact gaacgagcac 2640

ctggggctgc tgggacccta tatcagagct gaagtggagg ataacattat ggtcaccttc 2700

agaaatcagg catctaggcc ttacagtttt tattcaagcc tgatctctta cgaagaggac 2760

cagaggcagg gagcagaacc acgaaaaaac ttcgtgaagc ctaatgagac caaaacatac 2820

ttttggaagg tgcagcacca tatggcccca acaaaagacg aattcgattg caaggcatgg 2880

gcctattttt ctgacgtgga tctggagaag gacgtccaca gtggcctgat cgggccactg 2940

ctggtgtgtc atactaacac cctgaatccc gcacacggca ggcaggtcac tgtccaggaa 3000

ttcgccctgt tctttaccat ctttgatgag acaaaaagct ggtacttcac cgaaaacatg 3060

gagcgaaatt gccgggctcc atgtaatatt cagatggaag accccacatt caaggagaac 3120

taccgctttc atgccatcaa tgggtatatt atggatactc tgcccggact ggtcatggct 3180

caggaccaga gaatcaggtg gtacctgctg agcatggggt ccaacgagaa tatccactca 3240

attcatttca gcggacacgt gtttactgtc cggaagaaag aagagtataa aatggccctg 3300

tacaacctgt atcccggcgt gttcgaaacc gtcgagatgc tgcctagcaa ggcagggatc 3360

tggagagtgg aatgcctgat tggggagcac ctgcatgccg gaatgtctac cctgtttctg 3420

gtgtacagta ataagtgtca gacacccctg gggatggctt ccggacatat ccgggatttc 3480

cagattaccg catctggaca gtacggccag tgggccccta agctggctag actgcactat 3540

tccgggtcta tcaacgcttg gtccacaaaa gagcctttct cttggattaa ggtggacctg 3600

ctggcaccaa tgatcattca tggcatcaaa actcaggggg ccaggcagaa gttctcctct 3660

ctgtacatct cacagtttat catcatgtac agcctggatg gcaagaaatg gcagacatac 3720

cgcggcaata gcacagggac tctgatggtg ttctttggca acgtggacag ttcagggatc 3780

aagcacaaca ttttcaatcc ccctatcatt gctagataca tcaggctgca cccaacccat 3840

tattctattc gaagtacact gcggatggaa ctgatggggt gcgatctgaa cagttgttca 3900

atgcccctgg gaatggagtc caaggcaatc tctgacgccc agattaccgc tagctcctac 3960

ttcactaata tgtttgctac ctggagcccc tccaaagcac gactgcatct gcagggacga 4020

agcaacgcat ggcgaccaca ggtgaacaat cccaaggagt ggctgcaggt cgattttcag 4080

aaaactatga aggtgaccgg agtcacaact cagggcgtga aaagtctgct gacctcaatg 4140

tacgtcaagg agttcctgat ctctagttca caggacggcc accagtggac actgttcttt 4200

cagaacggaa aggtgaaagt cttccagggc aatcaggatt cctttacacc tgtggtcaac 4260

tctctggacc cacccctgct gactcgctac ctgcgaatcc acccacagtc ctgggtgcat 4320

cagattgcac tgagaatgga agtcctgggc tgcgaggccc aggacctgta ttga 4374

<210> 22

<211> 222

<212> DNA

<213> Artificial sequence

<220>

<223> Transthyretin promoter

<400> 22

gtctgtctgc acatttcgta gagcgagtgt tccgatactc taatctccct aggcaaggtt 60

catattgact taggttactt attctccttt tgttgactaa gtcaataatc agaatcagca 120

ggtttggagt cagcttggca gggatcagca gcctgggttg gaaggagggg gtataaaagc 180

cccttcacca ggagaagccg tcacacagat ccacaagctc ct 222

<210> 23

<211> 5066

<212> DNA

<213> Artificial sequence

<220>

<223> Full-length cassette sequence

<400> 23

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tacgcgtgtc tgtctgcaca tttcgtagag cgagtgttcc 180

gatactctaa tctccctagg caaggttcat attgacttag gttacttatt ctccttttgt 240

tgactaagtc aataatcaga atcagcaggt ttggagtcag cttggcaggg atcagcagcc 300

tgggttggaa ggagggggta taaaagcccc ttcaccagga gaagccgtca cacagatcca 360

caagctcctg ctagcaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg 420

gttatggccc ttgcgtgcct tgaattactg acactgacat ccactttttc tttttctcca 480

caggtttaaa cgccaccatg cagattgagc tgagcacctg cttcttcctg tgtctgctga 540

ggttctgctt ctctgccacc aggaggtatt acctgggggc tgtggagctg agctgggact 600

atatgcagtc tgacctgggg gagctgcctg tggatgctag gttccccccc agggtgccca 660

agagcttccc ctttaacact tctgtggtgt acaagaagac cctgtttgtg gagttcactg 720

accacctgtt caacattgcc aagcccaggc ccccctggat ggggctgctg gggcccacca 780

tccaggctga ggtgtatgac actgtggtga tcaccctgaa gaacatggcc agccaccctg 840

tgagcctgca tgctgtgggg gtgagctact ggaaggcttc tgagggggct gagtatgatg 900

accagactag ccagagggag aaggaggatg acaaggtgtt tcctgggggc agccatacct 960

atgtgtggca ggtgctgaag gagaatggcc ccatggcctc tgaccccctg tgcctgacct 1020

acagctacct gtctcatgtg gacctggtga aggacctgaa ctctggcctg attggggctc 1080

tgctggtgtg tagggagggc agcctggcta aggaaaagac ccagaccctg cataagttta 1140

tcctgctgtt tgctgtgttt gatgagggca agagctggca ctctgagacc aagaacagcc 1200

tgatgcagga tagggatgct gcctctgcca gggcttggcc taagatgcac actgtgaatg 1260

ggtatgtgaa taggagcctg cctggcctga ttggctgcca caggaagtct gtgtactggc 1320

atgtgattgg gatgggcacc acccctgagg tccatagcat cttcctggag ggccacactt 1380

tcctggtgag gaaccacaga caggcctctc tggagatctc tcccatcacc ttcctgactg 1440

ctcagactct gctgatggac ctgggccagt tcctgctgtt ttgccatatt agcagccacc 1500

agcatgatgg gatggaggcc tatgtgaagg tggatagctg ccctgaggag cctcagctga 1560

ggatgaagaa caatgaggag gctgaagact atgatgatga cctgactgat tctgagatgg 1620

atgtggtgag gtttgatgat gacaatagcc ccagcttcat tcagatcagg tctgtggcca 1680

agaaacaccc caagacctgg gtgcactaca ttgctgctga ggaagaggac tgggactatg 1740

ctcccctggt gctggcccct gatgataggt cttataagag ccagtacctg aacaatgggc 1800

cccagaggat tggcaggaag tacaagaagg tgaggttcat ggcctacact gatgaaacct 1860

tcaaaaccag ggaggccatt cagcatgagt ctggcatcct gggccctctg ctgtatgggg 1920

aggtggggga caccctgctg atcatcttca agaaccaggc cagcaggccc tacaacatct 1980

atcctcatgg catcactgat gtgaggcccc tgtacagcag gaggctgccc aagggggtga 2040

agcacctgaa agacttcccc atcctgcctg gggagatctt taagtataag tggactgtga 2100

ctgtggagga tggccctacc aagtctgacc ccaggtgtct gaccaggtac tattctagct 2160

ttgtgaacat ggagagggac ctggcctctg gcctgattgg gcccctgctg atctgctaca 2220

aggagtctgt ggaccagagg ggcaaccaga tcatgtctga caagaggaat gtgatcctgt 2280

tttctgtgtt tgatgagaat aggagctggt acctgactga gaacatccag aggtttctgc 2340

ccaatcctgc tggggtgcag ctggaggatc ctgagttcca ggccagcaat atcatgcata 2400

gcatcaatgg ctatgtgttt gacagcctgc agctgtctgt gtgcctgcat gaggtggcct 2460

actggtacat cctgagcatt ggggcccaga ctgactttct gtctgtgttc ttttctggct 2520

ataccttcaa gcacaagatg gtgtatgagg ataccctgac cctgttcccc ttctctgggg 2580

agactgtgtt catgagcatg gagaatcctg ggctgtggat cctggggtgc cacaactctg 2640

attttaggaa cagggggatg actgccctgc tgaaggtgtc tagctgtgat aagaacactg 2700

gggactacta tgaggacagc tatgaggaca tttctgctta tctgctgtct aagaataatg 2760

ccattgagcc cagaagcttc agccagaatc cccctgtgct gaagagacat cagagggaga 2820

tcaccagaac taccctgcag tctgatcagg aggagattga ctatgatgac actatctctg 2880

tggagatgaa gaaggaggac tttgacatct atgatgagga tgagaatcag tctcccagga 2940

gctttcagaa gaagaccaga cattacttca ttgctgctgt ggagaggctg tgggactatg 3000

gcatgagctc tagccctcat gtgctgagga acagggccca gtctggctct gtgccccagt 3060

tcaagaaggt ggtgttccag gaattcactg atggcagctt cacccagccc ctgtacaggg 3120

ggggagctgaa tgagcacctg ggcctgctgg ggccttatat cagggctgag gtggaggata 3180

atattatggt gactttcagg aaccaggcca gcaggcccta ctctttctat agcagcctga 3240

tctcttatga ggagatcag aggcaggggg ctgagcctag gaagaacttt gtgaagccca 3300

atgagactaa gacctacttc tggaaggtcc agcaccacat ggcccctacc aaggatgagt 3360

ttgactgcaa ggcctgggcc tatttctctg atgtggatct ggagaaggat gtccattctg 3420

ggctgattgg ccccctgctg gtgtgccaca ctaacactct gaatcctgcc catggcaggc 3480

aggtgactgt ccaggagttt gccctgttct tcactatctt tgatgagacc aagagctggt 3540

actttactga gaacatggag aggaactgca gagctccttg caatattcag atggaggacc 3600

ccaccttcaa ggagaattac aggttccatg ccattaatgg gtacatcatg gacaccctgc 3660

ctggcctggt gatggctcag gaccagagga tcaggtggta cctgctgagc atgggctcta 3720

atgagaatat ccacagcatc cacttctctg ggcatgtgtt cactgtgagg aagaaggagg 3780

agtacaagat ggctctgtat aatctgtacc ctggggtgtt tgaaactgtg gagatgctgc 3840

cctctaaggc tggcatctgg agggtggagt gcctgattgg ggagcacctg catgctggca 3900

tgagcaccct gttcctggtg tacagcaaca agtgccagac ccccctgggc atggcctctg 3960

gccacatcag ggacttccag atcactgcct ctggccagta tggccagtgg gcccccaagc 4020

tggccaggct gcactattct ggcagcatca atgcctggag caccaaggag cccttcagct 4080

ggatcaaggt ggacctgctg gcccccatga tcattcatgg catcaagacc caggggggcca 4140

ggcagaagtt cagctctctg tacatctctc agttcatcat catgtactct ctggatggga 4200

agaagtggca gacctacagg ggcaacagca ctggcaccct gatggtgttc tttgggaatg 4260

tggactcttc tggcatcaag cacaacatct tcaatccccc catcattgct aggtatatta 4320

ggctgcatcc cacccactac agcatcaggt ctaccctgag gatggagctg atgggctgtg 4380

acctgaactc ttgcagcatg cccctgggca tggagtctaa ggccatctct gatgcccaga 4440

ttactgccag cagctacttc accaacatgt ttgccacctg gagcccctct aaggccaggc 4500

tgcatctgca ggggagaggagc aatgcctgga ggcctcaggt gaacaacccc aaggagtggc 4560

tgcaggtgga tttccagaag accatgaagg tgactggggt gaccacccag ggggtcaaga 4620

gcctgctgac cagcatgtat gtgaaggagt tcctgatcag cagcagccag gatggccacc 4680

agtggactct gttctttcag aatgggaagg tgaaggtgtt tcagggcaat caggactctt 4740

tcacccctgt ggtgaacagc ctggaccccc ccctgctgac cagatacctg aggatccacc 4800

cccagtcttg ggtgcatcag attgccctga ggatggaggt gctgggctgt gaggctcagg 4860

atctgtactg agcggccgca ataaaagatc agagctctag agatctgtgt gttggttttt 4920

tgtgtaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac 4980

tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag 5040

cgagcgagcg cgcagctgcc tgcagg 5066

<210> 24

<211> 11976

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of full-length plasmid

<400> 24

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tacgcgtgtc tgtctgcaca tttcgtagag cgagtgttcc 180

gatactctaa tctccctagg caaggttcat attgacttag gttacttatt ctccttttgt 240

tgactaagtc aataatcaga atcagcaggt ttggagtcag cttggcaggg atcagcagcc 300

tgggttggaa ggagggggta taaaagcccc ttcaccagga gaagccgtca cacagatcca 360

caagctcctg ctagcaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg 420

gttatggccc ttgcgtgcct tgaattactg acactgacat ccactttttc tttttctcca 480

caggtttaaa cgccaccatg cagattgagc tgagcacctg cttcttcctg tgtctgctga 540

ggttctgctt ctctgccacc aggaggtatt acctgggggc tgtggagctg agctgggact 600

atatgcagtc tgacctgggg gagctgcctg tggatgctag gttccccccc agggtgccca 660

agagcttccc ctttaacact tctgtggtgt acaagaagac cctgtttgtg gagttcactg 720

accacctgtt caacattgcc aagcccaggc ccccctggat ggggctgctg gggcccacca 780

tccaggctga ggtgtatgac actgtggtga tcaccctgaa gaacatggcc agccaccctg 840

tgagcctgca tgctgtgggg gtgagctact ggaaggcttc tgagggggct gagtatgatg 900

accagactag ccagagggag aaggaggatg acaaggtgtt tcctgggggc agccatacct 960

atgtgtggca ggtgctgaag gagaatggcc ccatggcctc tgaccccctg tgcctgacct 1020

acagctacct gtctcatgtg gacctggtga aggacctgaa ctctggcctg attggggctc 1080

tgctggtgtg tagggagggc agcctggcta aggaaaagac ccagaccctg cataagttta 1140

tcctgctgtt tgctgtgttt gatgagggca agagctggca ctctgagacc aagaacagcc 1200

tgatgcagga tagggatgct gcctctgcca gggcttggcc taagatgcac actgtgaatg 1260

ggtatgtgaa taggagcctg cctggcctga ttggctgcca caggaagtct gtgtactggc 1320

atgtgattgg gatgggcacc acccctgagg tccatagcat cttcctggag ggccacactt 1380

tcctggtgag gaaccacaga caggcctctc tggagatctc tcccatcacc ttcctgactg 1440

ctcagactct gctgatggac ctgggccagt tcctgctgtt ttgccatatt agcagccacc 1500

agcatgatgg gatggaggcc tatgtgaagg tggatagctg ccctgaggag cctcagctga 1560

ggatgaagaa caatgaggag gctgaagact atgatgatga cctgactgat tctgagatgg 1620

atgtggtgag gtttgatgat gacaatagcc ccagcttcat tcagatcagg tctgtggcca 1680

agaaacaccc caagacctgg gtgcactaca ttgctgctga ggaagaggac tgggactatg 1740

ctcccctggt gctggcccct gatgataggt cttataagag ccagtacctg aacaatgggc 1800

cccagaggat tggcaggaag tacaagaagg tgaggttcat ggcctacact gatgaaacct 1860

tcaaaaccag ggaggccatt cagcatgagt ctggcatcct gggccctctg ctgtatgggg 1920

aggtggggga caccctgctg atcatcttca agaaccaggc cagcaggccc tacaacatct 1980

atcctcatgg catcactgat gtgaggcccc tgtacagcag gaggctgccc aagggggtga 2040

agcacctgaa agacttcccc atcctgcctg gggagatctt taagtataag tggactgtga 2100

ctgtggagga tggccctacc aagtctgacc ccaggtgtct gaccaggtac tattctagct 2160

ttgtgaacat ggagagggac ctggcctctg gcctgattgg gcccctgctg atctgctaca 2220

aggagtctgt ggaccagagg ggcaaccaga tcatgtctga caagaggaat gtgatcctgt 2280

tttctgtgtt tgatgagaat aggagctggt acctgactga gaacatccag aggtttctgc 2340

ccaatcctgc tggggtgcag ctggaggatc ctgagttcca ggccagcaat atcatgcata 2400

gcatcaatgg ctatgtgttt gacagcctgc agctgtctgt gtgcctgcat gaggtggcct 2460

actggtacat cctgagcatt ggggcccaga ctgactttct gtctgtgttc ttttctggct 2520

ataccttcaa gcacaagatg gtgtatgagg ataccctgac cctgttcccc ttctctgggg 2580

agactgtgtt catgagcatg gagaatcctg ggctgtggat cctggggtgc cacaactctg 2640

attttaggaa cagggggatg actgccctgc tgaaggtgtc tagctgtgat aagaacactg 2700

gggactacta tgaggacagc tatgaggaca tttctgctta tctgctgtct aagaataatg 2760

ccattgagcc cagaagcttc agccagaatc cccctgtgct gaagagacat cagagggaga 2820

tcaccagaac taccctgcag tctgatcagg aggagattga ctatgatgac actatctctg 2880

tggagatgaa gaaggaggac tttgacatct atgatgagga tgagaatcag tctcccagga 2940

gctttcagaa gaagaccaga cattacttca ttgctgctgt ggagaggctg tgggactatg 3000

gcatgagctc tagccctcat gtgctgagga acagggccca gtctggctct gtgccccagt 3060

tcaagaaggt ggtgttccag gaattcactg atggcagctt cacccagccc ctgtacaggg 3120

ggggagctgaa tgagcacctg ggcctgctgg ggccttatat cagggctgag gtggaggata 3180

atattatggt gactttcagg aaccaggcca gcaggcccta ctctttctat agcagcctga 3240

tctcttatga ggagatcag aggcaggggg ctgagcctag gaagaacttt gtgaagccca 3300

atgagactaa gacctacttc tggaaggtcc agcaccacat ggcccctacc aaggatgagt 3360

ttgactgcaa ggcctgggcc tatttctctg atgtggatct ggagaaggat gtccattctg 3420

ggctgattgg ccccctgctg gtgtgccaca ctaacactct gaatcctgcc catggcaggc 3480

aggtgactgt ccaggagttt gccctgttct tcactatctt tgatgagacc aagagctggt 3540

actttactga gaacatggag aggaactgca gagctccttg caatattcag atggaggacc 3600

ccaccttcaa ggagaattac aggttccatg ccattaatgg gtacatcatg gacaccctgc 3660

ctggcctggt gatggctcag gaccagagga tcaggtggta cctgctgagc atgggctcta 3720

atgagaatat ccacagcatc cacttctctg ggcatgtgtt cactgtgagg aagaaggagg 3780

agtacaagat ggctctgtat aatctgtacc ctggggtgtt tgaaactgtg gagatgctgc 3840

cctctaaggc tggcatctgg agggtggagt gcctgattgg ggagcacctg catgctggca 3900

tgagcaccct gttcctggtg tacagcaaca agtgccagac ccccctgggc atggcctctg 3960

gccacatcag ggacttccag atcactgcct ctggccagta tggccagtgg gcccccaagc 4020

tggccaggct gcactattct ggcagcatca atgcctggag caccaaggag cccttcagct 4080

ggatcaaggt ggacctgctg gcccccatga tcattcatgg catcaagacc caggggggcca 4140

ggcagaagtt cagctctctg tacatctctc agttcatcat catgtactct ctggatggga 4200

agaagtggca gacctacagg ggcaacagca ctggcaccct gatggtgttc tttgggaatg 4260

tggactcttc tggcatcaag cacaacatct tcaatccccc catcattgct aggtatatta 4320

ggctgcatcc cacccactac agcatcaggt ctaccctgag gatggagctg atgggctgtg 4380

acctgaactc ttgcagcatg cccctgggca tggagtctaa ggccatctct gatgcccaga 4440

ttactgccag cagctacttc accaacatgt ttgccacctg gagcccctct aaggccaggc 4500

tgcatctgca ggggagaggagc aatgcctgga ggcctcaggt gaacaacccc aaggagtggc 4560

tgcaggtgga tttccagaag accatgaagg tgactggggt gaccacccag ggggtcaaga 4620

gcctgctgac cagcatgtat gtgaaggagt tcctgatcag cagcagccag gatggccacc 4680

agtggactct gttctttcag aatgggaagg tgaaggtgtt tcagggcaat caggactctt 4740

tcacccctgt ggtgaacagc ctggaccccc ccctgctgac cagatacctg aggatccacc 4800

cccagtcttg ggtgcatcag attgccctga ggatggaggt gctgggctgt gaggctcagg 4860

atctgtactg agcggccgca ataaaagatc agagctctag agatctgtgt gttggttttt 4920

tgtgtaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac 4980

tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag 5040

cgagcgagcg cgcagctgcc tgcaggggca gcttgaagga aatactaagg caaaggtact 5100

gcaagtgctc gcaacattcg cttatgcgga ttattgccgt agtgccgcga cgccgggggc 5160

aagatgcaga gattgccatg gtacaggccg tgcggttgat attgccaaaa cagagctgtg 5220

ggggagagtt gtcgagaaag agtgcggaag atgcaaaggc gtcggctatt caaggatgcc 5280

agcaagcgca gcatatcgcg ctgtgacgat gctaatccca aaccttaccc aacccacctg 5340

gtcacgcact gttaagccgc tgtatgacgc tctggtggtg caatgccaca aagaagagtc 5400

aatcgcagac aacattttga atgcggtcac acgttagcag catgattgcc acggatggca 5460

acatattaac ggcatgatat tgacttattg aataaaattg ggtaaatttg actcaacgat 5520

gggttaattc gctcgttgtg gtagtgagat gaaaagaggc ggcgcttact accgattccg 5580

cctagttggt cacttcgacg tatcgtctgg aactccaacc atcgcaggca gagaggtctg 5640

caaaatgcaa tcccgaaaca gttcgcaggt aatagttaga gcctgcataa cggtttcggg 5700

attttttata tctgcacaac aggtaagagc attgagtcga taatcgtgaa gagtcggcga 5760

gcctggttag ccagtgctct ttccgttgtg ctgaattaag cgaataccgg aagcagaacc 5820

ggatcaccaa atgcgtacag gcgtcatcgc cgcccagcaa cagcacaacc caaactgagc 5880

cgtagccact gtctgtcctg aattcattag taatagttac gctgcggcct tttacacatg 5940

accttcgtga aagcgggtgg caggaggtcg cgctaacaac ctcctgccgt tttgcccgtg 6000

catatcggtc acgaacaaat ctgattacta aacacagtag cctggatttg ttctatcagt 6060

aatcgacctt attcctaatt aaatagagca aatccctta ttgggggtaa gacatgaaga 6120

tgccagaaaa acatgacctg ttggccgcca ttctcgcggc aaaggaacaa ggcatcgggg 6180

caatccttgc gtttgcaatg gcgtaccttc gcggcagata taatggcggt gcgtttacaa 6240

aaacagtaat cgacgcaacg atgtgcgcca ttatcgccta gttcattcgt gaccttctcg 6300

acttcgccgg actaagtagc aatctcgctt atataacgag cgtgtttatc ggctacatcg 6360

gtactgactc gattggttcg cttatcaaac gcttcgctgc taaaaaagcc ggagtagaag 6420

atggtagaaa tcaataatca acgtaaggcg ttcctcgata tgctggcgtg gtcggaggga 6480

actgataacg gacgtcagaa aaccagaaat catggttatg acgtcattgt aggcggagag 6540

ctatttactg attactccga tcaccctcgc aaacttgtca cgctaaaccc aaaactcaaa 6600

tcaacaggcg ccggacgcta ccagcttctt tcccgttggt gggatgccta ccgcaagcag 6660

cttggcctga aagacttctc tccgaaaagt caggacgctg tggcattgca gcagattaag 6720

gagcgtggcg ctttacctat gattgatcgt ggtgatatcc gtcaggcaat cgaccgttgc 6780

agcaatatct gggcttcact gccgggcgct ggttatggtc agttcgagca taaggctgac 6840

agcctgattg caaaattcaa agaagcgggc ggaacggtca gagagattga tgtatgagca 6900

gagtcaccgc gattatctcc gctctggtta tctgcatcat cgtctgcctg tcatgggctg 6960

ttaatcatta ccgtgataac gccattacct acaaagccca gcgcgacaaa aatgccagag 7020

aactgaagct ggcgaacgcg gcaattactg acatgcagat gcgtcagcgt gatgttgctg 7080

cgctcgatgc aaaatacacg aaggagttag ctgatgctaa agctgaaaat gatgctctgc 7140

gtgatgatgt tgccgctggt cgtcgtcggt tgcacatcaa agcagtctgt cagtcagtgc 7200

gtgaagccac caccgcctcc ggcgtggata atgcagcctc cccccgactg gcagacaccg 7260

ctgaacggga ttatttcacc ctcagagaga ggctgatcac tatgcaaaaa caactggaag 7320

gaacccagaa gtatattaat gagcagtgca gatagagttg cccatatcga tgggcaactc 7380

atgcaattat tgtgagcaat acacacgcgc ttccagcgga gtataaatgc ctaaagtaat 7440

aaaaccgagc aatccatta cgaatgtttg ctgggtttct gttttaacaa cattttctgc 7500

gccgccacaa attttggctg catcgacagt tttcttctgc ccaattccag aaacgaagaa 7560

atgatgggtg atggtttcct ttggtgctac tgctgccggt ttgttttgaa cagtaaacgt 7620

ctgttgagca catcctgtaa taagcagggc cagcgcagta gcgagtagca ttttttttcat 7680

ggtgttattc ccgatgcttt ttgaagttcg cagaatcgta tgtgtagaaa attaaacaaa 7740

ccctaaacaa tgagttgaaa tttcatattg ttaatattta ttaatgtatg tcaggtgcga 7800

tgaatcgtca ttgtattccc ggattaacta tgtccacagc cctgacgggg aacttctctg 7860

cgggagtgtc cgggaataat taaaacgatg cacacagggt ttagcgcgta cacgtattgc 7920

attatgccaa cgccccggtg ctgacacgga agaaaccgga cgttatgatt tagcgtggaa 7980

agatttgtgt agtgttctga atgctctcag taaatagtaa tgaattatca aaggtatagt 8040

aatatctttt atgttcatgg atatttgtaa cccatcggaa aactcctgct ttagcaagat 8100

tttccctgta ttgctgaaat gtgatttctc ttgatttcaa cctatcatag gacgtttcta 8160

taagatgcgt gtttcttgag aatttaacat ttacaacctt tttaagtcct tttattaaca 8220

cggtgttatc gttttctaac acgatgtgaa tattatctgt ggctagatag taaatataat 8280

gtgagacgtt gtgacgtttt agttcagaat aaaacaattc acagtctaaa tcttttcgca 8340

cttgatcgaa tatttcttta aaaatggcaa cctgagccat tggtaaaacc ttccatgtga 8400

tacgagggcg cgtagtttgc attatcgttt ttatcgtttc aatctggtct gacctccttg 8460

tgttttgttg atgatttatg tcaaatatta ggaatgtttt cacttaatag tattggttgc 8520

gtaacaaagt gcggtcctgc tggcattctg gagggaaata caaccgacag atgtatgtaa 8580

ggccaacgtg ctcaaatctt catacagaaa gatttgaagt aatattttaa ccgctagatg 8640

aagagcaagc gcatggagcg acaaaatgaa taaagaacaa tctgctgatg atccctccgt 8700

ggatctgatt cgtgtaaaaa atatgcttaa tagcaccatt tctatgagtt accctgatgt 8760

tgtaattgca tgtatagaac ataaggtgtc tctggaagca ttcagagcaa ttgaggcagc 8820

gttggtgaag cacgataata atatgaagga ttattccctg gtggttgact gatcaccata 8880

actgctaatc attcaaacta tttagtctgt gacagagcca acacgcagtc tgtcactgtc 8940

aggaaagtgg taaaactgca actcaattac tgcaatgccc tcgtaattaa gtgaatttac 9000

aatatcgtcc tgttcggagg gaagaacgcg ggatgttcat tcttcatcac ttttaattga 9060

tgtatatgct ctcttttctg acgttagtct ccgacggcag gcttcaatga cccaggctga 9120

gaaattcccg gacccttttt gctcaagagc gatgttaatt tgttcaatca tttggttagg 9180

aaagcggatg ttgcgggttg ttgttctgcg ggttctgttc ttcgttgaca tgaggttgcc 9240

ccgtattcag tgtcgctgat ttgtattgtc tgaagttgtt tttacgttaa gttgatgcag 9300

atcaattaat acgatacctg cgtcataatt gattatttga cgtggtttga tggcctccac 9360

gcacgttgtg atatgtagat gataatcatt atcactttac gggtcctttc cggtgatccg 9420

acaggttacg gggcggcgac ctgcctgatg cggtattttc tccttacgca tctgtgcggt 9480

atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg 9540

cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg 9600

ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 9660

taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 9720

aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 9780

ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 9840

tcaactctat ctcggggctat tcttttgatt tagacctgca ggcatgcaag cttggcactg 9900

gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 9960

gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 10020

tcccaacagt tgcgcagcct gaatggcgaa tgcgatttat tcaacaaagc cgccgtcccg 10080

tcaagtcagc gtaatgctct gccagtgtta caaccaatta accaattctg attagaaaaa 10140

ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt 10200

ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc 10260

aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt 10320

cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg 10380

tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg 10440

ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc 10500

gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg 10560

gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa 10620

tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt 10680

acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac 10740

catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg 10800

cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg 10860

agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcttcgagca 10920

agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga 10980

cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg 11040

agacacaacg tggctttgtt gaataaatcg aacttttgct gagttgaagg atcagatcac 11100

gcatcttccc gacaacgcag accgttccgt ggcaaagcaa aagttcaaaa tcaccaactg 11160

gtccacctac aacaaagctc tcatcaaccg tggctccctc actttctggc tggatgatgg 11220

ggcgattcag gcctggtatg agtcagcaac accttcttca cgaggcagac ctctcgacgg 11280

agttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 11340

ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 11400

tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 11460

taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 11520

caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 11580

agtcgtgtct taccggggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 11640

gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 11700

gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 11760

ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 11820

acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 11880

tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 11940

ggttcctggc cttttgctgg ccttttgctc acatgt 11976

<210> 25

<211> 1457

<212> protein

<213> Artificial sequence

<220>

<223> Option FVIII-BDD

<400> 25

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser

20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg

35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val

50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile

65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln

85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser

100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser

115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp

130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu

145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser

165 170 175

Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile

180 185 190

Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr

195 200 205

Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly

210 215 220

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp

225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr

245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val

260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile

275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser

290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met

305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His

325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro

340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp

355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser

370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr

385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro

405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn

420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met

435 440 445

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu

450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu

465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro

485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe

515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp

530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg

545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu

565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val

580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu

595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp

610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val

625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp

645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe

660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr

675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro

690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly

705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys

740 745 750

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu

755 760 765

Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln

770 775 780

Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu

785 790 795 800

Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe

805 810 815

Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp

820 825 830

Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln

835 840 845

Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr

850 855 860

Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His

865 870 875 880

Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile

885 890 895

Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser

900 905 910

Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg

915 920 925

Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val

930 935 940

Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp

945 950 955 960

Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu

965 970 975

Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His

980 985 990

Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe

995 1000 1005

Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn

1010 1015 1020

Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys

1025 1030 1035

Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr

1040 1045 1050

Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr

1055 1060 1065

Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe

1070 1075 1080

Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met

1085 1090 1095

Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met

1100 1105 1110

Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly

1115 1120 1125

Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser

1130 1135 1140

Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg

1145 1150 1155

Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro

1160 1165 1170

Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser

1175 1180 1185

Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro

1190 1195 1200

Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe

1205 1210 1215

Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp

1220 1225 1230

Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu

1235 1240 1245

Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn

1250 1255 1260

Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro

1265 1270 1275

Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly

1280 1285 1290

Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys

1295 1300 1305

Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn

1310 1315 1320

Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln

1325 1330 1335

Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu

1340 1345 1350

Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val

1355 1360 1365

Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys

1370 1375 1380

Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu

1385 1390 1395

Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp

1400 1405 1410

Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr

1415 1420 1425

Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala

1430 1435 1440

Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr

1445 1450 1455

<210> 26

<211> 2351

<212> protein

<213> Artificial sequence

<220>

<223> Wild-type FVIII with BDD

<400> 26

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser

20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg

35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val

50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile

65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln

85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser

100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser

115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp

130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu

145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser

165 170 175

Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile

180 185 190

Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr

195 200 205

Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly

210 215 220

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp

225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr

245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val

260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile

275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser

290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met

305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His

325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro

340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp

355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser

370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr

385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro

405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn

420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met

435 440 445

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu

450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu

465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro

485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe

515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp

530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg

545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu

565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val

580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu

595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp

610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val

625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp

645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe

660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr

675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro

690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly

705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys

740 745 750

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro

755 760 765

Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp

770 775 780

Ile Glu Lys Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys

785 790 795 800

Ile Gln Asn Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser

805 810 815

Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr

820 825 830

Glu Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn

835 840 845

Ser Leu Ser Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly

850 855 860

Asp Met Val Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu

865 870 875 880

Lys Leu Gly Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys

885 890 895

Val Ser Ser Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn

900 905 910

Leu Ala Ala Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met

915 920 925

Pro Val His Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys

930 935 940

Ser Ser Pro Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu

945 950 955 960

Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu

965 970 975

Ser Ser Trp Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe

980 985 990

Lys Gly Lys Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala

995 1000 1005

Leu Phe Lys Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser

1010 1015 1020

Asn Asn Ser Ala Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser

1025 1030 1035

Leu Leu Ile Glu Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu

1040 1045 1050

Ser Asp Thr Glu Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg

1055 1060 1065

Met Leu Met Asp Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met

1070 1075 1080

Ser Asn Lys Thr Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln

1085 1090 1095

Lys Lys Glu Gly Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met

1100 1105 1110

Ser Phe Phe Lys Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile

1115 1120 1125

Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro

1130 1135 1140

Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu

1145 1150 1155

Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys

1160 1165 1170

Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro

1175 1180 1185

Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu

1190 1195 1200

Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu

1205 1210 1215

Lys Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile

1220 1225 1230

His Thr Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu

1235 1240 1245

Leu Ser Thr Arg Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr

1250 1255 1260

Ala Pro Val Leu Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn

1265 1270 1275

Arg Thr Lys Lys His Thr Ala His Phe Ser Lys Lys Gly Glu Glu

1280 1285 1290

Glu Asn Leu Glu Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu

1295 1300 1305

Lys Tyr Ala Cys Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln

1310 1315 1320

Asn Phe Val Thr Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg

1325 1330 1335

Leu Pro Leu Glu Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp

1340 1345 1350

Asp Thr Ser Thr Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro

1355 1360 1365

Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala

1370 1375 1380

Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser

1385 1390 1395

Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser

1400 1405 1410

Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe

1415 1420 1425

Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys

1430 1435 1440

Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys

1445 1450 1455

Lys Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly

1460 1465 1470

Asp Gln Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser

1475 1480 1485

Val Thr Tyr Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp

1490 1495 1500

Leu Pro Lys Thr Ser Gly Lys Val Glu Leu Leu Pro Lys Val His

1505 1510 1515

Ile Tyr Gln Lys Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser

1520 1525 1530

Pro Gly His Leu Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr

1535 1540 1545

Glu Gly Ala Ile Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val

1550 1555 1560

Pro Phe Leu Arg Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser

1565 1570 1575

Lys Leu Leu Asp Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln

1580 1585 1590

Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys

1595 1600 1605

Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys

1610 1615 1620

Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys

1625 1630 1635

Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg

1640 1645 1650

Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu

1655 1660 1665

Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr

1670 1675 1680

Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile

1685 1690 1695

Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys

1700 1705 1710

Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr

1715 1720 1725

Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser

1730 1735 1740

Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr

1745 1750 1755

Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu

1760 1765 1770

His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp

1775 1780 1785

Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser

1790 1795 1800

Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly

1805 1810 1815

Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr

1820 1825 1830

Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu

1835 1840 1845

Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu

1850 1855 1860

Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His

1865 1870 1875

Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln

1880 1885 1890

Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp

1895 1900 1905

Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn

1910 1915 1920

Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His

1925 1930 1935

Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met

1940 1945 1950

Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser

1955 1960 1965

Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr

1970 1975 1980

Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr

1985 1990 1995

Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly

2000 2005 2010

Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly

2015 2020 2025

Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro

2030 2035 2040

Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala

2045 2050 2055

Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His

2060 2065 2070

Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser

2075 2080 2085

Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile

2090 2095 2100

Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser

2105 2110 2115

Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr

2120 2125 2130

Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn

2135 2140 2145

Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile

2150 2155 2160

Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg

2165 2170 2175

Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys

2180 2185 2190

Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln

2195 2200 2205

Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser

2210 2215 2220

Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp

2225 2230 2235

Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe

2240 2245 2250

Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys

2255 2260 2265

Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser

2270 2275 2280

Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys

2285 2290 2295

Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val

2300 2305 2310

Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His

2315 2320 2325

Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu

2330 2335 2340

Gly Cys Glu Ala Gln Asp Leu Tyr

2345 2350

<210> 27

<211> 736

<212> protein

<213> Artificial sequence

<220>

<223> AAV-LK03 VP1 capsid

<400> 27

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Gln Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly

145 150 155 160

Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Lys Leu Ser Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr

435 440 445

Gln Gly Thr Thr Ser Gly Thr Thr Asn Gln Ser Arg Leu Leu Phe Ser

450 455 460

Gln Ala Gly Pro Gln Ser Met Ser Leu Gln Ala Arg Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser Lys Thr Ala Asn Asp Asn

485 490 495

Asn Asn Ser Asn Phe Pro Trp Thr Ala Ala Ser Lys Tyr His Leu Asn

500 505 510

Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly

530 535 540

Lys Glu Gly Thr Thr Ala Ser Asn Ala Glu Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln

565 570 575

Tyr Gly Thr Val Ala Asn Asn Leu Gln Ser Ser Asn Thr Ala Pro Thr

580 585 590

Thr Arg Thr Val Asn Asp Gln Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Met Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 28

<211> 738

<212> protein

<213> Artificial sequence

<220>

<223> AAV-SPK VP1 capsid

<400> 28

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Ser Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro

180 185 190

Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser

210 215 220

Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ser Thr Asn Asp

260 265 270

Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn

275 280 285

Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn

290 295 300

Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn

305 310 315 320

Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala

325 330 335

Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln

340 345 350

Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe

355 360 365

Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn

370 375 380

Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr

385 390 395 400

Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr

405 410 415

Asn Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser

420 425 430

Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu

435 440 445

Ser Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Gln Gln Leu Leu

450 455 460

Phe Ser Gln Ala Gly Pro Asn Asn Met Ser Ala Gln Ala Lys Asn Trp

465 470 475 480

Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Leu Ser

485 490 495

Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His

500 505 510

Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr

515 520 525

His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met

530 535 540

Phe Gly Lys Gln Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Ser Val

545 550 555 560

Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr

565 570 575

Glu Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gln Gln Asn Ala Ala

580 585 590

Pro Ile Val Gly Ala Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val

595 600 605

Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile

610 615 620

Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe

625 630 635 640

Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val

645 650 655

Pro Ala Asp Pro Pro Thr Thr Phe Asn Gln Ala Lys Leu Ala Ser Phe

660 665 670

Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu

675 680 685

Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr

690 695 700

Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Glu

705 710 715 720

Gly Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg

725 730 735

Asn Leu

<210> 29

<211> 14

<212> protein

<213> Artificial sequence

<220>

<223> Option FVIII-BDD

<400> 29

Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg

1 5 10

<---

Claims (66)

1. A method of treating a person with hemophilia A, including the introduction of a recombinant vector based on adeno-associated virus (rAAV), where the vector genome includes a nucleic acid variant encoding factor VIII (FVIII) having a B-domain deletion (hFVIII-BDD), where the variant nucleic acid has 95% or greater identity with SEQ ID NO:7, where the individual has not demonstrated spontaneous bleeding for at least 1 year. 2. A method of treating a person with hemophilia A, including the introduction of a recombinant vector based on adeno-associated virus (rAAV), where the vector genome includes a nucleic acid variant encoding factor VIII (FVIII) having a B-domain deletion (hFVIII-BDD), where the variant nucleic acid contains no more than 2 cytosine-guanine dinucleotides (CpG), where the person has not demonstrated spontaneous bleeding for at least 1 year. 3. A method of treating a person with hemophilia A, including the introduction of a recombinant vector based on adeno-associated virus (rAAV), where the vector genome includes a nucleic acid encoding factor VIII (FVIII) or encoding factor VIII (FVIII) having a B-domain deletion (hFVIII -BDD), where the dose of rAAV vector administered to a person is less than 6 x 10 ¹² vector genomes per kilogram (vg/kg), and where the person does not exhibit spontaneous bleeding for at least 1 year. 4. The method of claim 1 or 2, wherein the dose of the rAAV vector administered to a human is from about 1×10 ⁹ to about 1×10 ¹⁴ vg/kg, inclusive. 5. Method according to any one of paragraphs. 1-3, wherein the dose of the rAAV vector administered to a human is from about 3×10 ¹¹ to about 7×10 ¹² vg/kg, inclusive. 6. Method according to any one of paragraphs. 1-5, wherein the amount of FVIII or hFVIII-BDD expressed in humans, as reflected in coagulation activity, is greater than predicted based on data obtained from studies in non-human primates injected with the rAAV vector. 7. Method according to any one of paragraphs. 1-6, where the amount of FVIII or hFVIII-BDD expressed in humans, as reflected in coagulation activity, is 1-4 times greater than the expression predicted based on the linear regression curve obtained from studies in non-human primates administered rAAV vector. 8. Method according to any one of paragraphs. 1-7, wherein the amount of FVIII or hFVIII-BDD expressed in the human, as reflected by coagulation activity, is approximately 3% or greater 14 or more days after administration of the rAAV vector, approximately 4% or greater 21 or more days after administration rAAV vector, approximately 5% or more at 21 or more days after rAAV vector administration, approximately 6% or more at 21 or more days after rAAV vector administration, approximately 7% or more at 21 or more days after rAAV vector administration, approximately 8 % or more 28 or more days after administration of the rAAV vector, approximately 9% or more 28 or more days after administration of the rAAV vector, approximately 10% or more 35 or more days after administration of the rAAV vector, approximately 11% or more after 35 or more days after administration of the rAAV vector, approximately 12% or more 35 or more days after administration of the rAAV vector. 9. Method according to any one of paragraphs. 1-7, wherein the amount of FVIII or hFVIII-BDD expressed in a person, as reflected by clotting activity, averages approximately 10% or more over a continuous 14-day period. 10. Method according to any one of paragraphs. 1, 2 and 4-9, wherein the rAAV vector is administered to a human at a dose of from about 1 x 10 ⁹ to about 1 x 10 ¹⁴ vg/kg, inclusive, wherein said FVIII or hFVIII-BDD is produced in the human at levels averaging from about 12% to about 100% activity, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 continuous days, weeks, or months after administration rAAV vector. 11. Method according to any one of paragraphs. 1-10, wherein FVIII or hFVIII-BDD is produced in a person in a steady state where the activity does not change by more than 5-50% over a period of 4, 6, 8 or 12 weeks or months. 12. Method according to any one of paragraphs. 1-10, wherein FVIII or hFVIII-BDD is produced in a person in a steady state where the activity does not change by more than 25-100% over a period of 4, 6, 8 or 12 weeks or months. 13. Method according to any one of paragraphs. 1-12, wherein anti-AAV antibodies are not detected in the individual prior to administration of the rAAV vector, or wherein the individual is seronegative for AAV. 14. Method according to any one of paragraphs. 1-12, wherein anti-AAV antibodies in humans are present at a titer of 1:5 or less prior to administration of the rAAV vector. 15. Method according to any one of paragraphs. 1-14, wherein said individual does not develop a cell-mediated immune response against the rAAV vector for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 continuous weeks or months after administration of the rAAV vector. 16. Method according to any one of paragraphs. 1-15, where the individual does not develop a humoral immune response against the rAAV vector sufficient to reduce or block the therapeutic effect of FVIII or hFVIII-BDD. 17. Method according to any one of paragraphs. 1-16, wherein the person does not produce detectable antibodies to the rAAV vector for at least about 1, 2, 3, 4, 5, or 6 months after administration of the rAAV vector. 18. Method according to any one of paragraphs. 1-17, further comprising administering an immunosuppressant agent. 19. Method according to any one of paragraphs. 1-17, further comprising administering an immunosuppressive agent following administration of the rAAV vector. 20. Method according to any one of paragraphs. 1-19, wherein the nucleic acid or nucleic acid variant has 95-100% sequence identity to SEQ ID NO:7. 21. Method according to any one of paragraphs. 1-20, wherein the nucleic acid or nucleic acid variant contains 20 or less, 15 or less, or 10 or less cytosine-guanine dinucleotides (CpGs). 22. Method according to any one of paragraphs. 1-20, where the nucleic acid or nucleic acid variant contains no more than 5 cytosine-guanine dinucleotides (CpG). 23. Method according to any one of paragraphs. 1-20, wherein the nucleic acid or nucleic acid variant contains 4, 3, 2, 1 or 0 cytosine-guanine dinucleotides (CpG). 24. Method according to any one of paragraphs. 1-20, wherein the nucleic acid or nucleic acid variant contains 1 cytosine-guanine dinucleotide (CpG). 25. Method according to any one of paragraphs. 1-24, wherein the nucleic acid or nucleic acid variant encodes SEQ ID NO:25 containing a deletion of one or more amino acids in the sequence SFSQNPPVLKRHQR (SEQ ID NO:29) or a deletion of the entire sequence SFSQNPPVLKRHQR. 26. Method according to any one of paragraphs. 1-24, wherein the nucleic acid or nucleic acid variant is encoded by SEQ ID NO:25. 27. Method according to any one of paragraphs. 1-24, wherein hFVIII-BDD is identical to hFVIII-BDD encoded by SEQ ID NO:19. 28. Method according to any one of paragraphs. 1-27, wherein said rAAV vector includes an AAV serotype or an AAV pseudotype, wherein said AAV pseudotype includes an AAV capsid serotype other than an ITR serotype. 29. Method according to any one of paragraphs. 1-28, wherein the vector genome further includes an intron, an expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs), and/or a filler polynucleotide sequence. 30. The method of claim 29, wherein the intron is present in the nucleic acid variant or flanks the nucleic acid variant. 31. The method of claim 29, wherein the expression control element is operably linked to the nucleic acid variant. 32. The method of claim 29, wherein the AAV ITR repeat(s) flank the 5' or 3' end of the nucleic acid variant. 33. The method of claim 29, wherein the filler polynucleotide sequence flanks the 5' or 3' end of the nucleic acid variant. 34. The method of claim 29, wherein the intron, expression control element, one or more adeno-associated virus inverted terminal repeats (ITRs), and/or filler polynucleotide sequence have been modified to reduce the content of cytosine-guanine dinucleotides (CpGs). 35. The method of claim 29, wherein the expression control element includes a constitutive or regulated control element, or a tissue-specific expression control element, or a promoter. 36. The method of claim 29, wherein the expression control element includes an element that ensures expression in the liver. 37. The method of claim 29, wherein the expression control element comprises a TTR promoter or a mutant TTR promoter. 38. The method of claim 37, wherein the mutant TTR promoter includes SEQ ID NO:22. 39. The method of claim 37, where the ITR includes one or more ITRs of any of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes or their combination. 40. Method according to any one of paragraphs. 1-39, where the vector genome includes an ITR, a promoter, a poly-A signal and/or an intronic sequence presented in SEQ ID NO:23. 41. Method according to any one of paragraphs. 1-40, wherein the rAAV vector includes a modified or variant AAV VP1, VP2 and/or VP3 capsid sequence or a wild-type AAV VP1, VP2 and/or VP3 capsid sequence. 42. Method according to any one of paragraphs. 1-41, wherein the rAAV vector includes a modified or variant AAV VP1, VP2 and/or VP3 capsid sequence having 90% or greater identity with the VP1, VP2 and/or VP3 sequences of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 , AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8. 43. Method according to any one of paragraphs. 1-41, wherein the rAAV vector includes a VP1, VP2, or VP3 capsid sequence selected from any of the AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV -2i8. 44. Method according to any one of paragraphs. 1-40, wherein the rAAV vector includes a capsid having 90% or greater sequence identity with the LK03 capsid (SEQ ID NO:27). 45. Method according to any one of paragraphs. 1-40, wherein the rAAV vector includes a capsid having 90% or greater sequence identity with the SPK capsid (SEQ ID NO:28). 46. Method according to any one of paragraphs. 1-40, where the rAAV vector includes the LK03 capsid (SEQ ID NO:27). 47. Method according to any one of paragraphs. 1-40, where the rAAV vector includes the SPK capsid (SEQ ID NO:28). 48. Method according to any one of paragraphs. 1-40, wherein the rAAV vector includes the nucleic acid variant of SEQ ID NO:7 and the LK03 capsid sequence (SEQ ID NO:27). 49. Method according to any one of paragraphs. 1-40, wherein the rAAV vector includes the nucleic acid variant SEQ ID NO:7 and the SPK capsid (SEQ ID NO:28). 50. Method according to any one of paragraphs. 1-49, wherein the rAAV vector includes a nucleic acid variant and one or more of a TTR promoter mutant (TTRmut), a synthetic intron, a poly-A, and an ITR in SEQ ID NO:23. 51. Method according to any one of paragraphs. 1-49, wherein the rAAV vector includes a nucleic acid variant and one or more of a TTR promoter mutant (TTRmut), a synthetic intron, a poly-A and an ITR in SEQ ID NO:23 and an LK03 capsid sequence (SEQ ID NO:27) or a capsid SPK (SEQ ID NO:28). 52. Method according to any one of paragraphs. 1-51, wherein the rAAV vector includes a pharmaceutical composition. 53. The method of claim 52, wherein the pharmaceutical composition includes a biocompatible carrier or excipient. 54. Method according to any one of paragraphs. 1-53, further comprising administering an empty AAV capsid, wherein, optionally, the empty AAV capsid is administered with the rAAV vector. 55. Method according to any one of paragraphs. 1-53, further comprising introducing an empty capsid of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and/or AAV-Rh74. 56. Method according to any one of paragraphs. 1-53, further comprising introducing an empty AAV capsid of the same serotype as the AAV vector being administered. 57. Method according to any one of paragraphs. 1-53, further comprising introducing an empty capsid having an LK03 capsid (SEQ ID NO:27) or an SPK capsid (SEQ ID NO:28). 58. Method according to any one of paragraphs. 53-57, wherein the ratio of said empty capsids to said rAAV vector is from about 2:1 to about 50:1. 59. Method according to any one of paragraphs. 1-58, wherein FVIII or hFVIII-BDD, encoded by the nucleic acid variant, is expressed in a cell, tissue or organ of the specified mammal. 60. The method of claim 59, wherein the cell comprises a secretory cell. 61. The method of claim 59, wherein the tissue or organ of said mammal comprises a liver. 62. Method according to any one of paragraphs. 1-61, wherein the rAAV vector is delivered to the specified person intravenously, intraarterially, intramuscularly, subcutaneously, intracavitarily, or by intubation or through a catheter. 63. Method according to any one of paragraphs. 1-62, where FVIII or hFVIII-BDD is expressed at levels without significantly increasing the risk of thrombosis. 64. Method according to any one of paragraphs. 1-63, wherein FVIII or hFVIII-BDD activity 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. 65. Method according to any one of paragraphs. 1-64, where the person does not require prophylaxis with FVIII protein 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. 66. Method according to any one of paragraphs. 1-65, further comprising testing in a person or monitoring a person for the presence or amount of antibodies to AAV, immune response against AAV, antibodies to FVIII or hFVIII-BDD, immune response against FVIII or hFVIII-BDD, amount of FVIII or hFVIII-BDD, activity FVIII or hFVIII-BDD, amounts or levels of one or more liver enzymes, or the frequency and/or severity or duration of bleeding episodes.

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