Cpg reduced factor viii variants, compositions and methods and uses for treatment of hemostasis disorders

Abstract

To provide nucleic acid variants encoding FVIII protein in the fields of recombinant coagulation factor production and the treatment of medical disorders associated with aberrant hemostasis.SOLUTION: CpG reduced nucleic acid variants encoding FVIII protein and methods of use thereof are disclosed. In particular embodiments, CpG reduced nucleic acid variants encoding FVIII are expressed more efficiently by cells, are secreted at increased levels by cells over wild-type Factor VIII proteins, exhibit enhanced expression and / or activity over wild-type Factor VIII proteins or are packaged more efficiently into viral vectors.SELECTED DRAWING: Figure 8

Description

[Technical Field]

[0001]

[0002] The present invention relates to the field of recombinant coagulation factor production and the treatment of medical disorders associated with hemostatic abnormalities. More particularly, the present invention provides nucleic acid variants (sequences) encoding factor VIII (FVIII) proteins, which optionally provide increased transcription and / or expression and / or activity compared to wild-type FVIII protein. [Background technology]

[0002]

[0003] Several publications and patent documents are cited throughout this specification to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as if set forth in its entirety.

[0003]

[0004] Hemophilia is an X-linked bleeding disorder that affects 1 in 5,000 males worldwide. Treatments aimed at increasing clotting factor levels to just above 1% of normal are associated with substantial improvement in severe disease phenotypes. Recent clinical trials of AAV-mediated gene transfer for hemophilia B (HB) demonstrated sustained, long-term expression of therapeutic levels of factor IX (FIX), but established that AAV vectors can be dose-limiting due to anti-AAV immune responses to the AAV capsid. While these data pertain to hemophilia B, 80% of all hemophilia cases are hemophilia A (HA), caused by FVIII deficiency.

[0004]

[0005] The current treatment for this disease is protein replacement therapy, which requires frequent infusions of factor VIII protein. It is urgently necessary to achieve sustained therapeutic expression levels of factor VIII so that patients no longer require such frequent protein treatments. Indeed, continuous expression of factor VIII is expected to prevent bleeding episodes and may ensure the establishment of immune tolerance to the protein.

[0005]

[0006] In summary, gene therapy for HA poses three distinct challenges: (1) the unique properties of human FVIII (hFVIII) make its expression more difficult than other proteins of similar size; (2) the relatively large size and sequence-specific effects of FVIII cDNA correlate with rearrangements, which impede AAV production; and (3) the high rate of anti-FVIII antibody (inhibitor) formation in response to protein therapy, which occurs in 25–30% of patients with severe (<1% FVIII) HA. Summary of the Invention

[0006]

[0007] The present invention provides cytosine-guanine dinucleotide (CpG)-reduced nucleic acid variants encoding factor VIII (FVIII) proteins. Such CpG-reduced nucleic acid variants differ from wild-type nucleic acids encoding FVIII, and may, for example, encode human FVIII proteins that optionally lack all or part of the FVIII B domain. Such CpG-reduced nucleic acid variants include variants that, when transfected into cells, exhibit increased expression (e.g., 1- to 5-fold increased expression) and result in increased secretion of FVIII protein, thereby resulting in increased activity, compared to a codon-optimized FVIII nucleic acid, such as FVIII-CO3 (SEQ ID NO: 21).

[0007]

[0008] In certain embodiments, CpG-reduced nucleic acid variants encoding FVIII with or without a full or partial deletion of the FVIII B domain can not only provide increased expression of FVIII and increased production of FVIII protein in mammals, but also achieve increased efficacy associated with gene transfer due to increased circulating levels of FVIII protein, achieving hemostasis for beneficial therapeutic outcomes.

[0008]

[0009] In certain embodiments, the nucleic acid variant encoding FVIII has a reduced CpG content compared to the wild-type nucleic acid encoding FVIII. In certain embodiments, the nucleic acid variant has at least 10 fewer CpGs than the wild-type nucleic acid encoding FVIII (SEQ ID NO: 19). In certain embodiments, the nucleic acid variant has four or fewer CpGs; three or fewer CpGs; two or fewer CpGs; or one or fewer CpGs. In certain embodiments, the nucleic acid variant has a maximum of four CpGs; three CpGs; two CpGs; or one CpG. In certain embodiments, the nucleic acid variant has no CpGs.

[0009]

[0010] In certain embodiments, a nucleic acid variant encoding FVIII has a reduced CpG content compared to a wild-type nucleic acid encoding FVIII, and such a CpG-reduced nucleic acid variant has 90% or more sequence identity to any of SEQ ID NOs: 1-18. In certain embodiments, a CpG-reduced nucleic acid variant has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more sequence identity to any of SEQ ID NOs: 1-18. In certain embodiments, a CpG-reduced nucleic acid variant has 90-95% sequence identity to any of SEQ ID NOs: 1-18. In certain embodiments, a CpG-reduced nucleic acid variant has 95%-100% sequence identity to any of SEQ ID NOs: 1-18. In certain embodiments, a CpG-reduced nucleic acid variant encoding FVIII is set forth in any of SEQ ID NOs: 1-18.

[0010]

[0011] In certain embodiments, the CpG-reduced nucleic acid variant is different from FVIII variant V3 (SEQ ID NO: 20) and / or FVIII variant CO3 (SEQ ID NO: 21).

[0011]

[0012] In certain embodiments, the CpG-reduced nucleic acid variants encoding FVIII proteins achieve higher expression and / or exhibit superior biological activity (e.g., as determined by plasma levels or clotting assays, or reduced bleeding in FVIII assays or FVIII deficiency models) compared to wild-type FVIII or wild-type FVIII comprising a B-domain deletion.

[0012]

[0013] In certain embodiments, the CpG-reduced nucleic acid variant encoding the FVIII protein is at least 75% identical to a wild-type human FVIII nucleic acid or a wild-type human FVIII nucleic acid comprising a B-domain deletion. In certain embodiments, the CpG-reduced nucleic acid variant encoding the FVIII protein is 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 a wild-type human FVIII nucleic acid or a wild-type human FVIII nucleic acid comprising a B-domain deletion.

[0013]

[0014] In certain embodiments, the CpG-reduced nucleic acid variant encoding the FVIII protein is mammalian, such as human. Such mammalian CpG-reduced nucleic acid variant encoding the FVIII protein includes human forms that can be based on human wild-type FVIII or human wild-type FVIII that includes a deletion of the B domain.

[0014]

[0015] The present invention also provides vectors and expression vectors comprising CpG-reduced nucleic acid variants encoding the FVIII proteins described herein. In certain embodiments, the vector or expression vector comprises an adeno-associated viral (AAV) vector, a retroviral vector, an adenoviral vector, a plasmid, or a lentiviral vector. In certain embodiments, the AAV vector comprises an AAV serotype or AAV pseudotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8 AAV. In certain embodiments, the expression vector comprises any of SEQ ID NOs: 1-18, or SEQ ID NO: 23 or 24.

[0015]

[0016] In certain embodiments, the expression control element comprises a constitutive or regulatable control element, or a tissue-specific expression control element or promoter. In certain embodiments, the expression control element comprises an element that confers expression in the liver. In certain embodiments, the expression control element comprises a TTR promoter or a mutant TTR promoter, such as SEQ ID NO: 22. In further particular aspects, the expression control element comprises a promoter set forth in PCT Publication WO 2016 / 168728 (U.S. Patent Application Nos. 62 / 148,696; 62 / 202,133; and 62 / 212,634), which are incorporated herein by reference in their entireties.

[0016]

[0017] The present invention further provides a viral vector comprising a CpG-reduced nucleic acid variant encoding a FVIII protein, or a vector or expression vector comprising a CpG-reduced nucleic acid variant encoding a FVIII protein. In certain embodiments, the viral vector comprises an AAV vector, a retroviral vector, an adenoviral vector, a plasmid, or a lentiviral vector.

[0017]

[0018] In certain embodiments, the AAV vector comprises an AAV pseudotype comprising an AAV capsid serotype that is distinct from the AAV serotype or ITR serotype. In additional specific aspects, the AAV vector comprises VP1, VP2, and / or VP3 capsid sequences that have 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.) to any of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8 AAV serotypes.

[0018]

[0019] Expression vectors may comprise additional components or elements. In certain embodiments, an expression vector, such as an AAV vector, further comprises an intron, an expression control element, one or more AAV inverted terminal repeats (ITRs) (e.g., any of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8 AAV serotypes, or a combination thereof), a filler polynucleotide sequence, and / or a polyA signal. In certain embodiments, the intron is within or adjacent to the CpG-reduced nucleic acid variant encoding FVIII, and / or the expression control element is operably linked to the CpG-reduced nucleic acid variant encoding FVIII, and / or the AAV ITR(s) are adjacent to the 5' or 3' end of the CpG-reduced nucleic acid variant encoding FVIII, and / or the filler polynucleotide sequence is adjacent to the 5' or 3' end of the CpG-reduced nucleic acid variant encoding FVIII.

[0019]

[0020] In certain embodiments, the expression control elements include constitutive or regulatable control elements, or tissue-specific expression control elements or promoters. In certain embodiments, the expression control elements include elements that confer expression in the liver (e.g., the TTR promoter or a mutant TTR promoter).

[0020]

[0021] The present invention additionally provides host cells comprising CpG-reduced nucleic acid variants encoding the FVIII proteins provided herein. In certain embodiments, the host cells comprise CpG-reduced nucleic acid variants encoding the FVIII proteins or expression vectors comprising CpG-reduced nucleic acid variants encoding the FVIII proteins. In certain embodiments, such host cells produce the FVIII proteins encoded by the nucleic acid variants, and the produced FVIII proteins are recovered. Such FVIII proteins produced by the cells, and optionally isolated and / or purified, can be administered to a subject.

[0021]

[0022] The present invention still additionally provides compositions comprising the CpG-reduced nucleic acid variants, vectors, and expression vectors encoding FVIII as described herein. In certain embodiments, the pharmaceutical composition comprises the vector, expression vector, or virus or AAV vector in a biologically compatible carrier or excipient. Such pharmaceutical compositions optionally comprise empty capsid AAV (e.g., lacking a vector genome comprising a nucleic acid variant encoding FVIII). In further specific embodiments, the CpG-reduced nucleic acid variants, vectors, expression vectors, or virus or AAV vectors encoding FVIII proteins are encapsulated in liposomes or mixed with phospholipids or micelles.

[0022]

[0023] The present invention still further provides a method for delivering or transferring a CpG-reduced nucleic acid variant encoding a FVIII protein into a mammal or mammalian cell. In one aspect, the method comprises administering to or contacting with the mammal or mammalian cell a CpG-reduced nucleic acid variant encoding FVIII, a vector comprising a CpG-reduced nucleic acid variant encoding a FVIII protein, an expression vector comprising a CpG-reduced nucleic acid variant encoding a FVIII protein, or a virus or AAV vector comprising a CpG-reduced nucleic acid variant encoding a FVIII protein. Such a method involves introducing a CpG-reduced nucleic acid variant encoding a FVIII protein into cultured mammalian cells or into a subject (e.g., a patient).

[0023]

[0024] The methods of the invention also include treating a mammalian subject (e.g., a patient), such as a human in need of FVIII (the human producing an insufficient amount of FVIII protein, or a defective or abnormal FVIII protein). In one aspect, a method of treating a mammal in need of FVIII includes providing a CpG-reduced nucleic acid variant encoding FVIII, or a vector comprising the CpG-reduced nucleic acid variant encoding FVIII; or an expression vector comprising the CpG-reduced nucleic acid variant encoding FVIII, or a virus or AAV vector comprising the CpG-reduced nucleic acid variant encoding FVIII; and administering to the mammalian subject an amount of the CpG-reduced nucleic acid variant encoding FVIII, or the vector comprising the CpG-reduced nucleic acid variant encoding FVIII, or the expression vector comprising the CpG-reduced nucleic acid variant encoding FVIII, or the virus or AAV vector comprising the CpG-reduced nucleic acid variant encoding FVIII, such that the FVIII encoded by the nucleic acid variant is expressed in the mammalian subject.

[0024]

[0025] In another embodiment, a method for treating a hemostasis-related disorder in a patient in need thereof (e.g., the patient produces insufficient amounts of FVIII protein, or defective or abnormal FVIII protein) comprises administering to the patient a therapeutically effective amount of a CpG-reduced nucleic acid variant encoding FVIII, or a vector comprising a CpG-reduced nucleic acid variant encoding FVIII, or an expression vector comprising a CpG-reduced nucleic acid variant encoding FVIII, or a virus or AAV vector comprising a CpG-reduced nucleic acid variant encoding FVIII, in a biologically acceptable carrier.

[0025]

[0026] In certain embodiments of the methods of the present invention, FVIII is expressed at a level and / or in a cell, tissue, or organ of a mammal that has a beneficial or therapeutic effect in the mammal. Such embodiments include the introduction of a CpG-reduced nucleic acid variant encoding FVIII into a tissue or organ, such as the liver. Such embodiments also include the introduction of a CpG-reduced nucleic acid variant encoding FVIII into a secretory cell. Such embodiments further include the introduction of a CpG-reduced nucleic acid variant encoding FVIII into an endocrine cell or an endothelial cell. Such embodiments additionally include the introduction of a CpG-reduced nucleic acid variant encoding FVIII into a hepatocyte, a sinusoidal endothelial cell, a megakaryocyte, a platelet, or a hematopoietic stem cell.

[0026]

[0027] Subjects (e.g., patients) and mammals (e.g., humans) who are candidates for administration (e.g., delivery) of a CpG-reduced nucleic acid variant encoding FVIII, or a vector comprising a CpG-reduced nucleic acid variant encoding FVIII, or an expression vector comprising a CpG-reduced nucleic acid variant encoding FVIII, or a virus or AAV vector comprising a CpG-reduced nucleic acid variant encoding FVIII, include subjects and mammals who have or are at risk of having disorders such as hemophilia A, von Willebrand's disease, and trauma-related bleeding, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC), or excessive anticoagulation treatment disorders.

[0027]

[0028] Subjects (e.g., patients) and mammals (e.g., humans) who are candidates for administration (e.g., delivery) of a CpG-reduced nucleic acid variant encoding FVIII, or a vector comprising a CpG-reduced nucleic acid variant encoding FVIII, or an expression vector comprising a CpG-reduced nucleic acid variant encoding FVIII, or a virus or AAV vector comprising a CpG-reduced nucleic acid variant encoding FVIII, include subjects and mammals that are AAV antibody seronegative, as well as subjects and mammals that have or are at risk of developing AAV antibodies. Such subjects (e.g., patients) and mammals (e.g., humans) may be seronegative or seropositive for the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV-Rh10, or AAV-Rh74 serotypes.

[0028]

[0029] Thus, the compositions and methods of the invention further comprise administering to the mammal or patient an empty capsid AAV, hi certain embodiments, empty capsids of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-Rh10, and / or AAV-Rh74 serotypes are further administered to the mammal or patient.

[0029]

[0030] Methods of administration (e.g., delivery) according to the present invention include any mode of contact or delivery, ex vivo or in vivo. In certain embodiments, administration (e.g., delivery) is intravenous, intraarterial, intramuscular, subcutaneous, intracavitary, intubated, or transcatheter.

[0030]

[0031] The present invention also provides methods for testing CpG-reduced nucleic acid variants encoding FVIII in small and large animal models tolerant to human FVIII to evaluate dosing and monitor the immunogenicity of the variants. The use of animal models provides a setting that allows the evaluation of humans currently undergoing protein replacement therapy with hFVIII-BDD, who do not have evidence of an anti-hFVIII antibody response, but who are likely to develop an immune response to such variants. [Brief explanation of the drawings]

[0031] [Figure 1] FIG. 1 shows human FVIII (hFVIII) levels 24 hours after hydrodynamic tail vein (HTV) injection of 50 μg of plasmid for 18 different clones (X01 to X18, corresponding to SEQ ID NOs: 1 to 18, respectively) and FVIII-CO3 (SEQ ID NO: 21). [Figure 2] Figures 2A-2C show FVIII levels in hemophilia A / CD4- / - mice after administration of FVIII (A) CO3 (SEQ ID NO: 21), X09 (SEQ ID NO: 9), X12 (SEQ ID NO: 12), and X16 (SEQ ID NO: 16); (B) CO3 (SEQ ID NO: 21), X01 (SEQ ID NO: 1), and X11 (SEQ ID NO: 11); or (C) CO3 (SEQ ID NO: 21), X07 (SEQ ID NO: 7), and X10 (SEQ ID NO: 10) via AAV vector. [Figure 3-1] Figures 3A-3B show the levels of hFVIII antigen in the plasma of NOD / SCID mice over 87 days after intravenous administration of either vehicle (circles), 4 x 10 (squares), 8 x 10 (triangles), or 1.6 x 10 vg / mouse (inverted triangles) of AAV-SPK-8005-hFVIII in ng / ml (B) or % of total antigen (C). Lines represent the mean ± SD of hFVIII in each cohort. Human FVIII plasma levels were assayed by ELISA, and FVIII (ng / ml) was converted to % of normal FVIII levels, assuming 150 ng / ml equals 100% activity. [Figure 3-2] Figure 3C shows plasma D-dimer levels in NOD / SCID mice after intravenous administration of either vehicle, 4x10, 8x10, or 1.6x10 vg / mouse of AAV-SPK-8005-hFVIII, as indicated by the x-axis (left to right). Bars represent the mean ± SD of mice in each cohort. D-dimer levels were assayed by ELISA. [Figure 4] FIG. 1 shows the NHP study design. [Figure 5]Figures 5A-5D show hFVIII antigen levels in NHPs after intravenous administration of AAV-SPK-8005 at either 2x10 (A), 5x10 (B), or 1x10 vg / kg (C). Lines represent individual animals. Plasma levels of human FVIII were assayed by ELISA. These levels represent repeated measurements obtained by serial sampling of the same group of animals during the study (n = 2-3 animals per cohort). Human FVIII levels measured from vehicle-treated animals are shown as open circles in all three graphs. ε = development of inhibitors to FVIII. [Figure 6] 6A-6C show ALT levels in NHPs at 2×10 12 (A), 5×10 12 (B), or 1×10 13 vg / kg (C) of AAV-SPK-8005. [Figure 7] 7A-7C show D-dimer levels in NHPs. Plasma D-dimer antigen concentrations in NHPs after intravenous administration of AAV-SPK-8005 at either 2 x 10 (A), 5 x 10 (B), or 1 x 10 vg / kg (C). The dotted line indicates 500 ng / ml, the upper normal limit for D-dimers in humans. [Figure 8] A summary of data on FVIII levels at three doses of AAV-SPK-8005 is shown. [Figure 9] 9A-9D show levels of hFVIII in cynomolgus monkey plasma after intravenous administration of AAV-SPK-8011 (LK03 capsid)-hFVIII at either 2×10 (A), 6×10 (B), or 2×10 (vg / kg) (C). Lines represent individual animals. Plasma levels of hFVIII were assayed by ELISA and represent repeated measurements obtained by serial bleeding of the same group of animals during the study (n=3 animals per cohort). Human FVIII levels measured in vehicle-treated animals are shown as open circles (n=2). ε=time at which the development of inhibitors to FVIII was detected in each individual animal. [Figure 10]Figure 1 shows a comparison of FVIII levels achieved with AAV-SPK-8011 (LK03 capsid)-hFVIII with reported levels of FVIII delivered by AAV vectors with AAV5 and AAV8 capsids. AAV5: http: / / www.biomarin.com / pdf / BioMarin_R&D_Day_4_20_2016.pdf, slide 16. AAV8: McIntosh J et al., Blood 2013;121(17):3335-44. [Figure 11] FIG. 1 shows the tissue distribution of AAV-SPK (SEQ ID NO: 28) and AAV-LK03 (SEQ ID NO: 27) in non-human primates, primarily in the kidney, spleen, and liver (third bar for each tissue). [Figure 12] FIG. 1 shows FVIII expression in the liver and spleen after systemic administration of AAV-SPK-8005 to mice. [Figure 13] Figure 1 shows the transduction efficiency of AAV-LK03 capsids analyzed in vitro, with the x-axis representing cynomolgus monkeys (left vertical bar) and humans (right vertical bar). [Figure 14] Figures 14A-14B show plasma concentrations of hFIX in rabbits after AAV administration. Rabbits were intravenously injected with the hFIX vectors AAV-SPK or AAV-LK03 at a dose of (A) 1 x 10 vg / kg (low dose, n = 4) or (B) 1 x 10 vg / kg (high dose, n = 3-5). Human FIX levels were compared between groups using a two-tailed Mann-Whitney test. No significant differences were observed. Animals 5 and 15 in the low-dose cohort were excluded from the analysis due to an injection error. Animals 9 and 10 developed neutralizing antibodies against human FIX, so these animals were also excluded from the graphs. [Figure 15] Figures 15A-15B show the time course of antibody formation to human FIX (anti-FIX). Rabbits were intravenously injected with the hFIX vectors AAV-SPK or AAV-LK03 at a dose of (A) 1 x 10 vg / kg (low dose, n = 4) or (B) 1 x 10 vg / kg (high dose, n = 3-5). Data are shown for each individual animal. DETAILED DESCRIPTION OF THE INVENTION

[0032]

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

[0033]

[0049] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to all forms of nucleic acids, 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 (si)RNA, trans-splicing RNA, or antisense RNA). Polynucleotides include naturally occurring, synthetic, and intentionally modified or altered polynucleotides (e.g., variant nucleic acids). Polynucleotides can be single-stranded, double-stranded, or triple-stranded, linear or circular, and can be of any length. When discussing polynucleotides, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0034]

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

[0035]

[0051] A "nucleic acid" or "polynucleotide" variant refers to a modified sequence that is genetically altered compared to the wild-type. A sequence can be genetically altered without changing the encoded protein sequence. Alternatively, a sequence may be genetically altered to encode a variant protein. A nucleic acid or polynucleotide variant can represent a combination sequence that has been codon-altered to encode a protein that still retains at least partial sequence identity with a reference sequence, such as a wild-type protein sequence, and also a combination sequence that has been codon-altered to encode a variant protein. For example, some codons in such a nucleic acid variant are altered so as not to change the amino acids of the protein (FVIII) encoded thereby, and in turn, some codons in the nucleic acid variant are altered so as to change the amino acids of the protein (FVIII) encoded thereby.

[0036]

[0052] The term "variant factor VIII (FVIII)" refers to a modified FVIII that has been genetically altered compared to unmodified wild-type FVIII (e.g., SEQ ID NO: 19) or FVIII-BDD. Such variants can be referred to as "nucleic acid variants encoding factor VIII (FVIII)." A particular example of a variant is a CpG-reduced nucleic acid encoding a FVIII or FVIII-BDD protein. The term "variant" need not appear every time a CpG-reduced nucleic acid encoding FVIII is referenced. Similarly, the term "variant" may be omitted from the term "CpG-reduced nucleic acid" or similar terms, but reference to a "CpG-reduced nucleic acid" is intended to include variants at the genetic level.

[0037]

[0053] FVIII constructs with reduced CpG content may represent an improvement over wild-type FVIII or FVIII-BDD that does not have reduced CpG content, and may actually represent an improvement without modifying the nucleic acid in a way that results in amino acid changes in the encoded FVIII or FVIII-BDD protein. When comparing expression, if the CpG-reduced nucleic acid encodes a FVIII protein that retains the B domain, it is appropriate to compare it to the expression of wild-type FVIII; if the CpG-reduced nucleic acid encodes a FVIII protein that lacks the B domain, it is compared to the expression of wild-type FVIII that also has a deletion of the B domain.

[0038]

[0054] "Variant factor VIII (FVIII)" can also refer to a FVIII protein that has been modified such that the modified FVIII protein has amino acid alterations compared to wild-type FVIII. Again, when comparing activity and / or stability, if the encoded variant FVIII protein retains the B domain, it is appropriate to compare the variant FVIII protein to wild-type FVIII; if the encoded variant FVIII protein has a deletion of the B domain, the variant FVIII protein is compared to wild-type FVIII that also has a deletion of the B domain.

[0039]

[0055] A variant FVIII may contain part of the B domain. Thus, a FVIII-BDD contains part of the B domain. In a FVIII-BDD, most of the B domain is generally deleted.

[0040]

[0056] A variant FVIII may comprise an "SQ" sequence, designated SFSQNPPVLKRHQR (SEQ ID NO: 29). Such variant FVIIIs with an SQ (FVIII / SQ) generally have a BDD, e.g., at least all or a portion of the BD is deleted. A variant FVIII, such as a FVIII-BDD, may have all or a portion of an "SQ" sequence, i.e., all or a portion of SEQ ID NO: 29. Thus, for example, a variant FVIII-BDD having an SQ sequence (SFSQNPPVLKRHQR, SEQ ID NO: 29) may have all or only a portion of the amino acid sequence SFSQNPPVLKRHQR. For example, a FVIII-BDD may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid residues of SFSQNPPVLKRHQR. Thus, variant FVIII proteins described herein include SFSQNPPVLKRHQR having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 internal deletions and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino or carboxy terminal deletions.

[0041]

[0057] "Polypeptides," "proteins," and "peptides" encoded by "nucleic acid" or "polynucleotide" sequences include not only the full-length naturally occurring (FVIII) sequence as in the naturally occurring wild-type protein, but also functional subsequences, modifications, or sequence variants, so long as such subsequences, modifications, or variants retain some functionality of the naturally occurring full-length protein. For example, a CpG-reduced nucleic acid encoding a FVIII protein may have a B-domain deletion as described herein and retain coagulation function. In the methods and uses of the present invention, such polypeptides, proteins, and peptides encoded by nucleic acid sequences may, but need not, be identical to the endogenous protein that is defective or poorly or deficiently expressed in the mammal being treated.

[0042]

[0058] Non-limiting examples of modifications include substitution of one or more nucleotides or amino acids (e.g., 1 to 3, 3 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 750, 750 to 850, or more nucleotides or residues). An example of a nucleic acid modification is CpG reduction. In certain embodiments, a CpG-reduced nucleic acid encoding FVIII, such as a human FVIII protein, has 10 or fewer CpGs compared to a wild-type sequence encoding human factor FVIII; or 5 or fewer CpGs compared to a wild-type sequence encoding human factor FVIII; or 5 or fewer CpGs in the CpG-reduced nucleic acid encoding FVIII.

[0043]

[0059] Exemplary amino acid modifications are conservative amino acid substitutions or deletions (e.g., subsequences or fragments) of a reference sequence, e.g., FVIII, such as FVIII with a B domain deletion. In certain embodiments, the modified or variant sequence retains at least part of the function or activity of the unmodified sequence.

[0044]

[0060] All mammalian and non-mammalian forms of nucleic acids encoding proteins, whether known or unknown, are expressly included, including other mammalian forms of CpG-reduced nucleic acids encoding FVIII and the FVIII proteins disclosed herein. Thus, the present invention includes genes and proteins from non-mammals, non-human mammals, and humans that function substantially similarly to the FVIII (e.g., human) genes and proteins described herein.

[0045]

[0061] The term "vector" refers to a small carrier nucleic acid molecule, a plasmid, a virus (e.g., an AAV vector), or other vehicle that can be manipulated by inserting or incorporating a nucleic acid. Such vectors can be used for genetic manipulation (i.e., "cloning vectors") to introduce / transfect a polynucleotide into a cell so that the inserted polynucleotide can be transcribed or translated within the cell. 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 nucleic acid sequence of a vector generally contains at least an origin of replication for propagation within the cell and optionally contains additional elements such as heterologous polynucleotide sequences, expression control elements (e.g., promoters, enhancers), introns, ITR(s), selectable markers (e.g., antibiotic resistance), polyadenylation signals, etc.

[0046]

[0062] Viral vectors are derived from or based on one or more nucleic acid elements comprising a viral genome. Specific viral vectors include lentiviruses, pseudotyped lentiviruses, and parvovirus vectors, such as adeno-associated virus (AAV) vectors. Also provided are vectors containing CpG-reduced nucleic acids encoding FVIII.

[0047]

[0063] The term "recombinant" as a modifier of vectors, such as recombinant viral vectors, e.g., lentiviral or parvoviral (e.g., AAV) vectors, and sequences, such as recombinant polynucleotides and polypeptides, means that the composition has been manipulated (i.e., engineered) in a manner not commonly found in nature. A particular example of a recombinant vector, such as an AAV vector, is when a polynucleotide not normally present in the genome of a wild-type virus (e.g., AAV) is inserted into the viral genome. An example of a recombinant polynucleotide is when a CpG-reduced nucleic acid encoding a FVIII protein is cloned into a vector, with or without the 5', 3', and / or intron regions with which this gene is normally associated in the viral (e.g., AAV) genome. The term "recombinant" is not necessarily used herein with reference to vectors, such as viral vectors and AAV vectors, and sequences, such as polynucleotides, but recombinant forms comprising the polynucleotide are expressly included despite any such omission.

[0048]

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

[0049]

[0065] Recombinant vector (e.g., lentivirus, parvovirus, AAV) sequences can be packaged, referred to herein as "particles," for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo. When recombinant vector sequences are encapsidated or packaged within an AAV particle, the particle may also be referred to as "rAAV." Such particles contain proteins that encapsidate or package the vector genome. Particular examples include viral envelope proteins, and in the case of AAV, capsid proteins.

[0050]

[0066] The "genome" of a vector refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form a viral (e.g., AAV) particle. When a recombinant plasmid is used to construct or produce a recombinant vector, the vector genome does not include the "plasmid" portion of the recombinant plasmid that does not correspond to the vector genome sequence. This non-vector genome portion of the recombinant plasmid is called the "plasmid backbone," which is important for the cloning and amplification of the plasmid, steps necessary for propagation and production of recombinant virus, but which is not itself packaged or encapsidated into a viral (e.g., AAV) particle. Thus, the "genome" of a vector refers to the nucleic acid that is packaged or encapsidated by the virus (e.g., AAV).

[0051]

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

[0052]

[0068] In a cell carrying a transgene, the transgene has been introduced / transferred by a vector, such as AAV, "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 be integrated into the genomic nucleic acid of the recipient cell. Once the introduced nucleic acid becomes integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it can be stably maintained in the cell or organism and further transferred or inherited to progeny cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid may be extrachromosomal in the recipient cell or host organism, or may simply be transiently present.

[0053]

[0069] A "transduced cell" is a cell into which a transgene has been introduced. Thus, a "transduced" cell (e.g., in a mammal, such as a cell or tissue or organ cell) refers to a genetic change in a cell following incorporation of an exogenous molecule, e.g., a nucleic acid (e.g., a transgene), into the cell. A "transduced" cell is thus a cell or its progeny into which an exogenous nucleic acid has been introduced. The cell(s) can proliferate and express the introduced protein or transcribe the nucleic acid. For gene therapy uses and methods, the transduced cell can be within a subject.

[0054]

[0070] "Expression control element" refers to a nucleic acid sequence(s) that influences expression of an operably linked nucleic acid. Control elements include expression control elements set forth herein, such as promoters and enhancers. A vector sequence, including an AAV vector, can contain one or more "expression control elements." Such elements are generally included to facilitate proper transcription, and, if appropriate, translation, of a heterologous polynucleotide (e.g., promoters, enhancers, splicing signals for introns, maintenance of the correct reading frame of a gene to allow in-frame translation of mRNA, and stop codons, etc.). Such elements are generally cis-acting and are referred to as "cis-acting" elements, but can also act in trans.

[0055]

[0071] Expression control can be at the level of transcription, translation, splicing, message stability, etc. Expression control elements that modulate transcription are generally proximal to the 5' end (i.e., "upstream") of the transcribed nucleic acid. Expression control elements can also be located at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., in an intron). Expression control elements can be located adjacent to the transcribed sequence or at significant distances (e.g., 1-10, 10-25, 25-50, 50-100, 100-500, or more nucleotides from the polynucleotide). However, due to length limitations of certain vectors, such as AAV vectors, expression control elements are generally within 1-1000 nucleotides of the transcribed nucleic acid.

[0056]

[0072] Functionally, expression of an operably linked nucleic acid can be at least partially controlled by an element (e.g., a promoter) such that the element modulates transcription of the nucleic acid and, appropriately, translation of the transcript. A particular example of an expression control element is a promoter that is typically located 5' of a transcribed sequence, e.g., a CpG-depleted nucleic acid encoding FVIII. A promoter generally increases the amount expressed from an operably linked nucleic acid compared to the amount expressed in the absence of the promoter.

[0057]

[0073] As used herein, "enhancer" can refer to a sequence located adjacent to a heterologous polynucleotide. Enhancer elements are generally located upstream of promoter elements, but can also function downstream or within a sequence (e.g., a CpG-reduced nucleic acid encoding FVIII). Thus, enhancer elements can be located 100, 200, or 300 or more base pairs upstream or downstream from a CpG-reduced nucleic acid encoding FVIII. Enhancer elements generally increase expression of an operably linked nucleic acid over that conferred by a promoter element.

[0058]

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

[0059]

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

[0060]

[0076] Expression control elements also include ubiquitous or promiscuous promoters / enhancers capable of driving expression of a polynucleotide in many different cell types. Such elements include, but are not limited to, the cytomegalovirus (CMV) immediate-early promoter / enhancer sequence, the Rous sarcoma virus (RSV) promoter / enhancer sequence, and other viral promoters / enhancers active in a variety of mammalian cell types, or synthetic elements that do not occur in nature (see, e.g., 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.

[0061]

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

[0062]

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

[0063]

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

[0064]

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

[0065]

[0081] Thus, additional elements for a vector include, but are not limited to, expression control (e.g., promoter / enhancer) elements, transcription termination signals or stop codons, 5' or 3' untranslated regions (e.g., polyadenylation (polyA) sequences) flanking sequences such as one or more copies of AAV ITR sequences, or introns.

[0066]

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

[0067]

[0083] Introns can also function as filler or stuffer polynucleotide sequences to achieve packaging length of the AAV vector into viral particles. Introns and intron fragments that function as filler or stuffer polynucleotide sequences can also enhance expression.

[0068]

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

[0069]

[0085] The term "isolated," when used as a modifier of a composition, means that the composition has been made by the hand of man or has been completely or at least partially separated from the in vivo environment it naturally resides in. Generally, an isolated composition is substantially free of one or more substances with which it is normally associated in nature, such as one or more proteins, nucleic acids, lipids, carbohydrates, and cell membranes.

[0070]

[0086] The term "isolated," in reference to nucleic acids of the invention, refers to a nucleic acid molecule that is separated from one or more sequences that immediately flank it (in the 5' and 3' directions) in the naturally occurring genome (genomic DNA) of the organism of origin. For example, an "isolated nucleic acid" can include DNA or cDNA inserted into a vector, such as a plasmid or viral vector, or integrated into the DNA of a prokaryotic or eukaryotic organism.

[0071]

[0087] The term "isolated" with respect to RNA molecules of the present invention primarily refers to RNA molecules encoded by isolated DNA molecules, as defined above. Alternatively, the term may refer to RNA molecules that have been sufficiently separated from RNA molecules with which they are associated in their natural state (i.e., in cells or tissues) such that they exist in "substantially pure" form (the term "substantially pure" is defined below).

[0072]

[0088] The terms "isolated protein" or "isolated and purified protein" are sometimes used herein in reference to proteins. This term primarily refers to a protein produced by expression of an isolated nucleic acid molecule. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it is naturally associated so that it is in "substantially pure" form.

[0073]

[0089] The term "isolated" does not exclude combinations produced by the hand of man, such as viral particles and pharmaceutical preparations that package or encapsidate recombinant vector (e.g., rAAV) sequences or vector genomes. The term "isolated" also does not exclude alternative physical forms of the composition, such as hybrid / chimeric, multimeric / oligomeric, modified (e.g., phosphorylated, glycosylated, lipidated) or derivatized forms, or forms expressed in host cells produced by the hand of man.

[0074]

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

[0075]

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

[0076]

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

[0077]

[0093] As used herein, the term "probe" refers to a nucleic acid, either an oligonucleotide, polynucleotide, or RNA or DNA, either naturally occurring, as in a purified restriction enzyme digest, or synthetically produced, that can anneal or specifically hybridize with a nucleic acid having a sequence complementary to the probe. Probes can be either single-stranded or double-stranded. The exact length of a probe depends on many factors, including temperature, the source of the probe, and the method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, oligonucleotide probes typically contain 15 to 25 or more nucleotides, although they may contain fewer nucleotides.

[0078]

[0094] Probes herein are selected to be "substantially" complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary to "specifically hybridize" or anneal with their respective target strands under a predetermined set of conditions. Thus, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences may be dispersed within the probe, provided that the probe sequence has sufficient complementarity to the target nucleic acid sequence to specifically anneal with it.

[0079]

[0095] The term "specifically hybridize" refers to the association between two single-stranded nucleic acid molecules that are sufficiently complementary in sequence (sometimes referred to as "substantially complementary") to permit such hybridization under predetermined conditions as commonly used in the art. In particular, this term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, substantially excluding hybridization of the oligonucleotide with a single-stranded nucleic acid of a non-complementary sequence.

[0080]

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

[0081]

[0097] Primers may vary in length depending on the requirements of specific conditions and applications. For example, in diagnostic applications, oligonucleotide primers are generally 15-25 nucleotides in length or longer. The primer must be sufficiently complementary to the desired template strand to anneal sufficiently to prime the synthesis of the desired extension product, i.e., to bring the 3' hydroxyl moiety of the primer into suitable proximity for use in initiating synthesis by a polymerase or similar enzyme. The primer sequence need not be an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer. Alternatively, non-complementary bases may be dispersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of extension products.

[0082]

[0098] The terms "identity," "homology," and grammatical variations thereof mean that two or more referenced entities are the same when these entities are "aligned sequences." Thus, by way of example, if two polypeptide sequences are identical, they have the same amino acid sequence over at least the referenced region or portion. If two polynucleotide sequences are identical, they have the same polynucleotide sequence over at least the referenced region or portion. Identity can extend over a given sequence range (region or domain). A "range" or "region" of identity represents a portion of the same two or more referenced entities. Thus, if two protein or nucleic acid sequences are identical over one or more sequence ranges or regions, the sequences share identity over that region. "Aligned" sequences represent multiple polynucleotide or protein (amino acid) sequences, often including modifications for missing or added bases or amino acids (gaps) compared to a reference sequence.

[0083]

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

[0084]

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

[0085]

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

[0086]

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

[0087]

[0103] The term "homologous" or "homology" means that two or more referenced entities share at least partial identity over a given region or portion. A "region, region, or domain" of homology or identity means that portions of two or more referenced entities share homology or are the same. Thus, if two sequences are identical over one or more sequence regions, the sequences share identity in those regions. "Substantial homology" means that the molecules are structurally or functionally conserved such that the molecule has or is predicted to have at least partial structure or function(s) of one or more of the structures or functions (e.g., biological function(s) or activity(ies)) of the reference molecule or related / corresponding regions or portions of the reference molecule with which it shares homology.

[0088]

[0104] The degree of identity (homology) or "percent identity" between two sequences can be determined using a computer program and / or a mathematical algorithm. For the purposes of the present invention, comparison of nucleic acid sequences is performed using the GCG Wisconsin Package version 9.1, available from the Genetics Computer Group in Madison, Wisconsin. For convenience, the default parameters specified by the program (gap creation penalty = 12, gap extension penalty = 4) are intended for use herein for comparing sequence identity. Alternatively, the Blastn 2.0 program provided by the National Center for Biotechnology Information (found on the World Wide Web at ncbi.nlm.nih.gov / blast / ; Altschul et al., 1990, J Mol Biol 215:403-410), using gapped alignment with default parameters, can be used to determine the level of identity and similarity between nucleic acid and amino acid sequences. For comparison of polypeptide sequences, the BLASTP algorithm is generally used in combination with a score such as PAM100, PAM250, BLOSUM62, or BLOSUM50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs 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 protein structural similarity using Delaunay-based topology mapping (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

[0089]

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

[0090]

[0106] The nucleic acids of the present invention can be maintained as DNA in any convenient cloning vector. In one embodiment, clones are maintained in a plasmid cloning / expression vector, such as pBluescript (Stratagene, La Jolla, CA), propagated in a suitable E. coli host cell. Alternatively, the nucleic acid can be maintained in a vector suitable for expression in mammalian cells. If post-translational modifications affect coagulation function, the nucleic acid molecule can be expressed in mammalian cells.

[0091]

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

[0092]

[0108] CpG-reduced nucleic acid variants encoding the FVIII of the present invention with deleted B domains and optionally amino acid substitutions, deletions or additions can be prepared in a variety of ways by known methods. The protein can be purified by immunoaffinity purification from a suitable source expressing the engineered FVIII, such as transformed bacteria or animal cell cultures or tissues.

[0093]

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

[0094]

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

[0095]

[0111] Vectors, such as those described herein, optionally contain regulatory elements required for expression of DNA in a host cell positioned in a manner that allows 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 described herein and known to those of skill in the art.

[0096]

[0112] FVIII encoded by CpG-reduced nucleic acid variants produced by gene expression in recombinant prokaryotic or eukaryotic systems can be purified by methods known in the art. In embodiments, commercially available expression / secretion systems can be used in which the recombinant protein is expressed and then secreted from the host cells for easy purification from the surrounding medium. If an expression / secretion vector is not used, alternative approaches involve purifying the recombinant protein by immunological interaction with an antibody that specifically binds the recombinant protein or affinity separation, such as by a nickel column for the isolation of recombinant proteins tagged with 6 to 8 histidine residues at the N- or C-terminus. Alternative tags can include the FLAG epitope, GST, or hemagglutinin epitope. Such methods are commonly used by those skilled in the art.

[0097]

[0113] FVIII proteins prepared by the above methods can be analyzed by standard procedures, for example, such proteins can be evaluated for altered clotting properties by known methods.

[0098]

[0114] Thus, the present invention also provides a method for producing a (disclosed) polypeptide, comprising expression from a nucleic acid encoding the polypeptide (generally a nucleic acid). This can be conveniently achieved by culturing a host cell containing such a vector under appropriate conditions that cause or allow production of the polypeptide. Polypeptides can also be produced in in vitro systems.

[0099]

[0115] The methods and uses of the present invention involve delivering (transducing) a nucleic acid (transgene) into a host cell, including dividing and / or non-dividing cells. The nucleic acids, recombinant vectors (e.g., rAAV), methods, uses, and pharmaceutical formulations of the present invention are additionally useful in methods of delivering, administering, or providing a protein to a subject in need thereof as a method of treatment. In this manner, the nucleic acid can be transcribed and the protein produced in vivo in the subject. The subject may benefit from or require the protein because they have a deficiency in the protein, or because producing the protein in the subject may confer some therapeutic effect as a method of treatment or otherwise.

[0100]

[0116] Vectors, recombinant viral particles, methods and uses comprising lentiviral or parvoviral vector (e.g., AAV) sequences can be used to deliver CpG-reduced nucleic acid variants encoding FVIII that have a biological effect for treating or ameliorating one or more symptoms associated with a FVIII deficiency or disorder. Recombinant lentiviral or parvoviral vector (e.g., AAV) sequences, plasmids, recombinant viral particles, methods and uses can be used to provide treatments for a variety of medical conditions associated with or caused by a FVIII deficiency or disorder.

[0101]

[0117] The nucleic acids, vectors, expression vectors (e.g., rAAV), and recombinant viral particles, methods, and uses of the present invention enable the treatment of genetic diseases, such as FVIII deficiency. For diseases with deficiency states, gene transfer can be used to introduce normal genes into affected tissues for replacement therapy, and antisense mutations can be used to create animal disease models. For imbalanced pathologies, gene transfer can be used to create pathology in a model system, which could then be used to combat the pathology. Site-specific integration of nucleic acid sequences can also be used to correct the deficiency.

[0102]

[0118] In certain embodiments, CpG-reduced nucleic acid variants encoding FVIII can be used, for example, as therapeutic and / or prophylactic agents (proteins or nucleic acids) or transgenes in genes that modulate the blood coagulation cascade. For example, CpG-reduced nucleic acid variants encoding FVIII can have coagulation activity similar to wild-type FVIII or altered coagulation activity compared to wild-type FVIII. Cell-based strategies allow for the continuous expression of CpG-reduced nucleic acid variants encoding FVIII in patients with hemophilia A. As disclosed herein, certain modifications of the FVIII molecule (nucleic acid and protein) result in increased expression at the nucleic acid level, increased coagulation activity, and thereby effectively improve hemostasis.

[0103]

[0119] CpG-reduced nucleic acid variants encoding FVIII can be used for various purposes according to the present invention. In one aspect, a nucleic acid delivery vehicle (i.e., expression vector) is provided for modulating blood coagulation, wherein the expression vector comprises a CpG-reduced nucleic acid variant encoding FVIII described herein. Administration of the FVIII-encoding expression vector to a patient results in expression of a FVIII protein that acts to alter the coagulation cascade. According to the present invention, expression of a CpG-reduced nucleic acid variant or functional fragment encoding a FVIII protein described herein enhances hemostasis.

[0104]

[0120] In further embodiments of the present invention, compositions and methods are provided for the administration of viral vectors comprising CpG-reduced nucleic acid variants encoding FVIII. In one aspect, the expression vector comprising CpG-reduced nucleic acid variants encoding FVIII is a viral vector.

[0105]

[0121] Expression vectors containing CpG-reduced nucleic acid variants encoding FVIII can be administered alone or in combination with other molecules useful for modulating hemostasis. According to the invention, the vector, expression vector, or therapeutic combination can be administered to a patient alone or in a pharmaceutically acceptable or biologically compatible composition.

[0106]

[0122] Viral vectors, such as lentivirus and parvovirus vectors, including AAV serotypes and variants thereof, provide a means for ex vivo, in vitro, and in vivo delivery of protein-encoding nucleic acids to cells so that the cells express the encoded proteins. AAV is useful as a gene therapy vector because it can penetrate cells and introduce nucleic acids / genetic material, thereby allowing the nucleic acid / genetic material to be stably maintained in the cell. In addition, these viruses can, for example, introduce nucleic acids / genetic material into specific sites. Because AAV is not associated with pathogenic diseases in humans, AAV vectors can deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and therapeutic agents) to human patients without causing substantial AAV pathogenesis or disease.

[0107]

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

[0108]

[0124] AAV and lentiviral particles can be advantageously used as vehicles for effective gene delivery. Such virions have several desirable characteristics for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with these vectors has also demonstrated a lack of persistent toxicity, with minimal or undetectable immune responses. AAV is known to infect a wide variety of cell types in vivo and in vitro via receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans, targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints, and hematopoietic stem cells. Non-viral vectors based on plasmid DNA or minicircles are also suitable gene transfer vectors for large genes, such as vectors encoding FVIII.

[0109]

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

[0110]

[0126] Thus, in various embodiments of the present invention, the vector includes a lentiviral vector, such as an adenoviral vector, or a parvoviral vector. In certain embodiments, the recombinant vector is a parvoviral vector. Parvoviruses are small viruses with a single-stranded DNA genome. "Adeno-associated virus" (AAV) is a member of the Parvoviridae family.

[0111]

[0127] Thus, the present invention provides a viral vector comprising a CpG-reduced nucleic acid variant encoding FVIII. For example, a recombinant AAV vector can comprise a CpG-reduced nucleic acid variant encoding FVIII, wherein the encoded FVIII protein optionally has a B-domain deletion. Thus, delivery or administration of the vector to a subject (e.g., a mammal) provides FVIII to the subject, such as a mammal (e.g., a human).

[0112]

[0128] Direct delivery of the vector or ex vivo transduction of human cells followed by infusion results in expression of FVIII, thereby exerting beneficial therapeutic effects on hemostasis. In the context of the Factor VIII of the present invention described herein, such administration enhances procoagulant activity.

[0113]

[0129] AAV vectors and lentiviral vectors generally do not contain viral genes associated with pathogenesis. Such vectors generally lack one or more wild-type AAV genes, such as the rep and / or cap genes, in whole or in part, but retain at least one functional flanking ITR sequence if necessary for rescue, replication, and packaging of the recombinant vector into AAV vector particles. For example, only essential portions of the vector, such as the ITR and LTR elements, are included. Thus, the AAV vector genome contains sequences (e.g., functional ITR sequences) required in cis for replication and packaging.

[0114]

[0130] The recombinant AAV vector and its methods and uses 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. Such vectors can be based on the same strain or serotype (or subgroup or variant), or can be different from each other. As a non-limiting example, recombinant AAV vectors based on one serotype genome can be identical in one or more of the capsid proteins that package the vector. In addition, the genome of the recombinant AAV vector can be based on the genome of an AAV (e.g., AAV2) serotype that is different from one or more of the AAV capsid proteins that package the vector. For example, the genome of the AAV vector may be based on AAV2, while at least one of the three capsid proteins may be, for example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8, or a variant thereof.

[0115]

[0131] 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, and variants thereof (e.g., capsid variants such as amino acid insertions, additions, substitutions, and deletions), e.g., as set forth in WO 2013 / 158879 (International Application PCT / US2013 / 037170), WO 2015 / 013313 (International Application PCT / US2014 / 047670), and U.S. Patent Application Publication No. 2013 / 0059732 (U.S. Patent No. 9,169,299 discloses LK01, LK02, LK03, etc.).

[0116]

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

[0117]

[0133] The serotypes (e.g., VP1, VP2, and / or VP3 sequences) of AAV and AAV variants (e.g., capsid variants) may or may not differ 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 AAV1-AAV12, Rh74, or Rh10 serotypes).

[0118]

[0134] As used herein, the term "serotype" refers to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological characteristics are determined based on the lack of cross-reactivity between antibodies and one AAV compared to another. Such differences in cross-reactivity are typically due to differences in the sequences / antigenic determinants of the capsid proteins (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). Although AAV variants, including capsid variants, may not be serologically distinct from a reference AAV or other AAV serotypes, they differ by at least one nucleotide or amino acid residue compared to the reference AAV or other AAV serotypes.

[0119]

[0135] In the traditional definition, a serotype means that the virus in question has been tested for neutralizing activity against sera specific to all existing, characterized serotypes, and no antibodies were found to neutralize the virus in question. As more naturally occurring virus isolates are discovered and / or capsid mutants are generated, a virus may or may not be serologically distinct from any of the currently existing serotypes. Thus, if a new virus (e.g., AAV) does not have serological differences, the new virus (e.g., AAV) is a subgroup or variant of the corresponding serotype. Mutant viruses with altered capsid sequences often have not yet been serologically tested for neutralizing activity to determine whether they are a separate serotype according to the traditional definition of serotype. Therefore, for convenience and to avoid repetition, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) and non-serologically distinct viruses (e.g., AAV) that may be included in a subgroup or variant of a given serotype.

[0120]

[0136] Thus, an AAV vector contains gene / protein sequences identical to those characteristic of a particular serotype. As used herein, an "AAV vector related to AAV1" refers to one or more AAV proteins (e.g., VP1, VP2, and / or VP3 sequences) that share substantial sequence identity with one or more polynucleotide or polypeptide sequences comprising AAV1. Similarly, an "AAV vector related to AAV8" refers to one or more AAV proteins (e.g., VP1, VP2, and / or VP3 sequences) that share substantial sequence identity with one or more polynucleotide or polypeptide sequences comprising AAV8. An "AAV vector related to AAV-Rh74" refers to one or more AAV proteins (e.g., VP1, VP2, and / or VP3 sequences) that share substantial sequence identity with one or more polynucleotide or polypeptide sequences comprising 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, are therefore also referred to as AAV vectors related to another serotype, for example AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 and AAV-2i8. AAV variants may have one or more different sequences from those described above, but may exhibit substantial sequence identity with one or more genes and / or proteins, and / or may have one or more functional characteristics (e.g., cell / tissue tropism, etc.) of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8. Non-limiting examples of AAV variants include capsid variants of any of VP1, VP2, and / or VP3.

[0121]

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

[0122]

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

[0123]

[0139] Recombinant AAV vectors, including hybrid and chimeric sequences related to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 and variants, can be constructed to contain one or more nucleic acid sequences (transgenes) flanked by one or more functional AAV ITR sequences using recombinant techniques known to those of skill in the art.

[0124]

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

[0125]

[0141] The appropriate composition for delivering FVIII can be determined by a physician taking into account various physiological variables, including but not limited to the patient's condition and hemodynamic status. Various compositions and routes of administration well suited for different applications are well known in the art and are described herein below.

[0126]

[0142] Preparations containing purified FVIII protein produced by expression of the CpG-reduced nucleic acid variants, vectors, and expression vectors of the present invention encoding FVIII of the present invention can be formulated as pharmaceutical preparations containing a physiologically acceptable matrix. The preparations can be formulated using substantially known prior art methods and can be mixed with a buffer containing salts such as NaCl, CaCl2, and amino acids such as glycine and / or lysine at a pH range of 6 to 8. The purified preparations containing FVIII can be stored in the form of a final solution or in lyophilized or frozen form until needed.

[0127]

[0143] The preparations can be stored in lyophilized form and dissolved into a clear solution using an appropriate reconstitution solution. Alternatively, the preparations according to the invention can be made available as liquid preparations or frozen liquids. The preparations according to the invention can optionally be particularly stable, i.e., can be left in dissolved form for long periods of time before administration or delivery.

[0128]

[0144] The preparations according to the invention can be made available as pharmaceutical preparations with FVIII activity in the form of mono- or multi-component preparations in combination with other factors. Before processing the purified protein into a pharmaceutical preparation, it is subjected to conventional quality control and shaped into a therapeutic presentation. In particular, during recombinant production, purified preparations are tested for the absence of cellular nucleic acids and nucleic acids derived from expression vectors, such as the expression vectors described in EP 0 714 987.

[0129]

[0145] Pharmaceutical protein preparations can be used at dosages of between 30 and 100 IU / kg (1 IU is 100 ng / ml) as a single daily injection or up to three times daily for several days. Patients can be treated immediately upon presentation to the office with bleeding. Alternatively, patients may receive bolus infusions of FVIII every 8 to 12 hours, or once daily infusions once sufficient improvement is observed.

[0130]

[0146] Thus, the nucleic acids, vectors, recombinant vectors (e.g., rAAV), and recombinant viral particles of the invention, as well as other compositions, agents, drugs, and biologics (proteins), can be incorporated into pharmaceutical compositions that are useful, inter alia, for in vivo or ex vivo administration and delivery to a subject.

[0131]

[0147] In certain embodiments, the pharmaceutical composition also contains a pharmaceutically acceptable carrier or excipient, including any pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and that can be administered without undue toxicity.

[0132]

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

[0133]

[0149] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugar, and ethanol. Pharmaceutically acceptable salts may also be included, such as inorganic salts of hydrochloride, hydrobromide, phosphate, sulfate, and the like; and organic salts of acetate, propionate, malonate, benzoate, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

[0134]

[0150] Pharmaceutical compositions may be supplied as salts, which can be formed with a number of acids, including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble 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 or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, in the pH range of 4.5-5.5, which is combined with a buffer solution prior to use.

[0135]

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

[0136]

[0152] Pharmaceutical compositions can be formulated to be compatible with particular routes of administration or delivery as described herein or known to those skilled in the art, and thus include carriers, diluents, or excipients suitable for administration by various routes.

[0137]

[0153] 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, Hank's solution, Ringer's solution, dextrose, fructose, ethanol, animal oils, vegetable oils, or synthetic oils. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.

[0138]

[0154] Additionally, suspension of active compound can be prepared as appropriate oily suspension injection.Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.The suspension can also optionally contain suitable stabilizers or agents that increase the solubility of the compound, allowing for the preparation of highly concentrated solutions.

[0139]

[0155] Cosolvents and adjuvants can be added to the formulation.Non-limiting examples of cosolvents include alcohols containing hydroxyl groups or other polar groups, such as isopropyl alcohol; glycols such as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ethers; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.Adjuvants include surfactants such as soybean lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

[0140]

[0156] After the pharmaceutical composition has been prepared, it can be placed in an appropriate container and labeled for treatment. For administration of a FVIII-containing vector or polypeptide, such labeling would include amount, frequency, and method of administration.

[0141]

[0157] Pharmaceutical compositions and delivery systems suitable for the compositions, methods and uses of the present invention are known in the art (e.g., Remington: The Science and Practice of Pharmacy (2003) 20th edition, Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18th edition, Mack Publishing Co., Easton, PA; The Merck Index (1996) 12th edition, 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), edited by R.L. Juliano, Oxford, NY, pp. 253-315).

[0142]

[0158] The present invention also provides methods for introducing a CpG-reduced nucleic acid variant encoding FVIII into a cell or an animal. In certain embodiments, the present invention provides a method for modulating hemostasis. In one aspect, the method comprises contacting an individual (a subject such as a patient or a mammal) with or administering to the individual a nucleic acid delivery vehicle (e.g., an AAV vector) comprising a CpG-reduced nucleic acid variant encoding FVIII, under conditions whereby the FVIII polypeptide is expressed in the individual. In another embodiment, the method comprises providing a nucleic acid delivery vehicle (e.g., an AAV vector) comprising a CpG-reduced nucleic acid variant encoding FVIII to a cell of the individual (a subject such as a patient or a mammal), under conditions whereby the FVIII polypeptide is expressed in the individual.

[0143]

[0159] From the above it can be seen that CpG-depleted nucleic acid variants encoding FVIII can be used in the treatment of disorders associated with defective, insufficient or abnormal blood clotting.

[0144]

[0160] Compositions of CpG-reduced nucleic acid variants encoding FVIII, including vectors, recombinant vectors (e.g., rAAV), and recombinant viral particles, can be administered to a subject in need thereof in sufficient or effective amounts to achieve the methods and uses of the present invention. An "effective amount" or "sufficient amount" refers to an amount, either single or multiple times, that alone or in combination with one or more other compositions (therapeutic agents such as drugs or immunosuppressants), treatments, protocols, or therapeutic regimens provides a detectable response, expected or desired result in, or benefit to, the subject, to any measurable or detectable extent, or for any period of time (e.g., minutes, hours, days, months, years, or cure) of any duration (long or short term).

[0145]

[0161] Dosages may vary and may depend on the type, onset, progression, severity, frequency, duration, or probability of the disease for which treatment is directed, the desired clinical endpoint, previous or concurrent treatments of the subject, the general health, age, sex, race, or immunocompetence of the subject, and other factors understood by those of skill in the art. The amount, number, frequency, or duration of administration may be proportionally increased or decreased as indicated by any adverse side effects of the treatment or therapy, complications, or other risk factors and the condition of the subject. Those of skill in the art will recognize factors that may affect the dosage and timing necessary to provide an amount sufficient to achieve therapeutic or prophylactic benefit.

[0146]

[0162] The dose to achieve a therapeutic effect, e.g., the dose as vector genome / kilogram body weight (vg / kg), will vary based on several factors, including, but not limited to, the route of administration, the expression level of the heterologous polynucleotide required to achieve a therapeutic effect, the specific disease being treated, any host immune response to the viral vector, the host immune response to the heterologous polynucleotide or expression product (protein), and the stability of the expressed protein. One of skill in the art will be able to determine the dose range of rAAV / vector genome for treating a patient with a particular disease or disorder based on the aforementioned and other factors. Generally, the dose to achieve a therapeutic effect will be at least 1 x 10 per kilogram of subject body weight. 8 More than 10, for example, 1 x 10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 or 1 x 10 14 In mice, the range is 1 × 10 10 ~1×10 11 , and 1 × 10 in dogs 12 ~1×10 13 A range of AAV doses was effective.

[0147]

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

[0148]

[0164] For treating such hemophilic subjects, a typical dose to achieve the desired therapeutic effect is at least 1 x 10 per kilogram of subject body weight. 10 vector genomes (vg) / (vg / kg), or approximately 1 x 10 per kg of subject body weight 10 ~1×10 11 vg, or approximately 1 x 10 per kg of subject body weight 11 ~1×10 12 vg, or approximately 1 x 10 per kg of subject body weight 12 ~1×10 13 The dose of the AAV vector may be generally at the lower end of the dose spectrum so that there is no substantial immune response to the FVIII or AAV vector.

[0149]

[0165] An "effective amount" or "sufficient amount" for treatment (e.g., to ameliorate or achieve a therapeutic benefit or improvement) is a dose that is generally effective to achieve a measurable response to one, more than one, or all adverse symptoms, outcomes, or complications of the disease, such as one or more adverse symptoms, disorders, illnesses, conditions, or complications caused by or associated with the disease, although a satisfactory result would be to reduce, alleviate, inhibit, suppress, limit, or control the progression or worsening of the disease.

[0150]

[0166] An effective or sufficient amount may, but need not, be given in a single dose and may require multiple doses, and may be administered alone or in combination with another composition (e.g., drug), treatment, protocol, or therapeutic regimen, but need not be. For example, the amount may be proportionally increased as indicated by the needs of the subject, the type, condition, and severity of the disease being treated, or the side effects, if any, of the treatment. In addition, an effective or sufficient amount need not be effective or sufficient when given in single or multiple doses without a second composition (e.g., another drug or agent), treatment, protocol, or therapeutic regimen, as additional doses, amounts, or durations beyond such doses, or additional compositions (e.g., drugs or agents), treatments, protocols, or therapeutic regimens, may be included to be considered effective or sufficient in a given subject. An amount considered effective also includes an amount that results in the reduced use of another treatment, therapeutic regimen, or protocol, such as the administration of a recombinant coagulation factor protein (e.g., FVIII) for the treatment of a coagulation disorder (e.g., hemophilia A).

[0151]

[0167] Thus, the methods and uses of the present invention also include, inter alia, methods and uses that result in a reduced need for or use of another compound, agent, drug, therapeutic regimen, treatment protocol, step, or therapeutic agent. For example, with respect to blood clotting disorders, a method or use of the present invention has a therapeutic benefit if it results in a given subject receiving less frequent or reduced doses or no administration of a recombinant clotting factor protein to replace a deficient or defective (abnormal or mutant) endogenous clotting factor in that subject. Thus, the present invention provides methods and uses that reduce the need for or use of another treatment or therapy.

[0152]

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

[0153]

[0169] The term "ameliorate" refers to a detectable or measurable improvement in a disease or its symptoms or underlying cellular response in a subject. A detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limitation, or control of the occurrence, frequency, severity, progression, or duration of a disease or a complication caused by or associated with a disease, or an improvement in the symptoms or underlying causes of a disease or the outcome of a disease, or regression of a disease. With respect to HemA, an effective amount is, for example, an amount that reduces the frequency or severity of acute bleeding episodes in a subject, or an amount that reduces clotting time as measured, for example, by a clotting assay.

[0154]

[0170] Thus, pharmaceutical compositions of the present invention include compositions in which the active ingredients are contained in an amount effective to achieve the intended therapeutic purpose, and it is well within the ability of a skilled physician to determine a therapeutically effective dose using the techniques and guidance provided herein.

[0155]

[0171] The therapeutic dose depends, among other factors, on the age and general condition of the subject, the severity of the abnormal blood coagulation phenotype, and the strength of the regulatory sequences regulating the expression level of the CpG-reduced nucleic acid variant encoding FVIII. Thus, the therapeutically effective amount in humans falls within a relatively broad range that can be determined by a physician based on the individual patient's response to vector-based FVIII treatment. Such doses may be administered alone or in combination with immunosuppressants or immunosuppressants.

[0156]

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

[0157]

[0173] The composition may be administered alone. In certain embodiments, the CpG-reduced nucleic acid variant, vector, expression vector / recombinant vector (e.g., rAAV), or recombinant viral particle encoding FVIII achieves a therapeutic effect without immunosuppressive agents. The therapeutic effect of FVIII optionally persists without administration of immunosuppressive agents for a period of, for example, 2 to 4, 4 to 6, 6 to 8, 8 to 10, 10 to 14, 14 to 20, 20 to 25, 25 to 30, or 30 to 50 days or more, for example, 50 to 75, 75 to 100, 100 to 150, or 150 to 200 days or more. Thus, in certain embodiments, the CpG-reduced nucleic acid variant, vector, expression vector / recombinant vector (e.g., rAAV), or recombinant viral particle encoding FVIII achieves a therapeutic effect without administration of immunosuppressive agents for a period of time.

[0158]

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

[0159]

[0175] In certain embodiments, the immunosuppressant is an anti-inflammatory agent. In certain embodiments, the immunosuppressant is a steroid. In certain embodiments, the immunosuppressant is a cyclosporine (e.g., cyclosporine A), mycophenolate, rituximab, or a derivative thereof. Additional specific agents include stabilizing compounds.

[0160]

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

[0161]

[0177] Factor VIII, alone or in combination with other agents, can be administered to, contacted with, or directly infused into a patient in a suitable biological carrier as described herein. To achieve, and optionally maintain for a period of time, prophylactically and / or therapeutically effective levels of FVIII polypeptides, vectors and expression vectors of the invention comprising CpG-reduced nucleic acid variants encoding FVIII can be administered to a patient by a variety of means. Those skilled in the art can readily determine specific protocols for using expression vectors of the invention encoding FVIII for therapeutic treatment of a particular patient.

[0162]

[0178] Protocols for the production of adenoviral vectors and their administration to patients are described in U.S. Patent Nos. 5,998,205; 6,228,646; 6,093,699; and 6,100,242, which are incorporated herein by reference in their entireties, and International Patent Applications WO 94 / 17810 and WO 94 / 23744. AAV vectors, for example, are employed, among others, to deliver Factor VIII (FVIII) encoded by CpG-reduced nucleic acid variants to patients in need thereof.

[0163]

[0179] Factor VIII (FVIII) encoded by a CpG-reduced nucleic acid variant delivered by an AAV vector of the invention can be administered to a patient by any means known in the art.

[0164]

[0180] The methods and uses of the present invention include delivery and administration systemically, regionally, or locally, or by any route, for example, by injection or infusion. In vivo delivery of pharmaceutical compositions can generally be achieved via injection using a conventional syringe, although other delivery methods, such as convection-enhanced delivery, are envisioned (see, e.g., U.S. Pat. No. 5,720,720). For example, compositions may be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intravenously, intrapleurally, intraarterially, orally, intrahepatically, via the portal vein, or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal application. Clinicians specializing in the treatment of patients with blood clotting disorders can determine the optimal route for administration of adenovirus-associated vectors containing CpG-reduced nucleic acid variants encoding FVIII based on several criteria, including, but not limited to, the patient's condition and the goal of treatment (e.g., enhanced or reduced blood clotting).

[0165]

[0181] The methods and uses of the invention can be combined with any compound, agent, drug, treatment, or other therapeutic regimen or protocol that has a desired therapeutic, beneficial, additional, synergistic, or complementary activity or effect. Exemplary combination compositions and treatments include second active agents, such as biologics (proteins), pharmaceuticals (e.g., immunosuppressants), and drugs. Such biologics (proteins), pharmaceuticals, drugs, treatments, and therapies can be administered or performed before, substantially contemporaneously with, or after any other method or use of the invention, such as a therapeutic method for treating a subject for a blood clotting disorder, such as HemA.

[0166]

[0182] The compound, agent, drug, treatment, or other therapeutic regimen or protocol can be administered as a combined composition, or can be administered separately, e.g., simultaneously, sequentially, or consecutively (before or after) delivery or administration of the nucleic acid, vector, recombinant vector (e.g., rAAV), or recombinant viral particle. Thus, the present invention provides combinations in which the methods or uses of the present invention are combined with any compound, agent, drug, therapeutic regimen, treatment protocol, step, therapeutic agent, or composition described herein or known to those of skill in the art. The compound, agent, drug, therapeutic regimen, treatment protocol, step, therapeutic agent, or composition can be administered or performed before, substantially contemporaneously with, or after administration of the nucleic acid, vector, recombinant vector (e.g., rAAV), or recombinant viral particle of the present invention to a subject.

[0167]

[0183] The present invention is useful for humans and animals, including veterinary applications. Accordingly, suitable subjects include mammals, such as humans and non-human mammals. The term "subject" refers to animals, generally mammals, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), livestock animals (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetal, neonatal, infant, juvenile, and adult subjects. Subjects also include animal disease models, e.g., mouse and other animal models of blood clotting disorders, such as HemA and others known to those skilled in the art.

[0168]

[0184] Suitable subjects for treatment according to the present invention include those at risk of having or producing an insufficient amount of a functional gene product (e.g., FVIII protein), or those with a deficiency thereof, or those producing an abnormal, partially functional, or non-functional gene product (e.g., FVIII protein) that may lead to disease. Suitable subjects for treatment according to the present invention also include those at risk of having or producing an abnormal or defective (mutant) gene product (protein) that leads to disease, such that reducing the amount, expression, or function of the abnormal or defective (mutant) gene product (protein) leads to treatment of the disease or reduces one or more symptoms or ameliorates the disease. Thus, target subjects include those with abnormal, insufficient, or absent blood clotting factor production, such as those with hemophilia (e.g., hemophilia A).

[0169]

[0185] Subjects can be tested for immune responses, such as antibodies against AAV. Thus, candidate hemophilia subjects can be screened before treatment with the methods of the present invention. Subjects can be tested for antibodies against AAV after treatment, and optionally monitored for a period of time after treatment. Subjects that develop antibodies can be treated with immunosuppressants or administered one or more additional doses of AAV vectors.

[0170]

[0186] Suitable subjects for treatment according to the present invention include those who have or are at risk of developing antibodies to AAV. AAV vectors can be administered or delivered to such subjects using several techniques. For example, empty capsid AAV (i.e., AAV lacking FVIII nucleic acid) can be delivered to transform the subject's cells with an AAV vector containing a CpG-reduced nucleic acid variant encoding FVIII by binding to AAV antibodies in the subject. The dosage of empty capsid AAV can be calibrated based on the amount of AAV antibody produced in a particular subject. The empty capsid can be any AAV serotype, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8.

[0171]

[0187] Alternatively or additionally, AAV vectors can be delivered by direct intramuscular injection (e.g., into one or more slow-twitch muscle fibers). In another alternative, AAV vectors can be delivered to the liver via the hepatic artery using a catheter introduced into the femoral artery. Non-surgical procedures, such as endoscopic retrograde cholangiopancreatography (ERCP), can also be employed to bypass the bloodstream and AAV antibodies by delivering AAV vectors directly to the liver. Other ductal systems, such as the submandibular duct, can also be used as entry points for delivering AAV vectors to subjects with developing or pre-existing anti-AAV antibodies.

[0172]

[0188] Administration or in vivo delivery to a subject can occur prior to the onset of adverse symptoms, conditions, complications, etc. caused by or associated with the disease. For example, screening (e.g., genetic) can be used to identify such subjects as candidates for the compositions, methods, and uses of the invention. Thus, such subjects include those who screen positive for an insufficient amount or deficiency of a functional gene product (e.g., FVIII protein), or those who produce an abnormal, partially functional, or non-functional gene product (e.g., FVIII protein).

[0173]

[0189] Administration to a subject or in vivo delivery according to the methods and uses of the invention as disclosed herein can be performed within 1 to 2, 2 to 4, 4 to 12, 12 to 24, or 24 to 72 hours after the subject is identified as having the disease targeted for treatment, after the subject has one or more symptoms of the disease, or after the subject is screened and identified as positive as described herein despite not having one or more symptoms of the disease. Of course, the methods and uses of the invention can be performed 1 to 7, 7 to 14, 14 to 21, 21 to 48, or more days, months, or years after the subject is identified as having the disease targeted for treatment, after the subject has one or more symptoms of the disease, or after the subject is screened and identified as positive as described herein.

[0174]

[0190] As used herein, a "unit dosage form" refers to a physically discrete unit suitable as a unitary dosage for a subject to be treated, each unit containing a predetermined amount, optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), calculated to produce a desired effect (e.g., a prophylactic or therapeutic effect) when administered one or more times. Unit dosage forms may be, for example, in ampoules and vials and may contain liquid compositions, or freeze-dried or lyophilized compositions; e.g., sterile liquid carriers that can be added prior to in vivo administration or delivery. Individual unit dosage forms may be included in multi-dose kits or containers. Recombinant vector (e.g., rAAV) sequences, recombinant viral particles, and pharmaceutical compositions thereof can be packaged in single-dose or multi-dose unit dosage forms for ease of administration and uniformity of dosage.

[0175]

[0191] To determine whether a subject is suitable for treatment with the methods of the present invention, such a subject can be tested for FVIII amount or FVIII activity. Candidate hemophilia subjects can be tested for FVIII amount or activity before treatment with the methods of the present invention. Subjects can be tested for FVIII amount or FVIII activity after treatment with the methods of the present invention. Such treated subjects can be monitored for FVIII amount or FVIII activity periodically after treatment, for example, every 1 to 4 weeks or every 1 to 6 months.

[0176]

[0192] Such subjects can be tested for one or more liver enzymes for adverse reactions or to determine whether the subject is suitable for treatment with the methods of the present invention. Thus, prior to treatment with the methods of the present invention, candidate hemophilia subjects can be screened for the amount of one or more liver enzymes. After treatment with the methods of the present invention, subjects can also be tested for the amount of one or more liver enzymes. Such treated subjects can be monitored periodically after treatment for elevated liver enzymes, for example, every 1 to 4 weeks or every 1 to 6 months.

[0177]

[0193] Exemplary liver enzymes include alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), although other enzymes indicative of liver damage can also be monitored. Normal levels of these enzymes in the circulation are generally defined as ranges with upper levels above which the enzyme levels are considered elevated and thus indicate liver damage. The normal range depends in part on the standard used by the clinical laboratory performing the assay.

[0178]

[0194] Such subjects can be monitored for bleeding episodes to determine whether they are eligible or responsive to treatment and / or to determine the amount or duration of response. Such subjects can be monitored for bleeding episodes to determine whether they require additional treatment, such as subsequent administration of an AAV vector or administration of an immunosuppressant, or more frequent monitoring. Thus, hemophilic subjects can be monitored for bleeding episodes before and after treatment with the methods of the invention. Subjects can also be tested for the frequency and severity of bleeding episodes during or after treatment with the methods of the invention.

[0179]

[0195] The present invention provides kits having packaging material and one or more components therein. The kits generally include a label or package insert containing a description of the components or instructions for in vitro, in vivo, or ex vivo use of the components therein. The kits can contain a collection of such components, such as a nucleic acid, a recombinant vector, a viral (e.g., AAV) vector, or a viral particle, and optionally a second active agent, such as another compound, agent, drug, or composition.

[0180]

[0196] A kit refers to a physical structure containing one or more components of the kit. The packaging can maintain the sterility of the components and can be made of materials commonly used for such purposes (e.g., paper, cardboard, glass, plastic, film, ampoules, vials, tubes, etc.).

[0181]

[0197] The label or package insert may include the identity of one or more components therein, the clinical pharmacology of the active ingredient(s), including dosage, mechanism of action, pharmacokinetics, and pharmacodynamics. The label or package insert may include information identifying the manufacturer, lot number, location and date of manufacture, and expiration date. The label or package insert may include information identifying the manufacturer's information, lot number, location and date of manufacture. The label or package insert may include information regarding the disease for which the components of the kit may be used. The label or package insert may include instructions for a clinician or subject to use one or more of the components of the kit in a method, use, or treatment protocol or therapeutic regimen. The instructions may include dosage, frequency, or duration, and instructions for practicing any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0182]

[0198] The label or package insert can include information regarding any benefit the components may provide, such as a prophylactic or therapeutic benefit. The label or package insert can include information regarding potential adverse side effects, complications, or reactions, such as alerting the subject or clinician regarding situations in which it may be inappropriate to use a particular composition. Adverse side effects or complications can also occur if the subject has received, will receive, or is currently receiving one or more other medications that may be incompatible with the composition, or if the subject has received, will receive, or is currently receiving another treatment protocol or therapeutic regimen that is expected to be incompatible with the composition; therefore, the instructions can include information regarding such incompatibilities.

[0183]

[0199] A label or package insert includes "printed matter," e.g., paper or cardboard, that is separate from or affixed to a component, kit, or packaging material (e.g., a box), or that is associated with an ampoule, tube, or vial containing a component of the kit. A label or package insert may additionally include a computer-readable medium, such as a bar-coded label, a disk, an optical disk such as a CD- or DVD-ROM / RAM, a DVD, an MP3, a magnetic tape, or an electronic storage medium such as RAM and ROM, or a hybrid thereof, such as a magnetic / optical storage medium, FLASH media, or a memory card.

[0184]

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

[0185]

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

[0186]

[0202] Various terms relating to the biological molecules of the present invention are used herein above and throughout the specification and claims.

[0187]

[0203] All of the features disclosed herein can be combined in any combination. Each feature disclosed herein can 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, vectors, plasmids, expression / recombinant vector (e.g., rAAV) sequences, or recombinant viral particles) are examples of classes of equivalent or similar features.

[0188]

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

[0189]

[0205] All numerical values ​​or ranges used herein include integers within such ranges and fractions of values ​​or integers within the ranges unless the context clearly dictates otherwise. Thus, by way of example, reference to 80% or greater identity includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, etc., and 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc.

[0190]

[0206] Reference to an integer greater than or less than includes any number greater than or less than, respectively, the referenced number. Thus, for example, reference to less than 100 includes 99, 98, 97, etc. all the way up to the number 1; less than 10 includes 9, 8, 7, etc. all the way up to the number 1.

[0191]

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

[0192]

[0208] Reference to a series of ranges includes ranges that combine the boundary values ​​of different ranges in the series. Thus, to illustrate a reference to a series of ranges, for example, 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 75, 75 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 400, 400 to 500, 500 to 750, 750 to 850 includes 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 10 to 30, 10 to 40, 10 to 50, 10 to 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.

[0193]

[0209] The present invention is generally disclosed herein using affirmative language to describe numerous embodiments and aspects. The present invention specifically includes embodiments in which certain subject matter, such as substances or materials, methods, steps and conditions, protocols, or procedures, is completely or partially excluded. For example, certain embodiments or aspects of the present invention exclude substances and / or method steps. Thus, the present invention is disclosed herein even in aspects that are not expressly excluded by the present invention, although the present invention is not generally expressed herein as not including such.

[0194]

[0210] Several embodiments of the present invention have been described. However, those skilled in the art can make various changes and modifications of the invention to adapt it to various uses and conditions without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate, but not in any way limit, the scope of the invention as claimed. [Example]

[0195] Example 1

[0211] Gene constructs for use in gene therapy for treating hemophilia are disclosed herein. In addition, these gene constructs encoding Factor VIII may be useful in vitro for setting up protein expression systems to produce recombinant FVIII protein for administration. Each gene construct may optionally include one or more of expression control (e.g., promoter) elements, the Factor VIII gene, and other regulatory features required for gene expression, such as introns, ITRs, stop codons, polyA signals, etc.

[0196] Example 2 CpG-reduced Factor VIII DNA sequence and certain vector constructs, plasmid constructs and AAV vector-producing cell lines

[0212] Eighteen different CpG-reduced nucleic acid variants encoding FVIII (SEQ ID NOs: 1-18) were produced and evaluated in expression assays. A mutant transthyretin (TTRmut) promoter (SEQ ID NO: 22) was used to generate a CpG-reduced human FVIII cDNA construct.

[0197]

[0213] The AAV-SPK-8011 expression cassette contains a CpG-reduced FVIII-X07 nucleic acid sequence and an LK03 capsid for packaging, which shares substantial homology with AAV3, a non-pathogenic, naturally replication-defective single-stranded DNA virus.

[0198]

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

[0199]

[0215] The cloned DNA used for gene transfer is a gene expression cassette encoding human coagulation factor VIII (hFVIII) under the control of a liver-specific promoter, packaged as a single-stranded genome in an AAV-LK03 capsid. This expression plasmid is called pAAV-TTRmut-hFVIII-X07. The expression plasmid was modified by introducing four point mutations into the TTR promoter, optimizing the coding region and increasing the expression of human FVIII. The AAV expression cassette contains the following elements: AAV2 ITR Transthyretin (TTR) promoter: A liver-specific transthyretin (TTR) promoter with four point mutations that increase 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: B-domain deleted and codon-optimized human FVIII coding sequence.

[0200] Rabbit beta globin poly A signal sequence (Levitt et al., 1989).

[0201] AAV2 ITR

[0216] Three DNA plasmid constructs are used to transfect human embryonic kidney 293 cells to produce the SPK-8011 vector by a helper virus-free process (Matsushita et al., 1998): The gene cassette (hFVIII coding sequence and associated regulatory elements) is cloned into a plasmid, giving rise to the vector plasmid pAAV-TTRmut-hFVIII-X07.

[0202] The AAV viral genome (rep and cap) lacking the viral ITRs was cloned into a plasmid to give the AAV packaging plasmid pLK03, which provides in trans the AAV2 rep gene and the AAV-LK03 cap gene required for packaging of AAV vectors. To prevent the formation of replication-competent AAV by non-homologous recombination, the viral promoter for the rep gene (p5) was relocated into the plasmid.

[0203] Three genes from adenovirus-2 are cloned into a third plasmid (pCCVC-AD2HP) to provide the helper virus genes necessary for vector production. Plasmid pCCVC-AD2HPv2 is an 11,832 bp plasmid construct carrying the RNAs of three adenovirus genes, E2A, E4, and VA, to provide the "helper" functions necessary for AAV vector replication and encapsidation. Plasmid pCCVC-AD2HPv2 is a derivative of pCCVC-AD2HP, and in this plasmid, Amp R The 1882 bp DrdI-DrdI restriction fragment containing the gene and part of the pUC ori sequence was removed and transformed into Kan R It has been replaced by a DrdI-DrdI fragment from plasmid pAAV2-hRPE65v2, which contains the entire gene and part of the pUC ori sequence.

[0204]

[0217] The cell substrate used for AAV vector production is a derivative of primary human embryonic kidney (HEK) 293 cells. The HEK293 cell line is a permanent line transformed with sheared human adenovirus type 5 (Ad5) DNA (Graham et al., 1977). The working cell bank was derived from a characterized HEK293 master cell bank from the Center for Cellular and Molecular Therapeutics (CCMT) at Children's Hospital of Philadelphia (CHOP).

[0205] Example 3 Study of AAV-hFVIII vectors in mice.

[0206]

[0218] A FVIII transgene construct (hFVIII) was packaged into an adeno-associated virus (AAV) vector and delivered to mice. Briefly, 8-10 week old hemophilia A / CD4 mice were treated with IVFVIII. - / - Mice, groups of four, received 4 × 10 AAV-hFVIII vectors 12 Immunodeficient mice were used to allow quantification of plasma levels of FVIII, since inhibitory antibodies against FVIII that form in normal mice would prevent long-term analysis of FVIII expression.

[0207]

[0219] The expression levels of FVIII were determined, and in some cases, the expression levels were higher than those mediated by the CO3 sequence (SEQ ID NO: 21) encoding hFVIII. As shown in Figure 2, vectors containing AAV-SPK-8005 expressed higher levels of hFVIII compared to the reference AAV-CO3 vector. This data surprisingly reveals that some of the DNA sequences expressed higher levels of FVIII than the codon-optimized sequence encoding FVIII (CO3, SEQ ID NO: 21).

[0208]

[0220] AAV-Spark8005 (also called SPK-8005) rather than AAV-LK03-hFVIII (also called AAV-LK03-hFVIII and SPK-8011) was used in this study to ensure efficient transduction of mouse hepatocytes (i.e., expression of the hFVIII transgene). Therefore, this study was designed to investigate the safety of sustained expression of hFVIII, rather than the safety of the AAV-LK03 capsid.

[0209]

[0221] Three doses of AAV-SPK-8005-hFVIII were used to generate hFVIII antigen levels of approximately 5–25, 25–75, and 50–150%, respectively (4 × 10 10 , 8×10 10 , 1.6×10 11 vg / mouse; approximately 1.6 × 10 based on a mouse weight of 25 g 12 , 3.2 × 1012 , 6.4×10 12 vg / kg). The study included 350 male NOD / SCID mice (Table 1) and was divided into two substudies: the main study (n=270) and the bioanalytical study (n=80). In the main study, 60 mice were treated with either vehicle or one of three doses of vector (4×10 10 , 8×10 10 , 1.6×10 11 vg / mouse). Ten mice were used for clinical chemistry evaluation on days 29 / 30, 10 for hematology, and the remaining 10 animals underwent coagulation evaluation. These 30 mice were sacrificed on days 29 or 30. Another group of 30 mice treated with either vehicle or one of the three vector doses was handled similarly at day 87 and sacrificed on day 87. At termination, gross pathology observations were performed on all animals in the main study, and comprehensive histopathology was performed on 10 animals / cohort per time point (a subset of hematology). A separate cohort of 30 naive mice was used for background control clinical pathology measurements.

[0210]

[0222] For bioanalysis studies, 20 mice were injected with vehicle or one of three vector doses. Blood was collected from these animals serially before injection of the test article and on days 15, 30, 60, and 87. The planned plasma volume collected for each sample was intended to be sufficient for determination of both hFVIII antigen and D-dimer levels. However, due to insufficient plasma volume collected, only one assay was performed on individual mouse plasma at all time points except the last. Thus, some mice were examined for circulating hFVIII antigen levels, while others were examined for D-dimer levels. Because a larger volume of plasma is required to perform the hFVIII ELISA (minimum 50 μL) than the D-dimer ELISA (minimum 20 μL), the volume of collected plasma determined the assay chosen.

[0211] [Table 1]

[0212]

[0223] Plasma FVIII antigen levels: As shown in Figures 3A-3B, a dose-response in circulating levels of hFVIII antigen was observed over 87 days. 10 In the 100-hFVIII (vg / mouse) study, mean hFVIII levels of 64±49 ng / ml were observed 60 days after injection, with 115±60 ng / ml and 273±58 ng / ml observed at the mid- and high-dose levels, respectively. These antigen levels correspond to 43, 77, and 182% of normal hFVIII antigen (150 ng / ml is equivalent to 100%). Therefore, in NOD / SCID mice with normal hemostasis, total (mouse + human) FVIII levels of 143%, 177%, and 282% of those expected at the three dose levels, respectively. Thus, sustained, supraphysiological levels of hFVIII were observed in the plasma of immunodeficient mice using AAV-SPK-8005-hFVIII, making this study suitable for evaluating the safety of long-term expression of hFVIII.

[0213]

[0224] D-dimer levels: To assess the thrombotic potential of persistent hFVIII expression in hemostatically normal but immunocompromised mice, D-dimer antigen levels were measured. The mean pre-dose D-dimer level among 50 naive mice was 8.8 ± 2.9 ng / ml. Data in Figure 3C represent the mean D-dimer levels in the four dose cohorts. There was no statistical difference in D-dimer levels between cohorts at all five time points (one-way ANOVA p = 0.46). We concluded that persistent hFVIII expression for at least 87 days at levels as high as 194% of normal (day 30) in this mouse strain was not associated with elevated D-dimer levels.

[0214]

[0225] Clinical Anatomopathology: Nine animals were euthanized early or found dead during the course of the study (six in the main study and three in the bioanalytical study).

[0215]

[0226] Six animals in the main study were examined histopathologically, and malignant lymphomas were observed in four of these six mice, including one mouse injected with the vehicle control (animal 7729 in group 1, animal 7871 in group 3, animal 7880 in group 3, and animal 7874 in group 3). The biological significance of the tumor findings was considered unclear. Statistical significance of individual group comparisons with the control group was considered unlikely. A high spontaneous frequency of thymic lymphomas and neoplastic extension of the spleen and lymph nodes is known to occur in this strain (Prochazka, Gaskins, Shultz, & Leiter, 1992).

[0216]

[0227] No test article-related non-neoplastic findings were present in these six mice. The microscopic findings observed were considered incidental, of a nature normally observed in mice of this strain and age, and / or were of similar incidence and severity in control and treated animals and therefore not related to AAV-SPK-8005-hFVIII administration.

[0217]

[0228] The remaining 234 mice included in the main study survived to the planned time point. No adverse or AAV-SPK-8005-hFVIII-related clinical observations were observed in the mice throughout the study. All clinical observations, including crusting, alopecia or thinning hair cover, and curled tail, were considered unrelated to AAV-SPK-8005-hFVIII administration because they were common in this mouse species and / or occurred across groups. Body weight and weight gain were similar between dose groups and were not affected by AAV-SPK-8005-hFVIII administration. The seemingly significant (p<0.05 or p<0.01) decrease in mean body weight in Group 4 from Day 32 to the end of the study was likely due to a redistribution of group weight after euthanasia on Day 29 / 30 (some heavier animals were euthanized compared to Group 1) and was not related to AAV-SPK-8005-hFVIII administration. Mice in Group 4 gained weight similarly to the other groups throughout the study. All other significant differences (p<0.05 or p<0.01) in mean body weight or weight gain were not considered related to AAV-SPK-8005-hFVIII, as gains and losses were sporadic, not dose-dependent, and were considered related to normal fluctuations in mouse weight.

[0218]

[0229] Clinical pathology was performed on animals in the main study. Clinical chemistry parameters were analyzed for 10 mice / cohort per time point (days 29 / 30 and 87). Coagulation assessments were performed on a separate group of 10 mice / cohort, and hematology measurements were performed on the remaining group of 10 mice / cohort. Gross pathology was performed on all animals, and histopathology was performed on the group of 10 mice used for hematology evaluation. There were no AAV-SPK-8005-hFVIII-related changes in hematology or clinical chemistry parameters in mice from either the euthanasia timepoints on days 29 / 30 and 87. In general, when significant differences (p<0.05 or p<0.01) were present in hematology and clinical chemistry parameters compared with control values, these differences were not associated with AAV-SPK-8005-hFVIII because the corresponding parameters were not affected and the observations were not dose-dependent. All changes in clinical chemistry and hematology parameters were sporadic, attributed to a single animal, were of a magnitude commonly observed in experimental animals, and / or fell within the clinical pathology parameters evaluated in naive animals.

[0219]

[0230] Changes in coagulation parameters were observed in mice administered AAV-SPK-8005-hFVIII. A dose-dependent decrease in mean aPTT was observed at day 29 / 30, with values ​​for groups 3 and 4 significantly different from control values ​​(p<0.05 or p<0.01). A significant decrease in mean aPTT values ​​(p<0.01) was also observed in all AAV-SPK-8005-hFVIII groups compared to the control group at day 87. A decrease in mean prothrombin time was also observed in the AAV-SPK-8005-hFVIII groups compared to the control group on days 29 / 30 and 87, but this decrease was only statistically significant for groups 2 and 3 on days 29 / 30 and for group 4 on day 87 (p<0.05 or p<0.01). Mean fibrinogen values ​​were similar between dose groups throughout the study. These effects are likely related to the pharmacological action of AAV-SPK-8005-hFVIII and are not considered adverse. As shown in Figures 3A-3C and described above, all mice injected with AAV-SPK-8005-hFVIII expressed the hFVIII antigen, so supraphysiological levels of total FVIII are expected to circulate in the plasma of these hemostatically normal mice. These levels are expected to have an effect on coagulation parameters such as decreased aPTT and prothrombin time.

[0220]

[0231] Groups of 120 mice (30 per cohort) in the main study were sacrificed on days 29 or 30 of the study. No gross pathology observations related to AAV-SPK-8005-hFVIII were made in these mice. Organ weight analysis revealed that absolute heart and kidney weights differed between the 10 control and vector-injected animals sacrificed on day 29; however, this was not observed between the 10 control and vector-injected animals sacrificed on day 30, so the significance of this finding is unclear. There was no microscopic correlation with the statistically significant increase in absolute heart and kidney weights (and these weights as a percentage of brain weight) observed on day 29. Furthermore, heart and kidney weights as a percentage of body weight were not significantly different from controls. There was a significant increase in mean absolute lung weight in Group 2 animals; however, this was not dose-dependent and was therefore considered incidental and unrelated to AAV-SPK-8005-hFVIII. No other organ weight changes were observed on days 29 / 30.

[0221]

[0232] When histopathological analysis was performed on days 29 / 30, five animals had tumor findings. Bronchioloalveolar adenocarcinoma was observed in one animal (7824) in group 2. Malignant lymphoma was observed in one animal (7838) in group 2, one animal (7885) in group 3, and one animal (7941) in group 4. An adenoma was observed in the stomach of one animal (7942) in group 4. No tumor findings were observed in group 1. The biological significance of the tumor findings is considered unclear. Statistical significance of individual group versus control comparisons is unlikely. However, it is noteworthy that tumor findings were observed only in treated animals on days 29 / 30. In the absence of historical control data for NOD SCID mice of similar age, these tumor findings are uncertain.

[0222]

[0233] Non-neoplastic microscopic findings related to the test article were observed and were considered incidental in nature and commonly observed in this strain and age of mice, and / or were considered unrelated to AAV-SPK-8005-hFVIII administration, as they were of similar incidence and severity in control and treated animals.

[0223]

[0234] Another group of 120 main study mice (30 mice / cohort) was sacrificed 87 days after injection and analyzed in a similar manner. No gross pathological findings considered related to AAV-SPK-8005-hFVIII were observed, although lesions were observed in four mice (1 enlarged thymus not histologically analyzed, 1 enlarged thymus correlated with malignant lymphoma, 1 enlarged spleen not histologically analyzed, and 1 discolored testis). In contrast to those observed on days 29 and 30, decreased heart weight and no increase in body weight were observed. In addition, decreased liver weight was observed. Statistically significant changes in heart weight were small and not clearly related to dose. Statistically significant changes in absolute liver weight were small, and liver weight relative to body and brain weight was similar between groups. Therefore, the minor changes were interpreted as incidental and unrelated to AAV-SPK-8005-hFVIII administration. No changes in other organ weights were observed on day 87.

[0224]

[0235] Histopathological examination of the mice 87 days after injection identified four animals with tumor findings. Malignant lymphoma was observed in one animal (7808) in Group 2 and three animals (7868, 7869, and 7870) in Group 3. No tumor findings were observed in Group 1. The biological significance of the tumor findings was considered unclear. Statistical significance of individual group versus control comparisons is unlikely. However, it is noteworthy that tumor findings were observed in treated animals only at day 87. In the absence of historical control data for NOD SCID mice of similar age, these tumor findings are inconclusive.

[0225]

[0236] Regarding non-neoplastic changes, microscopic findings unrelated to the test article were observed and were considered incidental in nature and normal for this strain and age of mice and / or unrelated to AAV-SPK-8005-hFVIII administration due to similar incidence in control and treated animals.

[0226]

[0237] Conclusion: Dose 4×10 10 , 8×1010 , or 1.6×10 11 A single intravenous injection of 1000 mg / mouse of AAV-SPK-8005-hFVIII or control product into male NOD / SCID mice was well tolerated. AAV-SPK-8005-hFVIII did not result in any test-product-related deaths, adverse clinical observations, or changes in body weight. There were no toxicologically significant differences in organ weights, hematology, or coagulation parameters in male mice at days 29 / 30 or 87, and no treatment-related gross or histopathological findings. The decreases in mean aPTT and prothrombin time observed at both euthanasia timepoints were likely related to the supraphysiological levels of FVIII expressed in these hemostatically normal mice and were not adverse. During the main study (terminal phase), malignancies were observed in 9 of 60 vector-injected mice, or 15% of the animals. Seven of these 9 mice had lymphoma, which was most commonly found in the lymph nodes. This immunodeficient mouse strain is known to have a high spontaneous incidence of lymphoma (Prochazka et al., 1992) and a lifespan of only 8.5 months. Therefore, the tumor incidence observed in this study is unlikely to be related to administration of AAV-SPK-8005-hFVIII. The purpose of this study was to investigate the safety of persistent expression of hFVIII over a period of approximately 3 months. This study was not designed to investigate the AAV-SPK capsid. AAV-SPK and an immunodeficient mouse strain were used to ensure high-level expression of hFVIII. Administration of AAV-SPK-8005-hFVIII to NOD / SCID mice resulted in persistently high levels of hFVIII. Therefore, this study was appropriate to evaluate the safety of long-term expression of hFVIII.

[0227] Example 4 Study of AAV-SPK-8005 and AAV-SPK-8011 (LK03 capsid, FVIII-X07 (SEQ ID NO: 7)) vectors in non-human primates (NHPs).

[0228]

[0238] Based on the results in mice, FVIII transgene constructs packaged in adeno-associated virus (AAV) vectors were delivered to non-human primates (NHPs).

[0229]

[0239] Briefly, a single intravenous infusion of AAV-SPK-8005 or AAV-SPK-8011 (LK03 capsid) was performed in a dose-ranging study in male cynomolgus monkeys. hFVIII expression was studied over an 8-week period. The respective animal groups and dose levels are shown in Figure 4.

[0230]

[0240] NHPs received intravenous infusions over approximately 30 minutes via the saphenous vein using a calibrated infusion pump. Macaques were prescreened for neutralizing antibodies against the AAV capsid. All treated animals were initially determined to have titers of <1:3 prior to vector administration. This was done to ensure successful liver transduction, as even low titers inhibit vector uptake by liver cells after systemic delivery (Jiang et al., 2006). All animals were negative for the presence of neutralizing antibodies against FVIII prior to gene transfer.

[0231]

[0241] Plasma levels of hFVIII were measured by a human-specific ELISA, which does not detect endogenous FVIII in cynomolgus macaques. All animals in this study, except for one macaque in the mid-dose cohort, expressed hFVIII after vector delivery. Human factor VIII antigen levels peaked approximately 1–2 weeks after vector administration. One week after gene transfer, 2 × 10 AAV-SPK-8005 12 NHPs transduced with 1000 vg / kg expressed hFVIII antigen levels of 13.2 ± 3% (mean ± standard error of the mean). One week after gene transfer, the next treatment cohort (5 × 10 12At the highest tested dose of 1 × 10, the mean hFVIII level in two of three animals was 27 ± 0.2%. No human FVIII could be detected in a third macaque from the cohort at any time point. Retesting of the baseline plasma sample determined that this animal was in fact positive for the presence of anti-AAV antibodies, rendering the initially determined titer of <1:3 inaccurate. Finally, at the highest tested dose of 1 × 10, 13 vg / kg, a peak hFVIII antigen level of 54.1 ± 15.6% was observed after AAV infusion.

[0232]

[0242] As expected from studies in NHPs expressing human FIX, human FVIII expression declined in approximately one-third of the animals by approximately four weeks. This is consistent with the appearance of inhibitory antibodies to hFVIII in three of these macaques (marked with the symbol ε in Figure 5). The development of species-specific antibodies to hFVIII has previously been described in nonhuman primates and may occur due to differences in several amino acid residues between the human transgene product and endogenous cynomolgus monkey FVIII (McIntosh, J. et al., Blood 121:3335-44 (2013)).

[0233]

[0243] To assess the potential thrombogenicity of continuous expression of human FVIII, D-dimer antigen levels were measured in this study. It should be noted that there are few reports relating to the clinical relevance or even normal values ​​of D-dimer antigen levels in cynomolgus monkeys; for reference, the normal range of D-dimer in humans is less than 500 ng / ml. Because animals express endogenous cynomolgus monkey FVIII, hFVIII production as a result of liver gene transfer leads to supraphysiological levels of FVIII activity.

[0234]

[0244] 5×10 12Animals receiving a dose of 1000 vg / kg but not expressing human FVIII had a peak of 863 ng / ml 2 weeks after AAV infusion. The remaining animals did not show any significant increase in D-dimer antigen levels compared to baseline values. Together, these results suggest that expression of human FVIII at the levels targeted in these studies is not associated with an increased risk of thrombosis.

[0235]

[0245] Four weeks after vector administration, no vector-related changes were evident. Liver function tests were normal with small variations, but these variations appeared unrelated to vector dose, as they were present before dosing in most cases (Figure 6).

[0236]

[0246] D-dimer levels for up to 5 weeks are shown in Figure 7. One animal in the high-dose cohort had a small (577 ng / ml) transient increase in D-dimer levels 1 week after vector administration, when circulating human FVIII peaked at approximately 100%, and D-dimer levels rapidly returned to normal after this single elevated measurement. Of note, there was no correlation between D-dimer levels and hFVIII antigen levels (Figure 7, bottom panel).

[0237]

[0247] For the AAV-SPK-8011 (LK03 capsid) vector, three cohorts of cynomolgus monkeys (n=3) were administered escalating doses of AAV-SPK-8011 (LK03 capsid) (2 × 10 12 , 6×10 12 and 2 x 10 13 (vg / kg; Figure 4). Animals were monitored for clinical observations, body weight, and clinical pathology (clinical chemistry, hematology, coagulation, and urinalysis). In addition, hFVIII antigen levels, FVIII inhibitory antibody levels, and D-dimer levels were assessed throughout the study.

[0238]

[0248] hFVIII antigen data are shown in Figure 9. Mean hFVIII antigen levels peaked at approximately 2-3 weeks. Using 150 ng / ml as 100% normal hFVIII antigen level, 22.3 ± 6.2% hFVIII was observed in the low-dose cohort, 61.6 ± 15.7% and 153 ± 58.1% in the mid- and high-dose cohorts, respectively (Figures 9A-9D). Thus, the LK03 AAV capsid serotype efficiently transduces NHP hepatocytes in vivo, unlike mouse liver.

[0239]

[0249] The FVIII expression levels achieved with AAV-SPK-8011 (LK03 capsid) were compared with the reported levels of FVIII achieved with AAV vectors based on AAV5 and AAV8 capsids for FVIII delivery. The comparison revealed that the FVIII levels reached with AAV-SPK-8011 (LK03 capsid) were greater than the reported levels of FVIII delivered by AAV vectors with AAV5 and AAV8 capsids (Figure 10).

[0240]

[0250] AAV-SPK-8011 (LK03 capsid) at 2 × 10 12 , 6×10 12 or 2 x 10 13 Humoral responses to hFVIII were measured in the plasma of cynomolgus monkeys after administration of either 0.01 mg / kg or 0.01 mg / kg of hFVIII. Animals were assessed for anti-hFVIII IgG antibodies by ELISA at baseline and at the indicated time points.

[0241]

[0251] Despite the therapeutic hFVIII levels observed immediately after gene transfer, levels began to decline in most animals by approximately 4 weeks. This correlated with an increase in anti-hFVIII antibodies, consistent with previous studies using other AAV-hFVIII vectors. Others have also observed the generation of anti-FVIII antibodies after AAV-hFVIII gene transfer into the livers of NHPs (McIntosh, J. et al., Blood 121:3335-44 (2013)).

[0242] Example 5 Biodistribution of AAV-LK03 capsids in non-human primates (NHPs)

[0252] A non-GLP study investigated the biodistribution of AAV-LK03 capsids in non-human primates. Intravenous administration of an AAV-LK03-encapsid vector encoding human coagulation factor IX (AAV-LK03-hFIX) demonstrated that the two primary target tissues were the liver and spleen (Figure 11). Spleen tropism is not a unique feature of AAV-LK03. For example, the AAV5 capsid, which has been used in several liver-directed gene therapy trials (e.g., NCT02396342, NCT02082860, NCT02576795) and has a strong safety record, targets the spleen with equal, if not greater, efficacy than the liver in non-human primates (Paneda et al., 2013). The SPK-8011 expression cassette uses the mouse transthyretin or TTR promoter, which is thought to be liver-specific (Costa, 1991). To further support the liver specificity of the promoter, PCR-based expression analysis measured vector-derived FVIII expression in the liver and spleen of mice after administration of another AAV vector (i.e., AAV-SPK-8005) packaging the same expression cassette as SPK-8011. As shown in Figure 12, human FVIII expression in the spleen is several orders of magnitude lower than hepatocyte-derived expression.

[0243]

[0253] Although trials have been conducted with other AAV vectors, including several for hemophilia B (NCT02396342, NCT01620801, NCT00076557, NCT02484092, NCT02618915, NCT00979238, NCT01687608) and one for hemophilia A (NCT02576795), this is the first clinical trial using AAV-LK03. The trial, conducted by St. Jude Children's Research Hospital in collaboration with University College London, utilized scAAV2 / 8-LP1-hFIXco, an AAV8 vector with a self-complementary genome encoding a codon-optimized human factor IX cDNA. Ten subjects who received the vector had stable factor IX levels of 1-6% over a median period of 3.2 years, and all participants discontinued or reduced their use of prophylactic factor replacement (Nathwani et al., 2014). A clinical trial for hemophilia A used an AAV5 capsid-encapsulated vector (NCT02576795) encoding human FVIII. Preliminary data published in 2016 demonstrated that in several subjects, increases in FVIII activity after gene transfer ranged from 2-60% with up to 16 weeks of follow-up. (BioMarin, April 2016)

[0244] Example 6 Transduction efficiency of AAV-LK03 capsid analyzed in an in vitro setting

[0254] Primary hepatocytes of cynomolgus monkey and human origin were transduced with the AAV-LK03 vector expressing luciferase at four different multiplicities of infection (MOI), ranging from 500 to 62,500 vector genomes per cell. Luciferase expression was analyzed 72 hours after transduction.

[0245]

[0255] The AAV-LK03 capsid uniquely demonstrated significantly higher efficiency in transducing cultured human hepatocytes. In a representative example shown in Figure 13, LK03 was approximately five-fold more efficient in transducing human hepatocytes compared to non-human primate hepatocytes in vitro. Importantly, these results were consistent across multiple MOIs and replicates.

[0246] Example 7 Assessment of germline transmission of vector coding sequences

[0256] Evaluation of the germline transmission potential of vector-encoded sequences is important for the clinical translation of gene transfer strategies. This study was designed to: (1) examine the dissemination of AAV-SPK and AAV-LK03 in semen and determine the vector clearance kinetics; and (2) ensure that AAV administration to rabbits was successful, as confirmed by analysis of human factor IX antigen and anti-FIX antibodies in plasma.

[0247]

[0257] In this study, we used a rabbit model to analyze the dissemination of two vector capsids, AAV-SPK and AAV-LK03, into semen (Table 2). AAV-SPK dissemination into semen showed both dose- and time-dependent kinetics, with higher doses resulting in higher levels of vector sequences in semen for longer periods. The kinetics were very similar to those previously observed with AAV8 vectors (Favaro P et al., Molecular Therapy 17:1022-1030 (2009)). In contrast, AAV-LK03 vectors showed limited dissemination into semen. Because the levels of hFIX expressed from AAV-LK03 were similar to or higher than those observed with AAV-SPK vectors, and the ability to mediate liver-derived hFIX expression can be used as a surrogate for gene transfer, it is unlikely that this was due to lower overall vector exposure in mice injected with AAV-LK03.

[0248] [Table 2]

[0249] method

[0258] Animals and Vectors: New Zealand White rabbits were obtained from Covance Research Products (Denver, PA) and treated at 6 months of age with AAV vectors produced at the Children's Hospital of Philadelphia Vector Core. Test and control articles were administered via the marginal ear vein.

[0250]

[0259] Semen Collection: An artificial vagina (AV) developed by researchers at Argus Research Lab, Inc. (Horsham, PA) was used for semen collection. The AV was lined with a condom, its tip removed, a collection tube connected, and filled with warm water (55°C). Semen samples were obtained from experienced bucks stimulated by test bucks. Samples were collected before injection and at 1, 2, 4, 6, 8, and 10 weeks and 3 to 8 months after injection. Semen samples were shipped to Charles River Laboratories (Reno, NV) for vector copy number analysis using a validated real-time quantitative PCR assay.

[0251]

[0260] Blood sample collection: Blood was collected by medial auricular artery or marginal ear vein puncture before AAV administration and at multiple time points (before injection, 1 week, and 1–6 months after injection). After collection, each sample was placed on ice, processed to plasma, and stored in a −80°C freezer until shipped to the sponsor, where samples were also stored in a −80°C freezer until assayed.

[0252]

[0261] Human Factor IX Levels: Human FIX (hFIX) protein levels in rabbit plasma were quantified using a sandwich-format FIX ELISA kit (Affinity Biologicals, FIX:EIA) as follows: First, microtiter plate wells were coated with a capture antibody that recognizes hFIX and does not cross-react with endogenous rabbit FIX (1:1000 dilution). A standard curve was generated by diluting reference plasma with known human hFIX concentrations (the highest standard [500 ng / ml] was serially diluted down to 7.8 ng / ml). Sample plasma was diluted according to the expected concentration to ensure absorbance values ​​fell within the range of the standard curve. After adding samples to the wells, the plate was incubated for 90 minutes at room temperature and then washed three times. A horseradish peroxidase (HRP)-conjugated secondary antibody against hFIX was added to the plate to bind to the captured FIX (1:100 dilution). After washing the plate to remove unbound conjugated antibody, peroxidase activity was measured after incubation with 1-Step Ultra TMB substrate (Thermo Scientific, catalog no. 34028). The reaction was stopped with 1 M sulfuric acid and read using a SpectraMax M2e microplate reader with an absorbance setting of 450 nm. The resulting absorbance value is proportional to the concentration of hFIX present in the sample.

[0253]

[0262] Anti-hFIX antibody levels: The anti-hFIX assay is conceptually and methodologically similar to the above-described hFIX ELISA. Briefly, plates were coated with 1 μg / ml recombinant hFIX (Benefix, Wyeth). After incubation of plasma samples, a goat anti-rabbit IgG HRP-conjugated antibody (SIGMA, A4914) was used for detection. Samples with IgG levels twice the baseline reading were considered positive.

[0254] result Vector inoculation into semen

[0263] New Zealand rabbits were injected with AAV-SPK or AAV-LK03 (n=5 / group) vectors expressing hFIX under the control of the ApoE / hAAT liver-specific promoter at two doses: 1 × 10 12 vg / kg (low dose) or 1 × 10 13 The rabbits were injected at a dose of 1000 mg / kg (high dose). Semen samples were obtained from all rabbits before injection and at 1, 2, 4, 6, 8, and 10 weeks and 3 to 8 months after injection. Genomic DNA was purified from the semen samples and analyzed for the presence of hFIX sequences using a quantitative polymerase chain reaction (Q-PCR) assay. Charles River Laboratories (Reno, NV) developed the validated assay. Semen samples were considered positive if they had detectable hFIX levels above the lower limit of quantification (LLOQ) (approximately 200 ng / reaction, 10 copies / reaction or 50 copies / μg). Semen samples from rabbits that were negative for hFIX vector sequences at at least three consecutive time points were not further analyzed.

[0255]

[0264] Pre-treatment semen DNA from all vector- and vehicle-injected animals was negative for hFIX sequences. 12 Semen from rabbits injected with 1000 mg / kg of 1000 mg ... -2 All samples collected after 4 weeks were generally negative for hFIX sequences, except for the high-dose AAV-SPK-hFIX (1 × 10 copies / haploid genome), and none of the samples collected after 4 weeks were positive for vector sequences (Table 3). 13At the 10-fold higher dose (vg / kg), higher levels of vector were present (up to 178,352 copies / µg DNA or 0.59 copies / haploid genome) and clearance took longer, up to 5 months, in five animals (Table 3). With the exception of one animal (at week 1), rabbits treated with the low dose of AAV-LK03-hFIX showed no shedding of hFIX sequences into semen (Table 3). In addition, very little vector shedding into semen was observed at the 10-fold higher dose, with three animals completely lacking hFIX sequences at all time points and two animals showing low levels at week 2 (maximum: 392 copies / µg DNA or 1.3 x 10 -3 Copies / haploid genome) but not at subsequent time points (Table 3). Of the two vehicle-injected animals, one had spurious findings at 1 week (56 copies / μg DNA) and 5 months (96 copies / μg DNA). These values ​​are close to the LLOQ and most likely represent contamination during semen collection or DNA preparation steps.

[0256] [Table 3]

[0257] Plasma human FIX antigen levels

[0265] Circulating hFIX levels were measured in plasma samples from the animals at the indicated time points (FIGS. 14A-14B and Table 4).

[0258] [Table 4]

[0259]

[0266] In the low-dose cohort, the AAV-LK03 vector appeared to be a more potent vector than AAV-SPK, as measured by circulating hFIX levels. Six months after treatment with AAV-LK03 or AAV-SPK, mean hFIX levels were 552 ± 217 ng / ml versus 164 ± 45 ng / ml, respectively (Figure 14A). However, this difference did not reach statistical significance, likely due to the limited number of animals. Interestingly, no hFIX expression was detected 7 days after administration of the AAV-SPK vector, whereas strong expression was observed with AAV-LK03 at the same time point. The low hFIX levels, barely detectable above background in two of the animals (rabbits #5 and #15), may be due to injection failure. Excluding these animals from the analysis did not change the lack of statistical significance.

[0260]

[0267] The two capsids appeared to be equally potent when tested at high doses. 13 Six months after treatment with either AAV-LK03 or AAV-SPK at 1000 mg / kg, the mean hFIX levels were 2226 ± 868 ng / ml versus 2052 ± 909 ng / ml, respectively (Figure 14B). Of note, two different hFIX coding sequences were used in the AAV-SPK group: three animals received AAV-SPK-hFIX.C16 and two animals were treated with AAV-SPK-hFIX.C19-Padua (PD). The main difference between the hFIX.C16 and hFIX.C19-PD transgenes is that the latter encodes a codon-optimized, high-specific-activity hFIX variant, which affects the protein's biological activity but not its antigen levels as measured by ELISA.

[0261] Anti-FIX antibody

[0268] Based on reports by others, approximately 20-40% of animals were expected to develop antibodies to the human FIX vector (Favaro P et al., Molecular Therapy 17:1022-1030 (2009)). Figures 15A-15B and Tables 5A and 5B summarize the anti-AAV IgG levels in this study. Interestingly, although three of five animals treated with low-dose AAV-LK03 tested positive for human FIX antibodies one month after vector administration, IgG levels decreased over time, with only one animal barely doubling baseline levels at the end of the study (Table 5B). The kinetics of the appearance and future disappearance of anti-FIX IgG in this group of rabbits correlated well with the sharp decrease in hFIX levels observed at day 28, followed by a "rebound" of circulating hFIX (Figure 14A). Also, the high antibody titers against hFIX in the two animals treated with AAV-SPK-hFIX.C19-Padua may explain the low expression levels in these two rabbits.

[0262] [Table 5] [Table 6]

[0263] conclusion

[0269] Semen dissemination of AAV-SPK and AAV-LK03 vectors was quantified using a validated assay over a period of up to 8 months. AAV-SPK vector sequences were detected in the semen of a total of five rabbits 1 week after administration of the high vector dose. By week 10, sequences had disappeared from most animals, with the last detectable positive sample occurring at month 5. This is similar to the time course of AAV8 vectors administered to rabbits at the same vector dose (Favaro P et al., Molecular Therapy 17:1022-1030 (2009)). In contrast, very limited distribution of this vector was observed after the high dose of AAV-LK03, with three of five animals showing no vector sequences in semen at any time point. It is unlikely that lower semen dissemination was due to lower overall exposure of AAV-LK03 in rabbits. Confirmation that rabbits were successfully injected with each AAV vector was demonstrated by measuring plasma levels of hFIX, a surrogate indicator of gene transfer. 13 At 1000 mg / kg (vg / kg), similar circulating levels of hFIX were observed in animals injected with AAV-LK03 and AAV-SPK, demonstrating that these vectors are equally potent in mediating hepatic gene transfer.

[0264]

[0270] Consistent with studies examining germline transmission of AAV2 and AAV8 vectors expressing the hFIX transgene, some animals develop anti-hFIX antibodies, likely due to amino acid differences between rabbit and human factor IX.

[0265]

[0271] These results add to the current body of data regarding the germline transmission potential of AAV vectors. AAV-SPK has a pattern similar to that of previously studied serotypes, AAV2 and AAV8 vectors (Favaro P et al., Molecular Therapy 17:1022-1030 (2009)). In other words, there is dose-dependent seeding of AAV vector sequences into semen, which disseminated completely over time. However, AAV-LK03 differs from AAV2, AAV8, and AAV-SPK in that very little vector was distributed into semen, making this vector capsid potentially safer than other capsids with respect to genotoxicity.

[0266] Example 8 A clinical trial will be conducted to determine the safety and kinetics of a single IV infusion of AAV-FVIII. The AAV capsid used for the AAV vector has demonstrated favorable safety and efficacy, with a dose of approximately 1 x 10 cells per 100 mg / kg of FVIII administered 1-3 months after vector infusion. 12 Preclinical studies have shown that this antibody has the ability to achieve clinically meaningful FVIII activity levels of ≥ 1000 mg / kg, as well as a prevalence of cross-reactive neutralizing antibodies (Abs) against the AAV capsid that is approximately 10% lower than that of AAV8. A representative clinical trial design may be as shown in Table 6.

[0267] [Table 7] TIFF2024170416000009.tif176149

[0268] Example 9 TTR promoter

[0272] The characterization of the transthyretin (TTR) promoter was originally described by Costa and Grayson 1991, Nucleic Acids Research 19(15):4139-4145. The TTR promoter sequence was altered from TATTTGTGTAG to TATTGACTTAG.

[0269] TTR promoter with four nucleotide mutations (TTRmut), SEQ ID NO: 22 GTCTGTCTGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCTAGGCAAGGTTCATATT GACT TAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAGCTCCT

[0270] Example 10 CpG-reduced FVIII encoding a transgene construct and an exemplary AAV capsid CpG-reduced nucleic acid variant X01 (SEQ ID NO: 1) encoding FVIII atgcagattg agctgtctac ctgcttcttc ctgtgcctgctgaggttctg cttctctgct accaggaggt actacctggg ggctgtggag ctgagctgggattacatgca gtctgacctg ggggagctgc ctgtggatgc caggtttccc cccagggtgcccaagagctt ccccttcaat acctctgtgg tgtataagaa gaccctgttt gtggagttcactgatcatct gttcaacatt gctaaaccca ggcccccctg gatggggctg ctgggccctaccatccaggc tgaggtgtat gacactgtgg tgatcactct gaagaacatg gctagccatcctgtgtctct gcatgctgtg ggggtgagct actggaaggc ttctgagggg gctgagtatgatgatcagac tagccagagg gagaaggagg atgacaaggt gttccctggg ggctctcacacctatgtctg gcaggtgctg aaggagaatg gccccatggc ctctgatcct ctgtgtctgacctatagcta cctgagccat gtggacctgg tgaaggacct gaactctggc ctgattggggccctgctggt gtgtagggag gggagcctgg ccaaggagaa gacccagacc ctgcacaagttcattctgct gtttgctgtg tttgatgagg gcaagagctg gcattctgaa accaagaacagcctgatgca ggacagggat gctgcctctg ctagggcctg gcccaagatg cacactgtgaatgggtatgt caataggtct ctgcctggcc tgattggctg ccacaggaag tctgtgtactggcatgtgat tgggatgggc accacccctg aggtgcacag catctttctg gagggccacaccttcctggt gaggaatcac agacaggcca gcctggagat cagccccatc accttcctgactgcccagac cctgctgatg gacctgggcc agtttctgct gttctgccac atctctagccaccagcatga tggcatggag gcctatgtga aggtggactc ctgccctgag gagccccagctgaggatgaa gaataatgag gaggctgagg actatgatga tgacctgact gactctgagatggatgtggt gagatttgat gatgacaatt ctcccagctt cattcagatc aggtctgtggccaagaagca tcccaagacc tgggtgcact acattgctgc tgaggaggag gactgggactatgcccccct ggtgctggcc cctgatgaca ggagctataa gagccagtac ctgaataatggcccccagag gattgggagg aagtataaga aggtgaggtt catggcctat actgatgaaaccttcaagac cagagaggcc atccagcatg agtctgggat cctggggccc ctgctgtatggggaggtggg ggacaccctg ctgatcatct tcaagaacca ggccagcagg ccctacaacatctaccctca tggcatcact gatgtgaggc ctctgtacag cagaaggctg cccaagggggtgaagcatct gaaggacttc cccattctgc ctggggagat tttcaagtac aagtggactgtgactgtgga ggatggccca accaagtctg accctaggtg cctgactagg tactacagcagctttgtgaa tatggagagg gacctggcct ctggcctgat tggccccctg ctgatctgctacaaggagtc tgtggatcag aggggcaacc agatcatgtc tgacaagagg aatgtgatcctgttctctgt gtttgatgag aacaggagct ggtacctgac tgagaacatt cagaggtttctgcccaaccc tgctggggtg cagctggagg accctgaatt ccaggcctct aacatcatgcacagcattaa tggctatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtggcctactggta cattctgagc attggggccc agactgactt cctgtctgtg ttcttctctggctacacctt taagcacaag atggtgtatg aggataccct gaccctgttt cctttctctggggagactgt gttcatgagc atggagaacc ctggcctgtg gatcctgggc tgccacaactctgacttcag gaacaggggg atgactgctc tgctgaaggt gagcagctgt gataagaacactggggacta ctatgaggac agctatgagg acatctctgc ctatctgctg agcaagaataatgctattga gcccaggagc ttctctcaga acccccctgt gctgaagagg caccagaggggagatcaccag aactactctg cagtctgacc aggaggagat tgactatgat gacaccatctctgtggagat gaagaaggag gattttgata tttatgatga ggatgaaaac cagagccccaggagctttca gaagaagact aggcactatt tcattgctgc tgtggagagg ctgtgggactatggcatgtc ttctagcccc catgtgctga ggaacagggc ccagtctggc tctgtgccccagttcaagaa ggtggtgttc caggagttca ctgatggcag cttcactcag cccctgtacaggggggagct gaatgagcac ctggggctgc tgggccctta tatcagggct gaggtggaggataacatcat ggtgaccttc aggaaccagg ccagcaggcc ctacagcttc tactctagcctgatcagcta tgaggaggac cagaggcagg gggctgagcc caggaagaac tttgtgaagcccaatgac caagacttat ttctggaagg tgcagcacca tatggcccccc accaaggatgagtttgattg caaagcctgg gcctacttct ctgatgtgga cctggagaag gatgtgcactctgggctgat tggccccctg ctggtgtgcc acaccaacac tctgaaccct gcccatggcaggcaggtgac tgtgcaggag tttgccctgt tcttcaccat ctttgatgag actaagagctggctacttcac tgagaacatg gaggagaact gcagggcccc ctgcaatatc cagatggaggaccccacctt taggaaaat tataggtttc atgccattaa tggctacatc atggacaccctgcctggcct ggtgatggcc caggaccaga ggatcaggtg gtacctgctg agcatgggcagcaatgagaa cattcacagc atccacttct ctggccatgt gttcactgtg aggaaaggaggagatcaa gatggccctg tataatctgt accctggggt gtttgagact gtggagatgctgcccagcaa ggctggcatc tggagggtgg agtgcctgat tggggagcac ctgcatgctggcatgagcac cctgttcctg gtgtattcta acaagtgtca gacccccctg ggcatggcctctggccatat cagggacttc cagatcactg cctctggcca gtatgggcag tgggcccccaagctggccag gctgcattac tctggcagca tcaatgcctg gagcaccaag gagccattcagctggattaa ggtggacctg ctggctccaa tgattatcca tggcatcaag acccagggggccaggcagaa gtttagcagc ctgtacatct ctcagtttat catcatgtac tctctggatggcaaaaagtg gcagacctac aggggcaatt ctactggcac tctgatggtg ttctttggcaatgtggacag ctctgggatc aagcacaaca tctttaaccc ccctatcatt gccaggtacattaggctgca ccccacccat tacagcatca ggagcaccct gaggatggag ctgatgggctgtgatctgaa cagctgcagc atgcccctgg gcatggagag caaggctatc tctgatgcccagattactgc cagcagctac ttcaccaata tgtttgccac ctggagcccc agcaaggccaggctgcacct gcagggcagg tctaatgcct ggaggcccca ggtgaacaac cccaaggagtggctgcaggt ggacttccag aagaccatga aggtgactgg ggtgaccacc cagggggtgaagagcctgct gactagcatg tatgtgaagg agttcctgat cagcagcagc caggatggccatcagtggac cctgttcttc cagaatggca aggtgaaggt gttccagggc aatcaggacagcttcacccc tgtggtgaac agcctggacc cccccctgct gaccagatac ctgaggatccacccccagag ctgggtgcat cagattgccc tgaggatgga ggtgctgggg tgtgaggcccaggacctgta ctga

[0271] CpG-reduced nucleic acid variant X02 (SEQ ID NO: 2) encoding FVIII atgcagattg agctgtctac ctgctttttc ctgtgtctgctgaggttctg cttctctgcc actaggaggt actacctggg ggctgtggag ctgtcttgggattacatgca gtctgatctg ggggagctgc ctgtggatgc caggtttcct cccagggtgcccaagtcttt ccccttcaat acctctgtgg tgtataagaa gaccctgttt gtggagtttactgatcacct gttcaacatt gccaagccca ggcccccttg gatgggcctg ctggggcccaccatccaggc tgaggtgtat gacactgtgg tgatcaccct gaagaacatg gcctctcaccctgtgagcct gcatgctgtg ggggtgagct actggaaggc ctctgagggg gctgagtatgatgaccagac cagccagagg gagaggagg atgataaggt gttccctggg gggagccacaacttatgtgtg gcaggtgctg aaggaatg gcccaatggc ctctgatccc ctgtgcctgacctattctta cctgagccat gtggacctgg tgaaggacct gaactctggc ctgattggggccctgctggt gtgcagggag ggctctctgg ctaaggaa gacccagacc ctgcacaagttcatcctgct gtttgctgtg tttgatgagg ggaagagctg gcactctgag accaagaacagcctgatgca ggacagggat gctgcctctg ccagggcctg gcccaaaatg cacactgtgaatggctatgt gaataggagc ctgcctggcc tgattggctg ccacaggaag tctgtgtattggcatgtgat tggcatgggc accacccctg aggtgcactc tatcttcctg gagggccatactttcctggt gaggaatcat aggcaggcca gcctggagat tagccccatt acctttctgactgcccagac cctgctgatg gacctgggcc agttcctgct gttttgccac atcagctctcaccagcatga tggcatggag gcctatgtga aggtggatag ctgccctgag gagccccagctgaggatgaa gaacaatgag gaggctgagg attatgatga tgatctgact gattctgaaatggatgtggt gaggtttgat gatgacaata gcccctcttt catccagatc aggtctgtggccaagaagca tcctaagacc tgggtgcact acattgctgc tgaggaggag gactgggactatgctcccct ggtgctggcc cctgatgaca ggtcttacaa gagccagtac ctgaacaatggcccccagag aattgggagg aagtataaga aggtgagatt catggcttac actgatgagaccttcaagac tagggaggcc atccagcatg agtctggcat tctgggcccc ctgctgtatggggaggtggg ggacaccctg ctgatcatct tcaagaacca ggcctctagg ccctacaatatttaccccca tgggatcact gatgtgaggc ccctgtacag caggaggctg cctaagggggtgaagcatct gaaggacttc cccatcctgc ctggggagat cttcaagtat aagtggactgtgactgtgga agatggcccc accaagtctg accctaggtg cctgaccagg tactactcttcttttgtgaa catggagagg gacctggcct ctggcctgat tggccccctg ctgatctgctacaaggagtc tgtggaccag agggggaacc agattatgtc tgacaagagg aatgtgattctgttctctgt gtttgatgag aacaggagct ggtatctgac tgagaacatc cagaggttcctgcccaatcc tgctggggtg cagctggagg accctgagtt ccaggccagc aacatcatgcacagcatcaa tgggtatgtg tttgattctc tgcagctgtc tgtgtgcctg catgaggtggcctactggta catcctgagc attggggctc agactgattt cctgtctgtg ttcttttctggctacacctt taagcataag atggtgtatg aggacactct gaccctgttt cccttctctggggagactgt gtttatgagc atggagaacc ctggcctgtg gatcctgggc tgccacaactctgatttcag gaacaggggc atgactgctc tgctgaaggt gtcttcttgt gacaagaacactggggacta ttatgaggac agctatgagg acatctctgc ctacctgctg agcaagaacaatgctattga gcccagatct ttcagccaga acccccctgt gctgaagagg caccagaggggagatcactag gaccaccctg cagtctgacc aggaggagat tgactatgat gacactatctctgtggagat gaagaaggag gactttgata tctatgatga ggatgagaac cagtctcccaggagcttcca gaaaaagacc aggcactact tcattgctgc tgtggagagg ctgtgggactatggcatgtc ttctagcccc catgtgctga ggaacagggc ccagtctggg tctgtgccccagttcaagaa ggtggtgttc caggagttca ctgatgggag cttcacccag cctctgtacaggggggagct gaatgagcac ctggggctgc tgggccctta tattagggct gaggtggaggacaacatcat ggtgactttc aggaatcagg cctctaggcc ctatagcttc tacagctctctgatcagcta tgaggaggat cagaggcagg gggctgagcc caggaagaac tttgtgaagcccaatgac caagacctac ttctggaagg tgcagcacca catggctcct accaaggatgagtttgactg caaggcctgg gcctacttt ctgatgtgga cctggagaag gatgtgcactctggcctgat tggccccctg ctggtgtgtc ataccaacac cctgaaccct gcccatggcaggcaggtgac tgtgcaggag tttgccctgt tcttcaccat ctttgatgag accaagagctggtactttac tgagaacatg gagaggaatt gcagagcccc ttgcaacatc cagatggaggacccaacctt caaagagaac tacaggttcc atgccatcaa tgggtacatc atggacaccctgcctggcct ggtgatggct caggaccaga ggatcaggtg gtatctgctg agcatgggcagcaatgagaa tatccatagc attcacttct ctggccatgt gttcactgtg aggaagaaggaggagtacaa gatggccctg tataacctgt accctggggt gtttgagact gtggagatgctgccaagcaa ggctgggatt tggagggtgg agtgcctgat tggggagcac ctgcatgctggcatgtctac cctgttcctg gtgtactcca ataagtgcca gacccccctg ggcatggcctctggccacat cagggacttc cagatcactg cctctggcca gtatgggcag tgggccccaaagctggccag gctgcactat tctgggagca tcaatgcttg gagcaccaag gagcctttcagctggattaa ggtggatctg ctggccccca tgatcattca tggcatcaaa acccagggggctagacagaa gttttctagc ctgtacatca gccagttcat catcatgtac agcctggatggcaagaagtg gcagacttac aggggcaata gcactggcac cctgatggtg ttttttggcaatgtggacag ctctggcatc aagcacaaca tctttaaccc ccccattatt gccaggtatatcaggctgca tcccacccac tattctatta ggtctactct gagaatggag ctgatgggctgtgacctgaa cagctgtagc atgcccctgg ggatggagag caaggctatc tctgatgcccagatcactgc cagctcttat ttcaccaata tgtttgccac ctggtctccc tctaaggccaggctgcacct gcagggcagg agcaatgctt ggaggcccca ggtgaataac cccaaggagtggctgcaggt ggacttccag aagaccatga aggtgactgg ggtgactacc cagggggtgaagtctctgct gactagcatg tatgtgaagg agttcctgat cagcagcagc caggatgggcatcagtggac tctgttcttc cagaatggca aggtgaaggt cttccagggg aaccaggatagcttcactcc tgtggtgaac tctctggacc cccccctgct gactaggtat ctgaggatccacccccagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaggcccaggacctgta ttga

[0272] CpG-reduced nucleic acid variant X03 (SEQ ID NO: 3) encoding FVIII atgcagattg aactgtctac ttgtttcttc ctgtgcctgctgaggttttg cttctctgct actaggaggt actatctggg ggctgtggag ctgtcttgggactatatgca gtctgacctg ggggagctgc ctgtggatgc taggtttccc cccagggtgcccaagagctt cccctttaac acctctgtgg tgtataagaa gactctgttt gtggagttcactgaccatct gttcaacatt gccaagccaa ggcccccctg gatgggcctg ctgggccccaccatccaggc tgaggtgtat gacactgtgg tgattactct gaagaacatg gccagccatcctgtgagcct gcatgctgtg ggggtgtctt actggaaggc ctctgagggg gctgagtatgatgaccagac ctctcagagg gagaaggagg atgacaaggt gttccctggg ggctctcatacctatgtgtg gcaggtcctg aaggaatg ggcccatggc ctctgacccc ctgtgcctgacctactctta tctgtctcat gtggacctgg tgaaggacct gaactctggc ctgattggggccctgctggt gtgcagggag ggcagcctgg ctaaggaa gacccagact ctgcacaagttcatcctgct gtttgctgtg tttgatgagg gcaagagctg gcactctgag accaagaacagcctgatgca ggacagggat gctgcccttg ctagggcctg gcccaagatg caacactgtgaatgggtatgt gaacaggagc ctgccaggcc tgattggctg ccataggaag tctgtgtattggcatgtgat tgggatgggg actacccctg aggtccacag cattttcctg gaggggcatacctttctggt gaggaaccac aggcaggcct ctctggagat ctctcccatt actttcctgactgcccagac cctgctgatg gacctgggcc agttcctgct gttctgccac atcagcagccaccagcatga tggcatggag gcctatgtga aggtggatag ctgccctgag gagccccagctgaggatgaa aaacaatgag gaggctgagg attatgatga tgacctgact gattctgagatggatgtggt gaggtttgat gatgataaca gccccagctt catccagatt aggtctgtggccaagaagca tcccaagacc tgggtgcact acattgctgc tgaggaggag gattgggactatgctcctct ggtgctggcc cctgatgaca ggagctacaa gagccagtac ctgaataatggcccccagag gattggcagg aagtataaga aggtgaggtt catggcctac actgatgagacctttaagac cagggaggcc atccagcatg aatctgggat cctgggcccc ctgctgtatggggaggtggg ggacaccctg ctgattatct ttaagaacca ggctagcagg ccctacaacatttaccccca tggcattact gatgtgaggc ccctgtacag caggaggctg cccaagggggtgaagcacct gaaggatttc cccattctgc ctggggagat ctttaagtac aaatggactgtgactgtgga ggatggccct actaagtctg atcccaggtg tctgaccaga tactacagcagctttgtgaa tatggagagg gacctggctt ctggcctgat tggccccctg ctgatctgctacaaggagtc tgtggaccag aggggcaatc agattatgtc tgacaagagg aatgtgatcctgttctctgt gtttgatgag aacagaagct ggtacctgac tgagaacatc cagaggttcctgcccaaccc tgctggggtg cagctggagg accctgagtt ccaggctagc aatatcatgcacagcattaa tggctatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtggcctattggta cattctgagc attggggccc agactgattt cctgtctgtg ttcttttctggctacacctt caagcacaag atggtgtatg aggatactct gaccctgttt cccttctctggggagactgt gttcatgagc atggagaacc ctggcctgtg gatcctgggc tgtcacaactctgacttcag gaacaggggc atgactgccc tgctgaaggt gagctcttgt gataagaacactggggacta ctatgaggac tcttatgagg acatctctgc ctacctgctg agcaagaacaatgctattga gcccaggagc ttctctcaga atccccctgt gctgaagagg catcagagggagatcactag gactaccctg cagtctgacc aggaagagat tgactatgat gacaccatctctgtggaaat gaagaaggag gactttgata tctatgatga ggatgaaaac cagagccccaggagcttcca gaagaagacc aggcattact tcattgctgc tgtggagagg ctgtgggactatgggatgag ctcttctccc catgtgctga ggaatagggc tcagtctggc tctgtcccacagttcaagaa ggtggtgttt caggagttca ctgatggcag cttcactcag cccctgtacaggggggagct gaatgagcat ctgggcctgc tggggcccta catcagggct gaggtggaggataacattat ggtgactttc aggaaccagg cctttaggcc ctacagcttc tacagcagcctgatcagcta tgaggaggac cagaggcagg gggctgagcc cagaagaac tttgtgaagcccaatgagac taagacctat ttctggaagg tgcagcatca catggctccc actaaagatgagtttgactg caaggcctgg gcctacttct ctgatgtgga tctggagaag gatgtgcattctgggctgat tggccctctg ctggtctgcc atactaacac cctgaatcct gcccatggcaggcaggtgac tgtgcaggag tttgccctgt tctttaccat ctttgatgag accaagtcttggtacttcac tgagaacatg gaggagaact gcagggcccc ctgtaacatc cagatggaggaccccacctt tagggaagaac tacaggttcc atgccatcaa tggctacatc atggacactctgcctggcct ggtgatggcc caggaccaga ggatcaggtg gtacctgctg tctatgggctctaatgagaa cattcattct atccacttct ctggccatgt gtttactgtg aggaagaaggaggtcaa gatggccctg tacaatctgt accctggggt gtttgaaact gtggagatgctgccctctaa ggctggcatc tggagggtgg agtgcctgat tggggaacac ctgcatgctggcatgagcac cctgttcctg gtctatagca ataagtgcca gacccccctg gggatggcctctgggcatat cagagacttc cagatcactg cctctggcca gtatggccag tgggcccccaagctggccag gctgcactac tctggcagca ttaatgcctg gagcaccaag gagcccttctcttggatcaa ggtggacctg ctggctccca tgatcatcca tgggatcaag acccagggggccaggcagaa gttcagcagc ctgtacatct ctcagttcat catcatgtac tctctggatggcaagaagtg gcagacctac aggggcaata gcactgggac cctgatggtg ttctttgggaatgtggacag ctctggcatc aagcacaata tcttcaaccc ccccatcatt gccaggtacatcagactgca ccccactcat tacagcatca ggagcactct gaggatggag ctgatgggctgtgacctgaa tagctgctct atgcccctgg gcatggagag caaggccatt tctgatgcccagattactgc ctcttcttac ttcactaata tgtttgccac ctggagcccc agcaaggccaggctgcatct gcaggggagg agcaatgcct ggaggcccca ggtgaacaac cccaaggagtggctgcaggt ggacttccag aagactatga aggtgactgg ggtgaccact cagggggtgaagagcctgct gaccagcatg tatgtgaagg agttcctgat ctcttctagc caggatgggcaccagtggac cctgtttttc cagaatggga aggtgaaggt gtttcagggc aatcaggacagctttactcc tgtggtgaac agcctggacc cccccctgct gactaggtac ctgaggattcacccccagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaggcccaggatctgta ctga

[0273] CpG-reduced nucleic acid variant X04 (SEQ ID NO: 4) encoding FVIII atgcagattg agctgtctac ctgcttcttt ctgtgcctgctgaggttctg tttctctgcc actaggaggt attatctggg ggctgtggag ctgtcctgggactacatgca gtctgatctg ggggagctgc ctgtggatgc caggttccct cccagggtgcccaagtcttt ccctttcaat acctctgtgg tgtacaagaa gactctgttt gtggagtttactgatcacct gtttaacatt gccaagccca ggcccccctg gatggggctg ctgggccccaccatccaggc tgaggtgtat gacactgtgg tgattactct gaagaatatg gcttctcaccctgtgagcct gcatgctgtg ggggtgagct actggaaggc ctctgagggg gctgagtatgatgaccagac cagccagagg gagaaggagg atgacaaggt gttccctggg ggcagccacacttatgtgtg gcaggtgctg aaggagaatg gcccaatggc ctctgacccc ctgtgcctgacctacagcta tctgagccat gtggatctgg tgaaggatct gaactctggc ctgattggggccctgctggt gtgcagggag ggctctctgg ccaaggagaa gactcagact ctgcacaagttcatcctgct gtttgctgtg tttgatgagg gcaagagctg gcactctgag accaagaactctctgatgca ggatagggat gctgcttctg ccagggcctg gcccaagatg cacactgtgaatgggtatgt gaataggagc ctgcctgggc tgattgggtg tcacaggaag tctgtgtactggcatgtgat tggcatgggc accactcctg aggtgcacag catctttctg gagggccacacttttctggt gaggaatcac aggcaggcca gcctggagat cagccccatc accttcctgactgcccagac cctgctgatg gatctgggcc agttcctgct gttttgccat atcagcagccatcagcatga tgggatggag gcttatgtga aggtggactc ttgccctgag gagcctcagctgaggatgaa gaataatgaa gaggctgagg actatgatga tgatctgact gactctgagatggatgtggt gaggtttgat gatgacaaca gccccagctt tatccagatt aggtctgtggccaagaagca ccccaagacc tgggtgcatt acattgctgc tgaggaagag gattgggactatgcccccct ggtgctggcc cctgatgaca ggagctacaa gtctcagtac ctgaacaatggccctcagag gattggcagg aagtacaaga aggtgaggtt catggcttac actgatgagaccttcaagac cagggaggcc attcagcatg aatctgggat cctgggcccc ctgctgtatggggaggtggg ggacaccctg ctgattattt tcaagaacca ggccagcagg ccctacaacatttatcctca tggcattact gatgtgagac ccctgtacag caggaggctg cctaagggggtgaagcacct gaaggacttc cccatcctgc ctggggagat cttcaagtac aagtggactgtgactgtgga ggatggcccc actaagtctg accccaggtg cctgactagg tactactccagctttgtgaa catggagagg gacctggcct ctggcctgat tggccccctg ctgatctgctacaaggagtc tgtggatcag aggggcaacc agatcatgtc tgacaagaga aatgtgatcctgttctctgt gtttgatgag aataggtctt ggtacctgac tgagaacatc cagaggtttctgcctaatcc tgctggggtg cagctggagg atcctgagtt ccaggcctct aacattatgcacagcatcaa tgggtatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtggcctactggta catcctgagc attggggccc agactgactt tctgtctgtg ttctctctggctacacctt taagcataag atggtgtatg aggacaccct gactctgttc cccttctctggggagactgt gttcatgagc atggagaacc caggcctgtg gatcctgggc tgccacaactctgatttcag gaataggggc atgactgcc tgctgaaggt gagcagctgt gataagaacactggggacta ttatgaggat agctatgagg acatctctgc ctacctgctg agcaagaacaatgccattga gcccaggagc ttcagccaga atcctcctgt gctgaagagg caccagaggggagatcaccag gaccaccctg cagtctgatc aggaggagat tgactatgat gacactatctctgtggagat gaagaaggag gactttgaca tctatgatga ggatgagaat cagagccccaggagcttcca gaagaagact agacactact ttattgctgc tgtggagagg ctgtgggactatggcatgag ctcttctccc catgtgctga gaaacagggc ccagtctggc tctgtgccccagttcaagaa ggtggtcttc caggagttca ctgatggctc tttcacccag cctctgtatagaggggagct gaatgagcac ctgggcctgc tgggccctta catcagggct gaggtggaggacaatatcat ggtgaccttc aggaaccagg ctagcaggcc ctactctttc tacagcagcctgatcagcta tgaggaggac cagaggcagg gggctgagcc taggaaat tttgtgaagcccaatgagac caagacctac ttctggaagg tgcagcacca catggctccc actaaggatgagtttgactg caaggcctgg gcctactttt ctgatgtgga cctggagaag gatgtgcattctggcctgat tggccccctg ctggtctgcc acaccaatac tctgaaccct gctcatgggagacaggtgac tgtgcaggag tttgccctgt tcttcaccat ctttgatgag accaagtcctggtactttac tgagaacatg gaggagaatt gcagggcccc ttgcaacatc cagatggaggaccccacctt caaggaaaaat tataggttcc atgccatcaa tggctacatc atggacaccctgcctggcct ggtgatggcc caggaccaga ggatcaggtg gtatctgctg tctatgggctctaatgagaa catccacagc atccatttct ctggccatgt gttcactgtg aggaaaggaggattaa gatggctctg tacaacctgt accctggggt ctttgagact gtggagatgctgcccagcaa ggctggcatt tggagggtgg agtgcctgat tggggaacac ctgcatgctgggatgagcac cctgttcctg gtgtactcta acaagtgcca gaccccactg ggcatggcttctggccacat cagggatttc cagattactg cctctggcca gtatggccag tgggctcccaagctggctag gctgcactac tctgggagca tcaatgcctg gtctactaag gagcctttctcttggatcaa agtggacctg ctggccccta tgatcatcca tgggatcaag actcagggggccaggcagaa gttcagcagc ctgtacatct ctcagttcat cattatgtac agcctggatggcaagaagtg gcagacctac aggggcaaca gcactggcac cctgatggtg ttctttgggaatgtggacag ctctgggatt aagcacaaca tctttaaccc ccccatcatt gccaggtatatcaggctgca ccctacccac tacagcatta ggagcaccct gaggatggag ctgatgggctgtgacctgaa cagctgcagc atgcccctgg ggatggagag caaggccatt tctgatgctcagatcactgc ttctagctac ttcactaaca tgtttgccac ctggtctccc agcaaggctagactgcacct gcaggggagg agcaatgcct ggaggcccca ggtgaataat cccaaggagtggctgcaggt ggatttccag aaaaccatga aggtgactgg ggtgactacc cagggggtgaagtctctgct gaccagcatg tatgtgaagg agttcctgat cagcagcagc caggatgggcatcagtggac cctgttcttt cagaatggga aggtgaaggt gtttcagggc aatcaggacagcttcacccc tgtggtgaac agcctggacc cccccctgct gaccaggtac ctgaggatccacccccagag ctgggtgcat cagattgccc tgaggatgga ggtgctgggc tgtgaggcccaggacctgta ctga

[0274] CpG-reduced nucleic acid variant X05 (SEQ ID NO: 5) encoding FVIII atgcagattg agctgtctac ttgcttcttc ctgtgcctgctgaggttctg cttctctgcc actaggaggt attacctggg ggctgtggag ctgagctgggactatatgca gtctgacctg ggggagctgc ctgtggatgc caggtttcct cccagggtgcctaagagctt ccccttcaac acctctgtgg tgtacaagaa gactctgttt gtggagtttactgatcatct gttcaacatt gccaagccca ggcctccttg gatggggctg ctgggccccaccatccaggc tgaggtgtat gacactgtgg tgattaccct gaagaatatg gccagccatcctgtgagcct gcatgctgtg ggggtgagct attggaaggc ctctgagggg gctgagtatgatgatcagac tagccagagg gagaaggagg atgacaaggt gttccctggg gggagccatacctatgtgtg gcaggtgctg aaggagaatg gccccatggc ctctgaccct ctgtgcctgacttatagcta cctgagccat gtggatctgg tgaaggacct gaactctggc ctgattggggccctgctggt gtgcagggag ggcagcctgg ccaaggagaa gactcagacc ctgcacaagttcatcctgct gtttgctgtg tttgatgagg ggaagtcctg gcactctgag actaagaacagcctgatgca ggatagggat gctgcttctg ccagggcctg gcctaagatg cacactgtgaatggctatgt gaataggagc ctgcctggcc tgattggctg ccataggaag tctgtgtactggcatgtgat tgggatgggc accacccctg aggtgcactc tattttcctg gagggccatactttcctggt gaggaaccat aggcaggcca gcctggagat cagccccatc actttcctgactgcccagac tctgctgatg gacctgggcc agttcctgct gttctgccac atcagcagccatcagcatga tggcatggag gcttatgtga aggtggacag ctgccctgag gagcctcagctgaggatgaa gaataatgag gaggctgagg actatgatga tgacctgact gactctgagatggatgtggt gaggtttgat gatgacaact ctccctcttt catccagatc aggtctgtggccaagaagca ccctaagacc tgggtgcact acattgctgc tgaggaggag gattgggactatgcccccct ggtgctggcc ccagatgaca ggagctacaa gtcccagtac ctgaacaatggcccccagag gattggcagg aagtacaaga aggtgaggtt catggcttat actgatgagactttcaagac cagggaggcc atccagcatg agtctggcat cctgggccct ctgctgtatggggaggtggg ggacaccctg ctgattatct tcaagaacca ggcttctagg ccctacaatatctaccctca tggcatcact gatgtgaggc ccctgtacag caggaggctg cccaagggggtgaagcatct gaaggatttc cccatcctgc ctggggagat ctttaagtat aagtggactgtgactgtgga ggatggcccc actaagtctg accccaggtg cctgaccagg tattacagcagctttgtgaa catggagagg gatctggctt ctgggctgat tggccccctg ctgatctgctacaaggagtc tgtggaccag aggggcaacc agatcatgtc tgacaagagg aatgtgatcctgttctctgt gtttgatgag aataggagct ggtacctgac tgagaacatc cagaggtttctgcccaatcc tgctggggtg cagctggagg atcctgagtt tcaggcctct aatatcatgcacagcatcaa tggctatgtg tttgactctc tgcagctgtc tgtgtgcctg catgaggtggcctattggta catcctgagc attggggccc agactgactt tctgtctgtg tttttttctggctacacctt caagcacaag atggtgtatg aggatactct gactctgttc cctttttctggggagactgt gttcatgtct atggagaacc ctgggctgtg gattctgggc tgccacaattctgacttcag gaacagaggc atgactgctc tgctgaaggt gagcagctgt gacaagaacactggggacta ctatgaggac tcttatgagg acatttctgc ctacctgctg agcaagaacaatgccattga gcccagaagc ttttctcaga acccccctgt gctgaagagg caccagagggagatcaccag gaccaccctg cagtctgacc aggaggagat tgactatgat gatactatttctgtggagat gaagaaggag gactttgaca tctatgatga ggatgagaac cagagccccaggtctttcca gaagaagact aggcactact ttattgctgc tgtggagagg ctgtgggactatgggatgtc tagctctcct catgtgctga ggaacagggc ccagtctggc tctgtgccccagtttaaaaa ggtggtgttc caggaattca ctgatggcag ctttacccag cctctgtacaggggggagct gaatgagcac ctggggctgc tggggcctta cattagggct gaggtggaggacaacatcat ggtgaccttc aggaatcagg ccagcaggcc ctactctttc tacagcagcctgatctctta tgaggaggac cagaggcagg gggctgaacc caggaagaac tttgtgaagcccaatgagac caagacctac ttctggaagg tgcagcacca catggctccc accaaggatgagtttgattg caaggcctgg gcttacttct ctgatgtgga tctggagaag gatgtgcactctgggctgat tggccccctg ctggtgtgcc acaccaacac tctgaaccct gcccatggcagacaggtgac tgtgcaggag tttgccctgt tcttcactat ctttgatgag actaagagctggctacttcac tgagaacatg gaggagaatt gcagggcccc ttgcaacatc cagatggaggaccccacctt tagggaagaac tacaggtttc atgccattaa tggctacatc atggacaccctgcctggcct ggtgatggcc caggaccaga ggatcaggtg gtacctgctg tctatggggagcaatgagaa catccacagc attcacttct ctggccatgt gttcactgtg aggaaaggaggagatcaa gatggccctg tacaacctgt accctggggt gtttgagact gtggagatgctgcccagcaa ggctgggatc tggagggtgg agtgcctgat tggggagcac ctgcatgctgggatgagcac cctgttcctg gtgtatagca acaagtgcca gacccccctg ggcatggcctctggccacat cagagacttt cagattactg cctctggcca gtatgggcag tgggcccccaagctggccag gctgcactat tctggctcta ttaatgcctg gagcactag gagcccttcagctggattaa ggtggacctg ctggctccca tgatcatcca tggcatcaag actcaggggggccaggcagaa gttctcttct ctgtacatca gccagttcat tatcatgtac tccctggatggcaagaagtg gcagacctat aggggcaaca gcactggcac cctgatggtg ttctttgggaatgtggacag ctctggcatc aagcataata tcttcaatcc ccccatcatt gctaggtacatcaggctgca ccccacccac tactctatta ggtctaccct gaggatggag ctgatgggctgtgacctgaa cagctgcagc atgcctctgg gcatggagag caaagccatc tctgatgcccagatcactgc cagcagctac tttaccaaca tgtttgctac ttggagcccc agcaaggccaggctgcacct gcaggggagg tctaatgcct ggaggcccca ggtgaacaac cccaaggagtggctgcaggt ggacttccag aagactatga aggtgactgg ggtgaccacc cagggggtgaagagcctgct gacctctatg tatgtgaagg agttcctgat tagcagcagc caggatggccaccagtggac cctgtttttc cagaatggga aggtgaaggt gtttcagggg aaccaggacagcttcactcc tgtggtgaac tctctggacc cccccctgct gaccaggtat ctgaggatccaccctcagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaggcccaggacctgta ctga

[0275] CpG-reduced nucleic acid variant X06 (SEQ ID NO: 6) encoding FVIII atgcagattg agctgagcac ctgcttcttc ctgtgcctgctgaggttttg cttctctgcc accaggaggt actacctggg ggctgtggag ctgagctgggattacatgca gtctgacctg ggggagctgc ctgtggatgc caggttccct cccagggtgcccaagtcttt ccccttcaac acttctgtgg tgtacaagaa gaccctgttt gtggagtttactgaccacct gttcaacatt gccaagccca ggcctccctg gatgggcctg ctgggccccaccattcaggc tgaggtgtat gacactgtgg tcatcaccct gaaaaatatg gctagccaccctgtgtctct gcatgctgtg ggggtgagct actggaaggc ctctgagggg gctgagtatgatgaccagac tagccagagg gagaaggagg atgacaaggt gttccctggg ggcagccacacttatgtgtg gcaggtgctg aaagagaatg gccccatggc ttctgatccc ctgtgtctgacctatagcta cctgagccat gtggatctgg tgaaggacct gaactctggc ctgattggggccctgctggt gtgcagggag ggcagcctgg ctaaggagaa gacccagacc ctgcataagttcatcctgct gtttgctgtg tttgatgagg gcaagagctg gcactctgag actaagaacagcctgatgca ggatagggat gctgcttctg ccagggcctg gcccaagatg cacactgtgaatgggtatgt gaacaggagc ctgcctggcc tgattggctg ccataggaag tctgtctattggcatgtgat tggcatgggc actactcctg aggtgcacag catctttctg gagggccacaccttcctggt gaggaaccac aggcaggcca gcctggagat ctctcccatc actttcctgactgctcagac cctgctgatg gacctgggcc agttcctgct gttctgtcac atctctagccaccagcatga tggcatggag gcctatgtga aggtggatag ctgccctgag gaaccccagctgaggatgaa gaacaatgag gaggctgagg attatgatga tgatctgact gattctgagatggatgtggt gaggtttgat gatgacaatt ctcctagctt cattcagatc agatctgtggccaaaaagca tcctaagact tgggtgcatt atattgctgc tgaggaggag gattgggattatgcccccct ggtgctggct cctgatgata ggagctacaa gtctcagtac ctgaataatgggccccagag gattggcagg aagtacaaga aggtgaggtt catggcctac actgatgagaccttcaagac cagggaggcc attcagcatg agtctgggat tctggggccc ctgctgtatggggaggtggg ggataccctg ctgatcattt tcaagaacca ggccagcagg ccctacaacatctaccccca tgggattact gatgtgaggc ccctgtactc taggaggctg cctaagggggtgaagcacct gaaggatttt cctatcctgc ctggggaaat cttcaagtac aagtggactgtgactgtgga ggatggcccc actaagtctg atcccaggtg tctgaccagg tattatagctcttttgtgaa catggagagg gatctggcct ctgggctgat tggccctctg ctgatctgctacaaggagtc tgtggaccag aggggcaacc agatcatgtc tgacaagagg aatgtgatcctgttctctgt gtttgatgag aacaggagct ggtatctgac tgagaacatc cagaggtttctgcccaatcc tgctggggtg cagctggagg atcctgagtt ccaggctagc aacatcatgcacagcatcaa tgggtatgtg tttgacagcc tgcagctgtc tgtgtgtctg catgaggtggcctactggta tatcctgtct attggggccc agactgactt cctgtctgtg tttttttctgggtatacttt taagcacaag atggtgtatg aggacaccct gactctgttc cccttctctggggagactgt gtttatgagc atggagaacc ctggcctgtg gatcctgggc tgccacaattctgacttcag gaataggggg atgactgccc tgctgaaggt gagcagctgt gataagaatactggggacta ctatgaggac tcttatgagg acatttctgc ctatctgctg tctaagaacaatgccattga acccaggagc ttctctcaga acccccctgt gctgaagagg caccagagggaaatcaccag aactactctg cagtctgatc aggaggaaat tgactatgat gacactatttctgtggagat gaagaaggag gactttgaca tctatgatga ggatgagaac cagagcccaaggagcttcca gaagaagact aggcactact tcattgctgc tgtggagagg ctgtgggactatggcatgag cagcagcccc catgtgctga gaaacagggc ccagtctggg tctgtgccccagttcaagaa ggtggtgttc caggagttca ctgatgggag cttcacccag cccctgtataggggggagct gaatgagcac ctgggcctgc tgggcccta tattagggct gaggtggaggacaacatcat ggtgaccttc aggaatcagg cctctaggcc ctacagcttc tacagcagcctgattagcta tgaggaggat cagaggcagg gggctgaacc caggaagaac tttgtgaagcccaatgac caagacctat ttctggaagg tgcagcatca catggccccc accaaggatgagtttgactg caaggcctgg gcctacttct ctgatgtgga tctggagaag gatgtgcactctggcctgat tggccccctg ctggtgtgcc acaccaacac cctgaaccct gctcatggcaggcaggtgac tgtgcaggag tttgccctgt tcttcaccat ctttgatgag actaagtcttggtacttcac tgagaatatg gagaggaatt gcagggcccc ctgcaatatt cagatggaagaccccacctt caaggagaat tacaggttcc atgccattaa tggctacatc atggataccctgcctggcct ggtgatggcc caggatcaga ggatcaggtg gtacctgctg agcatgggcagcaatgagaa catccactct atccacttct ctggccatgt gtttactgtg aggaagaaggaggagtataa gatggccctg tacaacctgt accctggggt ctttgagact gtggagatgctgccttctaa ggctggcatt tggagggtgg agtgcctgat tggggaacac ctgcatgctggcatgtctac cctgttcctg gtgtacagca ataagtgcca gacccccctg ggcatggcctctgggcatat cagggatttc cagatcactg cctctggcca gtatggccag tgggccccaaagctggctag gctgcactac tctgggagca tcaatgcctg gagcactaag gagcccttcagctggatcaa ggtggacctg ctggccccca tgattatcca tgggattaag actcagggggccaggcagaa gttcagcagc ctgtacatca gccagttcat tatcatgtac agcctggatggcaagaagtg gcagacctat aggggcaact ctactgggac cctgatggtg ttctttgggaatgtggatag ctctgggatc aagcacaata tcttcaaccc ccccatcatt gccaggtatatcaggctgca ccccacccac tacagcatta ggtctaccct gaggatggag ctgatgggctgtgatctgaa cagctgtagc atgcctctgg gcatggagtc taaggccatt tctgatgcccagattactgc tagcagctac ttcaccaaca tgtttgccac ctggtctccc agcaaggccaggctgcatct gcagggcagg tctaatgctt ggaggcccca ggtgaacaac ccaaaggagtggctgcaggt ggatttccag aagactatga aggtgactgg ggtgaccact cagggggtgaagtctctgct gacctctatg tatgtgaagg agttcctgat ctctagcagc caggatggccatcagtggac cctgttcttc cagaatggca aggtgaaagt gttccagggc aatcaggatagcttcactcc agtggtgaac agcctggatc cccctctgct gactaggtac ctgaggatccacccccagag ctgggtgcac cagattgcc tgaggatgga ggtgctgggc tgtgaggcccaggacctgta ctga

[0276] CpG-reduced nucleic acid variant X07 (SEQ ID NO: 7) encoding FVIII atgcagattg agctgagcac ctgctttctc ctgtgtctgctgaggttctg cttctctgcc accaggaggt attacctggg ggctgtggag ctgagctgggactatatgca gtctgacctg ggggagctgc ctgtggatgc taggttcccc cccagggtgcccaagagctt cccttaac acttctgtgg tgtacaagaa gaccctgttt gtggagttcactgaccacct gttcaacatt gccaagccca ggcccccctg gatggggctg ctggggcccaccatccaggc tgaggtgtat gacactgtgg tgatcaccct gaagaacatg gccagccaccctgtgagcct gcatgctgtg ggggtgagct actggaaggc ttctgagggg gctgagtatgatgaccagac tagccagagg gagaaggagg atgacaaggt gtttcctggg ggcagccatacctatgtgtg gcaggtgctg aaggagaatg gccccatggc ctctgacccc ctgtgcctgacctacagcta cctgtctcat gtggacctgg tgaaggacct gaactctggc ctgattggggctctgctggt gtgtagggag ggcagcctgg ctaaggaaaa gacccagacc ctgcataagtttatcctgct gtttgctgtg tttgatgagg gcaagagctg gcactctgag accaagaacagcctgatgca ggatagggat gctgcctctg ccagggcttg gcctaagatg cacactgtgaatgggtatgt gaataggagc ctgcctggcc tgattggctg ccacaggaag tctgtgtactggcatgtgat tgggatgggc accacccctg aggtccatag catcttcctg gagggccacactttcctggt gaggaaccac agacaggcct ctctggagat ctctcccatc accttcctgactgctcagac tctgctgatg gacctgggcc agttcctgct gttttgccat attagcagccaccagcatga tgggatggag gcctatgtga aggtggatag ctgccctgag gagcctcagctgaggatgaa gaacaatgag gaggctgaag actatgatga tgacctgact gattctgagatggatgtggt gaggtttgat gatgacaata gccccagctt cattcagatc aggtctgtggccaagaaaca ccccaagacc tgggtgcact acattgctgc tgaggaagag gactgggactatgctcccct ggtgctggcc cctgatgata ggtcttataa gagccagtac ctgaacaatgggccccagag gattggcagg aagtacaaga aggtgaggtt catggcctac actgatgaaaccttcaaaac cagggaggcc attcagcatg agtctggcat cctgggccct ctgctgtatggggaggtggg ggacaccctg ctgatcatct tcaagaacca ggccagcagg ccctacaacatctatcctca tggcatcact gatgtgaggc ccctgtacag caggaggctg cccaagggggtgaagcacct gaaagacttc cccatcctgc ctggggagat ctttaagtat aagtggactgtgactgtgga ggatggccct accaagtctg accccaggtg tctgaccagg tactattctagctttgtgaa catggagagg gacctggcct ctggcctgat tgggcccctg ctgatctgctacaaggagtc tgtggaccag aggggcaacc agatcatgtc tgacaagagg aatgtgatcctgttttctgt gtttgatgag aataggagct ggtacctgac tgagaacatc cagaggtttctgcccaatcc tgctggggtg cagctggagg atcctgagtt ccaggccagc aatatcatgcatagcatcaa tggctatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtggcctactggta catcctgagc attggggccc agactgactt tctgtctgtg ttcttttctggctatacctt caagcacaag atggtgtatg aggataccct gaccctgttc cccttctctggggagactgt gttcatgagc atggagaatc ctgggctgtg gatcctgggg tgccacaactctgattttag gaacaggggg atgactgccc tgctgaaggt gtctagctgt gataagaacactggggacta ctatgaggac agctatgagg acatttctgc ttatctgctg tctaagaataatgccattga gcccagaagc ttcagccaga atccccctgt gctgaagaga catcagagggagatcaccag aactaccctg cagtctgatc aggaggagat tgactatgat gacactatctctgtggagat gaagaaggag gactttgaca tctatgatga ggatgagaat cagtctcccaggagctttca gaagaagacc agacattact tcattgctgc tgtggagagg ctgtgggactatggcatgag ctctagccct catgtgctga ggaacagggc ccagtctggc tctgtgccccagttcaagaa ggtggtgttc caggaattca ctgatggcag cttcacccag cccctgtacaggggggagct gaatgagcac ctgggcctgc tggggcctta tatcagggct gaggtggaggataatattat ggtgactttc aggaaccagg ccagcaggcc ctactctttc tatagcagcctgatctctta tgaggaggat cagaggcagg gggctgagcc taggaaac tttgtgaagcccaatgagac taagcctac ttctggaagg tccagcacca catggcccct accaaggatgagtttgactg caaggcctgg gcctatttct ctgatgtgga tctggagaag gatgtccattctgggctgat tggccccctg ctggtgtgcc acactaacac tctgaatcct gcccatggcaggcaggtgac tgtccaggag tttgccctgt tcttcactat ctttgatgag accaagagctggtactttac tgagaacatg gaggagaact gcagagctcc ttgcaatatt cagatggaggaccccacctt caaggaat tacaggttcc atgccattaa tgggtacatc atggacaccctgcctggcct ggtgatggct caggaccaga ggatcaggtg gtacctgctg agcatgggctctaatgagaa tatccacagc atccacttct ctgggcatgt gttcactgtg aggaaaggaggagatcaa gatggctctg tataatctgt accctggggt gtttgaaact gtggagatgctgccctctaa ggctggcatc tggagggtgg agtgcctgat tggggagcac ctgcatgctggcatgagcac cctgttcctg gtgtacagca acaagtgcca gacccccctg ggcatggcctctggccacat cagggacttc cagatcactg cctctggcca gtatggccag tgggcccccaagctggccag gctgcactat tctggcagca tcaatgcctg gagcaccaag gagcccttcagctggatcaa ggtggacctg ctggccccca tgatcattca tggcatcaag acccagggggccaggcagaa gttcagctct ctgtacatct ctcagttcat catcatgtac tctctggatgggaagaagtg gcagacctac aggggcaaca gcactggcac cctgatggtg ttctttgggaatgtggactc ttctggcatc aagcacaaca tcttcaatcc ccccatcatt gctaggtatattaggctgca tcccacccac tacagcatca ggtctaccct gaggatggag ctgatgggctgtgacctgaa ctcttgcagc atgcccctgg gcatggagtc taaggccatc tctgatgcccagattactgc cagcagctac ttcaccaaca tgtttgccac ctggagcccc tctaaggccaggctgcatct gcaggggagg agcaatgcct ggaggcctca ggtgaacaac cccaaggagtggctgcaggt ggatttccag aagaccatga aggtgactgg ggtgaccacc cagggggtcaagagcctgct gaccagcatg tatgtgaagg agttcctgat cagcagcagc caggatggccaccagtggac tctgttcttt cagaatggga aggtgaaggt gtttcagggc aatcaggactctttcacccc tgtggtgaac agcctggacc cccccctgct gaccagatac ctgaggatccacccccagtc ttgggtgcat cagattgccc tgaggatgga ggtgctgggc tgtgaggctcaggatctgta ctga

[0277] CpG-reduced nucleic acid variant X08 (SEQ ID NO: 8) encoding FVIII atgcagattg agctgagcac ttgctttttt ctgtgcctgctgaggttttg tttttctgcc accaggaggt actacctggg ggctgtggag ctgagctgggactatatgca gtctgatctg ggggagctgc ctgtggatgc caggttcccc cccagggtgcccaagtcttt tcccttcaac acctctgtgg tgtataagaa gaccctgttt gtggagttcactgaccacct gttcaacatt gctaagccta ggcccccctg gatgggcctg ctgggccctaccattcaggc tgaggtgtat gacactgtgg tgatcaccct gaagaacatg gccagccatcctgtgagcct gcatgctgtg ggggtctctt actggaaggc ctctgagggg gctgagtatgatgaccagac cagccagaga gagaaggagg atgacaaggt cttccctggg ggctctcacacctatgtgtg gcaggtgctg aaggaaaatg gccccatggc ctctgacccc ctgtgcctgacctacagcta tctgagccat gtggatctgg tgaaggacct gaattctggc ctgattggggccctgctggt gtgcagggag ggcagcctgg ccaaggagaa gacccagacc ctgcacaagtttatcctgct gtttgctgtg tttgatgagg gcaagtcttg gcactctgag actaagaacagcctgatgca ggacagggat gctgcctctg ccagggcctg gcccaagatg cacactgtgaatggctatgt gaacaggagc ctgcctgggc tgattggctg ccacaggaag tctgtgtactggcatgtgat tggcatgggc accacccctg aggtgcacag catcttcctg gaaggccacactttcctggt gaggaaccat aggcaggcca gcctggagat cagccctatc accttcctgactgcccagac cctgctgatg gatctggggc agttcctgct gttctgccac atctctagccaccagcatga tgggatggag gcctatgtga aggtggacag ctgcccagag gagcctcagctgaggatgaa aaacaatgaa gaggctgagg attatgatga tgatctgact gactctgagatggatgtggt gagatttgat gatgacaata gccctagctt tattcagatc aggtctgtggctaagaagca ccccaagacc tgggtgcatt acattgctgc tgaggaggag gactgggattatgctcctct ggtgctggcc cctgatgata ggagctacaa gagccagtac ctgaataatggccctcagag gattggcagg aagtacaaga aggtgaggtt catggcttac actgatgagaccttcaagac tagggaggcc atccagcatg agtctgggat cctggggccc ctgctgtatggggaggtggg ggacaccctg ctgatcatct tcaagaacca ggctagcagg ccttacaacatctatcccca tgggatcact gatgtgagac ctctgtacag caggaggctg cccaagggggtcaagcatct gaaagacttc cccatcctgc ctggggagat ctttaagtat aagtggactgtgactgtgga ggatgggccc accaagtctg accccaggtg cctgaccagg tattacagcagctttgtgaa catggagagg gatctggcct ctgggctgat tggccccctg ctgatctgttacaaggaatc tgtggatcag aggggcaatc agatcatgtc tgacaagagg aatgtgatcctgttctctgt gtttgatgag aataggtctt ggtacctgac tgaaaacatc cagaggttcctgcccaaccc tgctggggtc cagctggagg atcctgagtt ccaggctagc aacatcatgcacagcatcaa tgggtatgtg tttgatagcc tgcagctgtc tgtgtgcctg catgaggtggcctactggta catcctgtct attggggccc agactgactt cctgtctgtg ttcttttctggctacacctt caagcacaag atggtgtatg aggacaccct gaccctgttc cccttctctggggagactgt ctttatgagc atggagaacc ctgggctgtg gatcctgggc tgccacaactctgatttcag gaataggggc atgactgctc tgctgaaggt gagctcttgt gacaagaacactggggatta ctatgaggac agctatgagg acatttctgc ctacctgctg agcaagaacaatgccattga gcctaggagc tttagccaga atcctcctgt cctgaagagg caccagagggagatcaccag gaccaccctg cagtctgacc aggaggagat tgactatgat gataccatctctgtggagat gaagaaggag gactttgaca tctatgatga ggatgagaat cagtctcccaggagcttcca gaagaagacc aggcactatt tcattgctgc tgtggagagg ctgtgggactatggcatgag cagctctcct catgtgctga ggaatagggc tcagtctggc tctgtgccccagttcaagaa agtggtgttt caggagttca ctgatggctc tttcacccag cctctgtataggggggagct gaatgagcac ctggggctgc tgggccccta tatcagggct gaggtggaggataacatcat ggtgaccttc aggaaccagg cctctaggcc ctacagcttc tatagcagcctgatcagcta tgaggaggac cagaggcagg gggctgagcc caggaagaac tttgtgaagcccaatgagac caagacttac ttctggaagg tgcagcatca catggccccc accaaggatgagtttgactg taaggcctgg gcctacttct ctgatgtgga tctggagaag gatgtgcactctggcctgat tggccccctg ctggtgtgcc ataccaatac tctgaaccct gctcatggcaggcaggtgac tgtgcaggag tttgctctgt tcttcactat ctttgatgag accaagtcttggtatttcac tgagaatatg gaggagaact gcagggcccc ctgcaacatc cagatggaggaccccacctt tagggaagaac tataggtttc atgccatcaa tggctacatc atggacaccctgcctggcct ggtgatggcc caggatcaga ggatcaggtg gtacctgctg agcatggggtctaatgagaa catccacagc atccacttct ctggccatgt gtttactgtg agaagaaggaggtcaa gatggctctg tacaatctgt accctggggt ctttgagact gtggagatgctgcctagcaa ggctgggatc tggagggtgg agtgcctgat tggggaacat ctgcatgctgggatgtctac tctgttcctg gtgtacagca acaagtgcca gacccccctg ggcatggcttctggccatat cagggacttt cagattactg cctctgggca gtatggccag tgggcccccaagctggctag gctgcattat tctggcagca tcaatgcctg gtctactaag gagcccttcagctggatcaa ggtggatctg ctggccccca tgatcatcca tggcatcaag acccaggggggccaggcagaa gtttagctct ctgtacatta gccagttcat catcatgtac agcctggatgggaagaagtg gcagacctac aggggcaatt ctactggcac cctgatggtg ttctttggcaatgtggacag ctctggcatc aagcacaaca tctttaaccc ccctatcatt gctaggtacatcaggctgca tcccacccat tacagcatca ggagcaccct gaggatggag ctgatgggctgtgacctgaa ctcttgcagc atgcccctgg gcatggagag caaggccatt tctgatgcccagattactgc cagcagctac ttcactaaca tgtttgccac ctggtctccc agcaaggccaggctgcacct gcagggcagg agcaatgcct ggaggcccca ggtgaacaac cccaaggagtggctgcaggt ggatttccag aagaccatga aggtgactgg ggtgaccacc cagggggtgaagagcctgct gactagcatg tatgtgaagg agttcctgat cagctctagc caggatggccaccagtggac tctgtttttc cagaatggca aggtgaaggt gttccagggc aaccaggactctttcactcc tgtggtgaac agcctggacc cccccctgct gaccaggtat ctgaggattcacccccagtc ttgggtgcat cagattgccc tgaggatgga ggtgctgggc tgtgaggcccaggatctgta ctga

[0278] CpG-reduced nucleic acid variant X09 (SEQ ID NO: 9) encoding FVIII atgcagattg agctgagcac ctgcttcttc ctgtgtctgctgagattttg cttttctgcc actaggaggt attacctggg ggctgtggag ctgtcttgggactacatgca gtctgatctg ggggagctgc ctgtggatgc caggttccca cctagggtgcctaagagctt tcccttcaat acctctgtgg tgtacaagaa gaccctgttt gtggagttcactgaccacct gttcaacatt gccaagccta ggcccccctg gatgggcctg ctgggccctaccatccaggc tgaagtgtat gacactgtgg tgatcaccct gaagaacatg gccagccaccctgtgagcct gcatgctgtg ggggtgtctt actggaaggc ctctgagggg gctgagtatgatgatcagac cagccagagg gagaaggaag atgacaaggt gttccctggg ggcagccacacctatgtctg gcaggtgctg aaggaatg gccccatggc ctctgatccc ctgtgcctgacctactctta cctgagccat gtggacctgg tgaaggatct gaattctggc ctgattggggccctgctggt gtgcagggag ggcagcctgg ccaaggaa gacccagacc ctgcataagttcatcctgct gtttgctgtg tttgatgaag ggaagagctg gcactctgag actaagaacagcctgatgca ggacagggat gctgcttctg ccagggcctg gcccaagatg caactgtgaatggctatgt gaatagaagc ctgcctggcc tgattgggtg ccacaggaag tctgtgtactggcatgtgat tgggatgggc actacccctg aggtgcatag catcttcctg gaaggccataccttcctggt gaggaatcat aggcaggctt ctctggaaat ttctcccatc actttcctgactgctcagac cctgctgatg gacctgggcc agttcctgct gttctgccac atcagctctcaccagcatga tgggatggag gcctatgtga aggtggacag ctgtcctgag gagccccagctgaggatgaa gaacaatgag gaggctgagg actatgatga tgacctgact gactctgagatggatgtggt caggtttgat gatgacaata gcccctcttt catccagatc aggtctgtggccaagaagca ccccaagact tgggtgcact acattgctgc tgaggaggag gattgggattatgcccctct ggtgctggcc cctgatgaca ggagctataa gtctcagtac ctgaataatggcccccagag gattgggagg aagtataaga aggtgaggtt tatggcctac actgatgagaccttcaagac cagggaggcc atccagcatg agtctggcat cctgggcccc ctgctgtatggggaggtggg ggataccctg ctgatcatct tcaagaacca ggcctctagg ccctacaatatctaccctca tggcatcact gatgtgagac ccctgtatag caggaggctg cctaagggggtgaagcacct gaaggacttc cccatcctgc ctggggagat cttcaagtat aagtggactgtgactgtgga ggatggcccc accaagtctg accccaggtg cctgaccagg tattacagctcttttgtgaa catggagagg gatctggcct ctgggctgat tggcccactg ctgatctgctacaaggagtc tgtggatcag aggggcaatc agatcatgtc tgacaagagg aatgtgatcctgttttctgt gtttgatgaa aataggtctt ggtatctgac tgagaacatc cagaggtttctgcccaatcc tgctggggtg cagctggagg atcctgagtt tcaggcctct aatatcatgcattctatcaa tggctatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtggcctactggta catcctgagc attggggctc agactgactt cctgtctgtg ttcttttctggctatacttt caagcacaag atggtgtatg aggacactct gaccctgttc cccttctctggggagactgt gttcatgtct atggaaaatc ctgggctgtg gattctgggc tgccacaattctgacttcag gaataggggg atgactgccc tgctgaaggt gtctagctgt gataagaacactggggatta ctatgaggac tcttatgaag atatctctgc ctatctgctg agcaagaacaatgccattga gcccaggagc ttcagccaga acccccctgt gctgaagagg caccagagggagatcaccag gaccactctg cagtctgatc aggaggagat tgactatgat gacactatctctgtggagat gaagaaggag gattttgaca tttatgatga ggatgagaac cagtctcccaggagcttcca gaagagacc aggcattact ttattgctgc tgtggagagg ctgtgggactatgggatgag cagctctcct catgtgctga ggaacagggc ccagtctggg tctgtgccccagttcaagaa ggtggtgttc caggagttca ctgatgggag cttcacccag cccctgttaggggggagct gaatgagcac ctgggcctgc tgggccccta catcagggct gaggtggaggataatatcat ggtgaccttc aggaaccagg ctagcaggcc ttacagcttt tacagcagcctgatctctta tgaagaagac cagaggcagg gggctgagcc cagaagaat tttgtgaagcctaatgagac caagacttat ttttggaagg tgcagcatca catggctcct accaaggatgagtttgactg caaggcctgg gcctactttt ctgatgtgga tctggagaag gatgtgcactctgggcctgat tggccctctg ctggtgtgcc atactaacac tctgaaccct gcccatgggaggcaggtgac tgtgcaggag tttgccctgt tcttcactat ttttgatgag accaagtcttggtatttcac tgagaacatg gaggagaact gcagggctcc ctgcaacatc cagatggagaaccccacctt caaggaaac tataggttcc atgccatcaa tgggtacatc atggataccctgcctggcct ggtgatggcc caggatcaga ggattaggtg gtatctgctg agcatgggctctaatgagaa catccacagc atccatttct ctggccatgt gttcactgtg aggaagaaggaggagtacaa gatggctctg tacaacctgt atcctggggt gtttgagact gtggagatgctgcccagcaa ggctggcatc tggagggtgg aatgcctgat tggggagcac ctgcatgctggcatgagcac tctgttcctg gtgtatagca acaagtgcca gacccccctg ggcatggcctctggccatat cagggatttc cagatcactg cttctggcca gtatggccag tgggcccccaagctggccag gctgcactat tctggcagca tcaatgcctg gagcactaag gagcctttttcttggatcaa ggtggacctg ctggccccta tgattattca tggcatcaag acccagggggccaggcagaa gttctctagc ctgtacatct ctcagttcat cattatgtat agcctggatggcaagaagtg gcagacctac aggggcaata gcactggcac cctgatggtg ttttttgggaatgtggactc ttctgggatc aagcacaaca tctttaaccc ccccatcatt gccaggtatattaggctgca ccccacccac tacagcatca ggagcaccct gaggatggag ctgatgggctgtgatctgaa ttcttgctct atgcccctgg gcatggagag caaggccatc tctgatgcccagatcactgc cagctcttac ttcaccaaca tgtttgccac ctggtctcct agcaaggccaggctgcatct gcagggcagg agcaatgcct ggaggcccca ggtgaacaac cccaaggagtggctgcaggt ggacttccag aagaccatga aggtgactgg ggtgaccact cagggggtgaagagcctgct gacctctatg tatgtgaagg agttcctgat cagcagcagc caggatggccaccagtggac tctgttcttc cagaatggga aggtgaaggt gttccagggc aaccaggatagctttacccc tgtggtgaac agcctggacc ctcctctgct gaccagatac ctgaggatccatcctcagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaggcccaggatctgta ctga

[0279] CpG-reduced nucleic acid variant X10 (SEQ ID NO: 10) encoding FVIII atgcagattg agctgagcac ttgcttcttc ctgtgcctgctgaggttctg cttttctgct actaggaggt actacctggg ggctgtggag ctgagctgggattacatgca gtctgacctg ggggagctgc cagtggatgc caggttcccc cccagggtgcccaagtcttt tcctttcaac acctctgtgg tgtacaagaa gaccctgttt gtggagttcactgaccacct gttcaacatt gccaagccca ggcccccctg gatggggctg ctggggcccaccatccaggc tgaggtgtat gacactgtgg tgattaccct gaagaacatg gctagccaccctgtgagcct gcatgctgtg ggggtgagct attggaaggc ctctgagggg gctgagtatgatgatcagac cagccagagg gaaaaggagg atgacaaggt gttccctggg ggcagccatacttatgtgtg gcaggtgctg aaggaatg ggcccatggc ctctgacccc ctgtgcctgacttacagcta tctgagccat gtggacctgg tgaaggatct gaactctggc ctgattggggctctgctggt gtgcagggag ggcagcctgg ctaaggaa gactcagact ctgcataagttcatcctgct gtttgctgtg tttgatgaag gcaagagctg gcactctgag accaagaactctctgatgca ggatagggat gctgcccttg ccagggcttg gcccaagatg caactgtgaatggctatgt gaacaggagc ctgcctggcc tgattgggtg ccacaggaag tctgtgtactggcatgtgat tggcatgggc accacccctg aggtgcacag cattttcctg gagggccacaccttcctggt gaggaatcac aggcaggcca gcctggagat cagcccccatc accttcctgactgcccagac cctgctgatg gacctggggc agtttctgct gttctgccac atcagcagccatcagcatga tggcatggag gcctatgtga aggtggactc ttgccctgag gagccccagctgaggatgaa gaaaatgag gaggctgagg attatgatga tgacctgact gactctgagatggatgtggt gaggtttgat gatgacaata gccccagctt catccagatt aggtctgtggccaagaagca ccctaagacc tgggtgcact acattgctgc tgaggaggag gattgggattatgcccccct ggtgctggct cctgatgaca ggtcttataa gagccagtac ctgaacaatgggccccagag gattggcagg aagtacaaga aggtgaggtt catggcttac actgatgagaccttcaagac tagggaggcc atccagcatg agtctggcat cctgggcccc ctgctgtatggggaggtggg ggataccctg ctgatcatct tcaagaacca ggccagcagg ccctacaacatttaccctca tggcatcact gatgtgaggc ccctgtacag caggagactg cccaagggggtgaagcacct gaaggatttt cccattctgc ctggggagat cttcaagtac aagtggactgtgactgtgga ggatggcccc accaagtctg atcccaggtg cctgactagg tactactcttcttttgtgaa tatggagagg gatctggcct ctggcctgat tggccccctg ctgatctgctacaaggagtc tgtggaccag aggggcaacc agatcatgtc tgacaagagg aatgtgatcctgttctctgt gtttgatgag aataggagct ggtacctgac tgagaatatc cagaggttcctgcctaatcc tgctggggtc cagctggagg atcctgagtt ccaggctagc aacattatgcacagcatcaa tggctatgtg tttgattctc tgcagctgtc tgtgtgcctg catgaggtggcttactggta catcctgtct attggggccc agactgattt cctgtctgtg ttcttctctggctacacttt caagcataag atggtgtatg aggataccct gaccctgttc cccttctctggggagactgt gttcatgtct atggagaacc ctggcctgtg gatcctgggc tgtcataactctgacttcag aaacaggggc atgactgccc tgctgaaggt gagcagctgt gacaagaacactggggacta ctatgaggac agctatgagg atatctctgc ttatctgctg agcaagaataatgccattga gcccaggagc ttcagccaga acccccctgt gctgaagagg caccagagggagatcactag gactaccctg cagtctgatc aggaggagat tgactatgat gacaccatctctgtggagat gaagaaggag gactttgaca tctatgatga ggatgagaac cagtcccccaggtctttcca gaagaagacc aggcactact tcattgctgc tgtggagagg ctgtgggactatggcatgag ctctagcccc catgtgctga ggaacagggc tcagtctggc tctgtgccccagttcaagaa ggtggtcttc caggagttca ctgatggctc ttttacccag cctctgtacagaggggagct gaatgagcac ctgggcctgc tgggccccta catcagggct gaggtggaggataatatcat ggtgaccttc agaaaccagg cctctaggcc ctacagcttc tacagcagcctgatctctta tgaggaggat cagaggcagg gggctgagcc caggaagaac tttgtgaagcccaatgagac caagacctac ttctggaagg tgcagcacca tatggcccct actaaggatgagtttgactg caaggcctgg gcttattttt ctgatgtgga cctggagaag gatgtgcactctgggctgat tggccccctg ctggtgtgcc acaccaacac cctgaaccct gcccatggcaggcaggtgac tgtgcaggag tttgccctgt tcttcactat ctttgatgag accaagagctggtacttcac tgagaacatg gagagaaatt gtagggctcc ctgcaatatc cagatggaggaccccacctt caaagaaaat tacagattcc atgccatcaa tgggtacatc atggataccctgcctgggct ggtgatggct caggaccaga ggatcaggtg gtacctgctg agcatggggtctaatgagaa catccactct atccatttct ctggccatgt gttcactgtg agaaagaaggaggagtataa gatggctctg tacaacctgt acccaggggt gtttgagact gtggaaatgctgcccagcaa agctgggatc tggagggtgg agtgcctgat tggggagcac ctgcatgctggcatgtctac cctgttcctg gtgtacagca acaagtgcca gactcccctg ggcatggcctctgggcacat cagggatttt cagatcactg cctctggcca gtatggccag tgggcccccaagctggccag gctgcactac tctggcagca ttaatgcttg gagcactaag gagcccttcagctggatcaa ggtggatctg ctggccccca tgatcatcca tggcatcaag acccagggggccaggcagaa gttctctagc ctgtacattt ctcagttcat catcatgtac agcctggatgggaagaagtg gcagacctac agggggaaca gcactgggac cctgatggtg ttctttggcaatgtggatag ctctggcatc aagcacaata tcttcaatcc ccccattatt gccaggtacattaggctgca tcctactcac tactctatta ggagcaccct gaggatggag ctgatggggtgtgacctgaa cagctgttct atgcccctgg gcatggagtc taaggctatc tctgatgcccagatcactgc cagcagctac ttcactaata tgtttgccac ctggagccct agcaaggccagactgcacct gcagggcagg agcaatgcct ggaggcccca ggtgaacaac cccaaggagtggctgcaggt ggacttccag aagaccatga aggtgactgg ggtgaccact cagggggtgaagagcctgct gaccagcatg tatgtgaagg agttcctgat cagcagcagc caggatggccaccagtggac cctgttcttc agaatggga aggtgaaggt gttccagggc aaccaggactctttcacccc tgtggtgaac agcctggatc ctcccctgct gaccaggtac ctgaggatccacccccagag ctgggtgcac cagattgctc tgaggatgga agtgctgggc tgtgaggcccaggatctgta ctga

[0280] CpG-reduced nucleic acid variant X11 (SEQ ID NO: 11) encoding FVIII atgcagattg agctgagcac ctgcttcttc ctgtgcctgctgaggttttg cttctctgct accaggaggt actacctggg ggctgtggag ctgagctgggaactatatgca gtctgacctg ggggagctgc ctgtggatgc taggttccct cccagggtgcccaagagctt cccctttaat acctctgtgg tgtacaagaa aaccctgttt gtggagttcactgaccatct gttcaacatt gccaagccca ggcccccttg gatgggcctg ctgggccccaccattcaggc tgaggtgtat gacactgtgg tcattaccct gagaacatg gcttctcaccctgtgagcct gcatgctgtg ggggtgagct actggaaggc ctctgagggg gctgagtatgatgaccagac cagccagagg gagaggagg atgataaggt gttccctggg ggcagccacacctatgtgtg gcaggtgctg aaggaatg gccccatggc ctctgatccc ctgtgcctgacctactctta tctgtctcat gtggacctgg tgaaggacct gaactctggc ctgattggggctctgctggt gtgcagggag ggctctctgg ccaaggagaa gacccagacc ctgcacaagtttattctgct gtttgctgtc tttgatgagg gcaagagctg gcattctgag accaagaacagcctgatgca ggacagggat gctgcctctg ccagggcctg gcccaaaatg cacactgtgaatggctatgt gaacaggagc ctgcctggcc tgattggctg ccacaggaag tctgtgtactggcatgtgat tggcatgggc accacccctg aggtgcacag catcttcctg gagggccacacctttctggt gaggaatcac aggcaggcca gcctggagat tagccccatc accttcctgactgcccagac cctgctgatg gacctgggcc agttcctgct gttctgccac atcagcagccaccagcatga tggcatggag gcctatgtga aggtggatag ctgccctgag gagccccagctgaggatgaa aaacaatgag gaggctgagg attatgatga tgacctgact gactctgagatggatgtggt gaggtttgat gatgacaata gccccagctt tattcagatt aggtctgtggctaagaagca ccccaagact tgggtgcact acattgctgc tgaggaggag gattgggactatgcccctct ggtcctggcc cctgatgata ggtcttacaa gagccagtat ctgaacaatggcccccagag gattggcagg aagtacaaga aggtgaggtt catggcctac actgatgagacctttaagac cagggaggcc attcagcatg agtctgggat cctgggcccc ctgctgtatgggggaggtggg ggacactctg ctgatcatct tcaagaacca ggccagcagg ccttataacatctaccctca tgggatcact gatgtgaggc ccctgtactc tagaaggctg cccaaggggtcaagcacct gaaggatttt cccatcctgc ctggggagat tttcaagtac aagtggactgtgactgtgga ggatggcccc accaagtctg accctaggtg cctgaccagg tactacagctcttttgtgaa catggagagg gacctggcct ctggcctgat tggccctctg ctgatttgctacaaggagtc tgtggaccag aggggcaacc agatcatgtc tgacaagagg aatgtgatcctgttttctgt gtttgatgag aacaggtctt ggtacctgac tgagaacatc cagaggttcctgcctaaccc agctggggtg cagctggagg atcctgagtt ccaggccagc aatattatgcatagcattaa tggctatgtg tttgatagcc tgcagctgtc tgtgtgcctg catgaggtggcctactggta catcctgagc attggggccc agactgactt tctgtctgtg ttcttctctggctacacctt caagcataag atggtgtatg aggacaccct gactctgttc cctttttctggggagactgt gtttatgagc atggagaatc ctggcctgtg gatcctgggc tgccataattctgacttcag gaacaggggc atgactgccc tgctgaaagt gagcagctgt gacaagaatactggggacta ctatgaagac agctatgagg acatctctgc ctacctgctg agcaagaacaatgccattga gcccaggagc ttcagccaga accccccagt gctgaagagg caccagagagagatcaccag gactaccctg cagtctgacc aggaggagat tgactatgat gacaccatttctgtggagat gaagaaggag gactttgaca tttatgatga ggatgagaat cagagccccaggagcttcca gaagaagact aggcactatt ttattgctgc tgtggagagg ctgtgggactatggcatgag cagctctccc catgtgctga ggaatagggc ccagtctggc tctgtgcctcagttcaagaa ggtggtgttc caggagttca ctgatggcag ctttacccag cccctgtataggggggagct gaatgagcac ctgggcctgc tgggccccta tatcagggct gaggtggaggacaatattat ggtgaccttt aggaaccagg ccagcaggcc ctactctttc tatagcagcctgatcagcta tgaggaggac cagaggcagg gggctgagcc caggaagaat tttgtgaagcctaatgagac caagacctac ttctggaagg tgcagcatca catggccccc accaaggatgagtttgactg caaggcttgg gcctatttct ctgatgtgga cctggagaag gatgtgcactctgggcctgat tggccccctg ctggtgtgcc acactaacac tctgaatcct gcccatggcaggcaggtgac tgtgcaggag tttgccctgt tcttcaccat ctttgatgag accaagagctggctacttcac tgagaacatg gaggagaact gcagggcccc ctgcaacatc cagatggaggatcccacctt caaggacac tacaggtttc atgccatcaa tggctacatc atggacactctgcctggcct ggtgatggcc caggatcaga ggatcaggtg gtacctgctg agcatgggctctaatgagaa tatccatagc atccacttct ctggccatgt gttcactgtc aggaaaggaggagatcaa gatggctctg tataatctgt accctggggt gtttgagact gtggagatgctgcccagcaa ggctggcatc tggagggtgg agtgcctgat tggggagcac ctgcatgctgggatgagcac cctgtttctg gtgtactcta acaagtgcca gacccccctg ggcatggcctctgggcacat cagggatttc cagatcactg cttctggcca gtatggccag tgggcccccaagctggccag gctccactac tctggcagca tcaatgcctg gtctaccaag gagcccttttcttggattaa ggtggacctg ctggccccca tgatcatcca tggcatcaag acccaggggggccaggcagaa gttcagcagc ctgtacatca gccagttcat catcatgtac agcctggatggcaaaaagtg gcagacctac aggggcaata gcactgggac tctgatggtg ttctttggcaatgtggacag ctctgggatc aagcacaata tcttcaaccc tcccatcatt gctaggtacatcaggctgca ccccacccac tatagcatca ggtctaccct gaggatggag ctgatgggctgtgacctgaa ctcttgcagc atgcccctgg gcatggagtc caaagctatc tctgatgcccagattactgc cagcagctac ttcaccaaca tgtttgccac ctggtctccc tctaaggccaggctgcacct gcagggcagg agcaatgcct ggaggcccca ggtgaacaat cccaaggagtggctgcaggt ggatttccag aaaactatga aggtgactgg ggtgaccacc cagggggtgaagtctctgct gaccagcatg tatgtgaagg agttcctgat ctcttctagc caggatggccaccagtggac tctgttcttc cagaatggca aggtgaaggt gttccagggc aaccaggacagcttcacccc tgtggtgaac tctctggatc cccccctgct gaccaggtac ctgaggattcatccccagag ctgggtgcac cagattgctc tgagaatgga ggtgctgggg tgtgaggctcaggacctgta ttga

[0281] CpG-reduced nucleic acid variant X12 (SEQ ID NO: 12) encoding FVIII atgcagattg agctgtctac ttgtttttt ctgtgcctgctgaggttctg cttctctgcc accaggaggt attacctggg ggctgtggag ctgagctgggattacatgca gtctgatctg ggggagctgc ctgtggatgc caggttcccc cccagggtgcccaagagctt ccccttcaac acctctgtgg tgtataagaa gaccctgttt gtggagttcactgatcatct gtttaacatt gccaagccca ggcccccctg gatgggcctg ctgggcccaactatccaggc tgaggtgtat gacactgtgg tcatcaccct gaagaatatg gccagccatcctgtgagcct gcatgctgtg ggggtgagct actggaaggc ctctgagggg gctgagtatgatgaccagac cagccagagg gagaaggagg atgacaaggt gttccctggg ggcagccacacctatgtgtg gcaggtgctg aaggaatg gccccatggc ctctgacccc ctgtgcctgactatagatcta cctgtctcat gtggacctgg tgaaggacct gaactctggc ctgattggggccctgctggt ctgtagggaa ggcagcctgg ccaaggaa gacccagacc ctgcacaagtttatctgct gtttgctgtg tttgatgaag gcaagagctg gcactctgag accaagaattctctgatgca ggatagggat gctgcccttg ccagggcctg gcccaagatg catactgtgaatggctatgt gaacaagaagc ctgcctggcc tgattggctg ccataggaag tctgtgtattggcatgtgat tgggatgggc actacccctg aagtgcacag cattttcctg gagggccacactttcctggt gaggaaccac aggcaggcct ctctggagat cagccccatt actttcctgactgcccagac cctgctgatg gatctgggcc agttcctgct gttctgccac atctctagccaccagcatga tggcatggag gcctatgtga aggtggacag ctgccctgag gagccccagctgaggatgaa gaataatgag gaggctgagg attatgatga tgacctgact gactctgagatggatgtggt gaggtttgat gatgataata gccccagctt catccagatc aggtctgtggccaagaagca tcccaagacc tgggtgcact atattgctgc tgaagaggag gactgggactatgcccctct ggtgctggct cctgatgaca ggagctataa gagccagtat ctgaacaatgggccccagag gattgggagg aagtacaaga aggtgaggtt catggcctac actgatgagacctttaagac cagggaggcc atccagcatg agtctggcat tctggggccc ctgctgtatggggaggtggg ggacactctg ctgatcattt tcaagaacca ggccagcagg ccctacaatatttaccccca tggcatcact gatgtgaggc ccctgtacag caggaggctg cccaagggggtgaagcacct gaaggacttc cccatcctgc ctggggagat cttcaagtac aagtggactgtgactgtgga ggatggccct accaagtctg accctaggtg tctgactagg tactacagcagctttgtgaa catggagaga gacctggctt ctggcctgat tggccccctg ctgatctgctacaaggagtc tgtggatcag aggggcaacc agattatgtc tgataagagg aatgtcatcctgttctctgt gtttgatgag aacaggagct ggtatctgac tgagaacatt cagaggttcctgcccaaccc tgctggggtg cagctggagg accctgagtt ccaggccagc aacatcatgcattctattaa tggctatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtggcctactggta catcctgagc attggggccc agactgactt tctgtctgtg tttttctctgggtacacctt caagcacaag atggtctatg aggacaccct gaccctgttc cccttttctggggaaactgt gtttatgagc atggagaacc ctgggctgtg gatcctgggc tgccacaactctgactttag gaataggggc atgactgccc tgctgaaggt gagcagctgt gacaagaatactggggatta ctatgaggac agctatgagg atatctctgc ctacctgctg agcaagaacaatgccattga gcctaggagc ttcagccaga acccccctgt gctgaagagg caccagagggagatcaccag gaccaccctg cagtctgatc aggagagat tgactatgat gacaccatctctgtggagat gaagaggag gactttgata tttatgatga ggatgagaac cagagccccaggagcttcca gaagagacc aggcactatt tcattgctgc tgtggagagg ctgtgggactatggcatgag ctctagcccc catgtgctga ggaacagggc ccagtctggc tctgtgccccagttcaagaa ggtggtgttc caggaattta ctgatggcag ctttacccag cccctgtacagaggggagct gaatgagcac ctgggcctgc tgggccccta catcagggct gaggtggaggataatatcat ggtgaccttt aggaaccagg cctttaggcc ctattctttt tacagcagcctgatcagcta tgaggaggac cagaggcagg gggctgagcc taggaaac tttgtgaagcccaatgagac caagacctac ttttggaaag tgcagcacca catggcccccc actaaggatgagtttgattg caaggcctgg gcctatttct ctgatgtgga cctggagaag gatgtgcactctgggcctgat tggccccctg ctggtgtgcc acaccaacac tctgaaccct gcccatggcaggcaggtgac tgtgcaggag tttgccctgt tctttaccat ctttgatgag actaagagctggtatttcac tgagaacatg gaggagaact gcagagcccc ttgcaacatc cagatggaggaccctacctt caaggaac tataggttcc atgccatcaa tgggtacatc atggataccctgcctggcct ggtgatggct caggaccaga ggatcaggtg gtacctgctg agcatggggagcaatgagaa cattcatagc atccacttct ctgggcatgt gttcactgtg aggaagaaggaggagtataa gatggccctg tacaacctgt accctggggt gtttgagact gtggagatgctgcccagcaa ggctggcatc tggagggtgg agtgcctgat tggggagcac ctgcatgctggcatgagcac tctgttcctg gtgtacagca acaagtgcca gacccccctg ggcatggcctctggccacat cagggacttc cagattactg cctctgggca gtatgggcag tgggcccccaagctggccag gctgcactac tctgggtcta tcaatgcttg gagcaccaag gagcctttcagctggatcaa ggtggatctg ctggccccca tgatcattca tgggatcaag acccagggggccaggcagaa gttcagcagc ctgtatattt ctcagttcat catcatgtat tctctggatggcaaaaagtg gcagacctat agagggaaca gcactgggac cctgatggtg ttttttggcaatgtggatag ctctggcatc aagcacaata tcttcaaccc ccccattatt gccaggtacatcaggctgca ccccacccac tactctatca ggagcaccct gaggatggag ctgatgggctgtgatctgaa cagctgctct atgcctctgg ggatggaaag caaggccatc tctgatgcccagatcactgc cagcagctat ttcaccaata tgtttgccac ttggagccct agcaaggctaggctgcatct gcagggcagg tctaatgcct ggaggcccca ggtgaacaac cccaaggagtggctgcaggt ggacttccag aagactatga aagtgactgg ggtgaccacc cagggggtgaaaagcctgct gaccagcatg tatgtgaagg agttcctgat tagcagcagc caggatggccaccagtggac cctgttcttc cagaatggga aggtgaaggt gtttcagggc aatcaggatagcttcacccc agtggtgaac agcctggacc cccccctgct gaccaggtac ctgaggatccacccccagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaggcccaggatctgta ctga

[0282] CpG-reduced nucleic acid variant X13 (SEQ ID NO: 13) encoding FVIII atgcagattg agctgagcac ctgctttttc ctgtgcctgctgaggttctg cttctctgct accaggaggt actacctggg ggctgtggag ctgtcttgggattacatgca gtctgacctg ggggagctgc ctgtggatgc caggtttccc cccagggtgcccaagtcttt cccctttaac acctctgtgg tgtataagaa gactctgttt gtggagttcactgatcacct gttcaatatt gccaagccca ggcccccttg gatgggcctg ctgggccccactatccaggc tgaggtgtat gacactgtgg tcatcaccct gaagaacatg gccagccaccctgtgagcct gcatgctgtg ggggtgagct actggaaggc ctctgagggg gctgagtatgatgaccagac cagccagagg gagaaggagg atgacaaggt gttcccaggg gggtctcacacttatgtgtg gcaggtgctg aaggaatg ggcccatggc ctctgaccct ctgtgcctgactatagatcta cctgtctcat gtggatctgg tgaaggacct gaactctggc ctgattggggccctgctggt gtgcagggag gggagcctgg ccaaggaga gacccagacc ctgcacaagttcatcctgct gtttgctgtg tttgatgagg ggaagagctg gcactctgag accaagaatagcctgatgca ggacagggat gctgcttctg ctagggcctg gcctaagatg caactgtgaatggctatgt gaacaggagc ctgcctggcc tgattgggtg tcacaggaag tctgtgtactggcatgtgat tggcatgggg actactccag aagtgcacag catcttcctg gaggggcacaccttcctggt gaggaatcac aggcaggcca gcctggagat ttctcccatc actttcctgactgcccagac cctgctgatg gatctggggc agttcctgct gttctgccac atcagcagccatcagcatga tgggatggag gcctatgtga aggtggacag ctgccctgag gagcctcagctgaggatgaa gaacaatgag gaggctgagg actatgatga tgatctgact gactctgagatggatgtggt gaggtttgat gatgacaact ctcccagctt catccagatc aggtctgtggccaagaagca ccccaagacc tgggtgcact acattgctgc tgaggaggag gattgggattatgctcccct ggtgctggct cctgatgata ggagctacaa gagccagtat ctgaataatgggccccagag gattggcagg aagtataaga aggtgaggtt catggcctac actgatgagacctttaagac cagggaggct attcagcatg agtctggcat cctgggcccc ctgctgtatggggaggtggg ggacaccctg ctgatcattt tcaagaacca ggccagcagg ccctataacatctatcccca tgggatcact gatgtgaggc ccctgtactc taggaggctg cccaagggggtcaagcacct gaaggacttc cccatcctgc ctggggagat cttcaagtac aagtggactgtgactgtgga ggatggcccc actaagtctg accccaggtg cctgactagg tactacagcagctttgtgaa catggagaga gatctggcct ctggcctgat tggccccctg ctgatctgctacaaagagtc tgtggatcag aggggcaacc agatcatgtc tgacaagagg aatgtgatcctgttctctgt gtttgatgag aacagaagct ggtacctgac tgagaacatt cagaggtttctgcccaaccc tgctggggtc cagctggagg accctgagtt tcaggccagc aacatcatgcacagcatcaa tgggtatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtggcctactggta tatcctgagc attggggccc agactgattt cctgtctgtg ttcttctctggctacacttt caagcacaag atggtgtatg aggataccct gaccctgttc cctttctctggggaaactgt gttcatgagc atggagaacc ctgggctgtg gatcctgggg tgccacaattctgatttcag gaacagaggc atgactgctc tgctgaaggt gtctagctgt gacaagaacactggggacta ctatgaggac agctatgagg acatctctgc ctacctgctg agcaagaacaatgctattga acccaggtct ttcagccaga acccccctgt gctgaagagg caccagagggagatcactag gaccaccctg cagtctgatc aggaggagat tgactatgat gacaccatctctgtggagat gaagaaggag gactttgaca tctatgatga ggatgagaat cagtctcccaggagcttcca gaagaagact aggcattact tcattgctgc tgtggagagg ctgtgggactatggcatgag ctctagccct catgtgctga ggaacagggc ccagtctggc tctgtgccccagttcaagaa ggtggtgttt caggagttca ctgatggcag cttcacccag cccctgtacaggggggagct gaatgagcat ctgggcctgc tgggccccta catcagggct gaggtggaggaacacatcat ggtgaccttc agaaatcagg ctagcaggcc ctacagcttc tacagcagcctgatctctta tgaggaggac cagaggcagg gggctgagcc cagaagaac tttgtgaagcccaatgagac caagacctat ttctggaagg tgcagcacca catggcccccc accaaggatgagtttgattg caaggcctgg gcctacttct ctgatgtgga cctggagaag gatgtgcattctgggctgat tggccctctg ctggtgtgcc acaccaacac cctgaatcct gcccatggcaggcaggtgac tgtgcaggag tttgccctgt tctttactat ctttgatgag accaagtcttggtattttac tgagaacatg gaggagaact gcagggcccc ctgcaacatc cagatggaggaccccacctt caaggaac tacagattcc atgccatcaa tggctacatt atggacactctgcctggcct ggtgatggcc caggaccaga ggatcaggtg gtacctgctg tctatgggcagcaatgagaa cattcactct atccacttct ctgggcatgt gttcactgtg aggaaaggaggagatcaa gatggccctg tacaacctgt accctggggt gtttgagact gtggagatgctgcctagcaa ggctgggatc tggagggtgg agtgcctgat tggggagcac ctgcatgctggcatgtctac cctgttcctg gtgtacagca acaagtgcca gacccccctg ggcatggcctctggccacat cagagatttt cagatcactg cctctggcca gtatggccag tgggctcctaagctggccag gctgcactac tctggcagca tcaatgcctg gagcaccaag gagccctttagctggatcaa ggtggacctg ctggccccca tgatcatcca tggcatcaag actcagggggccaggcagaa gttctctagc ctgtacatta gccagttcat catcatgtat agcctggatggcaagaagtg gcagacctac aggggcaaca gcactgggac cctgatggtg ttctttgggaatgtggacag ctctgggatc aagcacaata tcttcaaccc ccccattatt gccaggtatattaggctgca ccccactcac tacagcatta ggagcaccct gaggatggag ctgatgggctgtgatctgaa cagctgcagc atgcccctgg gcatggagtc taaggccatc tctgatgcccagatcactgc cagctcttac ttcaccaaca tgtttgccac ttggagcccc agcaaggccaggctgcacct gcagggcagg agcaatgcct ggaggcccca ggtgaacaac cccaaggagtggctgcaggt ggatttccag aagactatga aggtgactgg ggtgaccact cagggggtgaagagcctgct gactagcatg tatgtgaagg agttcctgat cagctctagc caggatggccaccagtggac cctgttcttt cagaatggca aggtgaaggt gttccagggc aaccaggactctttcacccc tgtggtgaat tctctggacc ctcccctgct gactaggtat ctgaggattcatccccagag ctgggtgcat cagattgccc tgaggatgga ggtgctggggc tgtgaggcccaggacctgta ttga

[0283] CpG-reduced nucleic acid variant X14 (SEQ ID NO: 14) encoding FVIII atgcagattg agctgagcac ctgcttcttc ctgtgcctgctgaggttttg cttttctgcc actaggaggt actacctggg ggctgtggag ctgtcttgggattacatgca gtctgacctg ggggagctgc cagtggatgc caggttcccc ccaagggtgcccaagtcttt tcccttcaat acctctgtgg tgtacaagaa gaccctgttt gtggagtttactgatcatct gtttaacatt gccaagccca ggcccccctg gatggggctg ctgggccccaccatccaggc tgaggtgtat gatactgtgg tgattaccct gaagaatatg gccagccatcctgtgtctct gcatgctgtg ggggtgtctt attggaaggc ctctgagggg gctgagtatgatgatcagac cagccagagg gagaggagg atgataaggt gttccctggg ggctctcacacctatgtgtg gcaggtgctg aaggagaatg ggcctatggc ctctgaccca ctgtgcctgacttacagcta tctgagccat gtggacctgg tgaaggacct gaactctggg ctgattggggccctgctggt gtgcagggag ggcagcctgg ccaaggagaa gactcagacc ctgcacaagttcatcctgct gtttgctgtg tttgatgagg gcaagtcttg gcactctgag accaagaacagcctgatgca ggatagggat gctgcctctg ccagggcctg gcccaagatg cacactgtgaatggctatgt gaacaggtct ctgcctggcc tgattggctg ccacaggaag tctgtgtactggcatgtgat tggcatgggc accacccctg aggtgcatag cattttcctg gagggccacaccttcctggt gaggaaccac aggcaggcta gcctggagat cagccccatc actttcctgactgcccagac cctgctgatg gacctgggcc agttcctgct gttctgccac atctctagccaccagcatga tggcatggag gcctatgtga aggtggactc ttgtcctgag gagccccagctgaggatgaa gaacaatgag gaggctgagg attatgatga tgatctgact gattctgagatggatgtggt gaggtttgat gatgacaaca gcccctcttt catccagatc aggtctgtggccaagaagca ccccaagacc tgggtgcact acattgctgc tgaggaggag gattgggattatgcccccct ggtgctggcc cctgatgaca ggagctataa gtctcagtac ctgaacaatggcccccagag aattggcagg aagtacaaga aggtgaggtt catggcctat actgatgagaccttcaaaac cagggaggcc attcagcatg agtctggcat cctggggccc ctgctgtatggggaggtggg ggacaccctg ctgatcatct tcaagaacca ggctagcagg ccttacaacatctaccccca tgggatcact gatgtgaggc ccctgtacag caggaggctg cctaagggggtgaagcacct gaaggacttt cccattctgc ctggggagat cttcaagtat aagtggactgtgactgtgga ggatgggccc accaagtctg accccaggtg cctgactagg tactactctagctttgtgaa catggagagg gacctggcct ctgggctgat tggccccctg ctgatctgttacaaggagtc tgtggaccag aggggcaacc agatcatgtc tgataagagg aatgtgatcctgttctctgt gtttgatgag aacaggagct ggtacctgac tgagaacatc cagagattcctgcccaaccc tgctggggtg cagctggagg atcctgagtt ccaggccagc aacatcatgcattctatcaa tgggtatgtg tttgatagcc tgcagctgtc tgtgtgtctg catgaggtggcctactggta cattctgagc attggggccc agactgactt cctgtctgtg ttcttctctggctacacttt caaacacaag atggtgtatg aggacaccct gaccctgttc cccttctctggggagactgt gtttatgagc atggagaacc ctgggctgtg gattctgggc tgccacaactctgacttcag aaacaggggc atgactgcc tgctgaaggt gtcttctttgt gataagaacactggggacta ttgaagac agctatgagg acatctctgc ctacctgctg agcaagaataatgctattga gcccaggtct ttctctcaga acccccctgt gctgaagagg caccagaggggagatcaccag gaccaccctg cagtctgatc aggaggagat tgactatgat gacactatttctgtggagat gaagaaggaa gactttgata tctatgatga ggatgagaac cagagccctaggagcttcca gaagaagact aggcattact tcattgctgc tgtggagagg ctgtgggactatggcatgag cagcagcccc catgtgctga ggaatagggc tcagtctggc tctgtgcctcagttcaagaa ggtggtgttc caggaattca ctgatggcag cttcactcag cccctgtacaggggggagct gaatgagcac ctggggctgc tgggccctta catcagggct gaggtggaggacaatatcat ggtgaccttt aggaaccagg cctctaggcc ttacagcttc tactctagcctgatctctta tgaagaggac cagaggcagg gggctgagcc caggaagaac tttgtgaagcccaatgac taagacttac ttctggaagg tgcagcacca catggctccc accaaggatgagtttgactg caaggcttgg gcctacttct ctgatgtgga cctggagaag gatgtgcactctgggctgat tgggcccctg ctggtgtgcc acactaacac tctgaatcct gcccatggcagacaggtgac tgtgcaggag tttgccctgt tttttaccat ctttgatgag actaagtcttggtacttcac tgagaacatg gaggagaact gcagggcccc ctgcaacatc cagatggaggatcccacctt caaggacac tacaggtttc atgccatcaa tggctacatc atggacaccctgcctggcct ggtgatggct caggaccaga ggattaggtg gtatctgctg agcatgggcagcaatgagaa tatccactct atccacttct ctgggcatgt gttcactgtg aggaaaggaggagatcaa gatggccctg tataacctgt atcctggggt gtttgagact gtggagatgctgcccagcaa ggctggcatc tggagagtgg agtgcctgat tggggagcac ctgcatgctggcatgagcac tctgtttctg gtgtatagca acaagtgtca gacccctctg ggcatggcctctgggcacat tagggacttt cagatcactg cttctggcca gtatgggcag tgggctcccaagctggccag gctccactat tctggcagca ttaatgcctg gagcaccaag gagcctttcagctggatcaa ggtggacctg ctggccccca tgatcatcca tgggatcaag acccagggggctaggcagaa gttcagcagc ctgtacatca gccagtttat catcatgtat tctctggatggcaagaagtg gcagacctac aggggcaatt ctactggcac tctgatggtg ttctttgggaatgtggatag ctctgggatc aagcataata tcttcaatcc ccccattatt gctaggtatatcaggctgca ccccacccac tatagcatca ggagcaccct gaggatggag ctgatggggtgtgacctgaa cagctgcagc atgcccctgg gcatggagag caaggctatt tctgatgcccagatcactgc cagcagctac tttactaata tgtttgccac ctggagcccc agcaaggccagactgcacct gcagggcagg tctaatgcct ggaggcctca ggtgaataac cccaaggagtggctgcaggt ggacttccag aaaaccatga aggtgactgg ggtgactacc cagggggtgaagtctctgct gaccagcatg tatgtgaagg agttcctgat ctcttctagc caggatggccaccagtggac cctgttcttt cagaatggga aggtgaaggt cttccagggc aaccaggatagcttcacccc tgtggtgaat agcctggatc ctcctctgct gaccaggtat ctgaggatccacccccagag ctgggtgcat cagattgccc tgaggatgga ggtgctgggc tgtgaggctcaggacctgta ctga

[0284] CpG-reduced nucleic acid variant X15 (SEQ ID NO: 15) encoding FVIII atgcagattg agctgagcac ctgtttcttc ctgtgcctgctgaggttctg tttctctgcc actaggaggt actacctggg ggctgtggag ctgagctgggaactatatgca gtctgacctg ggggagctgc ctgtggatgc caggttcccc cccagggtgcctaagagctt ccccttcaat acttctgtgg tgtacaagaa gactctgttt gtggagtttactgaccacct gttcaacatt gctaagccca ggcctccctg gatggggctg ctgggccccaccatccaggc tgaggtgtat gatactgtgg tgattaccct gaagaacatg gcctctcatccagtgagcct gcatgctgtg ggggtgagct actggaaggc ctctgaaggg gctgagtatgatgaccagac cagccagagg gagaaggagg atgacaaggt gttccctggg ggcagccacacctatgtgtg gcaggtgctg aaggaatg gcccaatggc ctctgacccc ctgtgcctgactatagatcta cctgagccat gtggatctgg tgaaggacct gaattctggc ctgattggggccctgctggt gtgcagagag ggctctctgg ctaaggaa gacccagact ctgcacaagttcatcctgct gtttgctgtg tttgatgagg gcaagagctg gcactctgag actaagaatagcctgatgca ggacagggat gctgcttctg ccagggcctg gcccaagatg catactgtgaatggctatgt gaacaggagc ctgcctggcc tgattggctg tcacaggaaa tctgtctactggcatgtgat tgggatgggc actacccctg aggtgcactc tatcttcctg gagggccataccttcctggt gaggaaccac aggcaggcca gcctggagat ctctcccatt accttcctgactgcccagac cctgctgatg gatctgggcc agttcctgct gttctgccac atcagcagccaccagcatga tgggatggag gcttatgtga aggtggatag ctgccctgag gagccccagctgaggatgaa gaacaatgag gaggctgagg actatgatga tgacctgact gactctgagatggatgtggt gaggtttgat gatgacaact ctcccagctt tattcagatc aggtctgtggctaagaagca ccccaagact tgggtgcact acattgctgc tgaggaggag gactgggactatgcccctct ggtgctggct cctgatgaca ggtcttacaa gtctcagtac ctgaataatggccctcagag gattggcagg aagtacaaga aggtgaggtt catggcctac actgatgagaccttcaagac cagggaggcc atccagcatg agtctggcat cctgggcccc ctgctgtatggggaggtggg ggataccctg ctgatcatct tcaagaatca ggccagcagg ccctacaacatctaccccca tggcatcact gatgtgaggc cactgtacag caggaggctg cccaagggggtgaagcatct gaaggacttc cccattctgc ctggggagat cttcaagtac aaatggactgtgactgtgga ggatggccct accaagtctg accccaggtg tctgaccagg tactacagcagctttgtgaa tatggagagg gacctggcct ctggcctgat tggccccctg ctgatctgctacaaggagtc tgtggaccag aggggcaatc agatcatgtc tgataagagg aatgtgattctgttctctgt gtttgatgag aacaggagct ggtacctgac tgagaacatc cagaggttcctgcccaatcc tgctggggtg cagctggagg accctgagtt ccaggccagc aatatcatgcacagcatcaa tggctatgtc tttgacagcc tgcagctgtc tgtgtgcctg catgaggtggcttactggta tattctgagc attggggccc agactgattt cctgtctgtg ttcttttctggctatacctt taagcacaag atggtgtatg aggacaccct gaccctgttc cccttctctggggagactgt gttcatgtct atggagaacc ctgggctgtg gatcctgggc tgccacaactctgacttcag gaacaggggg atgactgccc tgctgaaggt gtctagctgt gataagaacactggggacta ttatgaggac agctatgagg acatctctgc ttacctgctg agcaagaacaatgccattga gcccaggtct ttcagccaga atccccctgt gctgaagagg catcagagggagatcaccag gaccaccctg cagtctgatc aggaggagat tgattatgat gacactatctctgtggaaat gaagaaggag gactttgaca tctatgatga ggatgagaac cagagccccaggagcttcca gaagaagacc aggcactact tcattgctgc tgtggagagg ctgtgggattatggcatgag cagctctccc catgtgctga ggaacagagc ccagtctggc tctgtgcctcagttcaagaa ggtggtcttc caggagttca ctgatggctc tttcacccag cccctgtacaggggggagct gaatgagcac ctgggcctgc tggggcccta cattagggct gaggtggaggataacatcat ggtgactttc agaaaccagg ccagcaggcc ttacagcttt tactcttctctgattagcta tgaggaggat cagaggcagg gggctgagcc taggaagaac tttgtgaagcccaatgagac caagacctat ttctggaagg tgcagcacca catggctccc actaaggatgagtttgactg caaggcttgg gcctacttct ctgatgtgga cctggagaag gatgtgcactctggcctgat tgggcccctg ctggtgtgcc acaccaacac cctgaaccct gcccatggcaggcaggtgac tgtgcaggag tttgccctgt tcttcaccat ctttgatgag actaagagctggtacttcac tgagaacatg gaggagaact gcagggcccc ctgcaacatc cagatggaggaccccacctt caaggagaat tacaggttcc atgccatcaa tggctacatt atggacaccctgcctggcct ggtgatggcc caggatcaga ggatcaggtg gtatctgctg agcatgggctctaatgagaa catccacagc atccacttct ctggccatgt gtttactgtg aggaaaggagaataacaa gatggctctg tataacctgt accctggggt gtttgagact gtggagatgctgcccagcaa ggctgggatc tggagggtgg agtgcctgat tggggagcac ctgcatgctgggatgagcac cctgttcctg gtgtatagca ataagtgcca gacccccctg ggcatggcttctggccacat cagggatttc cagatcactg cttctggcca gtatggccag tgggctcccaagctggctag gctgcattac tctgggtcta tcaatgcctg gagcactag gagcccttcagctggatcaa ggtggacctg ctggccccca tgatcattca tggcatcaag acccaggggctaggcagaa gttcagcagc ctgtacatca gccagttcat cattatgtac agcctggatggcaagaagtg gcagacttac aggggcaata gcactgggac tctgatggtg ttctttggcaatgtggactc ttctggcatc aagcaaca tcttcaaccc tcccatcatt gccaggtacattaggctgca ccctacccac tactctatca ggagcaccct gaggatggag ctgatggggtgtgatctgaa ctcttgcagc atgcctctgg gcatggaaag caaagccatc tctgatgcccagatcactgc ctctagctat ttcaccaata tgtttgccac ctggagccct agcaaggccaggctgcacct gcagggcaga tctaatgcct ggaggcccca ggtgaacaat cccaaggagtggctgcaggt ggacttccag aagaccatga aggtgactgg ggtgaccact cagggggtgaagagcctgct gactagcatg tatgtgaagg agttcctgat ctcttctagc caggatggccaccagtggac cctgttcttc cagaatggca aggtgaaagt gttccagggc aaccaggatagcttcactcc tgtggtgaac tctctggacc ctcccctgct gactaggtac ctgaggattcatccccagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaggcccaggatctgta ctga

[0285] CpG-reduced nucleic acid variant X16 (SEQ ID NO: 16) encoding FVIII atgcagattg agctgagcac ctgcttcttc ctgtgcctgctgaggttctg cttctctgcc accaggaggt actacctggg ggctgtggag ctgtcttgggactatatgca gtctgacctg ggggagctgc cagtggatgc caggttcccc cccagggtgcccaagagctt tcctttcaac acttctgtgg tgtacaagaa gaccctgttt gtggagttcactgaccacct gttcaatatt gctaagccca ggccaccctg gatgggcctg ctgggccctaccattcaggc tgaggtgtat gacactgtgg tgattactct gaagaatatg gccagccaccctgtgagcct gcatgctgtg ggggtgtctt actggaaggc ctctgagggg gctgagtatgatgatcagac ttctcagagg gagaggagg atgataaggt gttccctggg ggctctcacacttatgtgtg gcaggtgctg aaggaatg gccccatggc ttctgatcca ctgtgcctgacctactctta cctgagccat gtggacctgg tgaaggacct gaactctggc ctgattggggccctgctggt gtgcagggag ggcagcctgg ccaaggaa gacccagacc ctgcataagttcatcctgct gtttgctgtg tttgatgagg ggaagagctg gcactctgag accaagaattctctgatgca ggacagggat gctgcccttg ccagggcctg gcctaagatg caactgtgaatggctatgt gaacaggtct ctgcctggcc tgattggctg ccacaggaag tctgtgtactggcatgtgat tggcatgggc actacccctg aggtgcacag cattttcctg gagggccacaccttcctggt caggaaccat aggcaggcct ctctggagat cagccccatc actttcctgactgcccagac cctgctgatg gacctgggcc agttcctgct gttctgccac attagcagccaccagcatga tggcatggag gcctatgtga aggtggactc ttgccctgag gagccccagctgaggatgaa gaacaatgag gaagctgagg attatgatga tgacctgact gactctgagatggatgtggt gaggtttgat gatgacaaca gccccagctt catccagatc aggtctgtggccaagaagca ccccaagacc tgggtgcact acattgctgc tgaggaggag gattgggactatgctcccct ggtgctggct cctgatgata ggagctacaa gtctcagtac ctgaataatggcccccagag gattggcagg aagtacaaga aggtgaggtt catggcctac actgatgagaccttcaagac cagagaggct atccagcatg agtctgggat cctggggccc ctgctgtatggggaggtggg ggacaccctg ctgatcatct tcaagaacca ggccagcaga ccctacaacatctaccccca tgggatcact gatgtgaggc ccctgtacag caggaggctg cctaagggggtgaagcacct gaaggacttc cccatcctgc ctggggagat cttcaagtat aagtggactgtgactgtgga ggatgggccc accaagtctg accctaggtg cctgactagg tactactctagctttgtgaa catggagagg gacctggcct ctggcctgat tggccccctg ctgatttgctacaaggagtc tgtggatcag aggggcaatc agatcatgtc tgacaagagg aatgtgatcctgttctctgt gtttgatgag aataggtctt ggtacctgac tgagaacatc cagaggttcctgcctaatcc tgctggggtg cagctggagg accctgagtt tcaggccagc aacatcatgcacagcatcaa tggctatgtg tttgactctc tgcagctgtc tgtgtgcctg catgaggtggcttactggta tatcctgagc attggggctc agactgactt cctgtctgtg ttcttttctggctacacttt taagcacaag atggtgtatg aggacaccct gaccctgttc cccttttctggggagactgt gttcatgtct atggagaacc ctgggctgtg gattctgggc tgtcacaactctgacttcag aaacaggggc atgactgccc tgctgaaggt gtctagctgt gacaagaatactggggacta ctatgaggac agctatgagg acatttctgc ctatctgctg agcaagaacaatgccattga gcccaggagc ttttctcaga atccccctgt gctgaagagg caccagagagagatcaccag gaccactctg cagtctgatc aggaggagat tgattatgat gacactatctctgtggagat gaagaaagag gactttgata tctatgatga ggatgagaat cagtctcccaggagcttcca gaagaagact agacactact tcattgctgc tgtggagagg ctgtgggactatggcatgag ctctagccct catgtgctga ggaacagggc ccagtctggg tctgtgccccagttcaagaa ggtggtgttc caggagttca ctgatggcag cttacccag cccctgtataggggggagct gaatgagcat ctgggcctgc tgggcccta tattagggct gaagtggaggacaacatcat ggtgaccttt aggaaccagg ccagcaggcc ctacagcttt tacagcagcctgattagcta tgaggaggat cagagacagg gggctgagcc caggaagaac tttgtgaagcccaatgac caagacctac ttctggaagg tgcagcacca catggcccct accaaggatgagtttgactg caaggcctgg gcttacttct ctgatgtgga cctggagaaa gatgtgcactctggcctgat tgggcccctg ctggtgtgcc acaccaacac cctgaaccct gcccatgggaggcaggtgac tgtgcaggag tttgccctgt tttcaccat ctttgatgag accaagagctggtacttcac tgagaacatg gagaggaact gcagggcccc ctgtaacatc cagatggaggatcctacttt caaggagaac tacaggttcc atgccattaa tgggtacatc atggacaccctgcctgggct ggtgatggcc caggatcaga ggattaggtg gtatctgctg tctatgggctctaatgaaa catccactct atccacttct ctggccatgt gttcactgtg aggaagaaggaggagtacaa gatggccctg tacaacctgt accctggggt gtttgaaact gtggagatgctgccctctaa agctgggatc tggagggtgg agtgcctgat tggggagcac ctgcatgctggcatgagcac cctgttcctg gtgtacagca ataagtgcca gactcccctg ggcatggcttctgggcacat cagggatttc cagatcactg cctctggcca gtatggccag tgggcccccaagctggctag gctgcactac tctggcagca tcaatgcctg gagcaccaag gagcccttctcttggattaa ggtggacctg ctggctccca tgatcattca tggcatcaag acccagggggccaggcagaa gttttctagc ctgtatatta gccagttcat catcatgtat agcctggatgggaagaagtg gcagacctac agggggaata gcactggcac cctgatggtg ttttttggcaatgtggattc ttctggcatc aagcataaca tcttcaatcc ccctatcatt gccaggtacattaggctgca tcccacccat tactctatca ggagcaccct gaggatggag ctgatggggtgtgatctgaa cagctgtagc atgcccctgg gcatggagtc caaggctatc tctgatgcccagatcactgc cagcagctac ttcaccaaca tgtttgccac ctggagcccc agcaaggccaggctgcacct gcagggcagg tctaatgcct ggaggcccca ggtgaacaat cccaaggagtggctgcaggt ggacttccag aagactatga aggtgactgg ggtgaccact cagggggtgaagagcctgct gaccagcatg tatgtgaagg agttcctgat ctcttctagc caggatgggcatcagtggac cctgtttttt cagaatggca aagtgaaggt gtttcagggg aatcaggacagctttacccc tgtggtgaac agcctggatc ctcctctgct gactagatac ctgaggatccacccccagag ctgggtccac cagattgctc tgaggatgga ggtgctgggg tgtgaggctcaggacctgta ctga

[0286] CpG-reduced nucleic acid variant X17 (SEQ ID NO: 17) encoding FVIII atgcagattg agctgagcac ctgcttcttt ctgtgcctgctgaggttctg cttctctgcc accaggaggt actacctggg ggctgtggaa ctgagctgggactatatgca gtctgacctg ggggagctgc ctgtggatgc caggttcccc cccagggtgcccaagtcttt cccctttaac acttctgtgg tgtacaagaa gaccctgttt gtggagtttactgaccacct gttcaatatt gccaagccca ggcccccctg gatgggcctg ctgggcccaaccatccaggc tgaggtgtat gatactgtgg tgatcaccct gaagaacatg gccagccaccctgtgagcct gcatgctgtg ggggtgagct attggaaggc ttctgagggg gctgagtatgatgaccagac tagccagagg gagaaggagg atgacaaggt gttccctggg gggtctcatacctatgtgtg gcaggtgctg aaggagaatg gccccatggc ctctgacccc ctgtgcctgacctattctta cctgagccat gtggacctgg tcaaggacct gaactctggc ctgattggggctctgctggt gtgcagggag ggcagcctgg ccaaggagaa gactcagact ctgcataagttcatcctgct gtttgctgtg tttgatgagg gcaagagctg gcactctgag accaagaactctctgatgca ggatagggat gctgcctctg ccagggcctg gcccaagatg cacactgtgaatggctatgt gaataggtct ctgcctggcc tgattggctg ccataggaag tctgtgtactggcatgtgat tggcatgggc actacccctg aggtgcactc tatcttcctg gaggggcacaccttcctggt gaggaaccac aggcaggcca gcctggagat ctctcccatc accttcctgactgcccagac tctgctgatg gacctgggcc agttcctgct gttctgccat atcagcagccaccagcatga tggcatggag gcctatgtga aggtggacag ctgcccagag gaaccccagctgaggatgaa gaacaatgag gaggctgagg actatgatga tgacctgact gactctgagatggatgtggt gaggtttgat gatgacaaca gccccagctt tattcagatc aggtctgtggccaagaagca ccccaagacc tgggtgcact acattgctgc tgaggaggag gactgggattatgcccccct ggtgctggcc cctgatgaca ggtcttacaa gtctcagtac ctgaacaatggcccccagag gattgggagg aagtacaaga aggtgaggtt catggcctac actgatgagaccttcaagac cagggaggcc atccagcatg agtctggcat cctggggccc ctgctgtatggggaggtggg ggataccctg ctgattatct tcaagaacca ggctagcagg ccctataacatctaccccca tggcattact gatgtgaggc ccctgtactc taggagactg cccaagggggtgaagcacct gaaagacttc cccatcctgc ctggggagat cttcaagtat aagtggactgtgactgtgga ggatggcccc actaagtctg accccaggtg cctgaccagg tattacagcagctttgtgaa tatggagagg gatctggctt ctggcctgat tgggcctctg ctgatttgctacaaggagtc tgtggatcag agggggaacc agattatgtc tgacaagagg aatgtgattctgttctctgt gtttgatgag aacaggagct ggtacctgac tgagaatatc cagaggttcctgcctaatcc tgctggggtg cagctggagg accctgagtt ccaggctagc aacattatgcacagcatcaa tggctatgtg tttgacagcc tgcagctgtc tgtgtgcctg catgaggtggcttactggta cattctgtct attggggccc agactgactt cctgtctgtg ttcttctctggctacacctt caagcacaag atggtgtatg aggacactct gaccctgttc cccttctctggggagactgt gttcatgagc atggagaatc ctgggctgtg gattctgggg tgccacaactctgatttcag gaacaggggc atgactgccc tgctgaaggt gagcagctgt gacaagaacactggggatta ttatgaggac agctatgagg acatttctgc ctacctgctg agcaagaacaatgccattga gcctaggagc ttcagccaga atccccctgt gctgaagaga caccagagggagatcactag gaccactctg cagtctgatc aggaggagat tgactatgat gacaccatttctgtggagat gaagaaggag gactttgata tttatgatga ggatgagaac cagagccccagaagcttcca gaagaagacc aggcactact tcattgctgc tgtggagagg ctgtgggattatggcatgtc ttctagcccc catgtgctga ggaacagggc tcagtctggc tctgtgcctcagttcaagaa ggtggtgttc caggagttca ctgatgggag cttcacccag cctctgtacaggggggagct gaatgaacat ctgggcctgc tggggcccta catcagggct gaggtggaggataatatcat ggtgactttc aggaatcagg cctctaggcc ctacagcttc tactctagcctgatcagcta tgaggaggac cagaggcagg gggctgagcc taggaagaat tttgtgaaacccaatgagac caacctac ttttggaagg tgcagcacca catggcccct accaaggatgagtttgactg taaggcctgg gcctacttct ctgatgtgga cctggagaag gatgtgcattctgggctgat tggccccctg ctggtgtgcc acaccaacac cctgaaccct gcccatggcaggcaggtgac tgtgcaggag tttgccctgt tcttcaccat ctttgatgag actaagagctggtatttcac tgagaacatg gaggagaact gtagggctcc ctgcaacatc cagatggaggatccaacttt caaggacac tacaggttcc atgccatcaa tggctacatc atggacaccctgcctggcct ggtgatggcc caggaccaga ggattaggtg gtacctgctg agcatgggctctaatgagaa catccactct atccacttct ctggccatgt gtttactgtg aggaagaaggaggatcaa gatggctctg tacaacctgt accctggggt gtttgagact gtggagatgctgcctagcaa ggctggcatt tggagagtgg agtgtctgat tggggagcac ctgcatgctgggatgtctac cctgttcctg gtgtactcta acaagtgcca gacccccctg gggatggcttctgggcacat cagagatttt cagattactg cttctgggca gtatggccag tgggctcccaagctggccag actgcattac tctggctcta ttaatgcttg gagcaccaag gagcctttcagctggatcaa ggtggacctg ctggctccca tgatcatcca tggcattaag actcagggggctaggcagaa gttcagcagc ctgtatattt ctcagtttat tatcatgtat tctctggatggcaagaagtg gcagacttac aggggcaaca gcactggcac cctgatggtg ttctttggcaatgtggacag ctctgggatc aagcataaca tcttcaaccc ccccattatt gccaggtacatcaggctgca ccccacccac tattctatca ggagcactct gaggatggag ctgatggggtgtgacctgaa cagctgctct atgcccctgg gcatggagag caaggccatc tctgatgcccagatcactgc cagctcttat ttcaccaaca tgtttgccac ctggagcccc agcaaggccaggctgcacct gcagggcaga agcaatgcct ggaggcccca ggtgaacaat cctaaggagtggctgcaggt ggacttccag aagactatga aggtgactgg ggtgactacc cagggggtgaagagcctgct gaccagcatg tatgtgaagg agttcctgat tagcagcagc caggatgggcatcagtggac cctgttcttc cagaatggga aggtgaaggt gttccagggc aatcaggacagcttcacccc tgtggtgaac agcctggacc cccccctgct gaccaggtac ctgaggatccatccccagag ctgggtgcac cagattgctc tgagaatgga ggtgctggggc tgtgaggcccaggacctgta ttga

[0287] CpG-reduced nucleic acid variant X18 (SEQ ID NO: 18) encoding FVIII atgcagattg agctgtctac ctgtttttt ctgtgcctgctgaggttctg cttctctgct accaggaggt attatctggg ggctgtggag ctgagctgggactacatgca gtctgacctg ggggagctgc ctgtggatgc caggtttcct cccagggtgcctaagagctt ccccttcaac acctctgtgg tgtacaagaa gactctgttt gtggagttcactgaccacct gttcaacatt gccaagccca ggcccccctg gatggggctg ctgggccccactatccaggc tgaggtgtat gatactgtgg tgattaccct gaagaacatg gcctctcaccctgtgtctct gcatgctgtg ggggtgagct actggaaggc ttctgagggg gctgaatatgatgatcagac ctctcagagg gagaaggagg atgacaaggt gtttcctggg ggcagccacacctatgtgtg gcaggtgctg aaggaatg ggcccatggc ctctgatccc ctgtgcctgacctacagcta cctgagccat gtggacctgg tgaaggacct gaactctggc ctgattggggccctgctggt gtgcagggag ggcagcctgg ccaaggaaaa gacccagacc ctgcataagttcatcctgct gtttgctgtg tttgatgagg gcaagtcttg gcactctgag accaagaacagcctgatgca ggacagggat gctgcctctg ctagggcctg gcccaagatg cacactgtgaatgggtatgt gaacagatct ctgcctggcc tgattggctg ccacaggaag tctgtgtactggcatgtgat tggcatgggg accacccctg aggtgcatag catcttcctg gaggggcacaccttcctggt gagaaatcat aggcaggcca gcctggagat tagccccatc accttcctgactgcccagac cctgctgatg gacctgggcc agttcctgct gttctgccac atttctagccaccagcatga tggcatggag gcctatgtga aggtggatag ctgccctgaa gagccccagctgaggatgaa gaacaatgag gaggctgagg attatgatga tgatctgact gactctgagatggatgtggt gaggtttgat gatgacaaca gccccagctt catccagatc aggtctgtggccaagaagca ccctaagacc tgggtgcact acattgctgc tgaagaggag gactgggactatgcccccct ggtgctggcc ccagatgaca ggtcttacaa gagccagtac ctgaataatggcccccagag gattgggagg aagtataaga aagtgaggtt catggcttac actgatgagacctttaagac tagggaggcc attcagcatg agtctgggat tctgggccct ctgctgtatggggaggtggg ggacaccctg ctgatcattt tcaagaacca ggccagcagg ccctataatatttatcccca tgggattact gatgtcaggc ccctgtacag caggaggctg cctaagggggtgaagcacct gaaggacttc cccattctgc ctggggagat cttcaagtat aagtggactgtgactgtgga ggatggcccc accaagtctg atcctaggtg cctgaccagg tactatagcagctttgtgaa catggagagg gacctggctt ctggcctgat tggccccctg ctgatctgctacaaggaatc tgtggaccag aggggcaacc agattatgtc tgacaagagg aatgtgatcctgttttctgt gtttgatgag aataggagct ggtatctgac tgagaacatc cagaggttcctgcccaatcc tgctggggtg cagctggagg accctgagtt ccaggcttct aacatcatgcatagcatcaa tgggtatgtg tttgactctc tgcagctgtc tgtgtgcctg catgaggtggcctattggta catcctgagc attggggccc agactgactt cctgtctgtg ttctctctggctacacctt caagcacaag atggtgtatg aggacaccct gaccctgttc cctttctctggggagactgt gttcatgagc atggagaacc ctggcctgtg gattctgggc tgccataattctgacttcag aaacaggggc atgactgctc tgctgaaggt gagcagctgt gacaagaatactggggacta ctatgaggac tcttatgagg atatttctgc ctacctgctg agcaagaacaatgctattga gcccaggagc ttcagccaga acccccctgt cctgaagagg catcagagggagatcactag gaccaccctg cagtctgatc aggagagat tgactatgat gacactatctctgtggaaat gaagaggag gactttgata tctatgatga ggatgagaac cagagccccaggtctttcca gaagagacc aggcactact tcattgctgc tgtggagagg ctgtgggactatggcatgtc tagcagcccc catgtgctga ggaacagagc ccagtctggc tctgtgccccagttcaagaa ggtggtgttt caggagttca ctgatgggag cttcactcag cccctgttaggggggagct gaatgagcat ctgggcctgc tggggcccta catcagggct gaggtggaggataacatcat ggtgaccttc aggaaccagg ccagcaggcc ctactctttc tactcttctctgatcagcta tgaggagat cagaggcagg gggctgagcc taggaaac tttgtcaagcctaatgagac taagcctac ttttggaagg tgcagcacca catggctccc actaaggatgagtttgattg caaggcctgg gcctacttct ctgatgtgga cctggagaag gatgtgcactctgggcctgat tggccccctg ctggtgtgtc acaccaatac cctgaaccct gcccatggcaggcaggtcac tgtgcaggag tttgccctgt ttttcactat ctttgatgag actaagtcttggtacttcac tgagaacatg gaaggaatt gcagggctcc ctgcaacatc cagatggaggaccccacctt caaggaac tacaggtttc atgccatcaa tggctacatc atggacaccctgcctggcct ggtgatggct caggatcaga ggattaggtg gtatctgctg agcatgggcagcaatgagaa catccacagc atccactttt ctggccatgt gttcactgtg aggaaaggaggagatcaa gatggctctg tacaatctgt accctggggt gtttgagact gtggagatgctgcccagcaa ggctgggatc tggagggtgg agtgcctgat tggggaacac ctgcatgctggcatgtctac cctgttcctg gtgtactcta acaagtgcca gactcccctg ggcatggcctctgggcacat cagggacttc cagatcactg cctctgggca gtatggccag tgggcccctaagctggctag gctgcattac tctggcagca tcaatgcctg gagcaccaag gagcccttcagctggatcaa ggtggacctg ctggccccta tgatcatcca tggcatcaag acccaggggggccagacaagaa gttctcttct ctgtacatct ctcagttcat catcatgtac tctctggatggcaagaagtg gcagacctac agggggaatt ctactggcac tctgatggtg ttctttgggaatgtggatag ctctgggatc aagcataata ttttcaaccc ccccattatt gctaggtacatcaggctgca cccaacccac tactctatta ggtctaccct gaggatggag ctgatgggctgtgacctgaa ctcttgtagc atgcccctgg gcatggagag caaggctatc tctgatgcccagatcactgc cagcagctac tttaccaaca tgtttgctac ttggagcccc agcaaggccaggctgcacct gcagggcagg agcaatgcct ggaggcccca ggtgaacaac cccaaggagtggctgcaggt ggattttcag aagaccatga aggtgactgg ggtgaccact cagggggtgaaaagcctgct gactagcatg tatgtgaagg agtttctgat cagcagctct caggatggccatcagtggac cctgttcttc cagaatggca aggtgaaggt gttccagggc aaccaggatagcttcacccc tgtggtgaat agcctggacc cccccctgct gaccaggtac ctgaggatccatccccagag ctgggtgcac cagattgccc tgaggatgga ggtgctgggc tgtgaagcccaggacctgta ctga

[0288] Wild-type Factor VIII-BDD cDNA (SEQ ID NO: 19) ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTTTGCGATTCTG CTTTAGTGCC ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGGACTATATGCA AAGTGATCTC GGTGAGCTGC CTGTGGACGC AAGATTTCCT CCTAGAGTGCCAAAATCTTT TCCATTCAAC ACCTCAGTCG TGTACAAAAA GACTCTGTTT GTAGAATTCACGGATCACCT TTTCAACATC GCTAAGCCAA GGCCACCCTG GATGGGTCTG CTAGGTCCTACCATCCAGGC TGAGGTTTAT GATACAGTGG TCATTACACT TAAGAACATG GCTTCCCATCCTGTCAGTCT TCATGCTGTT GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATGATGATCAGAC CAGTCAAAGG GAGAAAGAAG ATGATAAAGT CTTCCCTGGT GGAAGCCATACATATGTCTG GCAGGTCCTG AAAGAGAATG GTCCAATGGC CTCTGACCCA CTGTGCCTTACCTACTCATA TCTTTCTCAT GTGGACCTGG TAAAAGACTT GAATTCAGGC CTCATTGGAGCCCTACTAGT ATGTAGAGAA GGGAGTCTGG CCAAGGAAAA GACACAGACC TTGCACAAATTTATACTACT TTTTGCTGTA TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACTCCTTGATGCA GGATAGGGAT GCTGCATCTG CTCGGGCCTG GCCTAAAATG CACACAGTCAATGGTTATGT AAACAGGTCT CTGCCAGGTC TGATTGGATG CCACAGGAAA TCAGTCTATTGGCATGTGAT TGGAATGGGC ACCACTCCTG AAGTGCACTC AATATTCCTC GAAGGTCACACATTTCTTGT GAGGAACCAT CGCCAGGCGT CCTTGGAAAT CTCGCCAATA ACTTTCCTTACTGCTCAAAC ACTCTTGATG GACCTTGGAC AGTTTCTACT GTTTTGTCAT ATCTCTTCCCACCAACATGA TGGCATGGAA GCTTATGTCA AAGTAGACAG CTGTCCAGAG GAACCCCAACTACGAATGAA AAATAATGAA GAAGCGGAAG ACTATGATGA TGATCTTACT GATTCTGAAATGGATGTGGT CAGGTTTGAT GATGACAACT CTCCTTCCTT TATCCAAATT CGCTCAGTTGCCAAGAAGCA TCCTAAAACT TGGGTACATT ACATTGCTGC TGAAGAGGAG GACTGGGACTATGCTCCCTT AGTCCTCGCC CCCGATGACA GAAGTTATAA AAGTCAATAT TTGAACAATGGCCCTCAGCG GATTGGTAGG AAGTACAAAA AAGTCCGATT TATGGCATAC ACAGATGAAACCTTTAAGAC TCGTGAAGCT ATTCAGCATG AATCAGGAAT CTTGGGACCT TTACTTTATGGGGGAAGTTGG AGACACACTG TTGATTATAT TTAAGAATCA AGCAAGCAGA CCATATAACATCTACCCTCCA CGGAATCACT GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAGGTGTAAAACATTT GAAGGATTTT CCAATTCTGC CAGGAGAAT ATTCAAATAT AAATGGACAGTGACTGTAGA AGATGGGCCA ACTAAATCAG ATCCTCGGTG CCTGACCCGC TATTACTCTAGTTTCGTTAA TATGGAGAGA GATCTAGCTT CAGGACTCAT TGGCCCTCTC CTCATCTGCTACAAAGAATC TGTAGATCAA AGAGGAAACC AGATAATGTC AGACAAGGG AATGTCATCCTGTTTTCTGT ATTTGATGAG AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTCTCCCCAATCC AGCTGGAGTG CAGCTTGAGG ATCCAGAGTT CCAAGCCTCC AACATCATGCACAGCATCAA TGGCTATGTT TTTGATAGTT TGCAGTTGTC AGTTTGTTTG CATGAGGTGGCATACTGGTA CATTCTAAGC ATTGGAGCAC AGACTGACTT CCTTTCTGTC TTCTTCTCTGGATATACCTT CAAACACAAA ATGGTCTATG AAGACACACT CACCCTATTC CCATTCTCAGGAGAAACTGT CTTCATGTCG ATGGAAAACC CAGGTCTATG GATTCTGGGG TGCCACAACTCAGACTTTCG GAACAGAGGC ATGACCGCCT TACTGAAGGT TTCTAGTTGT GACAAACACTGGTGATTA TTACGAGGAC AGTTATGAAG ATATTTCAGC ATACTTGCTG AGTAAAAACAATGCCATTGA ACCAAGAAGC TTCTCCCAAA ACCCACCAGT CTTGAAACGC CATCAACGGGAAATAACTCG TACTACTCTT CAGTCAGATC REPEAT TREADCLASS REPEAT GATTTTGACA TTTATGATGA GGATGAAAAT CAGAGCCCCCGCAGCTTTCA AAGAAAAACA CGACACTATT TTATTGCTGC AGTGGAGAGG CTCTGGGATTATGGGATGAG TAGCTCCCCA CATGTTCTAA GAAACAGGGC TCAGAGTGGC AGTGTCCCTCAGTTCAAGAA AGTTGTTTTC CAGGAATTTA CTGATGGCTC CTTTACTCAG CCCTTATACCGTGGAGAACT AAATGAACAT TTGGGACTCC TGGGGCCATA TATAAGAGCA GAAGTTGAAGATAATATCAT GGTAACTTTC AGAAATCAGG CCTCTCGTCC CTATTCCTTC TATTCTAGCCTTATTTCTTA TGAGGAAGAT CAGAGGCAAG GAGCAGAACC TAGAAAAAAC TTTGTCAAGCCTAATGAAAC CAAAACTTAC TTTTGGAAAG TGCAACATCA TATGGCACCC ACTAAAGATGAGTTTGACTG CAAAGCCTGG GCTTATTTCT CTGATGTTGA CCTGGAAAAA GATGTGCACTCAGGCCTGAT TGGACCCCTT CTGGTCTGCC ACACTAACAC ACTGAACCCT GCTCATGGGAGACAAGTGAC AGTACAGGAA TTTGCTCTGT TTTTCACCAT CTTTGATGAG ACCAAAAGCTGGTACTTCAC TGAAAATATG GAAAGAAACT GCAGGGCTCC CTGCAATATC CAGATGGAAGATCCCACTTT TAAAGAGAAT TATCGCTTCC ATGCAATCAA TGGCTACATA ATGGATACACTACCTGGCTT AGTAATGGCT CAGGATCAAA GGATTCGATG GTATCTGCTC AGCATGGGCAGCAATGAAAA CATCCATTCT ATTCATTTCA GTGGACATGT GTTCACCGTA CGAAAAAAAGAGGAGTATAA AATGGCACTG TACAATCCT ATCCAGGTGT TTTTGAGACA GTGGAAATGTTACCATCCAA AGCTGGAATT TGGCGGGTGG AATGCCTTAT TGGCGAGCAT CTACATGCTGGGATGAGCAC ACTTTTCTG GTGTACAGCA ATAAGTGTCA GACTCCCCTG GGAATGGCTTCTGGACACAT TAGAGATTTT CAGATTACAG CTTCAGGACA ATATGGACAG TGGGCCCCAAAGCTGGCCAG ACTTCATTAT TCCGGATCAA TCAATGCCTG GAGCACCAAG GAGCCCTTTTCTTGGATCAA GGTGGATCTG TTGGCACCAA TGATTATTCA CGGCATCAAG ACCCAGGGTGCCCGTCAGAA GTTCTCCAGC CTCTACATCT CTCAGTTTAT CATCATGTAT AGTCTTGATGGGAAGAAGTG GCAGACTTAT CGAGGAAATT CCACTGGAAC CTTAATGGTC TTCTTTGGCAATGTGGATTC ATCTGGGATA AAACACAATA TTTTTAACCC TCCAATTATT GCTCGATACATCCGTTTGCA CCCAACTCAT TATAGCATTC GCAGCACTCT TCGCATGGAG TTGATGGGCTGTGATTTAAA TAGTTGCAGC ATGCCATTGG GAATGGAGAG TAAAGCAATA TCAGATGCACAGATTACTGC TTCATCCTAC TTTACCAATA TGTTTGCCAC CTGGTCTCCT TCAAAGCTCGACTTCACCT CCAAGGGAGG AGTAATGCCT GGAGACCTCA GGTGAATAAT CCAAAGAGTGGCTGCAAGT GGACTTCCAG AAGACAATGA AAGTCACAGG AGTAACTACT CAGGGAGTAAAATCTCTGCT TACCAGCATG TATGTGAAGG AGTTCCTCAT CTCCAGCAGT CAAGATGGCCATCAGTGGAC TCCTTTTTTT CAGAATGGCA AAGTAAAGGT TTTTCAGGGA AATCAAGACTCCTTCACACC TGTGGTGAAC TCTCTAGACC CACCGTTACT GACTCGCTAC CTTCGAATTCACCCCCAGAG TTGGGTGCAC CAGATTGCCC TGAGGATGGA GGTTCTGGGC TGCGAGGCACAGGACCTCTA CTGA

[0289] V3 Factor VIII cDNA (SEQ ID NO: 20)

[0290] CO3 Factor VIII cDNA (SEQ ID NO: 21) atgcagattg agctgtcaac ttgctttttc ctgtgcctgctgagattttg tttttccgct actagaagat actacctggg ggctgtggaa ctgtcttgggattacatgca gagtgacctg ggagagctgc cagtggacgc acgatttcca cctagagtccctaaatcatt ccccttcaac accagcgtgg tctataagaa aacactgttc gtggagtttactgatcacct gttcaacatc gctaagcctc ggccaccctg gatgggactg ctgggaccaacaatccaggc agaggtgtac gacaccgtgg tcattacact gaaaaacatg gcctcacaccccgtgagcct gcatgctgtg ggcgtcagct actggaaggc ttccgaaggg gcagagtatgacgatcagac ttcccagaga gaaaaagagg acgataaggt gtttcctggc gggtctcatacctatgtgtg gcaggtcctg aaagagaatg gccccatggc ttccgaccct ctgtgcctgacctactctta tctgagtcac gtggacctgg tcaaggatct gaacagcgga ctgatcggagcactgctggt gtgtagggaa gggagcctgg ctaaggagaa aacccagaca ctgcataagttcattctgct gttcgccgtg tttgacgaag gaaaatcatg gcacagcgag acaaagaatagtctgatgca ggaccgggat gccgcttcag ccagagcttg gcccaaaatg cacactgtgaacggctacgt caatcgctca ctgcctggac tgatcggctg ccaccgaaag agcgtgtattggcatgtcat cggaatgggc accacacctg aagtgcactc cattttcctg gaggggcatacctttctggt ccgcaaccac cgacaggcct ccctggagat ctctccaatt accttcctgacagctcagac tctgctgatg gatctgggac agttcctgct gttttgccac atcagctcccaccagcatga tggcatggag gcctacgtga aagtggacag ctgtcccgag gaacctcagctgaggatgaa gaacaatgag gaagctgaag actatgacga tgacctgacc gactccgagatggatgtggt ccgattcgat gacgataaca gcccctcctt tatccagatt agatctgtggccaagaaaca ccctaagaca tgggtccatt acatcgcagc cgaggaagag gactgggattatgcaccact ggtgctggca ccagacgatc gatcctacaa atctcagtat ctgaacaatggaccacagcg gattggcaga aagtacaaga aagtgaggtt catggcttat accgatgaaaccttcaagac tcgcgaagca atccagcacg agagcgggat tctgggacca ctgctgtacggagaagtggg ggacaccctg ctgatcattt ttaagaacca ggccagcagg ccttacaatatctatccaca tggaattaca gatgtgcgcc ctctgtacag ccggagactg ccaaagggcgtcaaacacct gaaggacttc ccaatcctgc ccggggaaat ttttaagtat aaatggactgtcaccgtcga ggatggcccc actaagacg accctaggtg cctgacccgc tactattcttagttcgtgaa tatggaaagg gatctggcca gcggactgat cggcccactg ctgatttgttacaaagagag cgtggatcag agaggcaacc agatcatgtc cgacaagagg aatgtgattctgttcagtgt cttgacgaa aaccggtcat ggtatctgac cgagaacatc cagagattcctgcctaatcc agccggagtg cagctggaag atcctgagtt tcaggcttct aacatcatgcatagtattaa tggctacgtg ttcgacagtc tgcagctgtc agtgtgtctg cacgaggtcgcttactggta tatcctgagc attggagcac agacagattt cctgagcgtg ttctttccggctacacttt taagcataaa atggtgtatg aggacacact gactctgttc cccttcagcggcgaaaccgt gtttatgtcc atggagaatc ccgggctgtg gatcctggga tgccacaacagcgatttcag gaatcgcggg atgactgcc tgctgaaagt gtcaagctgt gacaagaacccggagacta ctatgaagat tcatacgagg acatcagcgc atactgctg tccaaaaaaatgccattga acccaggtct tttagtcaga atcctccagt gctgaagagg caccagcgcgagatcacccg cactacctg cagagtgatc aggagaagat cgactacgac gatacaatttctgtggaaat gaaaagag gacttcgata tctatgacga agatgagaac cagagtcctcgatcattcca gaaaaacc cggcattact ttatgctgc agtggagcgc ctgtgggattatggcatgtc ctctagtcct cacgtgctgc gaaatcgggc ccagtcaggg agcgtcccacagttcaagaa agtggtcttc caggagttta cagacggatc ctttactcag ccactgtaccggggcgaact gaacgagcac ctggggctgc tgggacccta tatcagagct gaagtggaggataacattat ggtcaccttc agaaatcagg catctaggcc ttacagtttt tattcaagcctgatctctta cgaagaggac cagaggcagg gagcagaacc acgaaaaaac ttcgtgaagcctaatgagac caaaacatac ttttggaagg tgcagcacca tatggcccca acaaaagacgaattcgattg caaggcatgg gcctattttt ctgacgtgga tctggagaag gacgtccacagtggcctgat cgggccactg ctggtgtgtc atactaacac cctgaatccc gcacacggcaggcaggtcac tgtccaggaa ttcgccctgt tctttaccat ctttgatgag acaaaaagctggtacttcac cgaaaacatg gagcgaaatt gccgggctcc atgtaatatt cagatggagaaccccacatt caaggaaac taccgctttc atgccatcaa tgggtatatt atggatactctgcccggact ggtcatggct caggaccaga gaatcaggtg gtacctgctg agcatggggtccaacgagaa tatccactca attcatttca gcggacacgt gtttactgtc cggaagaagaagagataa aatggccctg tacaacctgt atcccggcgt gttcgaaacc gtcgagatgctgcctagcaa ggcagggatc tggagagtgg aatgcctgat tggggagcac ctgcatgccggaatgtctac cctgtttctg gtgtacagta ataagtgtca gacacccctg gggatggcttccggacatat ccgggatttc cagattaccg catctggaca gtacggccag tgggcccctaagctggctag actgcactat tccgggtcta tcaacgcttg gtccacaaaa gagcctttctcttggattaa ggtggacctg ctggcaccaa tgatcattca tggcatcaaa actcaggggggccaggcagaa gttctcctct ctgtacatct cacagtttat catcatgtac agcctggatggcaagaaatg gcagacatac cgcggcaata gcacagggac tctgatggtg ttctttggcaacgtggacag ttcagggatc aagcaaca ttttcaatcc ccctatcatt gctagagatacatcaggctgca cccaacccat tattctattc gaagtacact gcggatggaa ctgatggggtgcgatctgaa cagttgttca atgcccctgg gaatggagtc caaggcaatc tctgacgcccagattaccgc tagctcctac ttcactaata tgtttgctac ctggagcccc tccaaagcacgactgcatct gcagggacga agcaacgcat ggcgaccaca ggtgaacaat cccaaggagtggctgcaggt cgattttcag aaaactatga aggtgaccgg agtcacaact cagggcgtgaaaagtctgct gacctcaatg tacgtcaagg agttcctgat ctctagttca caggacggccaccagtggac actgttcttt cagaacggaa aggtgaaagt cttccagggc aatcaggattcctttacacc tgtggtcaac tctctggacc cacccctgct gactcgctac ctgcgaatccacccacagtc ctgggtgcat cagattgcac tgagaatgga agtcctgggc tgcgaggcccaggacctgta ttga

[0291] A full-length cassette (SEQ ID NO: 23) containing a mutant TTR promoter (TTRmut), a synthetic intron, a CpG-reduced Factor VIII cDNA, polyA, and ITRs. cctgcaggca gctgcgcgct cgctcgctca ctgaggccgcccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagcgcgcagagag ggagtggcca actccatcac taggggttcc tacgcgtgtc tgtctgcacatttcgtagag cgagtgttcc gatactctaa tctccctagg caaggttcat attgacttaggttacttatt ctccttttgt tgactaagtc aataatcaga atcagcaggt ttggagtcagcttggcaggg atcagcagcc tgggttggaa ggagggggta taaaagcccc ttcaccaggagaagccgtca cacagatcca caagctcctg ctagcaggta agtgccgtgt gtggttcccgcgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactg acactgacatccactttttc ttttctcca caggtttaaa cgccaccatg cagattgagc tgagcacctgcttcttcctg tgtctgctga ggttctgctt ctctgccacc aggaggtatt acctgggggctgtggagctg agctgggact atatgcagtc tgacctgggg gagctgcctg tggatgctaggttcccccc agggtgccca agagcttccc cttaacact tctgtggtgt acaagaagaccctgtttgtg gagttcactg accacctgtt caacattgcc aagcccaggc ccccctggatggggctgctg gggcccacca tccaggctga ggtgtatgac actgtggtga tcaccctgaagaacatggcc agccaccctg tgagcctgca tgctgtgggg gtgagctact ggaaggcttctgagggggct gagtatgatg accagactag ccagagggag aaggaggatg acaaggtgtttcctgggggc agccatacct atgtgtggca ggtgctgaag gagaatggcc ccatggcctctgaccccctg tgcctgacct acagctacct gtctcatgtg gacctggtga aggacctgaactctggcctg attggggctc tgctggtgtg tagggagggc agcctggcta aggaaaagacccagaccctg cataagttta tcctgctgtt tgctgtgttt gatgagggca agagctggcactctgagacc aagaacagcc tgatgcagga tagggatgct gcctctgcca gggcttggcctaagatgcac actgtgaatg ggtatgtgaa taggagcctg cctggcctga ttggctgccacaggaagtct gtgtactggc atgtgattgg gatgggcacc acccctgagg tccatagcatcttcctggag ggccacactt tcctggtgag gaaccacaga caggcctctc tggagatctctcccatcacc ttcctgactg ctcagactct gctgatggac ctgggccagt tcctgctgttttgccatatt agcagccacc agcatgatgg gatggaggcc tatgtgaagg tggatagctgccctgaggag cctcagctga ggatgaagaa caatgaggag gctgaagact atgatgatgacctgactgat tctgagatgg atgtggtgag gtttgatgat gacaatagcc ccagcttcattcagatcagg tctgtggcca agaaacaccc caagacctgg gtgcactaca ttgctgctgaggaagaggac tgggactatg ctcccctggt gctggcccct gatgataggt cttataagagccagtacctg aacaatgggc cccagaggat tggcaggaag tacaagaagg tgaggttcatggcctacact gatgaaacct tcaaaaccag ggaggccatt cagcatgagt ctggcatcctgggccctctg ctgtatgggg aggtggggga caccctgctg atcatcttca agaaccaggccagcaggccc tacaacatct atcctcatgg catcactgat gtgaggcccc tgtacagcaggaggctgccc aagggggtga agcacctgaa agacttcccc atcctgcctg gggagatctttaagtataag tggactgtga ctgtggagga tggccctacc aagtctgacc ccaggtgtctgaccaggtac tattctagct ttgtgaacat ggagagggac ctggcctctg gcctgattgggcccctgctg atctgctaca aggagtctgt ggaccagagg ggcaaccaga tcatgtctgacaagaggaat gtgatcctgt tttctgtgtt tgatgagaat aggagctggt acctgactgagaacatccag aggtttctgc ccaatcctgc tggggtgcag ctggaggatc ctgagttccaggccagcaat atcatgcata gcatcaatgg ctatgtgttt gacagcctgc agctgtctgtgtgcctgcat gaggtggcct actggtacat cctgagcatt ggggcccaga ctgactttctgtctgtgttc ttttctggct ataccttcaa gcacaagatg gtgtatgagg ataccctgaccctgttcccc ttctctgggg agactgtgtt catgagcatg gagaatcctg ggctgtggatcctggggtgc cacaactctg attttaggaa cagggggatg actgccctgc tgaaggtgtctagctgtgat aagaacactg gggactacta tgaggacagc tatgaggaca tttctgcttatctgctgtct aagaataatg ccattgagcc cagaagcttc agccagaatc cccctgtgctgaagagacat cagagggaga tcaccagaac taccctgcag tctgatcagg aggagattgactatgatgac actatctctg tggagatgaa gaaggaggac tttgacatct atgatgaggatgagaatcag tctcccagga gctttcagaa gaagaccaga cattacttca ttgctgctgtggagaggctg tgggactatg gcatgagctc tagccctcat gtgctgagga acagggcccagtctggctct gtgccccagt tcaagaaggt ggtgttccag gaattcactg atggcagcttcacccagccc ctgtacaggg gggagctgaa tgagcacctg ggcctgctgg ggccttatatcagggctgag gtggaggata atattatggt gactttcagg aaccaggcca gcaggccctactctttctat agcagcctga tctcttatga ggaggatcag aggcaggggg ctgagcctaggaagaacttt gtgaagccca atgagactaa gacctacttc tggaaggtcc agcaccacatggcccctacc aaggatgagt ttgactgcaa ggcctgggcc tatttctctg atgtggatctggagaaggat gtccattctg ggctgattg ccccctgctg gtgtgccaca ctaacactctgaatcctgcc catggcaggc aggtgactgt ccaggagttt gccctgttct tcactatctttgatgagacc aagagctggt actttactga gaacatggag aggaactgca gagctccttgcaatattcag atggaggacc ccaccttcaa ggaagaattac aggttccatg ccattaatgggtacatcatg gacaccctgc ctggcctggt gatggctcag gaccagagga tcaggtggtacctgctgagc atgggctcta atgagaatat ccacagcatc cacttcttg ggcatgtgttcactgtgagg aagaaggagg agtacaagat ggctctgtat aatctgtacc ctggggtgtttgaaactgtg gagatgctgc cctctaaggc tggcatctgg agggtggagt gcctgattggggagcacctg catgctggca tgagcaccct gttcctggtg tacagcaaca agtgccagacccccctgggc atggcctctg gccacatcag ggacttccag atcactgcct ctggccagtatggccagtgg gcccccaagc tggccaggct gcactattct ggcagcatca atgcctggagcaccaaggag cccttcagct ggatcaaggt ggacctgctg gcccccatga tcattcatggcatcaagacc cagggggcca ggcagaagtt cagctctctg tacatctctc agttcatcatcatgtactct ctggatggga agaagtggca gacctacagg ggcaacagca ctggcaccctgatggtgttc tttgggaatg tggactcttc tggcatcaag caacacatct tcaatccccccatcattgct aggtatatta gggctcatcc caccactac agcatcaggt ctaccctgaggatggagctg atgggctgtg acctgaactc ttgcagcatg cccctgggca tggagtctaaggccatctct gatgcccaga ttactgccag cagctacttc accaacatgt ttgccacctggagcccctct aaggccaggc tgcatctgca ggggaggagc aatgcctgga ggcctcaggtgaacaacccc aaggtggc tgcaggtgga tttccagaag accatgaagg tgactggggtgaccacccag ggggtcaaga gcctgctgac cagcatgtat gtgaaggagt tcctgatcagcagcagccag gatggccacc agtggactct gttctttcag aatgggaagg tgaaggtgtttcagggcaat caggactctt tcacccctgt ggtgaacagc ctggaccccc ccctgctgaccagatacctg aggatccacc cccagtcttg ggtgcatcag attgccctga ggatggaggtgctgggctgt gaggctcagg atctgtactg agcggccgca ataaaagatc agagctctagagatctgtgt gttggttttt tgtgtaggaa cccctagtga tggagttggc cactccctctctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggctttgcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcagg

[0292] A full-length plasmid (SEQ ID NO: 24) containing a mutant TTR promoter (TTRmut), a synthetic intron, a CpG-reduced Factor VIII cDNA, polyA, and ITRs. cctgcaggca gctgcgcgct cgctcgctca ctgaggccgcccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagcgcgcagagag ggagtggcca actccatcac taggggttcc tacgcgtgtc tgtctgcacatttcgtagag cgagtgttcc gatactctaa tctccctagg caaggttcat attgacttaggttacttatt ctccttttgt tgactaagtc aataatcaga atcagcaggt ttggagtcagcttggcaggg atcagcagcc tgggttggaa ggagggggta taaaagcccc ttcaccaggagaagccgtca cacagatcca caagctcctg ctagcaggta agtgccgtgt gtggttcccgcgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactg acactgacatccactttttc tttttctcca caggtttaaa cgccaccatg cagattgagc tgagcacctgcttcttcctg tgtctgctga ggttctgctt ctctgccacc aggaggtatt acctgggggctgtggagctg agctgggact atatgcagtc tgacctgggg gagctgcctg tggatgctaggttccccccc agggtgccca agagcttccc ctttaacact tctgtggtgt acaagaagaccctgtttgtg gagttcactg accacctgtt caacattgcc aagcccaggc ccccctggatggggctgctg gggcccacca tccaggctga ggtgtatgac actgtggtga tcaccctgaagaacatggcc agccaccctg tgagcctgca tgctgtgggg gtgagctact ggaaggcttctgagggggct gagtatgatg accagactag ccagagggag aaggaggatg acaaggtgtttcctgggggc agccatacct atgtgtggca ggtgctgaag gagaatggcc ccatggcctctgaccccctg tgcctgacct acagctacct gtctcatgtg gacctggtga aggacctgaactctggcctg attggggctc tgctggtgtg tagggagggc agcctggcta aggaaaagacccagaccctg cataagttta tcctgctgtt tgctgtgttt gatgagggca agagctggcactctgagacc aagaacagcc tgatgcagga tagggatgct gcctctgcca gggcttggcctaagatgcac actgtgaatg ggtatgtgaa taggagcctg cctggcctga ttggctgccacaggaagtct gtgtactggc atgtgattgg gatgggcacc acccctgagg tccatagcatcttcctggag ggccacactt tcctggtgag gaaccacaga caggcctctc tggagatctctcccatcacc ttcctgactg ctcagactct gctgatggac ctgggccagt tcctgctgttttgccatatt agcagccacc agcatgatgg gatggaggcc tatgtgaagg tggatagctgccctgaggag cctcagctga ggatgaagaa caatgaggag gctgaagact atgatgatgacctgactgat tctgagatgg atgtggtgag gtttgatgat gacaatagcc ccagcttcattcagatcagg tctgtggcca agaacaccc caagacctgg gtgcactaca ttgctgctgaggagaggac tgggactatg ctcccctggt gctggcccct gatgataggt cttataagagccagtacctg aacaatgggc cccagaggat tggcaggaag cccaagaagg tgaggttcatggcctacact gatgaaacct tcaaaaccag ggaggccatt cagcatgagt ctggcatcctggggccctctg ctgtatgggg aggtggggga caccctgctg atcatcttca agaaccaggccagcaggccc tacaacatct atcctcatgg catcactgat gtgaggcccc tgtacagcaggaggctgccc aagggggtga agcacctgaa agacttcccc atcctgcctg gggagatctttaagtataag tggactgtga ctgtggagga tggccctacc aagtctgacc ccaggtgtctgaccaggtac tattctagct ttgtgaacat ggagagggac ctggcctctg gcctgattgggcccctgctg atctgctaca aggagtctgt ggaccagagg ggcaaccaga tcatgtctgacaagaggaat gtgatcctgt tttctgtgtt tgatgagaat aggagctggt acctgactgagaaacatccag aggtttctgc ccaatcctgc tggggtgcag ctggaggatc ctgagttccaggccagcaat atcatgcata gcatcaatgg ctatgtgttt gacagcctgc agctgtctgtgtgcctgcat gaggtggcct actggtacat cctgagcatt ggggcccaga ctgactttctgtctgtgttc tttctggct ataccttcaa gcacaagatg gtgtatgagg ataccctgaccctgttcccc ttctctgggg agactgtgtt catgagcatg gagaatcctg ggctgtggatcctggggtgc cacaactctg attttaggaa caggggatg actgccctgc tgaaggtgtctagctgtgat aagaacactg gggactacta tgaggacagc tatgaggaca tttctgcttatctgctgtct aagaataatg ccattgagcc cagaagcttc agccagaatc cccctgtgctgaagagacat cagagggaga tcaccagaac taccctgcag tctgatcagg aggagattgactatgatgac actatctctg tggagatgaa gaaggaggac tttgacatct atgatgaggatgagaatcag tctcccagga gctttcagaa gaagaccaga cattacttca ttgctgctgtggagaggctg tgggactatg gcatgagctc tagccctcat gtgctgagga acagggcccagtctggctct gtgccccagt tcaagaaggt ggtgttccag gaattcactg atggcagcttcacccagccc ctgtacaggg gggagctgaa tgagcacctg ggcctgctgg ggccttatatcagggctgag gtggaggata atattatggt gactttcagg aaccaggcca gcaggccctactctttctat agcagcctga tctcttatga ggaggatcag aggcaggggg ctgagcctaggaagaacttt gtgaagccca atgagactaa gacctacttc tggaaggtcc agcaccacatggcccctacc aaggatgagt ttgactgcaa ggcctgggcc tatttctctg atgtggatctggagaaggat gtccattctg ggctgattgg ccccctgctg gtgtgccaca ctaacactctgaatcctgcc catggcaggc aggtgactgt ccaggagttt gccctgttct tcactatctttgatgagacc aagagctggt actttactga gaacatggag aggaactgca gagctccttgcaatattcag atggaggacc ccaccttcaa ggaagaattac aggttccatg ccattaatgggtacatcatg gacaccctgc ctggcctggt gatggctcag gaccagagga tcaggtggtacctgctgagc atgggctcta atgagaatat ccacagcatc cacttcttg ggcatgtgttcactgtgagg aagaaggagg agtacaagat ggctctgtat aatctgtacc ctggggtgtttgaaactgtg gagatgctgc cctctaaggc tggcatctgg agggtggagt gcctgattggggagcacctg catgctggca tgagcaccct gttcctggtg tacagcaaca agtgccagacccccctgggc atggcctctg gccacatcag ggacttccag atcactgcct ctggccagtatggccagtgg gcccccaagc tggccaggct gcactattct ggcagcatca atgcctggagcaccaaggag cccttcagct ggatcaaggt ggacctgctg gcccccatga tcattcatggcatcaagacc caggggcca ggcagaagtt cagctctctg tacatctctc agttcatcatcatgtactct ctggatggga agaagtggca gacctacagg ggcaacagca ctggcaccctgatggtgttc tttgggaatg tggactcttc tggcatcaag cacaacatct tcaatccccccatcattgct aggtatatta ggctgcatcc cacccactac agcatcaggt ctaccctgaggatggagctg atgggctgtg acctgaactc ttgcagcatg cccctgggca tggagtctaaggccatctct gatgcccaga ttactgccag cagctacttc accaacatgt ttgccacctggagcccctct aaggccaggc tgcatctgca ggggaggagc aatgcctgga ggcctcaggtgaacaacccc aaggtggc tgcaggtgga tttccagaag accatgaagg tgactggggtgaccacccag ggggtcaaga gcctgctgac cagcatgtat gtgaaggagt tcctgatcagcagcagccag gatggccacc agtggactct gttctttcag aatgggaagg tgaaggtgtttcagggcaat caggactctt tcacccctgt ggtgaacagc ctggaccccc ccctgctgaccagatacctg aggatccacc cccagtcttg ggtgcatcag attgccctga ggatggaggtgctgggctgt gaggctcagg atctgtactg agcggccgca ataaagatc agagctctagagatctgtgt gttggttttt tgtgtaggaa cccctagtga tggagttggc cactccctctctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgccccgacg cccgggctttgcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggca gcttgaaggaaatactaagg caaaggtact gcaagtgctc gcaacattcg cttatgcgga ttattgccgtagtgccgcga cgccgggggc aagatgcaga gattgccatg gtacaggccg tgcggttgatattgccaaaa cagagctgtg ggggagagt gtcgagaaag agtgcggaag atgcaaaggcgtcggctatt caaggatgcc agcaagcgca gcatatcgcg ctgtgacgat gctaatcccaaaccttacc aacccacctg gtcacgcact gttaagccgc tgtatgacgc tctggtggtgcaatgccaca aagaagagtc aatcgcagac aacattttga atgcggtcac acgttagcagcatgattgcc acggatggca acatattaac ggcatgatat tgacttattg aataaaattgggtaaatttg actcaacgat gggttaattc gctcgttgtg gtagtgagat gaaaagaggcggcgcttact accgattccg cctagttggt cacttcgacg tatcgtctgg aactccaaccatcgcaggca gagaggtctg caaaatgcaa tcccgaaaca gttcgcaggt atagttagagcctgcataa cggttcggg attttttata tctgcacaac aggtaagagc attgagtcgataatcgtgaa gagtcggcga gcctggttag ccagtgctct ttccgttgtg ctgaattaagcgaataccgg aagcagaacc ggatcaccaa atgcgtacag gcgtcatcgc cgcccagcaacagcacaacc caaactgagc cgtagccact gtctgtcctg aattcattag tatagttacgctgcggcct tttacacatg accttcgtga aagcgggtgg caggaggtcg cgctaacaacctcctgccgt tttgcccgtg catatcggtc acgaacaaat ctgattacta aacacagtagcctggatttg ttctatcagt aatcgacctt attcctaatt aaatagagca aatccccttattgggggtaa gacatgaaga tgccagaaaa acatgacctg ttggccgcca ttctcgcggcaaaggaacaa ggcatcgggg caatccttgc gtttgcaatg gcgtaccttc gcggcagatataatggcggt gcgtttacaa aaacagtaat cgacgcaacg atgtgcgcca ttatcgcctagttcattcgt gaccttctcg acttcgccgg actaagtagc aatctcgctt atataacgagcgtgtttatc ggctacatcg gtactgactc gattggttcg cttatcaaac gcttcgctgctaaaaaagcc ggagtagaag atggtagaaa tcaataatca acgtaaggcg ttcctcgatatgctggcgtg gtcggaggga actgataacg gacgtcagaa aaccagaaat catggttatgacgtcattgt aggcggagag ctatttactg attactccga tcaccctcgc aaacttgtcacgctaaaccc aaaactcaaa tcaacaggcg ccggacgcta ccagcttctt tcccgttggtgggatgccta ccgcaagcag cttggcctga aagacttctc tccgaaaagt caggacgctgtggcattgca gcagattaag gagcgtggcg ctttacctat gattgatcgt ggtgatatccgtcaggcaat cgaccgttgc agcaatct gggctcact gccggggcgct ggttatggtcagttcgagca taaggctgac agcctgattg caaattca agaagcgggc ggaacggtcagagattga tgtagagca gagtcaccgc gattatctcc gctctgtta tctgcatcgtctgcctg tcatgggctg ttaatcatta ccgtgataac gccattacct aaagcccagcgcgacaaaatgccagag aactgaagct ggcgaacgcg gcaattactg acatgcagatgcgtcagcgt gatgttgctg cgctcgatgc aaatacacg aaggagttag ctgatgctaaagctgaaat gatgctctgc gtgatgatgt tgccgctggt cgtcgtcggt tgcacatcaaagcagctgt cagtcagtgc gtgaagccac caccgcctcc ggcgtggata atgcagcctccccccgactg gcagacaccg ctgaacggga ttttcacc ctcagagaga ggctgatcactatgcaaaa caacggaag gaacccagaa gtatattaat gagcagtgca gatgagttgcccatatcga tgggcaactc atgcaattat tgtgagcaat acacacgcgc ttccagcggagtataaatgc ctaagtaat aaaaccgagc aatccatta cgaatgtttg ctggttctctgttttaacaa cattttctgc gccgccacaa atttggctg catcgacagt tttcttctgcccaattccag aaacgaagaa atgatgggtg atggtttcct ttggtgctac tgctgccggttttgttttgaa cagtaaacgt ctgttgagca catcctgtaa taagcagggc cagcgcagtagcgagtagca ttttttcat ggtgttattc ccgatgcttt ttgaagttcg cagaatcgtatgtgtgtagaaa attaaacaaa ccctaaacaa tgagttgaaa tttcatattg ttaatattattaatgtatg tcaggtgcga tgaatcgtca ttgtattccc ggattaacta tgtccacagccctgacgggg aacttctctg cggggagtgtc cgggaataat taaaacgatg cacacagggtttagcgcgta cacgtattgc attatgccaa cgccccggtg ctgacacgga agaaaccggacgttatgatt tagcgtggaa agatttgtgt agtgttctga atgctctcag taaatagtaatgaattatca aaggtatagt aatatcttt atgttcatgg atattgtaa cccatcggaaaactcctgct ttagcaagat tttccctgta ttgctgaaat gtgatttctc ttgatttcaacctatcatag gacgtttcta taagatgcgt gtttcttgag aatttaacat ttacaacctttttaagtcct tttattaaca cggtgttatc gttttctaac acgatgtgaa tattatctgtggctagatag taaatataat gtgagacgtt gtgacgtttt agttcagaat aaaacaattcacagtctaaa tctttcgca cttgatcgaa tatttcttta aaaatggcaa cctgagccattggtaaaacc ttccatgtga tacgaggcg cgtagtttgc attcgttt ttatcgtttcaatctggtct gacctccttg tgttttgttg atgatttatg tcaaatatta ggaatgttttcacttaatag tattggttgc gtaacaaagt gcggtcctgc tggcattctg gagggaaatacaaccgacag atgtatgtaa ggccaacgtg ctcaaatctt catacagaaa gatttgaagtaatattttaa ccgctagatg aagagcaagc gcatggagcg acaaaatgaa taaagaacaatctgctgatg atccctccgt ggatctgatt cgtgtaaaaa atatgcttaa tagcaccattcttgagtt acctgatgt tgtaattgca tgtatagaac ataaggtgtc tctggaagcattcagagcaa ttgaggcagc gttggtgaag cacgataata atatgaagga ttattccctggtggttgact gatcaccata actgctaatc attcaaacta tttagtctgt gacagagccaacacgcagtc tgtcactgtc aggaaagtgg taaaactgca actcaattac tgcaatgccctcgtaattaa gtgaatttac aatatcgtcc tgttcggagg gaagaacgcg ggatgttcattcttcatcac ttttaattga tgtatatgct ctcttttctg acgttagtct ccgacggcaggcttcaatga cccaggctga gaaattcccg gacccttttt gctcaagagc gatgttaatttgttcaatca tttggttagg aaagcggatg ttgcgggttg ttgttctgcg ggttctgttcttcgttgaca tgaggttgcc ccgtattcag tgtcgctgat ttgtattgtc tgaagttgtttttacgttaa gttgatgcag atcaattaat acgatacctg cgtcataatt gattatttgacgtggtttga tggcctccac gcacgttgtg atatgtagat gataatcatt atcactttacgggtcctttc cggtgatccg acaggttacg gggcggcgac ctgcctgatg cggtattttctccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgccctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacacttgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgccggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgctttacggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgccctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactcttgttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tagacctgcaggcatgcaag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcgttacccaact taatcgccctt gcagcacatc cccctttcgc cagctggcgt aatagcgaagaggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgcgatttattcaacaaagc cgccgtcccg tcaagtcagc gtaatgctct gccagtgtta caaccaattaaccaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatcaggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccgaggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaacatcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcaccatgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcattcttt ccagacttgttcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattcattcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaaacaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacctgaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagtaaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattccgtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgccatgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacctgattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaatttaatcgcg gcttcgagca agacgtttcc cgttgaatat ggctcataac accccttgtattactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgcaatgtaacatc agagattttg agacacaacg tggctttgtt gaataaatcg aacttttgctgagttgaagg atcagatcac gcatcttccc gacaacgcag accgttccgt ggcaaagcaaaagttcaaaa tcaccaactg gtccacctac aacaaagctc tcatcaaccg tggctccctcactttctggc tggatgatgg ggcgattcag gcctggtatg agtcagcaac accttcttcacgaggcagac ctctcgacgg agttccactg agcgtcagac cccgtagaaa agatcaaaggatcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccaccgctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaactggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggccaccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagtggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttaccggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcgaacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcccgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcacgagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacctctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgccagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt

[0293] FVIII-BDD encoded by the X01-X18 nucleic acid sequence [SQ sequence is bold / underlined] (SEQ ID NO: 25) MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPR SFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

[0294] Wild-type FVIII with BDD (SEQ ID NO: 26)

[0295] AAV-LK03 VP1 capsid (SEQ ID NO: 27) MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL

[0296] AAV-SPK VP1 capsid (SEQ ID NO: 28) used in AAV-SPK-8005 and AAV-SPK-hFIX MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPA A PSG V G PN TMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIA NNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGP NNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGAL PGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

[0297] [Table 8]

[0298] Specific definitions / abbreviations used BDD: Deletes all or at least part of the B domain (BD) FVIII-BDD: B domain deleted FVIII SQ:SFSQNPPVLKRHQR (SEQ ID NO: 29) FVIII / SQ: FVIII with SQ FVIIIX01-X18: Nucleic acid variants encoding CpG-reduced FVIII as set forth in SEQ ID NOs: 1 to 18, respectively TTRmut: TTR promoter with four mutations from TAmGTGTAG to TATTGACTTAG CO3: Codon-optimized FVIII nucleic acid variant shown in SEQ ID NO: 21 NHP: non-human primate ALT: alanine aminotransferase D-dimer protein fragment from the breakdown of a blood clot SPK-8005: AAV capsid (SEQ ID NO: 28) + TTRmut-hFVIII-X07; also referred to as AAV-SPK-8005 SPK-8011: AAV LK03 capsid (SEQ ID NO: 27) + TTRmut-hFVIII-X07; also referred to as AAV-SPK-8011

[0299]

[0273] Although several embodiments of the present invention have been described and exemplified in detail above, it is not intended that the invention be limited to such embodiments. Various modifications can be made to these embodiments without departing from the scope and spirit of the present invention as set forth in the following claims.

[0300] [Related Applications]

[0001] This patent application claims the benefit of U.S. patent application Ser. No. 62 / 249,001, filed October 30, 2015, Ser. No. 62 / 331,872, filed May 4, 2016, Ser. No. 62 / 349,532, filed June 13, 2016, and Ser. No. 62 / 357,874, filed July 1, 2016, all of which are expressly incorporated by reference in their entirety herein.

Claims

1. A nucleic acid variant encoding a factor VIII (FVIII) having a deletion of the B domain, said nucleic acid variant having 92% or greater sequence identity to SEQ ID NO:

7.

2. The nucleic acid variant of claim 1 having a sequence identity of 93% or more with SEQ ID NO:

7.

3. The nucleic acid variant of claim 1 having 94% or more sequence identity with SEQ ID NO:

7.

4. 2. The nucleic acid variant of claim 1 having 95% or more sequence identity with SEQ ID NO:

7.

5. 2. The nucleic acid variant of claim 1 having 95% to 100% sequence identity with SEQ ID NO:

7.

6. 2. The nucleic acid variant of claim 1 having no more than 20, no more than 15, or no more than 10 cytosine-guanine dinucleotides (CpG).

7. 2. The nucleic acid variant of claim 1 having five or fewer cytosine-guanine dinucleotides (CpG).

8. Not specified.

9. 2. The nucleic acid variant of claim 1 having 4, 3, 2, 1 or 0 cytosine-guanine dinucleotides (CpG).

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

11. 2. The nucleic acid variant of claim 1 , which encodes SEQ ID NO:

25.

12. A nucleic acid variant encoding a factor VIII (FVIII) having a B domain deletion (FVIII-BDD) having fewer cytosine-guanine dinucleotides (CpGs) than SEQ ID NO:

19.

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

14. A nucleic acid variant encoding human factor VIII having a B domain deletion (hFVIII-BDD) having no more than two cytosine-guanine dinucleotides (CpGs).

15. 15. The nucleic acid variant of claim 14 having one cytosine-guanine dinucleotide (CpG).

16. 15. The nucleic acid variant of claim 14, which has no cytosine-guanine dinucleotides (CpG).

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

19.

18. The nucleic acid variant according to any one of claims 1 to 17, which is different from FVIII-V3 (SEQ ID NO: 20) and CO3 (SEQ ID NO: 21).

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

20. A nucleic acid variant according to any one of claims 1 to 18, encoding SEQ ID NO:

25.

21. A vector comprising a nucleic acid variant according to any one of claims 1 to 20.

22. An expression vector comprising a nucleic acid variant according to any one of claims 1 to 20.

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

24. 24. The expression vector of claim 23, wherein the AAV vector comprises an AAV serotype or AAV pseudotype, the AAV pseudotype comprising an AAV capsid serotype that is different from the ITR serotype.

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

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

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

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

29. 26. The expression vector of claim 25, wherein the filler polynucleotide sequence is adjacent to the 5' or 3' end of the nucleic acid variant.

30. 30. The expression vector of any one of claims 25 to 29, wherein introns, expression control elements, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and / or filler polynucleotide sequences have been modified to have reduced cytosine-guanine dinucleotides (CpG).

31. 30. The expression vector of any one of claims 25 to 29, wherein the introns, expression control elements, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and / or filler polynucleotide sequences are modified to have 20 or less, 15 or less, 10 or less, 5 or less, or zero cytosine-guanine dinucleotides (CpGs).

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

33. 32. An expression vector according to any one of claims 25, 27, 30 and 31, wherein the expression control elements comprise elements that confer expression in the liver.

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

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

22.

36. 36. The expression vector of any one of claims 25-35, wherein the ITRs comprise 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 combinations thereof.

37. The expression vector according to any one of claims 24 to 30, comprising an ITR, a promoter, a polyA signal and / or an intron sequence as shown in SEQ ID NO:

23.

38. 38. An AAV vector comprising a nucleic acid variant according to any one of claims 1 to 20 or an expression vector according to any one of claims 25 to 37.

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

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

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

42. 39. The AAV vector of claim 38, comprising a capsid having 90% or greater sequence identity to the LK03 capsid (sequence number 27).

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

44. 39. The AAV vector of claim 38 comprising an LK03 capsid (sequence number 27).

45. 39. The AAV vector of claim 38, comprising an SPK capsid (sequence number 28).

46. 39. The AAV vector of claim 38, comprising a nucleic acid variant of SEQ ID NO:7 and an LK03 capsid sequence (SEQ ID NO:27).

47. 39. The AAV vector of claim 38, comprising a nucleic acid variant of SEQ ID NO:7 and an SPK capsid (SEQ ID NO:28).

48. 39. The AAV vector of claim 38, comprising a nucleic acid variant and one or more of a mutated TTR promoter (TTRmut), a synthetic intron, polyA and an ITR in SEQ ID NO:

23.

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

50. A host cell comprising a nucleic acid variant according to any one of claims 1 to 20 or a vector or expression vector according to any one of claims 21 to 37.

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

52. A host cell comprising an AAV vector according to any one of claims 38 to 49.

53. A host cell according to claim 52, which produces an AAV vector according to any one of claims 38 to 49.

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

55. 50. The nucleic acid variant of any one of claims 1 to 20, the vector or expression vector of any one of claims 21 to 37, or the AAV vector of any one of claims 38 to 49, encapsulated in a liposome or mixed with a phospholipid or micelle.

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

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

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

59. 56. The pharmaceutical composition of claim 54 or 55, wherein the empty capsid is an LK03 capsid (sequence number 27) or an SPK capsid (sequence number 28).

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

1.

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

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

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

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

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

66. 64. The method of claim 63, wherein the cells comprise hepatocytes, sinusoidal endothelial cells, megakaryocytes, platelets or hematopoietic stem cells.

67. 64. The method of claim 63, wherein the mammalian tissue or organ comprises a liver.

68. 68. The method of any one of claims 61 to 67, wherein the mammal produces insufficient amounts of factor VIII protein, or a defective or abnormal factor VIII protein.

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

70. 68. The method of any one of claims 61 to 67, wherein the Factor VIII encoded by the nucleic acid variant is expressed at a level that has a beneficial or therapeutic effect in a mammal.

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

72. 72. The method of claim 61, 62 or 71, wherein the mammal or patient has a disorder selected from the group consisting of hemophilia A, von Willebrand's disease and trauma-related bleeding, injury, thrombosis, thrombocytopenia, stroke, coagulation disorders, disseminated intravascular coagulation (DIC) and excessive anticoagulation treatment disorders.

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

74. The method of any one of claims 61 to 72, wherein FVIII is expressed at a level that does not substantially increase the risk of thrombosis.

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

76. The method of any one of claims 61-72, wherein FVIII is expressed at a level greater than 1% of the level of FVIII found in subjects without hemophilia A.

77. The method of any one of claims 61-72, wherein FVIII is expressed at a level greater than 3% of the level of FVIII found in subjects without hemophilia A.

78. 73. The method of any one of claims 61 to 72, wherein FVIII 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.

79. 73. The method of any one of claims 61-72, wherein FVIII is expressed at a level greater than 1% or 3% of the level of FVIII found in subjects without hemophilia A for at least 1, 2, 3, or 4 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or for at least 1 year.

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

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

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

83. AAV vector is administered in a dose of 1×10 per kilogram of mammal or patient. １２73. The method of any one of claims 61-72, wherein the FVIII is administered at a dose of less than about 20% of the activity or greater than 20% of the activity in the mammal or patient for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive days, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 months.

84. The AAV vector is about 5×10 per kilogram of mammal or patient. １１ 73. The method of any one of claims 61-72, wherein the FVIII is administered at a dose of about 20% or greater than 20% activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive days, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 months in the mammal or patient.

85. The method of any one of claims 61 to 84, wherein the mammal or patient is a human.

86. 86. The method of any one of claims 61 to 85, wherein the mammal, patient or human is seropositive or seronegative for AAV.

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

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

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

90. 90. The method of claim 89, wherein the empty capsid is an LK03 capsid (sequence number 27) or an SPK capsid (sequence number 28).

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

1.

92. 92. The method of any one of claims 61 to 91, further comprising administering an immunosuppressant.

93. 92. The method of any one of claims 61 to 91, further comprising the step of administering an immunosuppressant after the AAV vector is administered.

94. 92. The method of any one of claims 61-91, further comprising administering an immunosuppressant within 1 hour up to 45 days after the AAV vector is administered.

95. 95. The method of any one of claims 92 to 94, wherein the immunosuppressant comprises a steroid, a cyclosporine (e.g. cyclosporine A), mycophenolate, rituximab or a derivative thereof.

96. The AAV vector is about 1×10 per kilogram of mammal or patient body weight. ８ ~Approx. 1×10 １４ 96. The method of any one of claims 61 to 95, wherein the antibody is administered in the range of vector genomes (vg / kg).

97. The AAV vector is about 1×10 per kilogram of mammal or patient body weight. ９ ~Approx. 1×10 １３ 96. The method of any one of claims 61 to 95, wherein the antibody is administered in the range of vector genomes (vg / kg).

98. The AAV vector is about 1×10 per kilogram of mammal or patient body weight. １０ ~Approx. 1×10 １２ 96. The method of any one of claims 61 to 95, wherein the antibody is administered in the range of vector genomes (vg / kg).

99. The AAV vector is about 1×10 per kilogram of mammal or patient body weight. １１ ~Approx. 1×10 １２ 96. The method of any one of claims 61 to 95, wherein the antibody is administered in the range of vector genomes (vg / kg).

100. The AAV vector is about 1×10 per kilogram of mammal or patient body weight. １２ ~Approx. 1×10 １３ 96. The method of any one of claims 61 to 95, wherein the antibody is administered in the range of vector genomes (vg / kg).

101. The AAV vector is about 1×10 per kilogram of mammal or patient body weight. １３ ~Approx. 1×10 １４ 96. The method of any one of claims 61 to 95, wherein the antibody is administered in the range of vector genomes (vg / kg).

102. The AAV vector is about 5×10 per kilogram of mammal or patient body weight. １１ ~Approx. 1×10 １２ 96. The method of any one of claims 61 to 95, wherein the antibody is administered in the range of vector genomes (vg / kg).

103. The AAV vector is about 5×10 per kilogram of mammal or patient body weight. １１ 96. The method of any one of claims 61 to 95, wherein the vector is administered in a dose of 100 mg / kg of vector genomes (vg / kg).

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

105. A method for producing a FVIII protein, comprising expressing in a cell a nucleic acid variant according to any one of claims 1 to 20, or a vector or an expression vector according to any one of claims 21 to 37, and recovering the FVIII protein produced by the cell.

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

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