RU2818229C2 - Recovered nucleic acid which codes fviii-bdd-based fusion protein and heterologous signal peptide, and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an isolated nucleic acid coding a fused protein based on FVIII-BDD (coagulation factor VIII with a deleted B domain) and a heterologous signal peptide, and an expression cassette, a vector and a cell containing same is also provided. Also disclosed is a pharmaceutical composition containing said vector or cassette.

EFFECT: invention is effective for providing FVIII-BDD protein to a subject with hemophilia A, as well as for treating hemophilia A in a subject.

18 cl, 5 dwg, 3 ex

Description

Field of technology to which the invention relates

This application relates to the fields of genetics, gene therapy and molecular biology. More specifically, the present invention relates to a nucleic acid that encodes a fusion protein based on FVIII-BDD (blood clotting factor VIII with B domain deleted) and a heterologous signal peptide, an expression cassette and a vector based thereon, a host cell for producing the fusion protein at based on FVIII-BDD and heterologous signal peptide, as well as various applications of the above vector.

State of the art

Hemophilia is a recessive, X-linked, congenital disorder leading to a deficiency of one of the proteins involved in secondary hemostasis. Hemophilia A, or classical hemophilia, is the most common form of hemophilia; occurs in 1 in 5,000 newborn boys (Report on the annual global survey 2019, WFH https://www.wfh.org/en/our-work-research-data/annual-global-survey) and is caused by a deficiency of the coagulation factor VIII protein . According to the All-Russian Hemophilia Society, there are more than 6.5 thousand patients with hemophilia A in Russia (Report on the annual global survey 2019, WFH).

Coagulation factor VIII (FVIII) is a 280 kDa protein that is secreted into the blood primarily from sinusoidal epithelial cells of the liver (Fahs SA, Hille MT, Shi Q, Weiler H, Montgomery RR. A conditional knockout mouse model reveals endothelial cells as the principal and possibly exclusive source of plasma factor VIII/ Blood 2014 Jun 12;123(24):3706-13. doi: 10.1182/blood-2014-02-555151. Epub 2014 Apr 4. PMID: 24705491 and Everett LA, Cleuren AC , Khoriaty RN, Ginsburg D. Murine coagulation factor VIII is synthesized in endothelial cells/ Blood 2014 Jun 12;123(24):3697-705. doi: 10.1182/blood-2014-02-554501 Epub 2014 Apr 9. : 24719406). Activated FVIII circulates in the body as a heterodimer consisting of heavy (domains A1, A2, B) and light (domains A3, C1, C3) chains linked to each other through non-covalent metal-dependent interactions. As a result of FVIII processing, only domains A1-A3, C1, C2 are present in the activated form of the protein. This fact contributed to the creation of recombinant FVIII with the B domain deleted (FVIII-BDD), which is not inferior in its activity to full-length FVIII (Pittman DD, Alderman EM, Tomkinson KN, Wang JH, Giles AR, Kaufman RJ. Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII/ Blood. 1993 Jun 1; 81(11):2925-35. Unlike other proteins in the blood coagulation cascade, which are predominantly proteases, FVIII is a glycoprotein. However, it plays a critical role in the formation of the tenase complex, which is required for the formation of activated coagulation factor X (FXa), the first member of the final common coagulation pathway, which ultimately leads to the formation of cross-linked fibrin.

In more than half of the cases, severe hemophilia A is caused by inversions in intron 1 or 22 of the FVIII gene (Habart D, Kalabova D, Novotny M, Vorlova Z. Thirty-four novel mutations detected in factor VIII gene by multiplex CSGE: modeling of 13 novel amino acid substitutions/J ThrombHaemost. 2003 Apr; 1(4):773-81. doi: 10.1046/j.l538-7836.2003.00149.x. In other cases, abnormalities in the FVIII sequence are associated with various mutations, including antisense mutations, frameshifts, splice site mutations, deletions, and insertions.

The tendency to bleed in hemophilia A is related to detectable FVIII activity and is classified as mild (0.05-0.40 IU/ml), moderate (0.01-0.05 IU/ml) or severe (<0.01 IU/ml). ml). Patients with mild hemophilia typically experience abnormal bleeding only due to surgery or injury. In contrast, patients with moderate hemophilia experience prolonged bleeding reactions to relatively minor injuries, and patients with severe hemophilia often experience spontaneous bleeding. The severe form of hemophilia A is manifested by spontaneous hemarthrosis, soft tissue hematomas, retroperitoneal bleeding, intracerebral hemorrhage and delayed postoperative bleeding. Over time, complications from recurrent hemarthrosis and soft tissue hematomas include severe arthropathy, joint contractures, and pseudotumors leading to chronic disease. The proportion of patients with mild, moderate and severe forms of hemophilia A is not precisely established, but recent epidemiological studies suggest that approximately 60% of patients with hemophilia A have severe form (Report on the annual global survey 2019, WFH).

Today, the standard treatment regimen for patients suffering from hemophilia A is lifelong replacement therapy in the form of injections of recombinant FVIII (Report on the annual global survey 2019, WFH). Despite the success of hemophilia therapy, there are serious problems with this approach. Preventive replacement therapy for hemophilia A involves intravenous injections of recombinant FVIII every three days for the life of a patient with severe disease. This type of treatment is very expensive and does not guarantee the absence of complications associated primarily with hemarthrosis. In some cases, patients develop inhibitory antibodies. Inhibitory forms of hemophilia A are more often observed in patients with severe forms of the disease and require the use of alternative approaches to the treatment and prevention of the disease (Eckhardt CL, van der Bom JG, van der Naald M, Peters M, Kamphuisen PW, Fijnvandraat K. Surgery and inhibitor development in hemophilia A: a systematic review/ J Thromb Haemost. 2011 Oct; 9(10): 1948-58. doi: 10.1111/j.1538-7836.2011.04467.x.

Gene therapy for hemophilia A through adeno-associated virus (AAV)-based viral (expression) vectors encoding the FVIII gene has shown impressive results in a series of preclinical and clinical studies (Bunting S, Zhang L, Xie L, Bullens S, Mahimkar R, Fong S, Sandza K, Harmon D, Yates B, Handyside B, Sihn CR, Galicia N, Tsuruda L, O'Neill CA, Bagri A, Colosi P, Long S, Vehar G, Carter B. Gene Therapy with BMN 270 Results in Therapeutic Levels of FVIII in Mice and Primates and Normalization of Bleeding in Hemophilic Mice/ Mol Ther. 2018 Feb 7;26(2):496-509. and Peyvandi F, Garagiola I. Clinical advances in gene therapy updates on clinical trials of gene therapy in haemophilia/ Haemophilia 2019 Sep; doi: 10.1111/hae.13816 Epub 2019 Jul 8.PMID. : 31282050). In contrast to traditional approaches to the treatment of hemophilia, gene therapy using AAV allows the expression of introduced FVIII to be maintained at sufficient levels for several years after a single dose of the drug to patients (Long BR, Veron P, Kuranda K, Hardet R, Mitchell N, Hayes GM , Wong WY, Lau K, Li M, Hock MB, Zoog SJ, Vettermann C, Mingozzi F, Schweighardt B. Early Phase Clinical Immunogenicity of Valoctocogene Roxaparvovec, an AAV5-Mediated Gene Therapy for Hemophilia A/ Mol Ther 2021; 29(2):597-610. doi: 10.1016/j.ymthe.2020.12.008. Epub 2020 Dec 10. PMID: 33309883).

At the moment, there is not a single registered gene therapy drug in the world for the treatment of hemophilia A.

Thus, it is relevant to develop a gene therapy drug for the treatment of hemophilia A, as well as solutions that will improve the effectiveness of a gene therapy drug for the treatment of hemophilia A.

Disclosure of the invention

The inventors have unexpectedly discovered that the use of a nucleic acid encoding

1) a fusion protein based on FVIII-BDD and signal peptide FIX (SP-FIX), which has the amino acid sequence SEQ ID NO: 7 or

2) a fusion protein based on FVIII-BDD and immunoglobulin G Carr signal peptide (SP-IgGK), which has the amino acid sequence SEQ ID NO: 8 or

3) a fusion protein based on FVIII-BDD and Lactalbumin signal peptide (SP-Lactalbumin), which has the amino acid sequence SEQ ID NO: 9,

results in an increase in the level of production and activity of the FVIII-BDD protein compared to the use of a nucleic acid encoding the FVIII-BDD protein with a natural FVIII signal peptide (wild type).

Definitions and general methods

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention will have meanings that are commonly understood by those skilled in the art.

In addition, unless the context otherwise requires, terms in the singular include plural terms, and plural terms include singular terms. In general, the classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medicinal and pharmaceutical chemistry, and hybridization and protein and nucleic acid chemistry described herein are well known to those skilled in the art. widely used in this field. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as is typically carried out in the art, or as described herein.

The terms “naturally occurring,” “native,” or “wild type” are used to describe an object that can be found in nature to be different from that produced artificially. For example, a protein or nucleotide sequence that is present in an organism, including a virus, that can be isolated from a source in nature, and that has not been intentionally modified by a person skilled in the laboratory, is naturally occurring.

In the present specification and in the following claims, unless the context otherwise requires, the words “include” and “comprise” or variations thereof such as “includes”, “comprising”, “contains” or “comprising” are to be understood as including the specified whole or group of wholes, but not to the exclusion of any other whole or group of wholes.

Protein (Peptide)

As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

Nucleic acid molecules

The terms "nucleic acid", "nucleic sequence" or "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", as used interchangeably herein, refer to a distinct sequence of nucleotides, modified or unmodified , defining a fragment or region of nucleic acid, containing or not containing unnatural nucleotides and being either double-stranded DNA or RNA, or single-stranded DNA or RNA, or transcription products of these DNAs.

As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences produced by any methods available in the art, including, but not limited to, recombinant methods, i.e. cloning nucleic acid sequences from a recombinant library or cell genome, using conventional cloning and PCR technology, etc., and synthesis methods.

It should also be mentioned here that this invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in its natural state. The sequences of the present invention have been isolated and/or purified, i.e. were taken directly or indirectly, for example by copying, and their environment was at least partially modified. Thus, it should also be understood here as isolated nucleic acids obtained by genetic recombination, for example by host cells, or obtained by chemical synthesis.

The term nucleotide sequence covers its complement unless otherwise specified. Thus, a nucleic acid having a specific sequence should be understood as comprising its complementary strand with its complementary sequence.

Vector

The term “vector” as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In addition, the term “vector” as used herein means a recombinant viral particle capable of transporting a nucleic acid.

As used herein, the term “expression” is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

Application

“Gene therapy” is the insertion of genes into the cells and/or tissues of a subject to treat a disease, usually an inherited disease, whereby a defective mutant allele is replaced or supplemented with a functional allele.

“Treat,” “cure,” and “therapy” refer to a method of alleviating or eliminating a biological disorder and/or at least one of its associated symptoms.

The term “subject”, “patient”, “individual”, etc. are used interchangeably herein and refer to any animal that can be treated by the methods presented herein. In specific non-limiting embodiments, the subject, patient, or individual is a human. The above subject may be male or female of any age.

A "therapeutically effective amount" or "effective amount" is considered to be the amount of a therapeutic agent administered that will relieve, to a certain extent, one or more symptoms of the disease being treated.

Detailed Description of the Invention

Nucleic acid

In one aspect, the present invention provides an isolated nucleic acid that encodes a fusion protein of FVIII-BDD (blood clotting factor VIII with B domain deleted) and a heterologous signal peptide that includes an amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

An "isolated" nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule. The isolated nucleic acid molecule differs from the form or set in which it occurs naturally. Thus, the isolated nucleic acid molecule is different from the nucleic acid molecule that naturally exists in cells.

The signal peptide ensures the transport of the protein of interest within the cell to target organelles or promotes the secretion of the protein of interest into the intercellular space.

In some embodiments, the isolated nucleic acid encodes a fusion protein based on FVIII-BDD with the amino acid sequence of SEQ ID NO: 5 and a FIX signal peptide with the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the isolated nucleic acid encodes a fusion protein based on FVIII-BDD with the amino acid sequence of SEQ ID NO: 5 and an immunoglobulin G chain Carr signal peptide with the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the isolated nucleic acid encodes a fusion protein based on FVIII-BDD with the amino acid sequence of SEQ ID NO: 5 and a Lactalbumin signal peptide with the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the isolated nucleic acid encodes a fusion protein of FVIII-BDD and a heterologous signal peptide that has the amino acid sequence of SEQ ID NO: 7. The fusion protein of the amino acid sequence of SEQ ID NO: 7 includes FVIII-BDD of the amino acid sequence of SEQ ID NO: : 5 and signal peptide FIX with amino acid sequence SEQ ID NO: 2.

In some embodiments, the isolated nucleic acid encodes a fusion protein of FVIII-BDD and a heterologous signal peptide that has the amino acid sequence of SEQ ID NO: 8. The fusion protein of SEQ ID NO: 8 includes FVIII-BDD of the amino acid sequence of SEQ ID NO: 8. : 5 and the Carr signal peptide of the immunoglobulin G chain with the amino acid sequence SEQ ID NO: 3.

In some embodiments, the isolated nucleic acid encodes a fusion protein of FVIII-BDD and a heterologous signal peptide that has the amino acid sequence of SEQ ID NO: 9. The fusion protein of SEQ ID NO: 9 includes FVIII-BDD of the amino acid sequence of SEQ ID NO: : 5 and Lactalbumin signal peptide with amino acid sequence SEQ ID NO: 4.

In some embodiments, the isolated nucleic acid is the nucleotide sequence of SEQ ID NO: 11. The nucleic acid encodes a FVIII-BDD-FIX signal peptide fusion protein that has the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the isolated nucleic acid is the nucleotide sequence of SEQ ID NO: 12. The nucleic acid encodes a fusion protein based on FVIII-BDD and the Carr signal peptide of the immunoglobulin G chain, which has the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the isolated nucleic acid is the nucleotide sequence of SEQ ID NO: 13. The nucleic acid encodes a fusion protein of FVIII-BDD and the Lactalbumin signal peptide that has the amino acid sequence of SEQ ID NO: 9.

Expression cassette. Expression vector.

In one aspect, the present invention provides an expression cassette that includes the above nucleic acid that encodes a fusion protein based on FVIII-BDD and a heterologous signal peptide.

The term “cassette that expresses” or “expression cassette” as used herein specifically refers to a fragment of DNA that is capable, under appropriate circumstances, of driving the expression of a polynucleotide encoding a polypeptide of interest, the sequence of which is included in the expression cassette. When introduced into a host cell, the expression cassette is, among other things, capable of engaging cellular machinery to transcribe the polynucleotide encoding the polypeptide of interest into RNA, which is then typically further processed and finally translated into the polypeptide of interest. The expression cassette may be contained in an expression vector.

The expression cassette of the present invention contains a promoter element. The term "promoter" as used herein specifically refers to a DNA element that promotes transcription of a polynucleotide to which the promoter is operably linked. A promoter may also form part of a promoter/enhancer element. Although the physical boundaries between promoter and enhancer elements are not always clear, the term promoter generally refers to the location on a nucleic acid molecule to which RNA polymerase and/or associated factors bind and from which transcription is initiated. Enhancers enhance promoter activity temporally as well as spatially. There are many promoters known in the art that are transcriptionally active in a wide range of cell types. Promoters can be divided into two classes: those that function constitutively and those that are regulated by induction or release of repression. Both classes are suitable for protein expression. Promoters that are used to produce high levels of polypeptides in eukaryotic cells and, in particular, mammalian cells must be strong and preferably must be active in a wide range of cell types. Potent constitutive promoters that are capable of driving expression in many cell types are well known in the art and therefore need not be described in detail herein.

According to one embodiment of the invention, the HLP promoter is used in the expression cassette of the present invention.

In some embodiments, the expression cassette includes the following elements in a 5' to 3' direction:

left (first) ITR (inverted terminal repeats);

promoter;

any of the above nucleic acids that encodes a fusion protein based on FVIII-BDD and a heterologous signal peptide;

polyadenylation signal;

right (second) ITR.

The above structural elements of the expression cassette are functionally related to each other.

As used herein, the term "operably linked" refers to the linking of polynucleotide (or polypeptide) elements into a functional link. A nucleic acid is "operably linked" if it is in a functional relationship with another nucleic acid sequence. For example, a transcription control sequence is operably linked to a coding sequence if it affects the transcription of said coding sequence. The term "operably linked" means that the linked DNA sequences are generally contiguous and, where appropriate, junctions of two protein-coding regions are also contiguous and in frame.

In some embodiments, the expression cassette includes a left (first) ITR with the nucleotide sequence of SEQ ID NO: 14.

In some embodiments, the expression cassette includes an HLP promoter having the nucleotide sequence of SEQ ID NO: 15.

In some embodiments, the expression cassette includes a polyadenylation signal with the nucleotide sequence of SEQ ID NO: 16.

In some embodiments, the expression cassette includes a right (second) ITR with the nucleotide sequence of SEQ ID NO: 17.

In some embodiments, the expression cassette includes the following elements in a 5' to 3' direction:

left (first) ITR (inverted terminal repeats) with nucleotide sequence SEQ ID NO: 14;

promoter with nucleotide sequence SEQ ID NO: 15;

any of the above nucleic acids that encodes a fusion protein based on FVIII-BDD and a heterologous signal peptide;

polyadenylation signal with nucleotide sequence SEQ ID NO: 16;

right (second) ITR with nucleotide sequence SEQ ID NO: 17.

In one aspect, the present invention provides an expression vector that includes any of the above nucleic acids that encodes a fusion protein based on FVIII-BDD and a heterologous signal peptide, or any of the above expression cassettes.

In some embodiments, the vector is a plasmid, i.e. a circular, double-stranded piece of DNA into which additional DNA segments can be inserted.

In some embodiments, the vector is a viral (expression) vector in which additional DNA segments can be inserted into the viral genome.

In some embodiments, the vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal vectors). In other embodiments, vectors (eg, non-episomal vectors) can be integrated into the host cell genome upon introduction into the host cell, and thus replicate along with the host gene. Moreover, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors” (“expression vector” or “expression vector”)).

Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmid, YAC, EBV and the like. DNA molecules can be inserted into a vector such that the transcription and translation control sequences in the vector perform the intended function of regulating DNA transcription and translation. The expression vector and expression control sequences can be selected to be compatible with the expression host cell used. DNA molecules can be introduced into an expression vector by standard methods (eg, ligation of complementary restriction sites or blunt-end ligation if restriction sites are missing).

In some embodiments, the expression vector is a recombinant adeno-associated virus (AAV).

In some embodiments, the AAV is selected from the group consisting of the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, rAAV.rh8, rAAV. rhlO, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV. HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 or AAV.HSC16.

In some embodiments, the vector or cassette may include an expression control sequence. The term "expression control sequence" as used herein refers to polynucleotide sequences that are necessary to influence the expression and processing of the coding sequences to which they are inserted. Those skilled in the art will appreciate that the design of an expression vector or cassette, including the choice of expression control sequences, may depend on factors such as the choice of host cell type for transformation, the desired level of protein expression, etc. Expression control sequences include the corresponding transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (ie, the Kozak consensus sequence); sequences that increase protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. Preferred expression control sequences for a mammalian host expression cell include viral elements that provide high level expression of proteins in mammalian cells, such as promoters and/or enhancers derived from a retroviral LTR, cytomegalovirus (CMV) (eg, CMV promoter/enhancer), simian virus 40 (SV40) (eg, SV40 promoter/enhancer), adenovirus (eg, adenovirus large late promoter (AdMLP)), polyoma virus, as well as strong mammalian promoters such as the TTR promoter, native immunoglobulin promoter, actin promoter, and HLP promoter (hybrid liver-speciHc promoter). Expression control sequences include, at a minimum, all components whose presence is essential for expression and processing.

In addition to the above genes and expression control sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector or cassette has been introduced.

In one embodiment of the present invention, an expression vector refers to a vector containing one or more polynucleotide sequences of interest, genes or transgenes of interest that are flanked by parvovirus or inverted terminal repeat (ITR) sequences.

Neither the cassette nor the vector according to the invention contains nucleotide sequences of genes encoding non-structural proteins (Rep) and structural proteins (Cap) of the adeno-associated virus.

Host cell

In one aspect, the present invention relates to a host cell for producing a fusion protein based on FVIII-BDD and a heterologous signal peptide or for producing any of the above expression vectors, which contains any of the above nucleic acids that encodes a fusion protein based on FVIII-BDD and a heterologous signal peptide.

The term “host cell” as used herein means a cell into which a recombinant expression vector or cassette of the invention is introduced. The present invention relates to host cells, which may include, for example, a vector in accordance with the present invention described above. It should be understood that "host cell" means not only the specific cell claimed, but also the progeny of such a cell. Since modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term “host cell” as used herein.

Expression vectors or cassettes of the invention can be used to transfect a mammalian cell, plant cell, bacterial or yeast host cell. Transfection can occur by any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, nucleic acid-positively charged polymer complex transfection, nucleic acid-calcium phosphate precipitate transfection, polybrene-mediated transfection, protoplast fusion, transfection with liposome-encapsulated polynucleotides, and direct microinjection. DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells by viral (expression) vectors.

Mammalian cell lines used as hosts for transformation are well known in the art and include many immortalized cell lines available. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, BHK cells, African kidney cells vervet monkeys (COS), human hepatocellular carcinoma cells (eg Hep G2), A549 cells, SK-HEP1, HUH7, Hep-RG and a number of other cell lines. Cell lines are selected by determining which cell lines have high levels of expression and provide the desired characteristics of the protein produced. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells. When the recombinant expression vectors of the invention are introduced into mammalian host cells, the fusion protein is produced by culturing the host cells for a time sufficient to express the fusion protein in the host cells or, preferably, releasing the fusion protein into a growth medium in which the host cells are grown . The fusion protein can be isolated from the culture medium using standard protein purification methods. Plant host cells, for example, include Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Host bacterial cells include Escherichia and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

The above host cell does not refer to a host cell obtained using human embryos.

The above host cell does not refer to a host cell obtained by modifying the genetic integrity of human germline cells.

Pharmaceutical composition

In one aspect, the present invention relates to a pharmaceutical composition for delivering the FVIII-BDD gene to target cells, which includes any of the above expression vectors or cassettes.

In some embodiments, a pharmaceutical composition for delivering the FVIII-BDD gene to target cells includes any of the above expression vectors or cassettes in combination with one or more pharmaceutically acceptable excipients.

The active substance in the above composition is in an effective amount, for example, in a biologically effective amount.

"Pharmaceutical composition" means a composition comprising any of the above expression vectors of the invention and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, excipients, solvents, diluents, carriers, auxiliaries , distribution or delivery vehicles.

The pharmaceutical compositions of the present invention and methods for their manufacture will be readily apparent to those skilled in the art. The production of pharmaceutical compositions should preferably comply with GMP (Good Manufacturing Practices) requirements.

In some embodiments of the pharmaceutical composition, it may include a buffer composition, tonic agents (osmolytic or osmotic agent), stabilizers and/or solubilizers.

A "pharmaceutically acceptable" material is one that has no biological or other contraindications, e.g., the material can be administered to a subject without causing any undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, for ex vivo transfection of a cell or for in vivo administration of any of the above expression vectors of the invention directly to a subject.

The term "excipient" or "excipient" is used herein to describe any component other than those previously described in this invention. These are substances of inorganic or organic origin, used in the production process of medicines to give them the necessary physical and chemical properties.

The pharmaceutical composition according to the invention is stable.

A pharmaceutical composition is “stable” if the active agent retains its physical stability and/or chemical stability and/or biological activity through its stated shelf life at storage temperature, for example, 2-8°C. It is preferred that the active agent retains both physical and chemical stability as well as biological activity. The storage period is selected based on the results of stability studies during accelerated and natural storage.

In some embodiments, the pharmaceutical composition is a solution for intravenous administration.

In some embodiments, the pharmaceutical composition is a concentrate for the preparation of a solution for intravenous administration.

Application

In one aspect, the present invention relates to the use of any of the above expression cassettes or vectors or the above composition for delivering the FVIII-BDD gene to target cells.

In one aspect, the present invention relates to the use of any of the above expression cassettes or vectors or the above composition for providing the FVIII-BDD protein to a subject who has hemophilia A and/or does not have functional copies of the FVIII gene.

Absence of functional copies of the FVIII gene refers to inactivating mutations or deletions in all copies of the FVIII gene in the genome that result in loss or defective function of the FVIII gene.

In one aspect, the present invention provides a method of providing FVIII-BDD protein to a subject with hemophilia A, which comprises administering a therapeutically effective amount of any of the above expression vectors or the above composition to cells of a subject in need thereof.

In one aspect, the present invention relates to a method of delivering the FVIII-BDD gene to target cells of a subject with hemophilia A, which includes introducing any of the above expression vectors or the above composition into the cells of the subject.

By a subject requiring delivery of the FVIII-BDD gene to target cells or a subject requiring provision of the FVIII-BDD protein is meant a subject who has hemophilia A, or a subject who has a deficiency of coagulation factor FVIII, or a subject who has inactivating mutations or deletions in the FVIII gene that result in loss or defective function of the FVIII gene.

In one aspect, the present invention relates to the use of any of the above expression vectors or the above composition for the treatment of hemophilia A in a subject who has hemophilia A.

In one aspect, the present invention provides a method of treating hemophilia A in a subject, which includes administering a therapeutically effective amount of any of the above expression vectors or the above composition to a subject who has hemophilia A.

In some embodiments, hemophilia A is a severe form of hemophilia A (factor VIII activity <1%) or a moderate form of hemophilia A (factor VIII activity 1-5%).

Examples of routes of administration include topical, intranasal, inhalational, transmucosal, transdermal, enteral (eg, oral, rectal), parenteral (eg, intravenous, subcutaneous, intradermal, intramuscular) administration, and injection directly into a tissue or organ.

In some embodiments, any of the above expression vectors or the above composition is administered to a subject as an intravenous infusion.

Any of the above expression vectors is introduced into the body in an effective amount. Any of the above expression vectors is preferably administered to the body in a biologically effective amount. A "biologically effective" amount of an expression vector is an amount that is sufficient to cause cell transduction and expression of a nucleic acid sequence in the cell. If an expression vector is introduced into a cell in vivo, a "biologically effective" amount of the expression vector is an amount that is sufficient to cause transduction of the target cells and expression of the nucleic acid sequence in the target cell.

The dosages of any of the above expression vectors of this invention will depend on the method of administration of the particular vector, and can be determined by routine methods.

The cell for administering any of the above expression cassettes or vectors of this invention may be any cell type, including, but not limited to, epithelial cells (eg, skin, respiratory and intestinal epithelial cells), liver cells, muscle cells, spleen cells, fibroblasts , endothelial cells and the like.

Any of the above expression cassettes or vectors of this invention are not used to modify the genetic integrity of human germline cells.

In some embodiments, any of the above expression vectors according to this invention, as well as a drug based on it, are used in a monotherapy format.

In some embodiments, any of the above expression vectors of this invention, as well as a drug based thereon, are used in combination with replacement therapy with clotting factor concentrates, desmopressin and/or fibrinolysis inhibitors.

In some embodiments, any of the above expression vectors of this invention, as well as a drug based thereon, are used in combination with a monoclonal antibody (eg, emicizumab).

In some applications, any of the above expression vectors of this invention, as well as a drug based on it, is used in combination with drugs based on RNA interference technology (for example, fitusiran).

In some embodiments, any of the above expression vectors of this invention, as well as a drug based thereon, is administered to a subject in a single dose.

In some embodiments, any of the above expression vectors of this invention, as well as a drug based thereon, are administered to a subject multiple times.

Brief description of drawings

Figure 1 is a graph that shows the increase in the level of production of FVIII-BDD protein in the culture fluid after transfection with nucleic acids encoding a fusion protein based on FVIII-BDD (blood coagulation factor VIII with deleted B domain) and one of the heterologous signal peptides, compared with a nucleic acid encoding FVIII-BDD with a wild-type FVIII signal peptide.

1 - level of FVIII-BDD protein after transfection of cells with a nucleic acid that encodes a fusion protein based on human FVIII-BDD and natural FVIII signal peptide, which has the amino acid sequence SEQ ID NO: 6 (SP-FVIII-FVIII-BDD).

2 - level of FVIII-BDD protein after transfection of cells with a nucleic acid that encodes a fusion protein based on FVIII-BDD and the FIX signal peptide, which has the amino acid sequence SEQ ID NO: 7 (SP-FIX-FVIII-BDD).

3 - level of FVIII-BDD protein after transfection of cells with a nucleic acid that encodes a fusion protein based on human FVIII-BDD and the Lactalbumin signal peptide, which has the amino acid sequence SEQ ID NO: 9 (SP-Lactalbumin-FVIII-BDD).

4 - level of FVIII-BDD protein after transfection of cells with a nucleic acid that encodes a fusion protein based on human FVIII-BDD and the Carr signal peptide immunoglobulin G chain, which has the amino acid sequence SEQ ID NO: 8 (SP-IgGK-FVIII-BDD).

Figure 2 is a graph that shows the increase in the level of FVIII-BDD protein activity in culture fluid after transfection with nucleic acids encoding a FVIII-BDD (blood clotting factor VIII with B domain deleted) fusion protein and one of the heterologous signal peptides, compared with a nucleic acid encoding FVIII-BDD with a wild-type FVIII signal peptide.

1 - level of FVIII-BDD activity after transfection of cells with a nucleic acid that encodes a fusion protein based on human FVIII-BDD and natural FVIII signal peptide, which has the amino acid sequence SEQ ID NO: 6 (SP-FVIII-FVIII-BDD).

2 - level of FVIII-BDD activity after transfection of cells with a nucleic acid that encodes a fusion protein based on FVIII-BDD and the FIX signal peptide, which has the amino acid sequence SEQ ID NO: 7 (SP-FIX-FVIII-BDD).

3 - level of FVIII-BDD activity after transfection of cells with a nucleic acid that encodes a fusion protein based on human FVIII-BDD and the Lactalbumin signal peptide, which has the amino acid sequence SEQ ID NO: 9 (SP-Lactalbumin-FVIII-BDD).

4 - level of FVIII-BDD activity after transfection of cells with a nucleic acid that encodes a fusion protein based on human FVIII-BDD and the Carr chain signal peptide of immunoglobulin G, which has the amino acid sequence SEQ ID NO: 8 (SP-IgGK-FVIII-BDD).

Figure 3 is a graph that shows the increase in the level of FVIII-BDD protein production when nucleic acids are delivered in vitro as an rAAV expression vector containing a nucleic acid encoding a FVIII-BDD fusion protein and one of the heterologous signal peptides, compared with an rAAV-based expression vector containing a nucleic acid encoding FVIII-BDD with a wild-type FVIII signal peptide.

1 - level of FVIII-BDD protein after transduction of cells with expression vectors containing a nucleic acid that encodes a fusion protein based on human FVIII-BDD and natural FVIII signal peptide, which has the amino acid sequence SEQ ID NO: 6 (SP-FVIII-FVIII-BDD ).

2 - level of FVIII-BDD protein after transduction of cells with expression vectors containing a nucleic acid that encodes a fusion protein based on FVIII-BDD and the FIX signal peptide, which has the amino acid sequence SEQ ID NO: 7 (SP-FIX-FVIII-BDD).

3 - level of FVIII-BDD protein after transduction of cells with expression vectors containing a nucleic acid that encodes a fusion protein based on human FVIII-BDD and the Lactalbumin signal peptide, which has the amino acid sequence SEQ ID NO: 9 (SP-Lactalbumin FVIII-BDD).

Figure 4 is a graph that shows the increase in the level of FVIII-BDD protein activity when nucleic acids are delivered in vitro as an rAAV expression vector containing a nucleic acid encoding a FVIII-BDD fusion protein and one of the heterologous signal peptides, compared with an rAAV-based expression vector containing a nucleic acid encoding FVIII-BDD with a wild-type FVIII signal peptide.

1 - level of FVIII-BDD activity after transduction of cells with expression vectors containing a nucleic acid that encodes a fusion protein based on human FVIII-BDD and natural FVIII signal peptide, which has the amino acid sequence SEQ ID NO: 6 (SP-FVIII-FVIII-BDD ).

2 - level of FVIII-BDD activity after transduction of cells with expression vectors containing a nucleic acid that encodes a fusion protein based on FVIII-BDD and the FIX signal peptide, which has the amino acid sequence SEQ ID NO: 7 (SP-FIX-FVIII-BDD).

3 - level of FVIII-BDD activity after transduction of cells with expression vectors containing a nucleic acid that encodes a fusion protein based on human FVIII-BDD and the Lactalbumin signal peptide, which has the amino acid sequence SEQ ID NO: 9 (SP-Lactalbumin FVIII-BDD).

Figure 5 is a graph that shows the increase in FVIII-BDD protein levels when a nucleic acid encoding a fusion protein of FVIII-BDD and one of the heterologous signal peptides is delivered in vivo to B6.129S-F8tm1Smoc (HemA) mice as an expression vector based on rAAV.

1 - level of FVIII-BDD protein in the blood plasma of animals after injection of a control solution without AAV (negative control).

2 - level of FVIII-BDD protein in the blood plasma of animals after injection of an expression vector containing a nucleic acid that encodes a fusion protein based on FVIII-BDD and the FIX signal peptide, which has the amino acid sequence SEQ ID NO: 7 (SP-FIX-FVIII- BDD).

3 - level of FVIII-BDD protein in the blood plasma of animals after injection of an expression vector containing a nucleic acid that encodes a fusion protein based on human FVIII-BDD and the Lactalbumin signal peptide, which has the amino acid sequence SEQ ID NO: 9 (SP-Lactalbumin FVIII- BDD).

Examples

For a better understanding of the invention, the following examples are provided. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any form.

All publications, patents and patent applications referenced in this specification are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example in order to avoid ambiguity, those skilled in the art will appreciate from the teachings disclosed herein that certain changes and modifications may be made without departing from the spirit and scope of the appended embodiments. implementation of the invention.

Materials and general methods

Recombinant DNA Methods

For DNA manipulation, standard methods were used as described in Sambrook J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturers' instructions. Briefly, plasmid DNA was produced for further manipulation in E. coli cells grown under selective pressure with antibiotics to ensure that plasmids were not lost in the cell population. Plasmid DNA was isolated from cells using commercial kits, the concentration was measured, and used for cloning using restriction endonuclease treatment or PCR amplification methods. DNA fragments were ligated with each other using ligases and transformed into bacterial cells for clone selection and further development. All obtained genetic constructs were confirmed by restriction patterns and full Sanger sequencing.

Gene synthesis

The required gene segments were obtained from oligonucleotides created by chemical synthesis. Gene fragments ranging in length from 300 to 1000 bp, which are flanked by unique restriction sites, were assembled by renaturation of oligonucleotides on each other, followed by PCR amplification from extreme primers. As a result, a mixture of fragments was obtained, including the desired one. The fragments were cloned at restriction sites into intermediate vectors, after which the DNA sequences of the subcloned fragments were confirmed by DNA sequencing.

Determination of DNA sequences

DNA sequences were determined by Sanger sequencing. DNA and protein sequence analysis and sequence data processing were performed using SnapGene 4.2 and higher to generate, map, analyze, annotate, and illustrate sequences.

Cell culture cultivation

The cell lines HEK293 (Human Embryonic Kidney clone 293), HUH7 (human hepatocellular carcinoma cell lines) and HepG2 (human hepatocellular carcinoma cell lines) were used in the experiments. Suspension HEK293 cells used for AAV production were cultured under standard conditions at 37°C and 5% CO ₂ in complete nutrient medium without FBS and antibiotic. Adherent HUH7 and HepG2 cells, used to test the effectiveness of AAV drugs, were cultured under standard conditions at 37°C and 5% CO ₂ , in complete DMEM nutrient medium supplemented with 10% FBS, an antibiotic/antimycotic. Reseeding of HUH7 and HepG2 cells was carried out when 80-90% confluency was achieved. TrypLE Select enzyme (10x) was used to dissociate the cell monolayer. Cell viability was assessed using Trypan Blue staining and disposable cell counting chambers using a Countess II automated counter.

Cell culture transfections

To evaluate the functionality of new fusion protein variants during transfection, plasmids containing an expression cassette for expressing various variants of hFVIII-BDD transgenes were used. The HepG2 cell line was pre-seeded into the wells of 12-well plates with a density of 10,000 cells/ ^cm2 . A day later, plasmids were introduced in the same copy number as part of a complex with Lipofectamine 3000. On the 7th day after transfection, the content of the FVIII-BDD protein and its activity in the culture liquid were determined by ELISA and chromogenic test. Work to assess the level and activity of the FVIII-BDD protein in the culture liquid was carried out in 6 independent experiments. Intact HepG2 cells were used as a negative control.

Assembly and purification of AAV-based expression vectors

To assemble AAV-based expression vectors containing codon-optimized variants of the FVIII-BDD gene, HEK293 producer cells were used, which were transfected with 3 plasmids:

1) plasmids containing an AAV expression cassette for the expression of various variants of hFVIII-BDD transgenes;

2) plasmid for expression of the Sar gene of serotype AAV6 and the Rep gene of serotype AAV2. Each gene encodes multiple protein products using alternative reading frames;

3) a plasmid for the expression of Ad2 adenovirus genes necessary for the assembly and packaging of AAV capsids.

After 72 hours, the cells were lysed and the particles were purified and concentrated using filtration, chromatography and ultracentrifugation methods. The particle titer was determined using quantitative PCR with primers and a probe specific to a region of the recombinant viral genome and expressed as the number of copies of viral genomes per 1 ml.

Transduction of cell cultures

The HUH7 cell line was pre-seeded into the wells of 12-well plates with a density of 10,000 cells/ ^cm2 . After cell attachment to the adhesive substrate, AAV preparations were applied at an MOI of 500,000 vg/cell. On day 7 after transduction, the content of the FVIII-BDD protein and its activity in the culture liquid was determined by ELISA and chromogenic test. Work to assess the level and activity of the FVIII-BDD protein in the culture liquid was carried out in 6 independent experiments. Intact cells were used for negative control.

Determination of the amount of blood coagulation factor VIII BDD protein by ELISA

Assessment of the protein content of blood coagulation factor VIII-BDD in the culture fluid after transfection of HepG2 cells and transduction of HUH7 cells, as well as after injection of target candidates into the blood plasma of animals (mice), was carried out using the “sandwich” method of non-competitive enzyme-linked immunosorbent assay (ELISA). Briefly, samples diluted in dilution buffer were added to wells of a 96-well plate coated with primary coagulation factor VIII-BDD-specific antibodies. Standards for constructing a calibration curve and controls were added to the same tablet. The plate was incubated for 1 hour at 37°C. The wells of the plate were washed with washing buffer before adding biotinylated antibodies, a solution of streptavidin-peroxidase conjugate and TMB. A solution with specific biotinylated detection antibodies to factor VIII-BDD was added and the plate was incubated for 30 minutes at 37°C. Next, a solution of streptavidin-peroxidase conjugate was added to the resulting complex and the plate was incubated for 30 minutes at 37°C. To visualize the enzymatic reaction, a TMB solution was added. Once the desired degree of color intensity is achieved, a stop solution is added to all wells to stop the reaction. Next, the optical density of the solutions in the wells of the plate was measured. The concentration of blood coagulation factor VIII-BDD in the studied samples was determined using a calibration curve, taking into account preliminary dilution of the samples.

Determination of the level of protein activity of blood coagulation factor VIII BDD by ELISA

The activity of blood coagulation factor VIII protein in the culture fluid after transfection of HepG2 cells and transduction of HUH7 cells with target candidates was assessed using a chromogenic test. The essence of the test is that in the presence of calcium ions, phospholipids and factor IXa, factor X transforms into the activated form of Xa, factor VIII acts as a cofactor in this reaction, and the rate of activation of factor X is linearly related to the amount of factor VIII. Briefly, culture fluid samples, standard curve standards, and controls diluted in dilution buffer were added to the wells of a 96-well plate. The plate was incubated for 3 minutes at 37°C. A Factor reagent solution containing factor IXa, factor X, thrombin, CaCl ₂ and phospholipids was added to all wells of the plate. The plate was incubated for 4 minutes at 37°C. A solution of the chromogenic substrate S-2765+I-2581 was added to all wells of the plate. The plate was incubated for 7 minutes at 37°C. Once the desired degree of color intensity was achieved, a 20% acetic acid solution was added to all wells to stop the reaction. Next, the optical density of the solutions in the wells of the plate was measured. The activity of blood coagulation factor VIII in the studied samples was determined using a calibration curve, taking into account the preliminary dilution of the samples.

Conducting in vivo studies on laboratory animals

For the experiments, mice of the B6.129S-F8tm1Smoc (HemA) line deficient in FVIII (males 6-8 weeks old) were used. The drugs were administered to animals once by intravenous injection into the tail vein. A group of negative control animals was injected with a buffer solution that did not contain AAV. Blood plasma was collected on the day of injection before administration of the drugs, then on days 14 and 56 after administration of expression vectors.

Statistical data analysis

Results are presented as mean ± standard deviation (SD), and Dunnett's One-way ANOVA was used to compare experimental results and were determined to be statistically significant.

Example 1

In order to increase the level of secretion of the FVIII-BDD protein (coagulation factor VIII with the B domain deleted), the sequence SEQ ID NO: 6 (SP-FVIII-FVIII-BDD) was modified by replacing the signal peptide sequence of wild type FVIII with a signal peptide corresponding to the amino acid sequence specified in SEQ ID NO: 2 (SP-FIX) or SEQ ID NO: 3 (SP-IgGK) or SEQ ID NO: 4 (SP-Lactalbumin), resulting in fusion proteins corresponding to the amino acid sequences of SEQ ID NO : 7 (SP-FIX-FVIII-BDD), SEQ ID NO: 8 (SP-IgGK-FVIII-BDD) and SEQ ID NO: 9 (SP-LactalbuminFVIII-BDD).

Our obtained nucleic acids encoding fusion proteins based on FVIII-BDD and one of the heterologous signal peptides, which include an amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, were tested during cell transfection line in vitro as part of an expression cassette consisting of a left (first) ITR (inverted terminal repeats) corresponding to the sequence SEQ ID NO: 14, an HLP promoter (SEQ ID NO: 15), a gene of interest, a polyadenylation signal (SEQ ID NO: 16 ), right (second) ITR (SEQ ID NO: 17), where the genome of interest is one of the sequences SEQ ID NO: 10-13. A nucleic acid that encodes the human FVIII-BDD protein, including the natural FVIII-BDD signal peptide corresponding to the sequence SEQ ID NO 10 (SP-FVIII-FVIII-BDD), was used as a control.

The use of all nucleic acids SEQ ID NO: 11-13, encoding fusion proteins based on FVIII-BDD and one of the heterologous signal peptides with the sequences SEQ ID NO: 7-9, led to increased levels of production (Figure 1) and activity (Figure 2 ) FVIII-BDD protein compared to the use of the nucleic acid SEQ ID NO: 10 encoding the FVIII-BDD protein with the natural signal peptide SEQ ID NO: 6 (SP-FVIII-FVIII-BDD). At the same time, an unexpected increase in the level of production of the FVIII-BDD protein in the culture liquid obtained after transfection compared to the use of the nucleic acid SEQ ID NO: 10 (SP-FVIII-FVIII-BDD) by 4.3 times was shown by the sequence SEQ ID NO: 11 (SP -FIX-FVIII-BDD), 1.3 times showed the sequence SEQ ID NO: 12 (SP-IgGK-FVIII-BDD), and 4.9 times showed the sequence SEQ ID NO: 13 (SP-LactalbuminFVIII-BDD) . Similar data were observed for FVIII-BDD activity in the resulting culture fluid after transfection: an unexpected increase was observed when comparing nucleic acid SEQ ID NO: 10 (SP-FVIII-FVIII-BDD) with nucleic acid SEQ ID NO: 11 (SP-FIX-FVIII -BDD) (8.6 times increase), with SEQ ID NO: 12 (SP-IgGK-FVIII-BDD) (1.9 times increase) and with SEQ ID NO: 13 (SP-LactalbuminFVIII-BDD) ( an increase of 7.2 times). The observed correspondence between the results for the production and activity of the FVIII-BDD protein suggests that the replacement of the natural signal peptide FVIII-BDD with a signal peptide corresponding to the amino acid sequence specified in SEQ ID NO: 2 (SP-FIX), or SEQ ID NO: 3 (SP-IgGK), or SEQ ID NO: 4 (SP-Lactalbumin) leads to a significant increase in the production and activity of the FVIII-BDD protein, but does not affect the functionality of the FVIII-BDD protein.

Thus, the nucleic acids we have obtained encoding fusion proteins based on FVIII-BDD and one of the heterologous signal peptides, which include an amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, are capable of causing to the expression of the FVIII-BDD protein in vitro in the host cell and have a high potential for producing recombinant FVIII-BDD protein in producer cells for the treatment of Hemophilia A.

Example 2

rAAV-based expression vectors were prepared containing nucleic acids SEQ ID NO: 11-13 that encode FVIII-BDD fusion proteins and heterologous signal peptides that include an amino acid sequence selected from SEQ ID NO: 7-9. These expression vectors were tested for transduction of HUH7 cells in vitro. As a control, we used an rAAV-based expression vector containing the nucleic acid SEQ ID NO: 10, which encodes the human FVIII-BDD protein, including the natural FVIII signal peptide corresponding to the sequence SEQ ID NO: 6 (SP-FVIII-FVIII-BDD).

The use of expression vectors containing the nucleic acids SEQ ID NO: 11-13 resulted in an increase in the level of production (Figure 3) and activity (Figure 4) of the FVIII-BDD protein compared to the use of an expression vector containing the nucleic acid SEQ ID NO: 10 ( SP-FVIII-FVIII-BDD). At the same time, an unexpected increase in the level of production of the FVIII-BDD protein in the culture liquid obtained after transduction compared to the use of an expression vector with the nucleic acid SEQ ID NO: 10 (SP-FVIII-FVIII-BDD) was shown by an expression vector with the sequence SEQ ID NO: 11 (SP-FIX-FVIII-BDD), showed 2.4 times the expression vector with the sequence SEQ ID NO: 13 (SP-Lactalbumin-FVIII-BDD). Similar data were observed for FVIII-BDD activity in the culture fluid obtained after transduction. An unexpected increase was observed when comparing the nucleic acid expression vector SEQ ID NO: 10 (SP-FVIII-FVIII-BDD) with the nucleic acid expression vectors SEQ ID NO: 11 (SP-FIX-FVIII-BDD) (3.9 increase times) and with SEQ ID NO: 13 (SP-Lactalbumin-FVIII-BDD) (3.5 times increase). The results obtained are in agreement with the data obtained by transfecting HepG2 cells.

Thus, designed rAAV-based expression vectors encoding fusion proteins based on FVIII-BDD and one of the heterologous signal peptides that include an amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, capable of leading to the expression of the FVIII-BDD protein; in vitro.

Example 3

To conduct in vivo studies of rAAV expression vectors containing a nucleic acid selected from SEQ ID NO: 11 or SEQ ID NO: 13 that encode fusion proteins based on FVIII-BDD and one of the heterologous signal peptides that include an amino acid sequence, selected from SEQ ID NO: 7 or SEQ ID NO: 9, laboratory mice of the B6.129S-F8tm1Smoc (HemA) line deficient in FVIII were used. The study used a dose of rAAV drugs equal to 6×10 ¹³ vg/kg. A control solution without AAV was used as a negative control. The drugs were administered to animals once by intravenous hydrodynamic injection into the tail vein. Blood plasma was collected on the day of injection before drug administration, then on days 14 and 56 after drug administration. In blood plasma samples, the level of blood coagulation factor VIII-BDD protein was determined by ELISA, as described above.

As a result of in vivo studies (Figure 5), it was shown that when using both rAAV expression vectors containing a nucleic acid selected from SEQ ID NO: 11 or SEQ ID NO: 13, there was a significant increase in the amount of factor VIII-BDD in the blood plasma of animals on days 14 and 56 (Figure 5). When using an expression vector containing the nucleic acid SEQ ID NO: 11, FVIII-BDD expression was observed in the blood plasma of animals on days 14 and 56 after injection at a level of 296 and 504 ng/ml, respectively. When using an expression vector containing the nucleic acid SEQ ID NO: 13, FVIII-BDD expression was observed in the blood plasma of animals on days 14 and 56 after injection at a level of 270 and 381 ng/ml, respectively.

Thus, rAAV-based expression vectors are designed containing a nucleic acid selected from SEQ ID NO: 11 or SEQ ID NO: 13 that encode fusion proteins based on FVIII-BDD and one of the heterologous signal peptides that include the amino acid sequence selected from SEQ ID NO: 7 or SEQ ID NO: 9, are capable of leading to expression of the FVIII-BDD protein in vivo and have high potential for gene therapy for Hemophilia A.

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List of sequences

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FVIII-BDD and heterologous signal peptide and its application

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<170> BiSSAP 1.3.6

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<212>PRT

<213> Natural sequence

<220>

<223> FVIII signal peptide

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<212>PRT

<213> Natural sequence

<220>

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<210> 3

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<212>PRT

<213> Natural sequence

<220>

<223> Immunoglobulin G chain Kappa signal peptide

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Gly Ser Thr Gly

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<210> 4

<211> 19

<212>PRT

<213> Natural sequence

<220>

<223> signal peptide Lactalbumin

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Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala

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<211> 1438

<212>PRT

<213> Artificial sequence

<220>

<223> FVIII-BDD protein (blood clotting factor VIII with B deleted

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Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val

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Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser

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

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

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Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly

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

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Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg

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Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp

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Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe

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

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

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

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Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr

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Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile

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

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Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys

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

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

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

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

580 585 590

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

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Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser

610 615 620

Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu

625 630 635 640

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

645 650 655

Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro

660 665 670

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

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Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala

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Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu

705 710 715 720

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

725 730 735

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

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

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

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe

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

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

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Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr

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Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro

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

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

1025 1030 1035 1040

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

1045 1050 1055

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

1060 1065 1070

Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val

1075 1080 1085

Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val

1090 1095 1100

Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe

1105 1110 1115 1120

Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly

1125 1130 1135

His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp

1140 1145 1150

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

1155 1160 1165

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

1170 1175 1180

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

1185 1190 1195 1200

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

1205 1210 1215

Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275 1280

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

1285 1290 1295

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

1300 1305 1310

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

1315 1320 1325

Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met

1330 1335 1340

Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser

1345 1350 1355 1360

Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln

1365 1370 1375

Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn

1380 1385 1390

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

1395 1400 1405

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

1410 1415 1420

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

1425 1430 1435

<210> 6

<211> 1457

<212>PRT

<213> Artificial sequence

<220>

<223> FVIII-BDD protein with wild-type signal (SP-FVIII-FVIII-BDD)

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035 1040

Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly

1045 1050 1055

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

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130 1135

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

1140 1145 1150

Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr

1155 1160 1165

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

1170 1175 1180

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

1185 1190 1195 1200

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

1205 1210 1215

Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275 1280

Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu

1285 1290 1295

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

1300 1305 1310

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

1315 1320 1325

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

1330 1335 1340

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

1345 1350 1355 1360

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

1365 1370 1375

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

1380 1385 1390

Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe

1395 1400 1405

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

1410 1415 1420

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

1425 1430 1435 1440

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

1445 1450 1455

Tyr

<210> 7

<211> 1466

<212>PRT

<213> Artificial sequence

<220>

<223> fusion protein based on human FVIII-BDD and signaling

peptide FIX (SP-FIX-FVIII-BDD)

<400> 7

Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr

1 5 10 15

Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Ala Thr Arg Arg

20 25 30

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

35 40 45

Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro Arg Val Pro Lys

50 55 60

Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val

65 70 75 80

Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp

85 90 95

Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val

100 105 110

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

115 120 125

Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp

130 135 140

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

145 150 155 160

Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala

165 170 175

Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser His Val Asp Leu

180 185 190

Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg

195 200 205

Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile

210 215 220

Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr

225 230 235 240

Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp

245 250 255

Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly

260 265 270

Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His Val Ile Gly Met

275 280 285

Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu Gly His Thr Phe

290 295 300

Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr

305 310 315 320

Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu

325 330 335

Phe Cys His Ile Ser Ser His Gln His Asp Gly Met Glu Ala Tyr Val

340 345 350

Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn

355 360 365

Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp

370 375 380

Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg

385 390 395 400

Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His Tyr Ile Ala Ala

405 410 415

Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp

420 425 430

Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly

435 440 445

Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe

450 455 460

Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu

465 470 475 480

Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln

485 490 495

Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg

500 505 510

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

515 520 525

Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr

530 535 540

Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr

545 550 555 560

Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile

565 570 575

Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn

580 585 590

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

595 600 605

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

610 615 620

Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn

625 630 635 640

Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser

645 650 655

Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala

660 665 670

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

675 680 685

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

690 695 700

Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys

705 710 715 720

His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val

725 730 735

Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu

740 745 750

Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser

835 840 845

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

850 855 860

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

865 870 875 880

Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr

885 890 895

Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln

900 905 910

Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035 1040

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

1045 1050 1055

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

1060 1065 1070

Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His

1075 1080 1085

Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130 1135

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

1140 1145 1150

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

1155 1160 1165

Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu

1170 1175 1180

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

1185 1190 1195 1200

Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275 1280

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

1285 1290 1295

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

1300 1305 1310

Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser

1315 1320 1325

Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu

1330 1335 1340

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

1345 1350 1355 1360

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

1365 1370 1375

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

1380 1385 1390

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

1395 1400 1405

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

1410 1415 1420

Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu

1425 1430 1435 1440

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

1445 1450 1455

Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr

1460 1465

<210> 8

<211> 1458

<212>PRT

<213> Artificial sequence

<220>

<223> fusion protein based on human FVIII-BDD and signaling

Kappa peptide immunoglobulin G chain (SP-IgGK-FVIII-BDD)

<400> 8

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035 1040

Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro

1045 1050 1055

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

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130 1135

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

1140 1145 1150

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

1155 1160 1165

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

1170 1175 1180

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

1185 1190 1195 1200

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

1205 1210 1215

Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275 1280

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

1285 1290 1295

Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser

1300 1305 1310

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

1315 1320 1325

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

1330 1335 1340

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

1345 1350 1355 1360

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

1365 1370 1375

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

1380 1385 1390

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

1395 1400 1405

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

1410 1415 1420

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

1425 1430 1435 1440

His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp

1445 1450 1455

Leu Tyr

<210> 9

<211> 1457

<212>PRT

<213> Artificial sequence

<220>

<223> fusion protein based on human FVIII-BDD and signaling

Lactalbumin peptide (SP-Lactalbumin-FVIII-BDD)

<400> 9

Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035 1040

Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly

1045 1050 1055

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

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130 1135

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

1140 1145 1150

Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr

1155 1160 1165

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

1170 1175 1180

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

1185 1190 1195 1200

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

1205 1210 1215

Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275 1280

Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu

1285 1290 1295

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

1300 1305 1310

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

1315 1320 1325

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

1330 1335 1340

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

1345 1350 1355 1360

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

1365 1370 1375

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

1380 1385 1390

Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe

1395 1400 1405

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

1410 1415 1420

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

1425 1430 1435 1440

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

1445 1450 1455

Tyr

<210> 10

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid that encodes the FVIII-BDD protein with

signal wild type (SP-FVIII-FVIII-BDD)

<400> 10

atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60

accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120

ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180

acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240

gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300

gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360

ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420

gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480

aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540

gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600

gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660

tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720

gctgcatctg ctcggggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780

ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840

accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900

cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960

gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020

gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080

gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140

gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200

tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260

cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320

aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380

attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440

ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500

gatgtccgtc ctttgtattc aaggatta ccaaaaggtg taaaacattt gaaggatttt 1560

ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620

actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680

gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740

agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800

aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860

cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920

tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980

attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040

atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100

atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160

atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220

agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280

ttctctcaaa acccaccagt cttgaaacgc catcaacggg aaataactcg tactactctt 2340

cagtcagatc aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa 2400

gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca aaagaaaaca 2460

cgacactatt ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 2520

catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc 2580

caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact aaatgaacat 2640

ttgggactcc tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc 2700

agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta tgaggaagat 2760

cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 2820

ttttggaaag tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880

gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940

ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa 3000

tttgctctgt ttttcaccat ctttgatgag accaaaagct ggtacttcac tgaaaatatg 3060

gaaagaaact gcagggctcc ctgcaatatc cagatggaag atcccacttt taaagagaat 3120

tatcgcttcc atgcaatcaa tggctacata atggatacac tacctggctt agtaatggct 3180

caggatcaaa ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3240

attcatttca gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg 3300

tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa agctggaatt 3360

tggcgggtgg aatgccttat tggcgagcat ctacatgctg ggatgagcac actttttctg 3420

gtgtacagca ataagtgtca gactcccctg ggaatggctt ctggacacat tagagatttt 3480

cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540

tccggatcaa tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 3600

ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc 3660

ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg gcagacttat 3720

cgaggaaatt ccactggaac cttaatggtc ttctttggca atgtggattc atctgggata 3780

aaacacaata tttttaaccc tccaattatt gctcgataca tccgtttgca cccaactcat 3840

tatagcattc gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 3900

atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 3960

tttaccaata tgtttgccac ctggtctcct tcaaaagctc gacttcacct ccaagggagg 4020

agtaatgcct ggagaacctca ggtgaataat ccaaaagagt ggctgcaagt ggacttccag 4080

aagacaatga aagtcacagg agtaactact cagggagtaa aatctctgct taccagcatg 4140

tatgtgaagg agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt 4200

cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4260

tctctagacc caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 4320

cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctcta ctga 4374

<210> 11

<211> 4401

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid that encodes a fusion protein based on

human FVIII-BDD and FIX signal peptide

(SP-FIX-FVIII-BDD)

<400> 11

atgcagcgcg tgaacatgat catggcagaa tcaccaggcc tcatcaccat ctgcctttta 60

ggatatctac tcagtgctga atgtgccacc agaagatact acctgggtgc agtggaactg 120

tcatgggact atatgcaaag tgatctcggt gagctgcctg tggacgcaag atttcctcct 180

agagtgccaa aatcttttcc attcaacacc tcagtcgtgt acaaaaagac tctgtttgta 240

gaattcacgg atcacctttt caacatcgct aagccaaggc caccctggat gggtctgcta 300

ggtcctacca tccaggctga ggtttatgat acagtggtca ttacacttaa gaacatggct 360

tcccatcctg tcagtcttca tgctgttggt gtatcctact ggaaagcttc tgagggagct 420

gaatatgatg atcagaccag tcaaagggag aaagaagatg ataaagtctt ccctggtgga 480

agccatacat atgtctggca ggtcctgaaa gagaatggtc caatggcctc tgacccactg 540

tgccttacct actcatatct ttctcatgtg gacctggtaa aagacttgaa ttcaggcctc 600

attggagccc tactagtatg tagagaaggg agtctggcca aggaaaagac acagaccttg 660

cacaaattta tactactttt tgctgtattt gatgaaggga aaagttggca ctcagaaaca 720

aagaactcct tgatgcagga tagggatgct gcatctgctc gggcctggcc taaaatgcac 780

acagtcaatg gttatgtaaa caggtctctg ccaggtctga ttggatgcca caggaaatca 840

gtctattggc atgtgattgg aatgggcacc actcctgaag tgcactcaat attcctcgaa 900

ggtcacacat ttcttgtgag gaaccatcgc caggcgtcct tggaaatctc gccaataact 960

ttccttactg ctcaaacact cttgatggac cttggacagt ttctactgtt ttgtcatatc 1020

tcttcccacc aacatgatgg catggaagct tatgtcaaag tagacagctg tccagaggaa 1080

ccccaactac gaatgaaaaa taatgaagaa gcggaagact atgatgatga tcttactgat 1140

tctgaaatgg atgtggtcag gtttgatgat gacaactctc cttcctttat ccaaattcgc 1200

tcagttgcca agaagcatcc taaaacttgg gtacattaca ttgctgctga agaggaggac 1260

tgggactatg ctcccttagt cctcgccccc gatgacagaa gttataaaag tcaatatttg 1320

aacaatggcc ctcagcggat tggtaggaag tacaaaaaag tccgatttat ggcatacaca 1380

gatgaaacct ttaagactcg tgaagctatt cagcatgaat caggaatctt gggaccttta 1440

ctttatgggg aagttggaga cacactgttg attatattta agaatcaagc aagcagacca 1500

tataacatct accctcacgg aatcactgat gtccgtcctt tgtattcaag gagattacca 1560

aaaggtgtaa aacatttgaa ggattttcca attctgccag gagaaatatt caaatataaa 1620

tggacagtga ctgtagaaga tgggccaact aaatcagatc ctcggtgcct gacccgctat 1680

tactctagtt tcgttaatat ggagagagat ctagcttcag gactcattgg ccctctcctc 1740

atctgctaca aagaatctgt agatcaaaga ggaaaccaga taatgtcaga caagaggaat 1800

gtcatcctgt tttctgtatt tgatgagaac cgaagctggt acctcacaga gaatatacaa 1860

cgctttctcc ccaatccagc tggagtgcag cttgaggatc cagagttcca agcctccaac 1920

atcatgcaca gcatcaatgg ctatgttttt gatagtttgc agttgtcagt ttgtttgcat 1980

gaggtggcat actggtacat tctaagcatt ggagcacaga ctgacttcct ttctgtcttc 2040

ttctctggat ataccttcaa acacaaaatg gtctatgaag acacactcac cctattccca 2100

ttctcaggag aaactgtctt catgtcgatg gaaaacccag gtctatggat tctggggtgc 2160

cacaactcag actttcggaa cagaggcatg accgccttac tgaaggtttc tagttgtgac 2220

aagaacactg gtgattatta cgaggacagt tatgaagata tttcagcata cttgctgagt 2280

aaaaacaatg ccattgaacc aagaagcttc tctcaaaacc caccagtctt gaaacgccat 2340

caacgggaaa taactcgtac tactcttcag tcagatcaag aggaaattga ctatgatgat 2400

accatatcag ttgaaatgaa gaaggaagat tttgacattt atgatgagga tgaaaatcag 2460

agcccccgca gctttcaaaa gaaaacacga cactatttta ttgctgcagt ggagaggctc 2520

tgggattatg ggatgagtag ctccccacat gttctaagaa acagggctca gagtggcagt 2580

gtccctcagt tcaagaaagt tgttttccag gaatttactg atggctcctt tactcagccc 2640

ttataccgtg gagaactaaa tgaacatttg ggactcctgg ggccatatat aagagcagaa 2700

gttgaagata atatcatggt aactttcaga aatcaggcct ctcgtcccta ttccttctat 2760

tctagcctta tttcttatga ggaagatcag aggcaaggag cagaacctag aaaaaacttt 2820

gtcaagccta atgaaaccaa aacttacttt tggaaagtgc aacatcatat ggcacccact 2880

aaagatgagt ttgactgcaa agcctgggct tatttctctg atgttgacct ggaaaaagat 2940

gtgcactcag gcctgattgg accccttctg gtctgccaca ctaacacact gaaccctgct 3000

catggggagac aagtgacagt acaggaattt gctctgtttt tcaccatctt tgatgagacc 3060

aaaagctggt acttcactga aaatatggaa agaaactgca gggctccctg caatatccag 3120

atggaagatc ccacttttaa agagaattat cgcttccatg caatcaatgg ctacataatg 3180

gatacactac ctggcttagt aatggctcag gatcaaagga ttcgatggta tctgctcagc 3240

atgggcagca atgaaaacat ccattctatt catttcagtg gacatgtgtt cactgtacga 3300

aaaaaagagg agtataaaat ggcactgtac aatctctatc caggtgtttt tgagacagtg 3360

gaaatgttac catccaaagc tggaatttgg cgggtggaat gccttattgg cgagcatcta 3420

catgctggga tgagcacact ttttctggtg tacagcaata agtgtcagac tcccctggga 3480

atggcttctg gacacattag agattttcag attacagctt caggacaata tggacagtgg 3540

gccccaaagc tggccagact tcattattcc ggatcaatca atgcctggag caccaaggag 3600

cccttttctt ggatcaaggt ggatctgttg gcaccaatga ttattcacgg catcaagacc 3660

cagggtgccc gtcagaagtt ctccagcctc tacatctctc agtttatcat catgtatagt 3720

cttgatggga agaagtggca gacttatcga ggaaattcca ctggaacctt aatggtcttc 3780

tttggcaatg tggattcatc tgggataaaa cacaatattt ttaaccctcc aattattgct 3840

cgatacatcc gtttgcaccc aactcattat agcattcgca gcactcttcg catggagttg 3900

atgggctgtg atttaaatag ttgcagcatg ccattgggaa tggagagtaa agcaatatca 3960

gatgcacaga ttactgcttc atcctacttt accaatatgt ttgccacctg gtctccttca 4020

aaagctcgac ttcacctcca agggaggagt aatgcctgga gacctcaggt gaataatcca 4080

aaagagtggc tgcaagtgga cttccagaag acaatgaaag tcacaggagt aactactcag 4140

ggagtaaaat ctctgcttac cagcatgtat gtgaaggagt tcctcatctc cagcagtcaa 4200

gatggccatc agtggactct cttttttcag aatggcaaag taaaggtttt tcagggaaat 4260

caagactcct tcacacctgt ggtgaactct ctagacccac cgttactgac tcgctacctt 4320

cgaattcacc cccagagttg ggtgcaccag attgccctga ggatggaggt tctgggctgc 4380

gaggcacagg acctctactg a 4401

<210> 12

<211> 4377

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid that encodes a fusion protein based on

human FVIII-BDD and Kappa chain signal peptide

immunoglobulin G (SP-IgGK-FVIII-BDD)

<400> 12

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg gtcgaccggt 60

gccaccagaa gatactacct gggtgcagtg gaactgtcat gggactatat gcaaagtgat 120

ctcggtgagc tgcctgtgga cgcaagattt cctcctagag tgccaaaatc ttttccattc 180

aacacctcag tcgtgtacaa aaagactctg tttgtagaat tcacggatca ccttttcaac 240

atcgctaagc caaggccacc ctggatgggt ctgctaggtc ctaccatcca ggctgaggtt 300

tatgatacag tggtcattac acttaagaac atggcttccc atcctgtcag tcttcatgct 360

gttggtgtat cctactggaa agcttctgag ggagctgaat atgatgatca gaccagtcaa 420

agggaaaag aagatgataa agtcttccct ggtggaagcc atacatatgt ctggcaggtc 480

ctgaaagaga atggtccaat ggcctctgac ccactgtgcc ttacctactc atatctttct 540

catgtggacc tggtaaaaga cttgaattca ggcctcattg gagccctact agtatgtaga 600

gaagggagtc tggccaagga aaagacacag accttgcaca aatttatact actttttgct 660

gtatttgatg aagggaaaag ttggcactca gaaacaaaga actccttgat gcaggatagg 720

gatgctgcat ctgctcgggc ctggcctaaa atgcacacag tcaatggtta tgtaaacagg 780

tctctgccag gtctgattgg atgccacagg aaatcagtct attggcatgt gattggaatg 840

ggcaccactc ctgaagtgca ctcaatattc ctcgaaggtc acacatttct tgtgaggaac 900

catcgccagg cgtccttgga aatctcgcca ataactttcc ttactgctca aacactcttg 960

atggaccttg gacagtttct actgttttgt catatctctt cccaccaaca tgatggcatg 1020

gaagcttatg tcaaagtaga cagctgtcca gaggaacccc aactacgaat gaaaaataat 1080

gaagaagcgg aagactatga tgatgatctt actgattctg aaatggatgt ggtcaggttt 1140

gatgatgaca actctccttc ctttatccaa attcgctcag ttgccaagaa gcatcctaaa 1200

acttgggtac attacattgc tgctgaagag gaggactggg actatgctcc cttagtcctc 1260

gcccccgatg acagaagtta taaaagtcaa tatttgaaca atggccctca gcggattggt 1320

aggaagtaca aaaaagtccg atttatggca tacacagatg aaacctttaa gactcgtgaa 1380

gctattcagc atgaatcagg aatcttggga cctttacttt atggggaagt tggagacaca 1440

ctgttgatta tatttaagaa tcaagcaagc agaccatata acatctaccc tcacggaatc 1500

actgatgtcc gtcctttgta ttcaaggaga ttaccaaaag gtgtaaaaca tttgaaggat 1560

tttccaattc tgccaggaga aatattcaaa tataaatgga cagtgactgt agaagatggg 1620

ccaactaaat cagatcctcg gtgcctgacc cgctattact ctagtttcgt taatatggag 1680

agagatctag cttcaggact cattggccct ctcctcatct gctacaaaga atctgtagat 1740

caaagaggaa accagataat gtcagacaag aggaatgtca tcctgttttc tgtatttgat 1800

gagaaccgaa gctggtacct cacagagaat atacaacgct ttctccccaa tccagctgga 1860

gtgcagcttg aggatccaga gttccaagcc tccaacatca tgcacagcat caatggctat 1920

gtttttgata gtttgcagtt gtcagtttgt ttgcatgagg tggcatactg gtacattcta 1980

agcattggag cacagactga cttcctttct gtcttcttct ctggatatac cttcaaacac 2040

aaaatggtct atgaagacac actcacccta ttcccattct caggagaaac tgtcttcatg 2100

tcgatggaaa acccaggtct atggattctg gggtgccaca actcagactt tcggaacaga 2160

ggcatgaccg ccttactgaa ggtttctagt tgtgacaaga acactggtga ttattacgag 2220

gacagttatg aagatatttc agcatacttg ctgagtaaaa acaatgccat tgaaccaaga 2280

agcttctctc aaaacccacc agtcttgaaa cgccatcaac gggaaataac tcgtactact 2340

cttcagtcag atcaagagga aattgactat gatgatacca tatcagttga aatgaagaag 2400

gaagattttg acatttatga tgaggatgaa aatcagagcc cccgcagctt tcaaaagaaa 2460

acacgacact attttattgc tgcagtggag aggctctggg attatgggat gagtagctcc 2520

ccacatgttc taagaaacag ggctcagagt ggcagtgtcc ctcagttcaa gaaagttgtt 2580

ttccaggaat ttactgatgg ctcctttact cagcccttat accgtggaga actaaatgaa 2640

catttgggac tcctggggcc atatataaga gcagaagttg aagataatat catggtaact 2700

ttcagaaatc aggcctctcg tccctattcc ttctattcta gccttatttc ttatgaggaa 2760

gatcagaggc aaggagcaga acctagaaaa aactttgtca agcctaatga aaccaaaact 2820

tacttttgga aagtgcaaca tcatatggca cccactaaag atgagtttga ctgcaaagcc 2880

tgggcttatt tctctgatgt tgacctggaa aaagatgtgc actcaggcct gattggaccc 2940

cttctggtct gccacactaa cacactgaac cctgctcatg ggagacaagt gacagtacag 3000

gaatttgctc tgtttttcac catctttgat gagaccaaaa gctggtactt cactgaaaat 3060

atggaaagaa actgcagggc tccctgcaat atccagatgg aagatcccac ttttaaagag 3120

aattatcgct tccatgcaat caatggctac ataatggata cactacctgg cttagtaatg 3180

gctcaggatc aaaggattcg atggtatctg ctcagcatgg gcagcaatga aaacatccat 3240

tctattcatt tcagtggaca tgtgttcact gtacgaaaaa aagaggagta taaaatggca 3300

ctgtacaatc tctatccagg tgtttttgag acagtggaaa tgttaccatc caaagctgga 3360

atttggcggg tggaatgcct tattggcgag catctacatg ctgggatgag cacacttttt 3420

ctggtgtaca gcaataagtg tcagactccc ctgggaatgg cttctggaca cattagagat 3480

tttcagatta cagcttcagg acaatatgga cagtgggccc caaagctggc cagacttcat 3540

tattccggat caatcaatgc ctggagcacc aaggagccct tttcttggat caaggtggat 3600

ctgttggcac caatgattat tcacggcatc aagacccagg gtgcccgtca gaagttctcc 3660

agcctctaca tctctcagtt tatcatcatg tatagtcttg atgggaagaa gtggcagact 3720

tatcgaggaa attccactgg aaccttaatg gtcttctttg gcaatgtgga ttcatctggg 3780

ataaaacaca atatttttaa ccctccaatt attgctcgat acatccgttt gcacccaact 3840

cattatagca ttcgcagcac tcttcgcatg gagttgatgg gctgtgattt aaatagttgc 3900

agcatgccat tgggaatgga gagtaaagca atatcagatg cacagattac tgcttcatcc 3960

tactttacca atatgtttgc cacctggtct ccttcaaaag ctcgacttca cctccaaggg 4020

aggagtaatg cctggagacc tcaggtgaat aatccaaaag agtggctgca agtggacttc 4080

cagaagacaa tgaaagtcac aggagtaact actcagggag taaaatctct gcttaccagc 4140

atgtatgtga aggagttcct catctccagc agtcaagatg gccatcagtg gactctcttt 4200

tttcagaatg gcaaagtaaa ggtttttcag ggaaatcaag actccttcac acctgtggtg 4260

aactctctag acccaccgtt actgactcgc taccttcgaa ttcaccccca gagttgggtg 4320

caccagattg ccctgaggat ggaggttctg ggctgcgagg cacaggacct ctactga 4377

<210> 13

<211> 4374

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid, which is a fusion protein based on human

FVIII-BDD and Lactalbumin signal peptide

(SP-Lactalbumin-¬FVIII-BDD)

<400> 13

atgatgtcct ttgtctctct gctcctggtt ggcatcctgt tccatgccac ccaggccgcc 60

accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120

ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180

acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240

gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300

gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360

ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420

gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480

aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540

gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600

gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660

tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720

gctgcatctg ctcggggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780

ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840

accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900

cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960

gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020

gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080

gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140

gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200

tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260

cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320

aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380

attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440

ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500

gatgtccgtc ctttgtattc aaggatta ccaaaaggtg taaaacattt gaaggatttt 1560

ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620

actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680

gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740

agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800

aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860

cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920

tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980

attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040

atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100

atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160

atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220

agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280

ttctctcaaa acccaccagt cttgaaacgc catcaacggg aaataactcg tactactctt 2340

cagtcagatc aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa 2400

gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca aaagaaaaca 2460

cgacactatt ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 2520

catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc 2580

caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact aaatgaacat 2640

ttgggactcc tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc 2700

agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta tgaggaagat 2760

cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 2820

ttttggaaag tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880

gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940

ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa 3000

tttgctctgt ttttcaccat ctttgatgag accaaaagct ggtacttcac tgaaaatatg 3060

gaaagaaact gcagggctcc ctgcaatatc cagatggaag atcccacttt taaagagaat 3120

tatcgcttcc atgcaatcaa tggctacata atggatacac tacctggctt agtaatggct 3180

caggatcaaa ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3240

attcatttca gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg 3300

tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa agctggaatt 3360

tggcgggtgg aatgccttat tggcgagcat ctacatgctg ggatgagcac actttttctg 3420

gtgtacagca ataagtgtca gactcccctg ggaatggctt ctggacacat tagagatttt 3480

cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540

tccggatcaa tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 3600

ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc 3660

ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg gcagacttat 3720

cgaggaaatt ccactggaac cttaatggtc ttctttggca atgtggattc atctgggata 3780

aaacacaata tttttaaccc tccaattatt gctcgataca tccgtttgca cccaactcat 3840

tatagcattc gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 3900

atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 3960

tttaccaata tgtttgccac ctggtctcct tcaaaagctc gacttcacct ccaagggagg 4020

agtaatgcct ggagaacctca ggtgaataat ccaaaagagt ggctgcaagt ggacttccag 4080

aagacaatga aagtcacagg agtaactact cagggagtaa aatctctgct taccagcatg 4140

tatgtgaagg agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt 4200

cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4260

tctctagacc caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 4320

cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctcta ctga 4374

<210> 14

<211> 130

<212> DNA

<213> Natural sequence

<220>

<223> Left (first) ITR (inverted terminal repeats)

<400> 14

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct 130

<210> 15

<211> 252

<212> DNA

<213> Artificial sequence

<220>

<223> HLP promoter

<400> 15

tgtttgctgc ttgcaatgtt tgcccatttt agggtggaca caggacgctg tggtttctga 60

gccaggggggc gactcagatc ccagccagtg gacttagccc ctgtttgctc ctccgataac 120

tggggtgacc ttggttaata ttcaccagca gcctcccccg ttgcccctct ggatccactg 180

cttaaatacg gacgaggaca gggccctgtc tcctcagctt caggcaccac cactgacctg 240

ggacagtgaa tc 252

<210> 16

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Polyadenylation signal

<400> 16

cgcgaataaa agatctttat tttcattaga tctgtgtgtt ggttttttgt gtgatgcagc 60

<210> 17

<211> 141

<212> DNA

<213> Natural sequence

<220>

<223> Right ITR

<400> 17

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120

gagcgcgcag ctgcctgcag g 141

<---

Claims (23)

1. An isolated nucleic acid that encodes a fusion protein based on FVIII-BDD (blood clotting factor VIII with B domain deleted) and a heterologous signal peptide that includes an amino acid sequence selected from SEQ ID NO: 7 or SEQ ID NO: 9. 2. The isolated nucleic acid according to claim 1, wherein the fusion protein based on FVIII-BDD and a heterologous signal peptide has the amino acid sequence of SEQ ID NO: 7. 3. The isolated nucleic acid according to claim 1, wherein the fusion protein based on FVIII-BDD and a heterologous signal peptide has the amino acid sequence of SEQ ID NO: 9. 4. The isolated nucleic acid according to claim 2, which is the nucleotide sequence of SEQ ID NO: 11. 5. The isolated nucleic acid according to claim 3, which is the nucleotide sequence of SEQ ID NO: 13. 6. Expression cassette, which includes a nucleic acid according to any one of paragraphs. 1-5. 7. Expression cassette according to claim 6, including the following elements in the direction from the 5' end to the 3' end: left (first) ITR (inverted terminal repeats); promoter; nucleic acid according to any one of paragraphs. 1-5; polyadenylation signal; right (second) ITR. 8. An expression vector that includes a nucleic acid according to any one of paragraphs. 1-5 or an expression cassette according to any one of paragraphs. 6, 7. 9. The expression vector of claim 8, wherein the expression vector is a recombinant adeno-associated virus (AAV). 10. The expression vector of claim 9, wherein the AAV is selected from the group consisting of the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 AAV11, AAV12, AAV13, AAV14, AAV15, AAV16 , rAAV.rh8, rAAV.rhlO, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-l, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2 .5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 or AAV.HSC16. 11. A host cell for producing a fusion protein based on FVIII-BDD and a heterologous signal peptide, containing a nucleic acid according to any one of paragraphs. 1-5. 12. Pharmaceutical composition for delivery of the FVIII-BDD gene to target cells, including an expression vector or cassette according to any one of paragraphs. 8-10 in combination with one or more pharmaceutically acceptable excipients. 13. Use of an expression vector according to any one of paragraphs. 8-10 or compositions according to claim 12 for delivering the FVIII-BDD gene to target cells. 14. Use of an expression vector according to any one of paragraphs. 8-10 or the composition of claim 12 for providing the FVIII-BDD protein to a subject who has hemophilia A and/or does not have functional copies of the FVIII gene. 15. A method of providing the FVIII-BDD protein to a subject with hemophilia A, comprising administering a therapeutically effective amount of an expression vector according to any one of claims. 8-10 or compositions according to claim 12 into the cells of a subject in need thereof. 16. A method for delivering the FVIII-BDD gene to target cells of a subject with hemophilia A, including introducing an expression vector according to any one of claims. 8-10 or compositions according to claim 12 into the cells of the subject. 17. Use of an expression vector according to any one of paragraphs. 8-10 or compositions according to claim 12 for the treatment of hemophilia A in a subject who has hemophilia A. 18. A method of treating hemophilia A in a subject, which includes administering a therapeutically effective amount of an expression vector according to any one of claims. 8-10 or the composition according to claim 12 in a subject who has hemophilia A.