RU2808564C2 - Codon-optimized nucleic acid that encodes b-domain-deleted factor viii protein and its use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the recombinant production of blood coagulation factor VIII protein with B domain deleted (FVIII-BDD), and can be used in gene therapy for hemophilia A to deliver the FVIII-BDD gene to target cells. Codon-optimized variants of FVIII-BDD-encoding nucleic acids and expression constructs for their delivery to target cells have been proposed.

EFFECT: invention provides a medicinal product with high potential for gene therapy for hemophilia A.

9 cl, 6 dwg, 4 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 codon-optimized nucleic acid that encodes the FVIII-BDD protein, an expression cassette and a vector based thereon, a host cell for producing FVIII-BDD, and 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. RMY: 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. PMTD : 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 l;81(ll):2925-35. PMID: 8499631). 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 Thromb Haemost. 2003 Apr; 1(4):773-81. doi: 10.1046/j.1538-7836.2003.00149.x. PMID: 1287141/ In other cases, disturbances in the FVIII sequence are associated with various mutations, including antisense mutations, frameshifts, splice site mutations, deletions and insertions.

Bleeding tendency 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: 10.1ll1/j. 1538-7836.2011.04467.x. PMID: 21838755).

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. doi: 10.1016/j.ymthe. 2017.12.009. Epub 2017 Dec 14. PMID: 29292164 and Peyvandi F, Garagiola I. Clinical advances in gene therapy updates on clinical trials of gene therapy in haemophilia/ Haemophilia. 2019 Sep;25(5):738-746. doi: 10.1111/hae. 13816. Epub 2019 Jul 8.PMTD : 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 Feb 3; 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

One of the urgent goals of research in the development of effective gene therapy is codon optimization of genes of interest in vectors to obtain the maximum level of production of the protein of interest, which, in turn, will allow the use of lower doses of the vector to achieve a significant effect.

The inventors have unexpectedly discovered that a codon-optimized nucleic acid that encodes the FVIII-BDD protein with the amino acid sequence SEQ ID NO: 11 and includes a nucleotide sequence selected from the group: SEQ ID NO : 2 (variant FVIII-BDDco-v1), SEQ ID NO: 3 (variant FVIII-BDDco-v2), SEQ ID NO: 4 (variant FVIII-BDDco-v3) or SEQ ID NO: 5 (variant FVIII-BDDco- v4), unexpectedly leads to an increase in the expression level of the FVIII-BDD gene, an increase in the level of production of the blood coagulation factor VIII-BDD protein, as well as an increase in the level of activity of the blood coagulation factor VIII-BDD protein several times compared to the wild-type gene encoding the coagulation factor blood VIII-BDD (FVIII-BDD-wt). These variants of a codon-optimized nucleic acid according to the invention with a nucleotide sequence selected from the group: SEQ ID NO: 2 (option FVIII-BDDco-v1), SEQ ID NO: 3 (option FVIII-BDDco-v2), SEQ ID NO: 4 (variant FVIII-BDDco-v3) or SEQ ID NO: 5 (variant FVIII-BDDco-v4) are included in the expression cassette and the vector based on it.

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 codon-optimized nucleic acid that encodes a FVIII-BDD (Blood Domain Deleted Factor VIII) protein with the amino acid sequence of SEQ ID NO: 11 and includes a nucleotide sequence selected from the group: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

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.

One of the properties of the genetic code is degeneracy - the ability of different codons (trinucleotides) to encode the same amino acid. Such codons that give the same amino acid during translation are called synonymous. In natural sequences, the choice of one of the synonymous codons is carried out randomly in the process of evolution, but the frequencies of use of synonymous codons differ: for each amino acid there are more and less preferable ones. Codon optimization is a technique widely used in the world aimed at increasing the productivity of protein molecules, which consists in rationally matching each amino acid in the protein sequence with one of the suitable synonymous codons. One of the common principles of codon optimization involves the use of the most frequent codons; subsequently, other approaches were proposed, such as harmonization (reproducing the distribution of frequencies of used codons), but they do not always increase productivity. In addition to codon frequencies, the production efficiency can be influenced by the GC composition of the sequence (the ratio of the number of guanines and cytosines to the total length of the sequence); in particular, it has been shown that an increased GC composition is associated with an increase in the amount of mRNA in mammalian cells Grzegorz Kudla ET AL., High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells, June 2006, Volume 4, Issue 6, el80, pp. 933-942). It is also worth noting that stable elements of the secondary structure of mRNA, i.e. having low free energy of folding may reduce efficiency.

Various options for codon optimization of the sequence of the gene of interest can lead to the following (compared to the wild type gene):

a) the level of expression of genes of interest will be slightly increased;

b) the level of expression of genes of interest will be significantly increased;

c) the level of expression of genes of interest will remain approximately at the same level;

d) the level of expression of genes of interest will be reduced.

To obtain the above codon-optimized nucleic acid of the invention, codon optimization of the wild-type nucleic acid with the nucleotide sequence SEQ ID NO: 1 was carried out.

Codon optimization of the wild-type nucleic acid that encodes the FVIII-BDD protein with the nucleotide sequence SEQ ID NO: 1 resulted in a series of codon-optimized nucleic acids that were further tested for levels of protein production compared to the FVIII-BDD control -wt (wild type nucleic acid SEQ ID NO:1).

All codon-optimized nucleic acids resulted in increased levels of FVIII-BDD protein production compared to wild-type nucleic acid, while a number of codon-optimized nucleic acids resulted in a slight increase in FVIII-BDD protein production compared to wild-type nucleic acid. In turn, a codon-optimized nucleic acid according to the invention, which is selected from the group: SEQ ID NO: 2 (option FVIII-BDDco-v1), SEQ ID NO: 3 (option FVIII-BDDco-v2), SEQ ID NO: 4 (variant FVIII-BDDco-v3) or SEQ ID NO: 5 (variant FVIII-BDDco-v4), unexpectedly leads to an increase in the expression level of the FVIII-BDD gene, an increase in the production of blood clotting factor VIII-BDD protein, and an increase in the level of activity of the blood coagulation factor VIII-BDD protein several times compared to the wild-type gene (FVIII-BDD-wt).

Expression cassette. Expression vector.

In one aspect, the present invention provides an expression cassette that includes any of the above codon-optimized nucleic acids encoding the FVIII-BDD protein.

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;

a nucleic acid encoding a signal peptide;

any of the above codon-optimized nucleic acids encoding the FVIII-BDD protein;

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: 13.

In some embodiments, the expression cassette includes an HLP promoter having the nucleotide sequence of SEQ ID NO: 14.

In some embodiments, the expression cassette includes a polyadenylation signal with the nucleotide sequence of SEQ ID NO: 15.

In some embodiments, the expression cassette includes a right (second) ITR with the nucleotide sequence of SEQ ID NO: 16.

In some embodiments, the nucleic acid encoding the signal peptide has a nucleotide sequence selected from the group: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

In some embodiments, the codon-optimized nucleic acid encoding the FVIII-BDD protein with a signal peptide (FVIII-BDDco-v1 variant) has the nucleotide sequence of SEQ ID NO: 7.

In some embodiments, the codon-optimized nucleic acid encoding the FVIII-BDD protein with a signal peptide (FVIII-BDDco-v2 variant) has the nucleotide sequence of SEQ ID NO: 8.

In some embodiments, the codon-optimized nucleic acid encoding the FVIII-BDD protein with a signal peptide (FVIII-BDDco-v3 variant) has the nucleotide sequence of SEQ ID NO: 9.

In some embodiments, the codon-optimized nucleic acid encoding the FVIII-BDD protein with a signal peptide (FVIII-BDDco-v4 variant) has the nucleotide sequence of SEQ ID NO: 10.

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: 13;

promoter with nucleotide sequence SEQ ID NO: 14;

a nucleic acid encoding a signal peptide that has a nucleotide sequence selected from the group: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21;

any of the above codon-optimized nucleic acids encoding the FVIII-BDD protein;

polyadenylation signal with nucleotide sequence SEQ ID NO: 15;

right (second) ITR with nucleotide sequence SEQ ID NO: 16.

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: 13;

promoter with nucleotide sequence SEQ ID NO: 14;

a codon-optimized nucleic acid encoding a FVIII-BDD protein with a signal peptide, which has a nucleotide sequence selected from the group: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10;

polyadenylation signal with nucleotide sequence SEQ ID NO: 15;

right (second) ITR with nucleotide sequence SEQ ID NO: 16.

In one aspect, the present invention provides an expression vector that includes any of the above codon-optimized nucleic acids encoding the FVIII-BDD protein 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:

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-specific 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 provides a host cell for producing the FVIII-BDD protein or for producing any of the above expression vectors, which contains any of the above codon-optimized nucleic acids encoding the FVIII-BDD protein.

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, hamster kidney (HK) cells, African kidney cells green monkeys (COS), human hepatocellular carcinoma cells (for example, 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 FVIII-BDD 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 the growth medium in which the cells are grown -owners. 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 target cells and expression of a 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 diagram of plasmid pAAV-hFVIII-BDD, which contains the human coagulation factor VIII (FVIII) gene sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. The plasmid sequence also contains the following elements:

AmpR - beta-lactamase gene that provides resistance to ampicillin;

pUC origin - pUC origin of replication in bacteria;

ITR inverted terminal repeats;

HLP promoter - artificial tissue-specific promoter;

sPA is an artificial polyadenylation signal sequence to improve mRNA stability.

Figure 2 is a graph that shows the increase in the level of production of FVIII-BDD protein in the culture fluid after transfection with codon-optimized nucleic acids encoding FVIII-BDD protein (blood coagulation factor VIII with B domain deleted) compared to nucleic acid encoding FVIII protein -BDD, wild type.

FVIII-BDD protein level after cell transfection:

1 is a wild-type nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 1.

2 - a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 2.

3 - a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 3.

4 - a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 4.

5 - a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 5.

Figure 3 is a graph that shows the increase in the level of FVIII-BDD protein activity in culture fluid after transfection with codon-optimized nucleic acids encoding FVIII-BDD protein (coagulation factor VIII with B domain deleted) compared to nucleic acid encoding the protein FVIII-BDD, wild type.

Level of FVIII-BDD activity after cell transfection:

1 is a wild-type nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 1.

2 - a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 2.

3 - a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 3.

4 - a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 4.

5 - a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 5.

Figure 4 is a graph that shows the increase in the level of FVIII-BDD protein production when nucleic acids are delivered in vitro as an AAV-based expression vector containing a codon-optimized nucleic acid encoding the FVIII-BDD protein (coagulation factor VIII with B domain deleted ) compared to an AAV-based expression vector containing wild-type nucleic acid encoding the FVIII-BDD protein.

FVIII-BDD protein level after cell transduction:

1 - control solution without AAV (negative control).

2 - expression vectors based on AAV serotype 6 containing a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 2.

3 - expression vectors based on AAV serotype 6 containing a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 5.

4 - expression vectors based on AAV serotype 5 containing a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 2.

Figure 5 is a graph that shows the increase in the level of FVIII-BDD protein activity when delivered in vitro nucleic acids in the form of an AAV-based expression vector containing a codon-optimized nucleic acid encoding the FVIII-BDD protein (blood clotting factor VIII with B domain deleted ) compared to an AAV-based expression vector containing wild-type nucleic acid encoding the FVIII-BDD protein.

Level of FVIII-BDD activity after cell transduction:

1 - control solution without AAV (negative control).

2 - expression vectors based on AAV serotype 6 containing a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 2.

3 - expression vectors based on AAV serotype 6 containing a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 5.

4 - expression vectors based on AAV serotype 5 containing a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 2.

Figure 6 is a graph that shows the increase in FVIII-BDD protein levels when codon-optimized nucleic acid encoding FVIII-BDD protein is delivered in vivo to B6.129S-F8tmlSmoc (HemA) mice as an AAV-based expression vector.

The level of FVIII-BDD protein in the blood plasma of animals after injection:

1 - control solution without AAV (negative control).

2 - an expression vector based on AAV serotype 5 containing a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 2.

3 - an expression vector based on AAV serotype 6 containing a codon-optimized nucleic acid that encodes the FVIII-BDD protein, with the nucleotide sequence SEQ ID NO: 2.

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 from 300 to 1000 bp in length, which are flanked by unique restriction sites, were assembled by renaturation of oligonucleotides on each other, followed by HSR 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), SK-HEP1 (human adenocarcinoma endothelial cell line) and HUH7 (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 SK-HEP1 cells used to test the effectiveness of AAV drugs were cultured under standard conditions at 37°C and 5% CO ₂ in complete EMEM nutrient medium supplemented with 10% FBS, an antibiotic/antimycotic. Adherent HUH7 cells used to test the effectiveness of AAV drugs were cultured under standard conditions at 37°C and 5% CO ₂ in complete DMEM medium supplemented with 10% FBS, an antibiotic/antimycotic. Cell reseeding 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.

Transfection of cell cultures

To evaluate the functionality of new codon-optimized nucleic acids that encode the FVIII-BDD protein, plasmids containing a different expression cassette for the expression of various variants of hFVIII-BDD transgenes were used during transfection. Cells of the SK-HEP1 line were 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 SK-HEP1 cells were used as a negative control.

Assembly and purification of AAV-based expression vectors

To assemble expression vectors based on AAV containing 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 (Fig. 1);

2) a plasmid for the expression of the Sar gene of serotype AAV6 or AAV5 and the Rep gene of serotype AAV2. Each gene encodes multiple protein products using alternative reading frames;

3) a plasmid for the expression of Ad5 adenovirus genes necessary for the assembly and packaging of AAV capsids.

After 72 hours, the cells were lysed and the vectors were purified and concentrated using filtration, chromatography and ultracentifugation methods. The titer of viral vectors was determined using quantitative 1TCR with primers and 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 FVilli 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 cells with target candidates was carried out using the “sandwich” method of non-competitive enzyme-linked immunosorbent assay (ELISA). Briefly, culture fluid 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 the cell line 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 GCa, 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

C57BL/6 mice (males, 6-8 weeks old) were used for the experiments. 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 35 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: Modification of the hFVIII-BDD gene sequence using the developed codon optimization algorithm

The developed genetic constructs are nucleotide sequences encoding an expression cassette with the human blood coagulation factor VIII gene with the B domain deleted (FVIII-BDD).

The final expression cassette contains all the necessary elements for both gene expression and assembly into the recombinant AAV genome (Figure 1):

1) terminal ITR repeats at the ends of the sequence that is encapsidated into the expression vector;

2) elements for expression of the target gene (promoter, transgene, polyadenylation site);

3) the origin of bacterial replication and the antibiotic resistance gene for the production of plasmid DNA in bacterial cells.

The wild-type nucleic acid was a nucleic acid that encodes the human coagulation factor VIII protein with the B domain deleted and includes the nucleotide sequence SEQ ID NO: 1. This nucleic acid is used as a control in the expression cassette.

Additionally, in order to increase the efficiency of expression, the natural nucleotide sequence of the blood coagulation factor VIII gene with the deleted B domain was modified using a codon optimization algorithm.

As a result of codon optimization of the nucleic acid corresponding to the sequence of SEQ ID NO: 1, a number of codon-optimized nucleic acids of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 were obtained that were further tested for the level of protein expression of blood coagulation factor VIII-BDD and its activity in comparison with the control (nucleic acid SEQ ID NO: 1) in the expression cassette (Figure 1).

Example 2. Testing the performance of codon-optimized variants of the FVIII gene sequence with the B domain deleted in vitro

The codon-optimized nucleic acids we obtained, selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, encoding the FVIII-BDD protein, were tested by transfecting SK-HEP1 cells in vitro as part of an expression cassette consisting of a left (first) ITR (inverted terminal repeats), a tissue-specific promoter, a transgene, a polyadenylation signal, a right (second) ITR, where the transgene is one of the sequences SEQ ID NO: 2-5. As a control, the transgene was an unoptimized nucleic acid that encodes a human FVIII protein with the B domain deleted, corresponding to the sequence SEQ ID NO:l.

Use of all nucleic acids selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 encoding the FVIII-BDD protein resulted in increased levels of production (Figure 2) and activity (Figure 3) FVIII-BDD protein compared to using nucleic acid SEQ ID NO: 1 without optimization.

In addition, our codon-optimized nucleic acids encoding the coagulation factor VIII protein with the B domain deleted (SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5) can lead to increasing the expression of FVIII-BDD protein in vitro in a host cell compared to using the nucleic acid SEQ ID NO:l without optimization. Thus, the codon-optimized nucleic acids of the invention have high potential for producing recombinant FVIII-BDD protein for the treatment of Hemophilia A.

Example 3. Creation and performance testing of expression vectors carrying codon-optimized variants of the FVIII-BDD gene sequence in vitro

In order to demonstrate the efficiency of delivery of the developed codon-optimized nucleic acids using an expression vector in vitro, plasmids encoding the target genetic constructs along with other necessary plasmids were used to develop expression vectors based on adeno-associated virus serotype 5 carrying the nucleotide sequence SEQ ID NO: 2 (hereinafter referred to as AAV5-FVIII-BDDco-vl), and based on an adeno-associated virus serotype 6 carrying the nucleotide sequence SEQ ID NO:2 (hereinafter referred to as AAV6-FVIII-BDDco-vl) or the nucleotide sequence SEQ ID NO:5 (hereinafter referred to as AAV6-FVIII-BDDco-v4). The purified preparations of AAV5-FVIII-BDDco-vl, AAV6-FVIII-BDDco-vl and AAV6-FVIII-BDDco-v4 used for in vitro and in vivo studies were prepared using standard buffers and excipients that are safe and not change the properties of AAV.

The formulations AAV5-FVIII-BDDco-vl, AAV6-FVIII-BDDco-vl and AAV6-FVIII-BDDco-v4 were tested in vitro using the adherent cell line HUH7 (Figures 4 and 5). Line cells with a density of 10 000 cells/cm2 were seeded into the wells of 12-well plates. 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 were determined by ELISA. All samples were supplied in triplicates. Intact cells were used for negative control.

It was shown that the expression vectors AAV5-FVIII-BDDco-v1, AAV6-FVIII-BDDco-v1 and AAV6-FVIII-BDDco-v4 developed by us, carrying codon-optimized versions of the blood coagulation factor VIII-BDD gene, allow efficient delivery of the factor transgene blood coagulation VIII-BDD into HUH7 cells and ensure the production of the target protein, which is confirmed by analyzing the culture liquid by ELISA (Figure 4) and analyzing the activity (Figure 5) of the blood coagulation factor VIII-BDD protein. Delivery of SEQ ID NO:2 using an AAV serotype 5 expression vector results in the production of FVIII-BDD protein in the culture fluid at a level of 129 ng/ml and an activity of 97%. Delivery of SEQ ID NO:2 using an AAV serotype 6 expression vector results in production of FVIII-BDD protein in the culture fluid at a level of 104 ng/ml and an activity of 91%. Delivery of SEQ ID NO:5 using an AAV serotype 6 expression vector results in production of FVIII-BDD protein in the culture fluid at a level of 61 ng/ml and an activity of 68%.

Example 4. Testing the performance of drugs AAV5-FVIII-BDDco-vl and AAV6-FVIII-BDDco-v1 in vivo

In order to demonstrate the delivery efficiency of the designed codon-optimized nucleic acids when delivered as part of an expression vector in vivo, the selected drugs AAV5-FVIII-BDDco-v1 and AAV6-FVIII-BDDco-v1 were administered to C57BL/6 laboratory mice. The study used a dose of AAV drugs equal to 6x1013 vg/mouse. 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 (day 0), then on days 35 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, it was shown that in the case of using both drugs AAV5-FVIII-BDDco-vl and AAV6-FVIII-BDDco-vl, containing a codon-optimized sequence of the blood clotting factor VIII-BDD gene SEQ ID NO: 2, a significant increase in the amount of factor VIII-BDD in the blood plasma of animals on days 35 and 56 (Figure 6). In the case of using the drug AAV5-FVIII-BDDco-vl, containing the codon-optimized sequence of the gene for blood coagulation factor VIII-BDD SEQ ID NO: 2, expression of FVIII-BDD at levels 304 and 279 was observed in the blood plasma of animals on days 35 and 56 after injection ng/ml, respectively. In the case of using the drug AAV6-FVIII-BDDco-v1 containing the codon-optimized sequence of the gene for blood coagulation factor VIII-BDD SEQ ID N0:2 in the blood plasma of animals on days 35 and 56 after injection, FVIII-BDD expression was observed at levels 415 and 459 ng/ml, respectively.

Thus, the developed expression vectors based on AAV5 and AAV6, carrying codon-optimized versions of the coagulation factor VIII gene with the B domain deleted (AAV5-FVIII-BDDco-vl and AAV6-FVIII-BDDco-vl) are capable of leading to the expression of the FVIII- BDD in vivo and have high potential for gene therapy for Hemophilia A.

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List of sequences

<110> JSC "BIOCAD"

<120> Codon-optimized nucleic acid that encodes

coagulation factor VIII protein with deleted B domain, and its

application

<160> 22

<170> BiSSAP 1.3.6

<210> 1

<211> 4314

<212> DNA

<213> Artificial sequence

<220>

<223> nucleic acid encoding the FVIII-BDD protein (factor

blood coagulation VIII with deleted B domain) (wild

type)

<400> 1

gccaccagaa gatactacct gggtgcagtg gaactgtcat gggactatat gcaaagtgat

60

ctcggtgagc tgcctgtgga cgcaagattt cctcctagag tgccaaaatc ttttccattc

120

aacacctcag tcgtgtacaa aaagactctg tttgtagaat tcacggatca ccttttcaac

180

atcgctaagc caaggccacc ctggatgggt ctgctaggtc ctaccatcca ggctgaggtt

240

tatgatacag tggtcattac acttaagaac atggcttccc atcctgtcag tcttcatgct

300

gttggtgtat cctactggaa agcttctgag ggagctgaat atgatgatca gaccagtcaa

360

agggagaaag aagatgataa agtcttccct ggtggaagcc atacatatgt ctggcaggtc

420

ctgaaagaga atggtccaat ggcctctgac ccactgtgcc ttacctactc atatctttct

480

catgtggacc tggtaaaaga cttgaattca ggcctcattg gagccctact agtatgtaga

540

gaagggagtc tggccaagga aaagacacag accttgcaca aatttatact actttttgct

600

gtatttgatg aagggaaaag ttggcactca gaaacaaaga actccttgat gcaggatagg

660

gatgctgcat ctgctcgggc ctggcctaaa atgcacacag tcaatggtta tgtaaacagg

720

tctctgccag gtctgattgg atgccacagg aaatcagtct attggcatgt gattggaatg

780

ggcaccactc ctgaagtgca ctcaatattc ctcgaaggtc acacatttct tgtgaggaac

840

catcgccagg cgtccttgga aatctcgcca ataactttcc ttactgctca aacactcttg

900

atggaccttg gacagtttct actgttttgt catatctctt cccaccaaca tgatggcatg

960

gaagcttatg tcaaagtaga cagctgtcca gaggaacccc aactacgaat gaaaaataat

1020

gaagaagcgg aagactatga tgatgatctt actgattctg aaatggatgt ggtcaggttt

1080

gatgatgaca actctccttc ctttatccaa attcgctcag ttgccaagaa gcatcctaaa

1140

acttgggtac attacattgc tgctgaagag gaggactggg actatgctcc cttagtcctc

1200

gcccccgatg acagaagtta taaaagtcaa tatttgaaca atggccctca gcggattggt

1260

aggaagtaca aaaaagtccg atttatggca tacacagatg aaacctttaa gactcgtgaa

1320

gctattcagc atgaatcagg aatcttggga cctttacttt atggggaagt tggagacaca

1380

ctgttgatta tatttaagaa tcaagcaagc agaccatata acatctaccc tcacggaatc

1440

actgatgtcc gtcctttgta ttcaaggaga ttaccaaaag gtgtaaaaca tttgaaggat

1500

tttccaattc tgccaggaga aatattcaaa tataaatgga cagtgactgt agaagatggg

1560

ccaactaaat cagatcctcg gtgcctgacc cgctattact ctagtttcgt taatatggag

1620

agagatctag cttcaggact cattggccct ctcctcatct gctacaaaga atctgtagat

1680

caaagaggaa accagataat gtcagacaag aggaatgtca tcctgttttc tgtatttgat

1740

gagaaccgaa gctggtacct cacagagaat atacaacgct ttctccccaa tccagctgga

1800

gtgcagcttg aggatccaga gttccaagcc tccaacatca tgcacagcat caatggctat

1860

gtttttgata gtttgcagtt gtcagtttgt ttgcatgagg tggcatactg gtacattcta

1920

agcattggag cacagactga cttcctttct gtcttcttct ctggatatac cttcaaacac

1980

aaaatggtct atgaagacac actcacccta ttcccattct caggagaaac tgtcttcatg

2040

tcgatggaaa acccaggtct atggattctg gggtgccaca actcagactt tcggaacaga

2100

ggcatgaccg ccttactgaa ggtttctagt tgtgacaaga acactggtga ttattacgag

2160

gacagttatg aagatatttc agcatacttg ctgagtaaaa acaatgccat tgaaccaaga

2220

agcttctctc aaaacccacc agtcttgaaa cgccatcaac gggaaataac tcgtactact

2280

cttcagtcag atcaagagga aattgactat gatgatacca tatcagttga aatgaagaag

2340

gaagattttg acatttatga tgaggatgaa aatcagagcc cccgcagctt tcaaaagaaa

2400

acacgacact attttattgc tgcagtggag aggctctggg attatgggat gagtagctcc

2460

ccacatgttc taagaaacag ggctcagagt ggcagtgtcc ctcagttcaa gaaagttgtt

2520

ttccaggaat ttactgatgg ctcctttact cagcccttat accgtggaga actaaatgaa

2580

catttgggac tcctggggcc atatataaga gcagaagttg aagataatat catggtaact

2640

ttcagaaatc aggcctctcg tccctattcc ttctattcta gccttatttc ttatgaggaa

2700

gatcagaggc aaggagcaga acctagaaaa aactttgtca agcctaatga aaccaaaact

2760

tacttttgga aagtgcaaca tcatatggca cccactaaag atgagtttga ctgcaaagcc

2820

tgggcttatt tctctgatgt tgacctggaa aaagatgtgc actcaggcct gattggaccc

2880

cttctggtct gccacactaa cacactgaac cctgctcatg ggagacaagt gacagtacag

2940

gaatttgctc tgtttttcac catctttgat gagaccaaaa gctggtactt cactgaaaat

3000

atggaaagaa actgcagggc tccctgcaat atccagatgg aagatcccac ttttaaagag

3060

aattatcgct tccatgcaat caatggctac ataatggata cactacctgg cttagtaatg

3120

gctcaggatc aaaggattcg atggtatctg ctcagcatgg gcagcaatga aaacatccat

3180

tctattcatt tcagtggaca tgtgttcact gtacgaaaaa aagaggagta taaaatggca

3240

ctgtacaatc tctatccagg tgtttttgag acagtggaaa tgttaccatc caaagctgga

3300

atttggcggg tggaatgcct tattggcgag catctacatg ctgggatgag cacacttttt

3360

ctggtgtaca gcaataagtg tcagactccc ctgggaatgg cttctggaca cattagagat

3420

tttcagatta cagcttcagg acaatatgga cagtgggccc caaagctggc cagacttcat

3480

tattccggat caatcaatgc ctggagcacc aaggagccct tttcttggat caaggtggat

3540

ctgttggcac caatgattat tcacggcatc aagacccagg gtgcccgtca gaagttctcc

3600

agcctctaca tctctcagtt tatcatcatg tatagtcttg atgggaagaa gtggcagact

3660

tatcgaggaa attccactgg aaccttaatg gtcttctttg gcaatgtgga ttcatctggg

3720

ataaaacaca atatttttaa ccctccaatt attgctcgat acatccgttt gcacccaact

3780

cattatagca ttcgcagcac tcttcgcatg gagttgatgg gctgtgattt aaatagttgc

3840

agcatgccat tgggaatgga gagtaaagca atatcagatg cacagattac tgcttcatcc

3900

tactttacca atatgtttgc cacctggtct ccttcaaaag ctcgacttca cctccaaggg

3960

aggagtaatg cctggagacc tcaggtgaat aatccaaaag agtggctgca agtggacttc

4020

cagaagacaa tgaaagtcac aggagtaact actcagggag taaaatctct gcttaccagc

4080

atgtatgtga aggagttcct catctccagc agtcaagatg gccatcagtg gactctcttt

4140

tttcagaatg gcaaagtaaa ggtttttcag ggaaatcaag actccttcac acctgtggtg

4200

aactctctag acccaccgtt actgactcgc taccttcgaa ttcaccccca gagttgggtg

4260

caccagattg ccctgaggat ggaggttctg ggctgcgagg cacaggacct ctac

4314

<210> 2

<211> 4314

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleotide sequence,

encoding the FVIII-BDD protein (variant FVIII-BDDco-v1)

<400> 2

gccaccagaa ggtactacct gggcgccgtg gagctgagct gggactacat gcagagcgac

60

ctgggcgagc tgcctgtgga cgccagattc ccccccagag tgcctaagag cttccccttc

120

aacaccagcg tggtgtacaa gaagaccctg ttcgtggagt tcaccgacca cctgttcaac

180

atcgccaagc ccagaccccc ctggatgggc ctgctgggcc ctaccatcca ggccgaggtg

240

tacgacaccg tggtgatcac cctgaagaac atggccagcc accccgtgag cctgcacgcc

300

gtgggcgtga gctactggaa ggccagcgag ggcgccgagt acgacgacca gaccagccag

360

agagagaagg aggacgacaa ggtgttcccc ggcggcagcc acacctacgt gtggcaggtg

420

ctgaaggaga acggccccat ggccagcgac cccctgtgcc tgacctacag ctacctgagc

480

cacgtggacc tggtgaagga cctgaacagc ggcctgatcg gcgccctgct ggtgtgcaga

540

gagggcagcc tggccaagga gaagaccag accctgcaca agttcatcct gctgttcgcc

600

gtgttcgacg agggcaagag ctggcacagc gagaccaaga acagcctgat gcaggacaga

660

gacgccgcca gcgccagagc ctggcccaag atgcacaccg tgaacggcta cgtgaacagg

720

agcctgcccg gcctgatcgg ctgccacaga aagagcgtgt actggcacgt gatcggcatg

780

ggcaccaccc ccgaggtgca cagcatcttc ctggagggcc acaccttcct ggtgaggaac

840

cacagacagg ccagcctgga gatcagcccc atcaccttcc tgaccgccca gaccctgctg

900

atggacctgg gccagttcct gctgttctgc cacatcagca gccaccagca cgacggcatg

960

gaggcctacg tgaaggtgga cagctgcccc gaggagcccc agctgagaat gaagaacaac

1020

gaggaggccg aggactacga cgacgacctg accgacagcg agatggacgt ggtgagattc

1080

gacgacgaca acagccccag cttcatccag atcaggagcg tggccaagaa gcaccccaag

1140

acctgggtgc actacatcgc cgccgaggag gaggactggg actacgcccc cctggtgctg

1200

gcccccgacg acagaagcta caagagccag tacctgaaca acggccccca gaggatcggc

1260

agaaagtaca agaaggtgcg gttcatggcc tacaccgacg agaccttcaa gaccagggag

1320

gccatccagc acgagagcgg catcctgggc cctctgctgt acggcgaggt gggcgacacc

1380

ctgctgatca tcttcaagaa ccaggccagc agaccctaca acatctaccc ccacggcatc

1440

accgacgtga gacccctgta cagcagaaga ctgcccaagg gcgtgaagca cctgaaggac

1500

ttccccatcc tgcccggcga gatcttcaag tacaagtgga ccgtgaccgt ggaggacggc

1560

cccaccaaga gcgaccccag atgcctgacc agatactaca gcagcttcgt gaacatggag

1620

cgggacctgg ccagcggcct gatcggcccc ctgctgatct gctacaagga gagcgtggac

1680

cagagaggca accagatcat gagcgacaag cggaacgtga tcctgttcag cgtgttcgac

1740

gagaaccgga gctggtacct gaccgagaac atccagaggt tcctgcccaa ccccgccggc

1800

gtgcagctgg aggaccccga gttccaggcc agcaacatca tgcacagcat caacggctac

1860

gtgttcgaca gcctgcagct gagcgtgtgc ctgcacgagg tggcctactg gtacatcctg

1920

agcatcggcg cccagaccga cttcctgagc gtgttcttca gcggctacac cttcaagcac

1980

aagatggtgt acgaggacac cctgaccctg ttccccttca gcggcgagac cgtgttcatg

2040

agcatggaga accccggcct gtggatcctg ggctgccaca acagcgactt cagaaacaga

2100

ggcatgaccg ccctgctgaa ggtgagcagc tgcgacaaga acaccggcga ctactacgag

2160

gacagctacg aggacatcag cgcctacctg ctgagcaaga acaacgccat cgagcccagg

2220

agcttcagcc agaacccccc cgtgctgaag agacaccaga gagagatcac caggaccacc

2280

ctgcagagcg accaggagga gatcgactac gacgacacca tcagcgtgga gatgaagaag

2340

gaggacttcg acatctacga cgaggacgag aaccagagcc ccagaagctt ccagaagaag

2400

accaggcact acttcatcgc cgccgtggag agactgtggg actacggcat gagcagcagc

2460

ccccacgtgc tgagaaacag agcccagagc ggcagcgtgc cccagttcaa gaaggtggtg

2520

ttccaggagt tcaccgacgg cagcttcacc cagcccctgt acagaggcga gctgaacgag

2580

cacctgggcc tgctgggccc ctacatcaga gccgaggtgg aggacaacat catggtgacc

2640

ttccggaacc aggccagcag accctacagc ttctacagca gcctgatcag ctacgaggag

2700

gaccagaggc agggcgccga gcccagaaag aacttcgtga agcccaacga gaccaagacc

2760

tacttctgga aggtgcagca ccacatggcc cccaccaagg acgagttcga ctgcaaggcc

2820

tgggcctact tcagcgacgt ggacctggag aaggacgtgc acagcggcct gatcggcccc

2880

ctgctggtgt gccacaccaa caccctgaac cccgcccacg gcagacaggt gaccgtgcag

2940

gagttcgccc tgttcttcac catcttcgac gagaccaaga gctggtactt caccgagaac

3000

atggagcgga actgcagagc cccctgcaac atccagatgg aggaccccac cttcaaggag

3060

aactacaggt tccacgccat caacggctac atcatggaca ccctgcccgg cctggtgatg

3120

gcccaggacc agagaatcag atggtacctg ctgagcatgg gcagcaacga gaacatccac

3180

agcatccact tcagcggcca cgtgttcacc gtgaggaaga aggaggagta caagatggcc

3240

ctgtacaacc tgtaccccgg cgtgttcgag accgtggaga tgctgcccag caaggccggc

3300

atctggagag tggagtgcct gatcggcgag cacctgcacg ccggcatgag caccctgttc

3360

ctggtgtaca gcaacaagtg ccagaccccc ctgggcatgg ccagcggcca catcagagac

3420

ttccagatca ccgccagcgg ccagtacggc cagtgggccc ccaagctggc cagactgcac

3480

tacagcggca gcatcaacgc ctggagcacc aaggagccct tcagctggat caaggtggac

3540

ctgctggccc ccatgatcat ccacggcatc aagacccagg gcgccagaca gaagttcagc

3600

agcctgtaca tcagccagtt catcatcatg tacagcctgg acggcaagaa gtggcagacc

3660

tacagaggca acagcaccgg caccctgatg gtgttcttcg gcaacgtgga cagcagcggc

3720

atcaagcaca acatcttcaa cccccccatc atcgcccggt acatcaggct gcaccccacc

3780

cactacagca tcaggagcac cctgagaatg gagctgatgg gctgcgacct gaacagctgc

3840

agcatgcccc tgggcatgga gagcaaggcc atcagcgacg cccagatcac cgccagcagc

3900

tacttcacca acatgttcgc cacctggagc cccagcaagg ccagactgca cctgcagggc

3960

agaagcaacg cctggagacc ccaggtgaac aaccccaagg agtggctgca ggtggacttc

4020

cagaagacca tgaaggtgac cggcgtgacc acccagggcg tgaagagcct gctgaccagc

4080

atgtacgtga aggagttcct gatcagcagc agccaggacg gccaccagtg gaccctgttc

4140

ttccagaacg gcaaggtgaa ggtgttccag ggcaaccagg acagcttcac ccccgtggtg

4200

aacagcctgg acccccccct gctgaccaga tacctgagaa tccaccccca gagctgggtg

4260

caccagatcg ccctgagaat ggaggtgctg ggctgcgagg cccaggacct gtac

4314

<210> 3

<211> 4314

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleotide sequence,

encoding the FVIII-BDD protein (variant FVIII-BDDco-v2)

<400> 3

gccacaagaa gatactatct gggcgccgtg gagctgagct gggattatat gcagtccgac

60

ctgggcgagc tgccagtgga tgctagattc cctcctagag tgcctaagag ctttccattc

120

aatacctccg tggtgtacaa gaagaccctg ttcgtggagt ttaccgacca cctgttcaac

180

atcgccaaac ctagaccacc ttggatggga ctgctgggcc caactattca ggccgaagtg

240

tacgataccg tggtgatcac tctgaagaat atggcctccc accctgtgag cctgcatgca

300

gttggcgtga gctattggaa ggccagcgag ggagccgaat acgatgacca gaccagccag

360

agggaagg aagacgacaa ggtgtttccc ggaggaagcc acacctatgt gtggcaggtg

420

ctgaaggaga atggacctat ggccagcgat ccactgtgcc tgacctactc ttatctgagc

480

cacgtggacc tggtgaagga cctgaatagc ggcctgattg gcgcactgct ggtgtgtaga

540

gagggatctc tggccaaaga gaagaccag accctgcaca agtttatcct gctgtttgcc

600

gtgtttgacg agggcaaatc ttggcacagc gagaccaaaa acagcctgat gcaggataga

660

gacgcagcct ctgctagagc ttggcctaag atgcacaccg tgaatggcta cgtgaacaga

720

tccctgcccg gcctgattgg atgtcacaga aagagcgtgt actggcacgt gatcggaatg

780

ggaactaccc cagaagtgca cagcatcttt ctggagggac acacctttct ggtgcggaat

840

catagacagg ccagcctgga aatcagccct attacctttc tgaccgccca gacactgctg

900

atggatctgg gacagtttct gctgttctgc cacatcagct ctcaccagca tgacggaatg

960

gaagcctacg tgaaggtgga cagctgccct gaggaaccac agctgaggat gaagaataac

1020

gaggaggccg aggattacga cgatgacctg accgattctg agatggacgt ggtgagattc

1080

gacgacgata actctccctc cttcatccag atcagatccg tggccaaaaa gcaccctaaa

1140

acctgggtgc actatatcgc cgcagaggaa gaagactggg attacgcacc tctggtgctg

1200

gcccctgatg acagatctta taagtctcag tacctgaaca acggccctca gagaatcggc

1260

aggaagtaca agaaggtgag attcatggcc tacacagacg agacattcaa gaccagagag

1320

gccattcagc acgagtccgg cattctggga ccactgctgt acggagaggt gggcgataca

1380

ctgctgatca tcttcaaaaa tcaggccagc aggccctaca acatctaccc acatggcatc

1440

agacgtga ggcctctgta cagcagaaga ctgcctaaag gcgtgaagca cctgaaggac

1500

ttccccattc tgccaggcga gattttcaag tacaagtgga cagtgaccgt ggaggatgga

1560

cccacaaagt ctgaccctag atgcctgacc aggtactata gctccttcgt gaacatggaa

1620

agggatctgg cctctggact gattggccca ctgctgatct gctacaagga atccgtggac

1680

cagagaggaa accagatcat gagcgataag aggaatgtga tcctgttcag cgtgttcgat

1740

gagaacagga gctggtatct gaccgagaac atccagagat tcctgccaaa tccagccggc

1800

gtgcagctgg aagatcctga atttcaggcc tccaatatca tgcactccat caatggctac

1860

gtgtttgaca gcctgcagct gtctgtgtgt ctgcacgaag tggcctactg gtacatcctg

1920

agcattggag cccagaccga ctttctgagc gtgtttttct ctggctacac cttcaagcac

1980

aagatggtgt acgaggacac cctgacactg ttcccattct caggcgaaac cgtgtttatg

2040

agcatggaga acccaggact gtggattctg ggctgccaca actccgactt caggaataga

2100

ggaatgaccg ccctgctgaa agtgagcagc tgcgataaga acaccggcga ttattacgag

2160

gactcctacg aggatatcag cgcctacctg ctgtccaaaa acaatgccat cgagcccagg

2220

agcttctccc agaatccacc tgtgctgaaa agacaccaga ggggagatcac cagaaccaca

2280

ctgcagtctg atcaggagga gatcgactat gacgacacca tcagcgtgga gatgaagaag

2340

gaggactttg atatctacga cgaggatgag aaccagagcc ccaggagctt tcagaagaag

2400

accaggcact acttcatcgc cgcagtggag agactgtggg actacggaat gtcttcctcc

2460

cctcacgtgc tgagaaatag agcccagtct ggaagcgtgc cacagtttaa gaaggtggtg

2520

ttccaggagt ttaccgacgg ctctttcacc cagccactgt atagaggcga actgaacgaa

2580

cacctgggcc tgctgggacc ttatatcaga gccgaagtgg aggacaacat catggtgacc

2640

ttcagaaacc aggccagcag accttacagc ttctactcca gcctgatcag ctacgaagag

2700

gatcagagac agggcgccga acctaggaaa aattttgtga agccaaacga gaccaagacc

2760

tacttttgga aggtgcagca tcacatggcc cccaccaagg atgagttcga ttgtaaagcc

2820

tgggcctact tctccgatgt ggacctggaa aaggatgtgc acagcggact gatcggacca

2880

ctgctggtgt gccacacaaa taccctgaac cctgcccacg gaagacaggt gacagtgcag

2940

gaatttgccc tgttcttcac catctttgac gagaccaagt cctggtactt caccgagaac

3000

atggagagaa actgcagagc cccttgcaac attcagatgg aggacccaac attcaaggag

3060

aactacaggt tccacgccat caatggctac atcatggata cactgcctgg cctggtgatg

3120

gcccaggatc agagaattag gtggtacctg ctgtctatgg gctccaatga gaatatccac

3180

tccatccact tcagcggcca cgtgttcacc gtgagaaaga aagaggagta caagatggcc

3240

ctgtacaacc tgtacccagg cgtgtttgag accgtggaga tgctgccttc taaagctggc

3300

atctggagag tggagtgcct gatcggcgag catctgcatg ctggaatgtc taccctgttt

3360

ctggtgtact ccaacaagtg cgacaacct ctgggcatgg cttctggaca tatcagagac

3420

tttcagatca ccgcctccgg ccagtacgga caatgggctc caaagctggc cagactgcat

3480

tactctggct ctattaatgc ctggagcacc aaggagccct tttcctggat caaggtggat

3540

ctgctggccc ccatgattat ccacggcatc aaaacccagg gcgccaggca gaagttttct

3600

tccctgtaca tcagccagtt catcatcatg tacagcctgg atggcaagaa gtggcagacc

3660

tacagaggca atagcaccgg aaccctgatg gtgtttttcg gcaacgtgga ctcctccggc

3720

attaagcaca atatcttcaa cccacctatc atcgccaggt acatcagact gcaccccacc

3780

cactactcta tcagaagcac cctgaggatg gaactgatgg gctgtgacct gaattcctgt

3840

agcatgcctc tgggaatgga gtctaaagcc atcagtgacg cccagattac cgccagcagc

3900

tactttacca acatgtttgc tacatggtcc ccctctaagg ccagactgca cctgcaggga

3960

agatctaacg cctggagacc acaggtgaac aaccccaaag aatggctgca ggtggatttt

4020

cagaagacca tgaaagtgac cggcgtgaca acacagggcg tgaaatctct gctgacctct

4080

atgtacgtga aggagtttct gatcagcagc agccaggacg gccatcagtg gaccctgttt

4140

tttcagaatg gcaaggtgaa ggtgttccag ggcaatcagg actcattcac tccagtggtg

4200

aacagcctgg acccacctct gctgaccaga tacctgagaa ttcacccaca gtcctgggtg

4260

catcagatcg ccctgagaat ggaagtgctg ggatgtgagg cacaggacct gtac

4314

<210> 4

<211> 4314

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleotide sequence,

encoding the FVIII-BDD protein (variant FVIII-BDDco-v3)

<400> 4

gccacaagga ggtactatct gggcgccgtg gaactgagct gggactatat gcagagcgac

60

ctggggagagc tgcctgtgga tgccagattt ccacctaggg tgcccaagag cttccccttc

120

aatacaagcg tggtgtacaa gaagaccctg ttcgtggagt ttaccgacca cctgttcaac

180

atcgccaagc ctagacctcc ttggatggga ctgctgggcc ctaccattca ggccgaagtg

240

tacgacacag tggtgatcac cctgaagaat atggccagcc atccagtgtc cctgcacgca

300

gtgggagtgt cttattggaa ggcctctgag ggcgccgagt acgatgatca gaccagccag

360

agagagaagg aggacgacaa ggtgttcccc ggaggaagcc acacctacgt gtggcaggtg

420

ctgaaagaga atggcccaat ggcctccgac cccctgtgtc tgacatacag ctacctgagc

480

cacgtggacc tggtgaagga cctgaactct ggcctgatcg gagctctgct ggtgtgtaga

540

gaaggcagcc tggccaagga gaagaccag accctgcaca agttcatcct gctgtttgcc

600

gtgttcgatg agggcaaaag ctggcattcc gagaccaaga attccctgat gcaggatagg

660

gacgctgcca gcgctagagc ctggcctaag atgcacactg tgaatggcta cgtgaaccgg

720

agcctgccag gcctgatcgg ctgtcacaga aagagcgtgt actggcacgt gatcggcatg

780

ggcaccactc ctgaggtgca ctctatcttt ctggaaggcc acacctttct ggtgcggaac

840

catagacagg cctccctgga aatcagcccc atcacctttc tgaccgccca gaccctgctg

900

atggatctgg gacagttcct gctgttttgc cacatctcct ctcaccagca cgacggaatg

960

gaggcctacg tgaaagtgga cagctgtcct gaagaacctc agctgcggat gaagaataac

1020

gaggaggccg aggactacga cgatgacctg accgatagcg agatggacgt ggtgagattc

1080

gacgacgata acagccccag cttcatccag atcagatccg tggccaagaa gcaccccaag

1140

acatgggtgc actatatcgc cgcagaggag gaggattggg actacgcacc tctggtgctg

1200

gctcctgacg atagatccta caagtcccag tacctgaaca acggccccca gagaatcggc

1260

agaaagtaca agaaggtgag attcatggcc tacaccgacg agacctttaa gaccagagag

1320

gccatccagc acgagagcgg cattctggga ccactgctgt atggagaggt gggcgataca

1380

ctgctgatca tcttcaagaa tcaggcctcc cggccataca acatctatcc acacggcatc

1440

accgacgtga gaccactgta ctccaggaga ctgcccaagg gcgtgaagca cctgaaggac

1500

ttccctattc tgccaggcga gatcttcaag tacaagtgga cagtgaccgt ggaagacgga

1560

cctaccaaaa gcgaccccag atgcctgacc cggtactact ccagcttcgt gaacatggag

1620

agagatctgg ccagcggact gattggacct ctgctgattt gttacaagga gagcgtggac

1680

cagaggggca accagattat gagcgacaag cggaatgtga tcctgttctc cgtgttcgac

1740

gagaaccgca gctggtatct gaccgagaat atccagaggt tcctgccaaa ccccgccggc

1800

gttcagctgg aagatccaga gtttcaggcc tccaacatca tgcacagcat caatggctac

1860

gtgttcgaca gcctgcagct gtccgtgtgt ctgcatgaag tggcctactg gtatatcctg

1920

agcatcggcg cccagaccga ttttctgtcc gtgtttttct ccggctacac cttcaagcac

1980

aagatggtgt acgaggacac cctgaccctg tttcctttta gcggcgaaac cgtgttcatg

2040

agcatggaga acccaggact gtggattctg ggctgtcaca acagcgactt cagaaatcgg

2100

ggcatgaccg ctctgctgaa ggtgagctct tgtgacaaaa ataccggcga ctactacgag

2160

gacagctacg aggatatcag cgcctacctg ctgagcaaga acaacgccat cgagccaaga

2220

agcttcagcc agaatccccc cgtgctgaag agacatcaga gggagatcac cagaaccacc

2280

ctgcagagcg accaggagga gatcgattac gacgacacca tcagcgtgga gatgaagaag

2340

gaggatttcg acatctacga cgaggacgag aatcagagcc caagggagctt tcagaagaag

2400

acccggcact acttcatcgc tgccgtggag agactgtggg attatggcat gagctccagc

2460

cctcacgtgc tgagaaatag agcccagtct ggctccgtgc cccagttcaa gaaagtggtg

2520

ttccaggaat tcaccgacgg cagctttacc cagcccctgt atagaggcga actgaatgaa

2580

cacctgggcc tgctgggacc ttacatcaga gccgaagtgg aggacaacat catggtgacc

2640

tttaggaatc aggccagcag accctacagc ttctatagct ccctgatctc ctacgaggag

2700

gaccagagac agggcgctga gccaagaaag aatttcgtga agcccaacga gaccaagacc

2760

tacttctgga aagtgcagca ccacatggcc cctaccaagg atgagttcga ctgtaaagcc

2820

tgggcctact tcagcgacgt ggacctggag aaggacgtgc actccggact gatcggacca

2880

ctgctggtgt gccacacaaa caccctgaac cctgcccatg gaagacaggt gactgtgcag

2940

gaattcgccc tgttctttac catcttcgac gagaccaagt cctggtactt caccgagaat

3000

atggagagga attgcagagc cccctgtaac atccagatgg aggaccccac ctttaaggag

3060

aattacaggt ttcacgccat caacggctac atcatggaca cactgccagg cctggtgatg

3120

gcacaggatc agaggatcag atggtatctg ctgagcatgg gctccaacga gaacatccac

3180

tccatccact tcagcggcca cgtgtttacc gtgagaaaga aggagggata caagatggcc

3240

ctgtacaacc tgtatcccgg cgtgttcgaa accgtggaga tgctgccttc caaagccgga

3300

atctggagag tggagtgcct gattggcgag cacctgcacg ctggaatgtc caccctgttc

3360

ctggtgtata gcaacaagtg ccagaccccc ctgggcatgg ctagcggaca cattagagac

3420

tttcagatca ccgcctccgg ccagtacgga cagtgggccc caaaactggc taggctgcat

3480

tacagcggct ccatcaatgc ctggtccacc aaggagccat tcagctggat caaagtggat

3540

ctgctggccc ccatgatcat ccacggcatt aagacacagg gcgccaggca gaagttcagc

3600

tctctgtaca tctcccagtt catcatcatg tactccctgg acggcaagaa gtggcagacc

3660

tacaggggca atagcaccgg cacactgatg gtgtttttcg gcaacgtgga ctcttccggc

3720

atcaagcaca acatctttaa cccccctatc atcgccaggt acatcaggct gcaccccact

3780

cactacagca tcagatccac cctgagaatg gagctgatgg gatgcgacct gaacagctgc

3840

agcatgcccc tgggcatgga gagcaaggcc atttctgacg cccagatcac cgccagcagc

3900

tatttcacca atatgttcgc tacctggagc cccagcaagg ccagactgca tctgcagggc

3960

agaagcaacg cctggagacc acaggtgaac aatcctaaag agtggctgca ggtggacttc

4020

cagaagacca tgaaggtgac cggcgtgaca acccagggcg tgaaatctct gctgacctcc

4080

atgtacgtga aggagttcct gatctcctcc agccaggatg gccatcagtg gaccctgttc

4140

ttccagaacg gcaaggtgaa agtgttccag ggcaaccagg acagctttac ccctgtggtg

4200

aactctctgg atccccccct gctgaccagg tacctgagaa ttcaccccca gagctgggtg

4260

caccagatcg ccctgagaat ggaagtgctg ggatgcgagg cacaggacct gtac

4314

<210> 5

<211> 4314

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleotide sequence,

encoding the FVIII-BDD protein (variant FVIII-BDDco-v4)

<400> 5

gctacccgga ggtactacct gggcgctgtg gagctgagct gggactacat gcagagcgat

60

ctgggcgagc tgcccgtgga tgccagattt cccccaagag tgcctaagtc cttccccttc

120

aacaccagcg tggtgtacaa gaagaccctg ttcgtggagt tcaccgacca cctgttcaac

180

atcgccaagc caaggccccc ctggatggga ctgctgggac ctacaattca ggccgaggtg

240

tacgataccg tggtgatcac cctgaagaac atggcctccc acccagtgtc cctgcatgcc

300

gtgggcgtga gctactggaa ggcttctgaa ggcgccgagt acgacgacca gaccagccag

360

agggaagg aggacgacaa ggtgttccca ggcggatctc acacctacgt gtggcaggtg

420

ctgaaggaga atggcccaat ggccagcgac ccactgtgtc tgacctacag ctacctgtcc

480

cacgtggacc tggtgaagga cctgaactcc ggcctgatcg gcgccctgct ggtgtgtaga

540

gaaggatctc tggccaagga gaagaccag accctgcaca agttcatcct gctgttcgcc

600

gtgtttgacg agggcaagag ctggcattcc gagaccaaga acagcctgat gcaggacaga

660

gacgcagcca gcgctagagc ctggcccaag atgcacaccg tgaatggcta cgtgaaccgg

720

tccctgccag gcctgatcgg ctgtcacaga aaaagcgtgt actggcacgt gatcggcatg

780

ggcaccacac ccgaggtgca cagcatcttt ctggagggcc acaccttcct ggtgagaaac

840

cacaggcagg cctccctgga gatctccccc atcacctttc tgaccgccca gaccctgctg

900

atggatctgg gccagttcct gctgttctgc cacatcagct cccaccagca cgacggaatg

960

gaggcctacg tgaaggtgga tagctgcccc gaggagcccc agctgagaat gaagaacaac

1020

gaggaggccg aggactacga cgatgacctg accgattccg agatggacgt ggtgcgcttc

1080

gacgacgata atagccccag cttcatccag atcagatccg tggccaagaa gcacccaaag

1140

acctgggtgc actacatcgc tgccgaggag gaggactggg attacgcccc actggtgctg

1200

gcccctgatg acagatccta caagagccag tacctgaaca acggccccca gagaatcggc

1260

cggaagtaca agaaggtgag attcatggcc tacaccgacg agaccttcaa gaccagggaa

1320

gccatccagc acgagagcgg catcctggga ccactgctgt acggagaagt gggcgatacc

1380

ctgctgatca tcttcaagaa ccaggcctcc cggccctaca acatctaccc ccacggcatc

1440

accgacgtga ggccactgta cagcaggagg ctgcccaagg gcgtgaagca cctgaaggac

1500

ttccccatcc tgcccggcga gatctttaag tacaagtgga ccgtgaccgt ggaggacggc

1560

ccaaccaaga gcgacccaag atgcctgacc aggtactact cctccttcgt gaacatggag

1620

cgggatctgg ccagcggact gattggccct ctgctgatct gctacaagga gagcgtggac

1680

cagcgcggca atcagatcat gagcgacaag agaaacgtga tcctgttcag cgtgttcgac

1740

gagaatcgca gctggtacct gaccgagaac atccagaggt tcctgcccaa cccagccggc

1800

gtgcagctgg aagatcctga atttcaggcc agcaacatca tgcactccat caacggctac

1860

gtgttcgact ccctgcagct gagcgtgtgt ctgcacgaag tggcctactg gtacatcctg

1920

agcatcggcg cccagaccga cttcctgtcc gtgttctttt ccggctacac cttcaagcac

1980

aagatggtgt acgaggacac cctgaccctg ttccccttta gcggcgagac cgtgttcatg

2040

agcatggaga acccaggcct gtggattctg ggctgccaca acagcgactt ccgcaatagg

2100

ggcatgaccg ccctgctgaa agtgtccagc tgcgataaga acaccggcga ctactacgag

2160

gacagctacg aggacatctc cgcctacctg ctgagcaaga acaacgccat cgagcccaga

2220

tccttcagcc agaaccctcc agtgctgaag aggcaccagc gcgagatcac cagaaccacc

2280

ctgcagtctg atcaggagga gatcgactac gacgacacca tcagcgtgga gatgaagaag

2340

gaggacttcg acatctacga cgaggacgag aaccagagcc ctaggtcctt ccagaagaag

2400

acccggcact acttcatcgc cgcagtggag aggctgtggg attacggcat gagcagctcc

2460

ccacacgtgc tgagaaatag ggcccagagc ggcagcgtgc cccagttcaa aaaggtggtg

2520

ttccaggagt tcaccgacgg ctccttcacc cagcccctgt acagaggcga gctgaatgag

2580

cacctgggcc tgctgggacc ctacatcaga gcagaggtgg aggacaacat catggtgacc

2640

ttcaggaacc aggccagcag accctacagc ttctacagca gcctgatcag ctacgaggag

2700

gaccagagac agggagccga gccacgcaag aacttcgtga agcccaacga gaccaagacc

2760

tacttctgga aggtgcagca ccacatggcc cccaccaagg acgagttcga ttgtaaagcc

2820

tgggcctact tcagcgacgt ggacctggag aaggacgtgc acagcggcct gatcggacct

2880

ctgctggtgt gtcataccaa caccctgaac cccgcccacg gcagacaggt gactgtgcag

2940

gagtttgccc tgttcttcac catcttcgac gagaccaaga gctggtactt caccgagaac

3000

atggagagaa actgcagggc cccatgcaac atccagatgg aagaccccac cttcaaggag

3060

aactaccggt tccacgccat caacggctac atcatggata ccctgcctgg cctggtgatg

3120

gcccaggatc agagaatcag gtggtacctg ctgagcatgg gctccaacga gaacatccac

3180

agcatccact tctccggcca cgtgttcacc gtgaggaaaa aggaggagta caagatggcc

3240

ctgtacaacc tgtaccccgg cgtgtttgag accgtggaga tgctgccatc taaggccggc

3300

atttggagag tggagtgcct gatcggcgag cacctgcacg ccggaatgtc taccctgttc

3360

ctggtgtaca gcaacaagtg ccagaccccc ctgggcatgg ccagcggaca catcagagac

3420

ttccagatca ccgccagcgg ccagtacgga cagtgggctc ctaaactggc ccgcctgcac

3480

tatagcggca gcattaacgc ctggtccacc aaggagccat tcagctggat caaggtggat

3540

ctgctggccc ccatgatcat ccacggcatc aaaacacagg gcgccagaca gaagttctcc

3600

agcctgtaca tctcccagtt catcatcatg tactccctgg acggcaagaa gtggcagacc

3660

tacagaggca actccaccgg cacactgatg gtgttcttcg gcaacgtgga cagcagcggc

3720

atcaagcaca acatcttcaa ccccccatc atcgcccggt acatcagact gcacccaacc

3780

cactactcca tcaggtccac cctgaggatg gagctgatgg gctgcgatct gaattcctgc

3840

agcatgcccc tgggcatgga gtccaaagcc atctctgacg cccagatcac cgccagctcc

3900

tacttcacca acatgttcgc cacttggagc cccagcaagg ccagactgca cctgcagggc

3960

agaagcaatg cctggagacc ccaggtgaac aacccaaagg agtggctgca ggtggacttc

4020

cagaagacca tgaaggtgac cggcgtgacc acacagggcg tgaaaagcct gctgaccagc

4080

atgtacgtga aggagttcct gatcagctcc agccaggatg ggcaccagtg gaccctgttc

4140

tttcagaacg gcaaggtgaa ggtgttccag ggcaatcagg actcattcac ccctgtggtg

4200

aactctctgg acccccccct gctgacccgg tacctgagaa tccacccaca gtcttgggtg

4260

caccagatcg ccctgaggat ggaagtgctg ggctgcgagg ctcaggatct gtac

4314

<210> 6

<211> 4371

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding the FVIII-BDD protein with a signal

peptide (wild type)

<400> 6

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 c

4371

<210> 7

<211> 4371

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleotide sequence,

encoding the FVIII-BDD protein with a signal peptide (variant

FVIII-BDDco-v1)

<400> 7

atgcagatcg agctgagcac ctgcttcttc ctgtgcctgc tgaggttctg cttcagcgcc

60

accagaaggt actacctggg cgccgtggag ctgagctggg actacatgca gagcgacctg

120

ggcgagctgc ctgtggacgc cagattcccc cccagagtgc ctaagagctt ccccttcaac

180

accagcgtgg tgtacaagaa gaccctgttc gtggagttca ccgaccacct gttcaacatc

240

gccaagccca gacccccctg gatgggcctg ctgggcccta ccatccaggc cgaggtgtac

300

gacaccgtgg tgatcaccct gaagaacatg gccagccacc ccgtgagcct gcacgccgtg

360

ggcgtgagct actggaaggc cagcgagggc gccgagtacg acgaccagac cagccagaga

420

gagaaggagg acgacaaggt gttccccggc ggcagccaca cctacgtgtg gcaggtgctg

480

aaggagaacg gccccatggc cagcgacccc ctgtgcctga cctacagcta cctgagccac

540

gtggacctgg tgaaggacct gaacagcggc ctgatcggcg ccctgctggt gtgcagagag

600

ggcagcctgg ccaaggagaa gacccagacc ctgcacaagt tcatcctgct gttcgccgtg

660

ttcgacgagg gcaagagctg gcacagcgag accaagaaca gcctgatgca ggacagagac

720

gccgccagcg ccagagcctg gcccaagatg cacaccgtga acggctacgt gaacaggagc

780

ctgcccggcc tgatcggctg ccacagaaag agcgtgtact ggcacgtgat cggcatgggc

840

accacccccg aggtgcacag catcttcctg gagggccaca ccttcctggt gaggaaccac

900

agacaggcca gcctggagat cagccccatc accttcctga ccgcccagac cctgctgatg

960

gacctgggcc agttcctgct gttctgccac atcagcagcc accagcacga cggcatggag

1020

gcctacgtga aggtggacag ctgccccgag gagccccagc tgagaatgaa gaacaacgag

1080

gaggccgagg actacgacga cgacctgacc gacagcgaga tggacgtggt gagattcgac

1140

gacgacaaca gccccagctt catccagatc aggagcgtgg ccaagaagca ccccaagacc

1200

tgggtgcact acatcgccgc cgaggaggag gactgggact acgcccccct ggtgctggcc

1260

cccgacgaca gaagctacaa gagccagtac ctgaacaacg gcccccagag gatcggcaga

1320

aagtacaaga aggtgcggtt catggcctac accgacgaga ccttcaagac cagggaggcc

1380

atccagcacg agagcggcat cctgggccct ctgctgtacg gcgaggtggg cgacaccctg

1440

ctgatcatct tcaagaacca ggccagcaga ccctacaaca tctaccccca cggcatcacc

1500

gacgtgagac ccctgtacag cagaagactg cccaagggcg tgaagcacct gaaggacttc

1560

cccatcctgc ccggcgagat cttcaagtac aagtggaccg tgaccgtgga ggacggcccc

1620

accaagagcg accccagatg cctgaccaga tactacagca gcttcgtgaa catggagcgg

1680

gacctggcca gcggcctgat cggccccctg ctgatctgct acaagggagag cgtggaccag

1740

agaggcaacc agatcatgag cgacaagcgg aacgtgatcc tgttcagcgt gttcgacgag

1800

aaccggagct ggtacctgac cgagaacatc cagaggttcc tgcccaaccc cgccggcgtg

1860

cagctggagg accccgagtt ccaggccagc aacatcatgc acagcatcaa cggctacgtg

1920

ttcgacagcc tgcagctgag cgtgtgcctg cacgaggtgg cctactggta catcctgagc

1980

atcggcgccc agaccgactt cctgagcgtg ttcttcagcg gctacacctt caagcacaag

2040

atggtgtacg aggacaccct gaccctgttc cccttcagcg gcgagaccgt gttcatgagc

2100

atggagaacc ccggcctgtg gatcctgggc tgccacaaca gcgacttcag aaacagaggc

2160

atgaccgccc tgctgaaggt gagcagctgc gacaagaaca ccggcgacta ctacgaggac

2220

agctacgagg acatcagcgc ctacctgctg agcaagaaca acgccatcga gcccaggagc

2280

ttcagccaga acccccccgt gctgaagaga caccagagag agatcaccag gaccaccctg

2340

cagagcgacc aggaggagat cgactacgac gacaccatca gcgtggagat gaagaaggag

2400

gacttcgaca tctacgacga ggacgagaac cagagcccca gaagcttcca gaagaagacc

2460

aggcactact tcatcgccgc cgtggagaga ctgtgggact acggcatgag cagcagcccc

2520

cacgtgctga gaaacagagc cccagcggc agcgtgcccc agttcaagaa ggtggtgttc

2580

caggagttca ccgacggcag cttcacccag cccctgtaca gaggcgagct gaacgagcac

2640

ctgggcctgc tgggccccta catcagagcc gaggtggagg acaacatcat ggtgaccttc

2700

cggaaccagg ccagcagacc ctacagcttc tacagcagcc tgatcagcta cgaggaggac

2760

cagaggcagg gcgccgagcc cagaaagaac ttcgtgaagc ccaacgagac caagacctac

2820

ttctggaagg tgcagcacca catggccccc accaaggacg agttcgactg caaggcctgg

2880

gcctacttca gcgacgtgga cctggagaag gacgtgcaca gcggcctgat cggccccctg

2940

ctggtgtgcc acaccaacac cctgaacccc gcccacggca gacaggtgac cgtgcaggag

3000

ttcgccctgt tcttcaccat cttcgacgag accaagagct ggtacttcac cgagaacatg

3060

gagcggaact gcagagcccc ctgcaacatc cagatggagg accccacctt caaggagaac

3120

tacaggttcc acgccatcaa cggctacatc atggacaccc tgcccggcct ggtgatggcc

3180

caggaccaga gaatcagatg gtacctgctg agcatgggca gcaacgagaa catccacagc

3240

atccacttca gcggccacgt gttcaccgtg aggaagaagg aggagtacaa gatggccctg

3300

tacaacctgt accccggcgt gttcgagacc gtggagatgc tgcccagcaa ggccggcatc

3360

tggagagtgg agtgcctgat cggcgagcac ctgcacgccg gcatgagcac cctgttcctg

3420

gtgtacagca acaagtgcca gacccccctg ggcatggcca gcggccacat cagagacttc

3480

cagatcaccg ccagcggcca gtacggccag tgggccccca agctggccag actgcactac

3540

agcggcagca tcaacgcctg gagcaccaag gagcccttca gctggatcaa ggtggacctg

3600

ctggccccca tgatcatcca cggcatcaag acccagggcg cgacagaa gttcagcagc

3660

ctgtacatca gccagttcat catcatgtac agcctggacg gcaagaagtg gcagacctac

3720

agaggcaaca gcaccggcac cctgatggtg ttcttcggca acgtggacag cagcggcatc

3780

aagcacaaca tcttcaaccc ccccatcatc gcccggtaca tcaggctgca ccccacccac

3840

tacagcatca ggagcaccct gagaatggag ctgatgggct gcgacctgaa cagctgcagc

3900

atgcccctgg gcatggagag caaggccatc agcgacgccc agatcaccgc cagcagctac

3960

ttcaccaaca tgttcgccac ctggagcccc agcaaggcca gactgcacct gcagggcaga

4020

agcaacgcct ggagacccca ggtgaacaac cccaaggagt ggctgcaggt ggacttccag

4080

aagaccatga aggtgaccgg cgtgaccacc cagggcgtga agagcctgct gaccagcatg

4140

tacgtgaagg agttcctgat cagcagcagc caggacggcc accagtggac cctgttcttc

4200

cagaacggca aggtgaaggt gttccagggc aaccaggaca gcttcacccc cgtggtgaac

4260

agcctggacc cccccctgct gaccagatac ctgagaatcc acccccagag ctgggtgcac

4320

cagatcgccc tgagaatgga ggtgctgggc tgcgaggccc aggacctgta c

4371

<210> 8

<211> 4371

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleotide sequence,

encoding the FVIII-BDD protein with a signal peptide (variant

FVIII-BDDco-v2)

<400> 8

atgcagatcg agctgagcac ctgttttttt ctgtgcctgc tgaggttctg cttctccgcc

60

acaagaagat actatctggg cgccgtggag ctgagctggg attatatgca gtccgacctg

120

ggcgagctgc cagtggatgc tagattccct cctagagtgc ctaagagctt tccattcaat

180

acctccgtgg tgtacaagaa gaccctgttc gtggagttta ccgaccacct gttcaacatc

240

gccaaaccta gaccaccttg gatgggactg ctgggcccaa ctattcaggc cgaagtgtac

300

gataccgtgg tgatcactct gaagaatatg gcctcccacc ctgtgagcct gcatgcagtt

360

ggcgtgagct attggaaggc cagcgaggga gccgaatacg atgaccagac cagccagagg

420

gagaaggaag acgacaaggt gtttcccgga ggaagccaca cctatgtgtg gcaggtgctg

480

aaggagaatg gacctatggc cagcgatcca ctgtgcctga cctactctta tctgagccac

540

gtggacctgg tgaaggacct gaatagcggc ctgattggcg cactgctggt gtgtagagag

600

ggatctctgg ccaaagagaa gacccagacc ctgcacaagt ttatcctgct gtttgccgtg

660

tttgacgagg gcaaatcttg gcacagcgag accaaaaaca gcctgatgca ggatagac

720

gcagcctctg ctagagcttg gcctaagatg cacaccgtga atggctacgt gaacagatcc

780

ctgcccggcc tgattggatg tcacagaaag agcgtgtact ggcacgtgat cggaatggga

840

actaccccag aagtgcacag catctttctg gagggacaca cctttctggt gcggaatcat

900

agacaggcca gcctggaaat cagccctatt acctttctga ccgcccagac actgctgatg

960

gatctgggac agtttctgct gttctgccac atcagctctc accagcatga cggaatggaa

1020

gcctacgtga aggtggacag ctgccctgag gaaccacagc tgaggatgaa gaataacgag

1080

gaggccgagg attacgacga tgacctgacc gattctgaga tggacgtggt gagattcgac

1140

gacgataact ctccctcctt catccagatc agatccgtgg ccaaaaagca ccctaaaacc

1200

tgggtgcact atatcgccgc agaggaagaa gactgggatt acgcacctct ggtgctggcc

1260

cctgatgaca gatcttataa gtctcagtac ctgaacaacg gccctcagag aatcggcagg

1320

aagtacaaga aggtgagatt catggcctac agacgaga cattcaagac cagagaggcc

1380

attcagcacg agtccggcat tctgggacca ctgctgtacg gagaggtggg cgatacactg

1440

ctgatcatct tcaaaaatca ggccagcagg ccctacaaca tctacccaca tggcatcaca

1500

gacgtgaggc ctctgtacag cagaagactg cctaaaggcg tgaagcacct gaaggacttc

1560

cccattctgc caggcgagat tttcaagtac aagtggacag tgaccgtgga ggatggaccc

1620

acaaagtctg accctagatg cctgaccagg tactatagct ccttcgtgaa catggaaagg

1680

gatctggcct ctggactgat tggcccactg ctgatctgct acaaggaatc cgtggaccag

1740

agaggaaacc agatcatgag cgataagagg aatgtgatcc tgttcagcgt gttcgatgag

1800

aacaggagct ggtatctgac cgagaacatc cagagattcc tgccaaatcc agccggcgtg

1860

cagctggaag atcctgaatt tcaggcctcc aatatcatgc actccatcaa tggctacgtg

1920

tttgacagcc tgcagctgtc tgtgtgtctg cacgaagtgg cctactggta catcctgagc

1980

attggagccc agaccgactt tctgagcgtg tttttctctg gctacacctt caagcacaag

2040

atggtgtacg aggacaccct gacactgttc ccattctcag gcgaaaccgt gtttatgagc

2100

atggagaacc caggactgtg gattctgggc tgccacaact ccgacttcag gaatagagga

2160

atgaccgccc tgctgaaagt gagcagctgc gataagaaca ccggcgatta ttacgaggac

2220

tcctacgagg atatcagcgc ctacctgctg tccaaaaaca atgccatcga gcccaggagc

2280

ttctcccaga atccacctgt gctgaaaaga caccagaggg agatcaccag aaccacactg

2340

cagtctgatc aggaggagat cgactatgac gacaccatca gcgtggagat gaagaaggag

2400

gactttgata tctacgacga ggatgagaac cagagcccca ggagctttca gaagaagacc

2460

aggcactact tcatcgccgc agtggagaga ctgtgggact acggaatgtc ttcctcccct

2520

cacgtgctga gaaatagagc ccagtctgga agcgtgccac agtttaagaa ggtggtgttc

2580

caggagttta ccgacggctc tttcacccag ccactgtata gaggcgaact gaacgaacac

2640

ctgggcctgc tgggacctta tatcagagcc gaagtggagg acaacatcat ggtgaccttc

2700

agaaaccagg ccagcagacc ttacagcttc tactccagcc tgatcagcta cgaagaggat

2760

cagagacagg gcgccgaacc taggaaaaat tttgtgaagc caaacgagac caagacctac

2820

ttttggaagg tgcagcatca catggccccc accaaggatg agttcgattg taaagcctgg

2880

gcctacttct ccgatgtgga cctggaaaag gatgtgcaca gcggactgat cggaccactg

2940

ctggtgtgcc acacaaatac cctgaaccct gcccacggaa gacaggtgac agtgcaggaa

3000

tttgccctgt tcttcaccat ctttgacgag accaagtcct ggtacttcac cgagaacatg

3060

gagagaaact gcagagcccc ttgcaacatt cagatggagg acccaacatt caaggagaac

3120

tacaggttcc acgccatcaa tggctacatc atggatacac tgcctggcct ggtgatggcc

3180

caggatcaga gaattaggtg gtacctgctg tctatgggct ccaatgagaa tatccactcc

3240

atccacttca gcggccacgt gttcaccgtg agaaagaaag aggagtacaa gatggccctg

3300

tacaacctgt acccaggcgt gtttgagacc gtggagatgc tgccttctaa agctggcatc

3360

tggagagtgg agtgcctgat cggcgagcat ctgcatgctg gaatgtctac cctgtttctg

3420

gtgtactcca acaagtgcca gacacctctg ggcatggctt ctggacatat cagagacttt

3480

cagatcaccg cctccggcca gtacggacaa tgggctccaa agctggccag actgcattac

3540

tctggctcta ttaatgcctg gagcaccaag gagccctttt cctggatcaa ggtggatctg

3600

ctggccccca tgattatcca cggcatcaaa acccagggcg ccaggcagaa gttttcttcc

3660

ctgtacatca gccagttcat catcatgtac agcctggatg gcaagaagtg gcagacctac

3720

agaggcaata gcaccggaac cctgatggtg tttttcggca acgtggactc ctccggcatt

3780

aagcacaata tcttcaaccc acctatcatc gccaggtaca tcagactgca ccccacccac

3840

tactctatca gaagcaccct gaggatggaa ctgatgggct gtgacctgaa ttcctgtagc

3900

atgcctctgg gaatggagtc taaagccatc agtgacgccc agattaccgc cagcagctac

3960

tttaccaaca tgtttgctac atggtccccc tctaaggcca gactgcacct gcagggaaga

4020

tctaacgcct ggagaccaca ggtgaacaac cccaaagaat ggctgcaggt ggattttcag

4080

aagaccatga aagtgaccgg cgtgacaaca cagggcgtga aatctctgct gacctctatg

4140

tacgtgaagg agtttctgat cagcagcagc caggacggcc atcagtggac cctgtttttt

4200

cagaatggca aggtgaaggt gttccagggc aatcaggact cattcactcc agtggtgaac

4260

agcctggacc cacctctgct gaccagatac ctgagaattc acccacagtc ctgggtgcat

4320

cagatcgccc tgagaatgga agtgctgga tgtgaggcac aggacctgta c

4371

<210> 9

<211> 4371

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleotide sequence,

encoding the FVIII-BDD protein with a signal peptide (variant

FVIII-BDDco-v3)

<400> 9

atgcagatcg agctgagcac ctgtttcttc ctgtgcctgc tgaggttctg tttcagcgcc

60

acaaggaggt actatctggg cgccgtggaa ctgagctggg actatatgca gagcgacctg

120

ggagagctgc ctgtggatgc cagatttcca cctagggtgc ccaagagctt ccccttcaat

180

acaagcgtgg tgtacaagaa gaccctgttc gtggagttta ccgaccacct gttcaacatc

240

gccaagccta gacctccttg gatgggactg ctgggcccta ccattcaggc cgaagtgtac

300

gacacagtgg tgatcaccct gaagaatatg gccagccatc cagtgtccct gcacgcagtg

360

ggagtgtctt attggaaggc ctctgagggc gccgagtacg atgatcagac cagccagaga

420

gagaaggagg acgacaaggt gttccccgga ggaagccaca cctacgtgtg gcaggtgctg

480

aaagagaatg gcccaatggc ctccgacccc ctgtgtctga catacagcta cctgagccac

540

gtggacctgg tgaaggacct gaactctggc ctgatcggag ctctgctggt gtgtagagaa

600

ggcagcctgg ccaaggagaa gacccagacc ctgcacaagt tcatcctgct gtttgccgtg

660

ttcgatgagg gcaaaagctg gcattccgag accaagaatt ccctgatgca ggatagggac

720

gctgccagcg ctagagcctg gcctaagatg cacactgtga atggctacgt gaaccggagc

780

ctgccaggcc tgatcggctg tcacagaaag agcgtgtact ggcacgtgat cggcatgggc

840

accactcctg aggtgcactc tatctttctg gaaggccaca cctttctggt gcggaaccat

900

agacaggcct ccctggaaat cagccccatc acctttctga ccgcccagac cctgctgatg

960

gatctgggac agttcctgct gttttgccac atctcctctc accagcacga cggaatggag

1020

gcctacgtga aagtggacag ctgtcctgaa gaacctcagc tgcggatgaa gaataacgag

1080

gaggccgagg actacgacga tgacctgacc gatagcgaga tggacgtggt gagattcgac

1140

gacgataaca gccccagctt catccagatc agatccgtgg ccaagaagca ccccaagaca

1200

tgggtgcact atatcgccgc agaggaggag gattgggact acgcacctct ggtgctggct

1260

cctgacgata gatcctacaa gtcccagtac ctgaacaacg gcccccagag aatcggcaga

1320

aagtacaaga aggtgagatt catggcctac accgacgaga cctttaagac cagagaggcc

1380

atccagcacg agagcggcat tctgggacca ctgctgtatg gagaggtggg cgatacactg

1440

ctgatcatct tcaagaatca ggcctcccgg ccatacaaca tctatccaca cggcatcacc

1500

gacgtgagac cactgtactc caggagactg cccaagggcg tgaagcacct gaaggacttc

1560

cctattctgc caggcgagat cttcaagtac aagtggacag tgaccgtgga agacggacct

1620

accaaaagcg accccagatg cctgacccgg tactactcca gcttcgtgaa catggagaga

1680

gatctggcca gcggactgat tggacctctg ctgatttgtt acaagggagag cgtggaccag

1740

aggggcaacc agattatgag cgacaagcgg aatgtgatcc tgttctccgt gttcgacgag

1800

aaccgcagct ggtatctgac cgagaatatc cagaggttcc tgccaaaccc cgccggcgtt

1860

cagctggaag atccagagtt tcaggcctcc aacatcatgc acagcatcaa tggctacgtg

1920

ttcgacagcc tgcagctgtc cgtgtgtctg catgaagtgg cctactggta tatcctgagc

1980

atcggcgccc agaccgattt tctgtccgtg tttttctccg gctacacctt caagcacaag

2040

atggtgtacg aggacaccct gaccctgttt ccttttagcg gcgaaaccgt gttcatgagc

2100

atggagaacc caggactgtg gattctgggc tgtcacaaca gcgacttcag aaatcggggc

2160

atgaccgctc tgctgaaggt gagctcttgt gacaaaaata ccggcgacta ctacgaggac

2220

agctacgagg atatcagcgc ctacctgctg agcaagaaca acgccatcga gccaagaagc

2280

ttcagccaga atccccccgt gctgaagaga catcagaggg agatcaccag aaccaccctg

2340

cagagcgacc aggaggagat cgattacgac gacaccatca gcgtggagat gaagaaggag

2400

gatttcgaca tctacgacga ggacgagaat cagagcccaa ggagctttca gaagaagacc

2460

cggcactact tcatcgctgc cgtggagaga ctgtgggatt atggcatgag ctccagccct

2520

cacgtgctga gaaatagagc ccagtctggc tccgtgcccc agttcaagaa agtggtgttc

2580

caggaattca ccgacggcag ctttacccag cccctgtata gaggcgaact gaatgaacac

2640

ctgggcctgc tgggacctta catcagagcc gaagtggagg acaacatcat ggtgaccttt

2700

aggaatcagg ccagcagacc ctacagcttc tatagctccc tgatctccta cgaggaggac

2760

cagagacagg gcgctgagcc aagaaagaat ttcgtgaagc ccaacgagac caagacctac

2820

ttctggaaag tgcagcacca catggcccct accaaggatg agttcgactg taaagcctgg

2880

gcctacttca gcgacgtgga cctggagaag gacgtgcact ccggactgat cggaccactg

2940

ctggtgtgcc acacaaacac cctgaaccct gcccatggaa gacaggtgac tgtgcaggaa

3000

ttcgccctgt tctttaccat cttcgacgag accaagtcct ggtacttcac cgagaatatg

3060

gagaggaatt gcagagcccc ctgtaacatc cagatggagg accccacctt taaggagaat

3120

tacaggtttc acgccatcaa cggctacatc atggacacac tgccaggcct ggtgatggca

3180

caggatcaga ggatcagatg gtatctgctg agcatgggct ccaacgagaa catccactcc

3240

atccacttca gcggccacgt gtttaccgtg agaaagaagg aggagtacaa gatggccctg

3300

tacaacctgt atcccggcgt gttcgaaacc gtggagatgc tgccttccaa agccggaatc

3360

tggagagtgg agtgcctgat tggcgagcac ctgcacgctg gaatgtccac cctgttcctg

3420

gtgtatagca acaagtgcca gacccccctg ggcatggcta gcggacacat tagagacttt

3480

cagatcaccg cctccggcca gtacggacag tgggccccaa aactggctag gctgcattac

3540

agcggctcca tcaatgcctg gtccaccaag gagccattca gctggatcaa agtggatctg

3600

ctggccccca tgatcatcca cggcattaag acacagggcg ccaggcagaa gttcagctct

3660

ctgtacatct cccagttcat catcatgtac tccctggacg gcaagaagtg gcagacctac

3720

aggggcaata gcaccggcac actgatggtg tttttcggca acgtggactc ttccggcatc

3780

aagcacaaca tctttaaccc ccctatcatc gccaggtaca tcaggctgca ccccactcac

3840

tacagcatca gatccaccct gagaatggag ctgatgggat gcgacctgaa cagctgcagc

3900

atgcccctgg gcatggagag caaggccatt tctgacgccc agatcaccgc cagcagctat

3960

ttcaccaata tgttcgctac ctggagcccc agcaaggcca gactgcatct gcagggcaga

4020

agcaacgcct ggagaccaca ggtgaacaat cctaaagagt ggctgcaggt ggacttccag

4080

aagaccatga aggtgaccgg cgtgacaacc cagggcgtga aatctctgct gacctccatg

4140

tacgtgaagg agttcctgat ctcctccagc caggatggcc atcagtggac cctgttcttc

4200

cagaacggca aggtgaaagt gttccagggc aaccaggaca gctttacccc tgtggtgaac

4260

tctctggatc cccccctgct gaccaggtac ctgagaattc acccccagag ctgggtgcac

4320

cagatcgccc tgagaatgga agtgctgga tgcgaggcac aggacctgta c

4371

<210> 10

<211> 4371

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleotide sequence,

encoding the FVIII-BDD protein with a signal peptide (variant

FVIII-BDDco-v4)

<400> 10

atgcagatcg agctgtccac ctgcttcttc ctgtgcctgc tgcgcttctg tttctctgct

60

acccggaggt actacctggg cgctgtggag ctgagctggg actacatgca gagcgatctg

120

ggcgagctgc ccgtggatgc cagatttccc ccaagagtgc ctaagtcctt ccccttcaac

180

accagcgtgg tgtacaagaa gaccctgttc gtggagttca ccgaccacct gttcaacatc

240

gccaagccaa ggcccccctg gatgggactg ctgggaccta caattcaggc cgaggtgtac

300

gataccgtgg tgatcaccct gaagaacatg gcctcccacc cagtgtccct gcatgccgtg

360

ggcgtgagct actggaaggc ttctgaaggc gccgagtacg acgaccagac cagccagagg

420

gagaaggagg acgacaaggt gttcccaggc ggatctcaca cctacgtgtg gcaggtgctg

480

aaggagaatg gcccaatggc cagcgaccca ctgtgtctga cctacagcta cctgtcccac

540

gtggacctgg tgaaggacct gaactccggc ctgatcggcg ccctgctggt gtgtagagaa

600

ggatctctgg ccaaggagaa gacccagacc ctgcacaagt tcatcctgct gttcgccgtg

660

tttgacgagg gcaagagctg gcattccgag accaagaaca gcctgatgca ggacagagac

720

gcagccagcg ctagagcctg gcccaagatg cacaccgtga atggctacgt gaaccggtcc

780

ctgccaggcc tgatcggctg tcacagaaaa agcgtgtact ggcacgtgat cggcatgggc

840

accacacccg aggtgcacag catctttctg gagggccaca ccttcctggt gagaaaccac

900

aggcaggcct ccctggagat ctcccccatc acctttctga ccgcccagac cctgctgatg

960

gatctgggcc agttcctgct gttctgccac atcagctccc accagcacga cggaatggag

1020

gcctacgtga aggtggatag ctgccccgag gagccccagc tgagaatgaa gaacaacgag

1080

gaggccgagg actacgacga tgacctgacc gattccgaga tggacgtggt gcgcttcgac

1140

gacgataata gccccagctt catccagatc agatccgtgg ccaagaagca cccaaagacc

1200

tgggtgcact acatcgctgc cgaggaggag gactgggatt acgccccact ggtgctggcc

1260

cctgatgaca gatcctacaa gagccagtac ctgaacaacg gcccccagag aatcggccgg

1320

aagtacaaga aggtgagatt catggcctac accgacgaga ccttcaagac cagggaagcc

1380

atccagcacg agagcggcat cctgggacca ctgctgtacg gagaagtggg cgataccctg

1440

ctgatcatct tcaagaacca ggcctcccgg ccctacaaca tctaccccca cggcatcacc

1500

gacgtgaggc cactgtacag caggaggctg cccaagggcg tgaagcacct gaaggacttc

1560

cccatcctgc ccggcgagat ctttaagtac aagtggaccg tgaccgtgga ggacggccca

1620

accaagagcg acccaagatg cctgaccagg tactactcct ccttcgtgaa catggagcgg

1680

gatctggcca gcggactgat tggccctctg ctgatctgct acaaggagag cgtggaccag

1740

cgcggcaatc agatcatgag cgacaagaga aacgtgatcc tgttcagcgt gttcgacgag

1800

aatcgcagct ggtacctgac cgagaacatc cagaggttcc tgcccaaccc agccggcgtg

1860

cagctggaag atcctgaatt tcaggccagc aacatcatgc actccatcaa cggctacgtg

1920

ttcgactccc tgcagctgag cgtgtgtctg cacgaagtgg cctactggta catcctgagc

1980

atcggcgccc agaccgactt cctgtccgtg ttcttttccg gctacacctt caagcacaag

2040

atggtgtacg aggacaccct gaccctgttc ccctttagcg gcgagaccgt gttcatgagc

2100

atggagaacc caggcctgtg gattctgggc tgccacaaca gcgacttccg caataggggc

2160

atgaccgccc tgctgaaagt gtccagctgc gataagaaca ccggcgacta ctacgaggac

2220

agctacgagg acatctccgc ctacctgctg agcaagaaca acgccatcga gccgatcc

2280

ttcagccaga accctccagt gctgaagagg caccagcgcg agatcaccag aaccaccctg

2340

cagtctgatc aggaggagat cgactacgac gacaccatca gcgtggagat gaagaaggag

2400

gacttcgaca tctacgacga ggacgagaac cagagcccta ggtccttcca gaagaagacc

2460

cggcactact tcatcgccgc agtggagagg ctgtgggatt acggcatgag cagctcccca

2520

cacgtgctga gaaatagggc cccagcggc agcgtgcccc agttcaaaaa ggtggtgttc

2580

caggagttca ccgacggctc cttcacccag cccctgtaca gaggcgagct gaatgagcac

2640

ctgggcctgc tgggacccta catcagagca gaggtggagg acaacatcat ggtgaccttc

2700

aggaaccagg ccagcagacc ctacagcttc tacagcagcc tgatcagcta cgaggaggac

2760

cagagacagg gagccgagcc acgcaagaac ttcgtgaagc ccaacgagac caagacctac

2820

ttctggaagg tgcagcacca catggccccc accaaggacg agttcgattg taaagcctgg

2880

gcctacttca gcgacgtgga cctggagaag gacgtgcaca gcggcctgat cggacctctg

2940

ctggtgtgtc ataccaacac cctgaacccc gcccacggca gacaggtgac tgtgcaggag

3000

tttgccctgt tcttcaccat cttcgacgag accaagagct ggtacttcac cgagaacatg

3060

gagagaaact gcagggcccc atgcaacatc cagatggaag accccacctt caaggagaac

3120

taccggttcc acgccatcaa cggctacatc atggataccc tgcctggcct ggtgatggcc

3180

caggatcaga gaatcaggtg gtacctgctg agcatgggct ccaacgagaa catccacagc

3240

atccacttct ccggccacgt gttcaccgtg aggaaaaagg aggagtacaa gatggccctg

3300

tacaacctgt accccggcgt gtttgagacc gtggagatgc tgccatctaa ggccggcatt

3360

tggagagtgg agtgcctgat cggcgagcac ctgcacgccg gaatgtctac cctgttcctg

3420

gtgtacagca acaagtgcca gacccccctg ggcatggcca gcggacacat cagagacttc

3480

cagatcaccg ccagcggcca gtacggacag tgggctccta aactggcccg cctgcactat

3540

agcggcagca ttaacgcctg gtccaccaag gagccattca gctggatcaa ggtggatctg

3600

ctggccccca tgatcatcca cggcatcaaa acacagggcg cgacagaa gttctccagc

3660

ctgtacatct cccagttcat catcatgtac tccctggacg gcaagaagtg gcagacctac

3720

agaggcaact ccaccggcac actgatggtg ttcttcggca acgtggacag cagcggcatc

3780

aagcacaaca tcttcaaccc ccccatcatc gcccggtaca tcagactgca cccaacccac

3840

tactccatca ggtccaccct gaggatggag ctgatgggct gcgatctgaa ttcctgcagc

3900

atgcccctgg gcatggagtc caaagccatc tctgacgccc agatcaccgc cagctcctac

3960

ttcaccaaca tgttcgccac ttggagcccc agcaaggcca gactgcacct gcagggcaga

4020

agcaatgcct ggagacccca ggtgaacaac ccaaaggagt ggctgcaggt ggacttccag

4080

aagaccatga aggtgaccgg cgtgaccaca cagggcgtga aaagcctgct gaccagcatg

4140

tacgtgaagg agttcctgat cagctccagc caggatgggc accagtggac cctgttcttt

4200

cagaacggca aggtgaaggt gttccagggc aatcaggact cattcacccc tgtggtgaac

4260

tctctggacc cccccctgct gacccggtac ctgagaatcc acccacagtc ttgggtgcac

4320

cagatcgccc tgaggatgga agtgctgggc tgcgaggctc aggatctgta c

4371

<210> 11

<211> 1438

<212>PRT

<213> Artificial sequence

<220>

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

B

domain)

<400> 11

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

1 5 10 15

Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro

20 25 30

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

35 40 45

Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

595 600 605

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

675 680 685

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

690 695 700

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

740 745 750

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

755 760 765

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

770 775 780

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

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> 12

<211> 1457

<212>PRT

<213> Artificial sequence

<220>

<223> FVIII-BDD protein with signal peptide

<400> 12

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> 13

<211> 130

<212> DNA

<213> Natural sequence

<220>

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

<400> 13

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt

60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact

120

aggggttcct

130

<210> 14

<211> 252

<212> DNA

<213> Artificial sequence

<220>

<223> HLP promoter

<400> 14

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> 15

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Polyadenylation signal

<400> 15

cgcgaataaa agatctttat tttcattaga tctgtgtgtt ggttttttgt gtgatgcagc

60

<210> 16

<211> 141

<212> DNA

<213> Natural sequence

<220>

<223> Right ITR

<400> 16

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc

120

gagcgcgcag ctgcctgcag g

141

<210> 17

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding a signal peptide

<400> 17

atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagt

57

<210> 18

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding a signal peptide

<400> 18

atgcagatcg agctgagcac ctgcttcttc ctgtgcctgc tgaggttctg cttcagc

57

<210> 19

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding a signal peptide

<400> 19

atgcagatcg agctgagcac ctgttttttt ctgtgcctgc tgaggttctg cttctcc

57

<210> 20

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding a signal peptide

<400> 20

atgcagatcg agctgagcac ctgtttcttc ctgtgcctgc tgaggttctg tttcagc

57

<210> 21

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding a signal peptide

<400> 21

atgcagatcg agctgtccac ctgcttcttc ctgtgcctgc tgcgcttctg tttctct

57

<210> 22

<211> 19

<212>PRT

<213> Artificial sequence

<220>

<223> signal peptide

<400> 22

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

1 5 10 15

Cys Phe Ser

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Claims (15)

1. An isolated codon-optimized nucleic acid encoding the FVIII-BDD protein (blood clotting factor VIII with B domain deleted) with the amino acid sequence SEQ ID NO: 11, which includes a nucleotide sequence selected from the group: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. 2. An expression cassette, which includes the nucleic acid according to claim 1. 3. Expression cassette according to claim 2, including the following elements in the direction from the 5' end to the 3' end: left (first) ITR (inverted terminal repeats); promoter; a nucleic acid encoding a signal peptide; nucleic acid according to claim 1; polyadenylation signal; right (second) ITR. 4. An expression vector, which includes a nucleic acid according to claim 1 or an expression cassette according to any one of paragraphs. 2, 3. 5. The expression vector according to claim 4, where the expression vector is a recombinant adeno-associated virus (AAV). 6. The expression vector according to claim 5, where 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.rh10, 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. 7. Pharmaceutical composition for delivery of the FVIII-BDD gene to target cells, including an isolated codon-optimized nucleic acid according to claim 1, an expression vector according to any one of paragraphs. 4-6 or a cassette according to any one of paragraphs. 2, 3 in combination with one or more pharmaceutically acceptable excipients. 8. Use of an isolated codon-optimized nucleic acid according to claim 1, an expression vector according to any one of paragraphs. 4-6, cassettes according to any one of paragraphs. 2, 3 or compositions according to claim 7 for delivering the FVIII-BDD gene to target cells. 9. A method for delivering the FVIII-BDD gene to target cells, including introducing an isolated codon-optimized nucleic acid according to claim 1, an expression vector according to any one of claims. 4-6, cassettes according to any one of paragraphs. 2, 3 or compositions according to claim 7 into target cells.