Isolated nucleic acid encoding a fusion protein based on the FVIII-BDD and a heterologous signal peptide

Abstract

This application relates to the fields of genetics, gene therapy and molecular biology.More specifically, the present invention relates to a nucleic acid encoding a fusion protein based on FVIII-BDD (B domain deleted coagulation factor VIII) and a heterologous signal peptide, an expression cassette and vector based thereon, a host cell for producing a fusion protein based on FVIII-BDD and a heterologous signal peptide, and further various uses of said vector.

Description

[Technical field]

[0001] This application relates to the fields of genetics, gene therapy and molecular biology.More specifically, the present invention relates to a nucleic acid encoding a fusion protein based on FVIII-BDD (B domain deleted coagulation factor VIII) and a heterologous signal peptide, an expression cassette and vector based thereon, a host cell for producing a fusion protein based on FVIII-BDD and a heterologous signal peptide, and further various uses of said vector. [Background technology]

[0002] Hemophilia is an X-linked recessive genetic disorder that causes a defect in one of the proteins involved in secondary hemostasis. Hemophilia A, or classical hemophilia, is the most common variant of hemophilia; it occurs in 1 in 5000 newborn males (WFH 2019 Annual Global Survey Report, https: / / www.wfh.org / en / our-work-research-data / annual-global-survey) and is caused by a defect in the coagulation factor VIII protein. According to the Russian Society of Hemophilia, there are more than 6,500 patients with hemophilia A in Russia (WFH 2019 Annual Global Survey Report).

[0003] Coagulation factor VIII (FVIII) is a 280 kDa protein secreted primarily into the blood by hepatic sinusoidal epithelial cells (Fahs SA, Hille MT, Shi Q, Weiler H, Montgomery RR. A conditional knockout mouse model reveals endothelial cells as the principal and possibly exclusive source of plasma factor VIII / Blood. 2014 Jun 12;123(24):3706-13. doi:10.1182 / blood-2014-02-555151. Epub 2014 Apr 4. PMID:24705491 and Everett LA, Cleuren AC, Khoriaty RN, Ginsburg D. Murine coagulation factor VIII is synthesized in endothelial cells (FVIII). cells / Blood. 2014 Jun 12;123(24):3697-705. doi:10.1182 / blood-2014-02-554501. Epub 2014 Apr 9. PMID:24719406). Activated FVIII circulates in the organism as a heterodimer consisting of a heavy chain (A1, A2, B domains) and a light chain (A3, C1, C3 domains) bound to each other via non-covalent metal-dependent interactions. As a result of FVIII processing, only the A1-A3, C1, and C2 domains are present in the activated form of the protein. This fact contributed to the generation of B-domain-deleted recombinant FVIII (FVIII-BDD), whose activity is not inferior to that of full-length FVIII (Pittman DD, Alderman EM, Tomkinson KN, Wang JH, Giles AR, Kaufman RJ. Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII / Blood. 1993 Jun 1;81(11):2925-35. PMID:8499631).Unlike other proteins of the blood coagulation cascade, which mainly belong to the proteases, FVIII is a glycoprotein. However, it plays a crucial role in the formation of the tenase complex, which is necessary for the generation of activated coagulation factor X (FXa), the first member of the final common coagulation pathway that ultimately leads to the formation of cross-linked fibrin.

[0004] Inversions in intron 1 or intron 22 of the FVIII gene are responsible for more than half of the cases of severe hemophilia A (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, abnormalities in the FVIII sequence are associated with a variety of mutations, including antisense mutations, reading frameshifts, splice site mutations, deletions and insertions.

[0005] Bleeding tendency in hemophilia A correlates with defined 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). Patients with mild hemophilia typically experience abnormal bleeding only in association with medical interventions or trauma. In contrast, patients with moderate hemophilia have prolonged bleeding responses to relatively minor trauma, and patients with severe disease often have spontaneous bleeding. Severe hemophilia A manifests with spontaneous hemarthrosis, soft tissue hematomas, retroperitoneal hemorrhage, intracerebral hemorrhage, and delayed postoperative bleeding. Over time, complications from recurrent hemarthrosis and soft tissue hematomas can result in severe arthropathy, joint contractures, and pseudotumors, which can lead to chronic disease. The proportion of patients with mild, moderate, and severe variants of hemophilia A is not precisely known, but recent epidemiological studies have reported that approximately 60% of patients with hemophilia A have the severe variant (WFH 2019 Annual Global Surveillance Report).

[0006] To date, lifelong replacement therapy in the form of recombinant FVIII infusions is the standard of care for patients with hemophilia A (WFH 2019 Annual Global Survey Report). Despite the successes achieved in hemophilia therapy, this approach has serious challenges. Prophylactic replacement therapy for hemophilia A involves intravenous infusion of recombinant FVIII every 3 days throughout the life of patients with severe variants of the disease. Such a treatment approach is very expensive and does not guarantee the absence of complications, mainly associated with hemarthrosis. In some cases, patients develop inhibitory antibodies. Inhibitory variants of hemophilia A are more typically observed in patients with severe disease and necessitate the use of alternative approaches to disease treatment and prevention (Eckhardt CL, van der Bom JG, van der Naald M, Peters M, Kamphuisen PW, Fijnvandraat K. Surgery and inhibitor development in haemophilia A: a systematic review / J Thromb Haemost. 2011 October;9(10):1948-58. doi:10.1111 / j.1538-7836.2011.04467.x. PMID:21838755).

[0007] Gene therapy for hemophilia A using adeno-associated virus (AAV)-based viral (expression) vectors encoding the FVIII gene has shown excellent 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. PMID:31282050).Unlike conventional approaches to the treatment of hemophilia, gene therapy using AAV allows the expression of exogenous FVIII to be maintained at sufficient levels for several years after a single dose of therapeutic agent is administered to the patient (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 Haemophilia A / Mol Ther. 2021 Feb 3;29(2):597-610. doi:10.1016 / j.ymthe.2020.12.008. Epub 2020 Dec 10. PMID:33309883).

[0008] To date, no gene therapy products are registered worldwide for the treatment of hemophilia A. Thus, there is a need to develop gene therapy products for the treatment of hemophilia A and for solutions that will improve the efficacy of gene therapy products for the treatment of hemophilia A. Summary of the Invention [Means for solving the problem]

[0009] The authors of the present invention 1) a fusion protein based on the FVIII-BDD and the FIX signal peptide (SP-FIX) having the amino acid sequence of SEQ ID NO: 7; or 2) a fusion protein based on FVIII-BDD and immunoglobulin G kappa chain signal peptide (SP-IgGK) having the amino acid sequence of SEQ ID NO: 8; or 3) A fusion protein based on FVIII-BDD and lactalbumin signal peptide (SP-lactalbumin) having the amino acid sequence of SEQ ID NO: 9. It has surprisingly been found that use of a nucleic acid encoding the FVIII-BDD protein results in increased levels of FVIII-BDD protein production and activity when compared to use of a nucleic acid encoding a FVIII-BDD protein containing the naturally occurring FVIII signal peptide (wild-type). Definitions and general methods Unless otherwise defined herein, all technical and scientific terms used in connection with the present invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0010] Further, unless otherwise required by context, singular terms shall include plural terms and plural terms shall include the singular terms. Typically, the current classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid hybridization and chemistry described herein are well known by those of skill in the art and are widely used in the art. Enzymatic reactions and purification methods are performed according to manufacturer's guidelines as common in the art or as described herein.

[0011] The terms "naturally occurring," "native," or "wild-type" are used to describe objects that can be found in nature as distinct from those that are artificially produced. For example, a protein or nucleotide sequence present in an organism, including in a virus, that can be isolated from a natural source and has not been intentionally modified by a person in a laboratory, is naturally occurring.

[0012] As used in this description and the claims which follow, unless the context dictates otherwise, the words "include" and "comprise" or variations thereof, such as "includes," "including," "comprises," or "comprising," will be understood to imply the inclusion of a specified integer or group of integers but not the exclusion of any other integer or group of integers. Proteins (peptides) As used in this description, 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 combinations thereof. nucleic acid molecule The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence" are used interchangeably in this description and refer to the exact sequence of nucleotides, modified or not, that determine a fragment or region of a nucleic acid and that may or may not contain non-naturally occurring nucleotides and that are either double-stranded DNA or RNA, single-stranded DNA or RNA, or the transcription product of said DNA.

[0013] As used in this description, polynucleotides include, by way of non-limiting example, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cellular genomes using conventional cloning techniques and PCR, etc., and by synthetic means.

[0014] It should also be included here that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in the natural state. The sequences of the present invention are isolated and / or purified, i.e. sampled directly or indirectly, for example by copying, and their environment is at least partially modified. Thus, isolated nucleic acids obtained, for example, by recombinant genetics using a host cell or by chemical synthesis, should also be mentioned here.

[0015] Unless otherwise indicated, a term nucleotide sequence includes its complement, i.e., a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. vector As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Additionally, the term "vector" refers to a recombinant viral particle capable of transporting a nucleic acid.

[0016] As used in this description, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter. use "Gene therapy" is the insertion of a gene into a subject's cells and / or tissues to treat a disease, typically a genetic disease, in which a defective mutant allele is replaced by a functional allele.

[0017] "Treating," "treatment," and "therapy" refer to a method of alleviating or reversing a biological disorder and / or at least one of its attendant symptoms. The terms "subject", "patient", "individual" and the like are used interchangeably in this description and refer to any animal suitable for the methods described in this description. In certain non-limiting embodiments, the subject, patient or individual is a human. The subject may be male or female of any age.

[0018] By "therapeutically effective amount" or "effective amount" is meant that amount of the therapeutic agent being administered that will relieve to some extent one or more of the symptoms of the disease being treated. Detailed Description of the Invention nucleic acid In one aspect, the present invention relates to an isolated nucleic acid encoding a fusion protein based on FVIII-BDD (B domain deleted coagulation factor VIII) and a heterologous signal peptide, comprising an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

[0019] An "isolated" nucleic acid molecule is one that is identified and separated from at least one nucleic acid molecule impurity.An isolated nucleic acid molecule is different from the form or set in which it is found under natural conditions.That is, an isolated nucleic acid molecule is different from the nucleic acid molecule that exists in a cell under natural conditions.

[0020] Signal peptides provide for the transport of a protein of interest within a cell to a target organelle or facilitate the secretion of a protein of interest into the intercellular space. In some embodiments, the isolated nucleic acid encodes a fusion protein based on a FVIII-BDD having the amino acid sequence of SEQ ID NO:5 and a FIX signal peptide having the amino acid sequence of SEQ ID NO:2.

[0021] In some embodiments, the isolated nucleic acid encodes a fusion protein based on a FVIII-BDD having the amino acid sequence of SEQ ID NO:5 and an immunoglobulin G kappa chain signal peptide having the amino acid sequence of SEQ ID NO:3.

[0022] In some embodiments, the isolated nucleic acid encodes a fusion protein based on a FVIII-BDD having the amino acid sequence of SEQ ID NO:5 and a lactalbumin signal peptide having the amino acid sequence of SEQ ID NO:4.

[0023] In some embodiments, the isolated nucleic acid encodes a fusion protein based on a FVIII-BDD and a heterologous signal peptide having the amino acid sequence of SEQ ID NO: 7. A given fusion protein having the amino acid sequence of SEQ ID NO: 7 comprises a FVIII-BDD having the amino acid sequence of SEQ ID NO: 5 and a FIX signal peptide having the amino acid sequence of SEQ ID NO: 2.

[0024] In some embodiments, the isolated nucleic acid encodes a fusion protein based on a FVIII-BDD and a heterologous signal peptide having the amino acid sequence of SEQ ID NO: 8. A given fusion protein having the amino acid sequence of SEQ ID NO: 8 comprises a FVIII-BDD having the amino acid sequence of SEQ ID NO: 5 and an immunoglobulin G kappa chain signal peptide having the amino acid sequence of SEQ ID NO: 3.

[0025] In some embodiments, the isolated nucleic acid encodes a fusion protein based on a FVIII-BDD and a heterologous signal peptide having the amino acid sequence of SEQ ID NO: 9. A given fusion protein having the amino acid sequence of SEQ ID NO: 9 comprises a FVIII-BDD having the amino acid sequence of SEQ ID NO: 5 and a lactalbumin signal peptide having the amino acid sequence of SEQ ID NO: 4.

[0026] In some embodiments, the isolated nucleic acid is the nucleotide sequence of SEQ ID NO: 11. The given nucleic acid encodes a fusion protein based on the FVIII-BDD and FIX signal peptide having the amino acid sequence of SEQ ID NO:7.

[0027] In some embodiments, the isolated nucleic acid is the nucleotide sequence of SEQ ID NO: 12. The given nucleic acid encodes a fusion protein based on FVIII-BDD and immunoglobulin G kappa chain signal peptide having the amino acid sequence of SEQ ID NO:8.

[0028] In some embodiments, the isolated nucleic acid is the nucleotide sequence of SEQ ID NO: 13. The given nucleic acid encodes a fusion protein based on the FVIII-BDD and lactalbumin signal peptide having the amino acid sequence of SEQ ID NO:9. Expression cassette.Expression vector. In one aspect, the invention relates to an expression cassette comprising the above-described nucleic acid encoding a fusion protein based on the FVIII-BDD and a heterologous signal peptide.

[0029] As used herein, the term "expression cassette" or "expression cassette" refers in particular to a DNA fragment capable of causing expression of a polynucleotide encoding a polypeptide of interest, the sequence of which is included in the expression cassette, in an appropriate setting. When introduced into a host cell, the expression cassette is capable of, among other things, causing the cellular machinery to transcribe the polynucleotide encoding the polypeptide of interest into RNA, which is typically then further processed and ultimately translated into the polypeptide of interest. The expression cassette can be included in an expression vector.

[0030] The expression cassette of the present invention includes a promoter as an element. As used herein, the term "promoter" refers specifically to a DNA element that promotes the transcription of a polynucleotide to which the promoter is operably linked. A promoter may further form part of a promoter / enhancer element. The physical boundary between "promoter" and "enhancer" elements is not always clear, and the term "promoter" typically refers to the site on a nucleic acid molecule to which RNA polymerase and / or any associated factors bind and transcription is initiated. Enhancers enhance promoter activity in time as well as space. A large number of promoters are known in the art to be 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 derepression. Both classes are suitable for protein expression. Promoters used for high-level production of polypeptides in eukaryotic cells, particularly mammalian cells, should be strong and preferably active in a wide range of cell types. Strong constitutive promoters capable of driving expression in a large number of cell types are well known in the art, and therefore it is not necessary to describe them in detail here.

[0031] According to one embodiment of the invention, the HLP promoter is used in the expression cassette of the invention. In some embodiments, the expression cassette comprises the following elements from the 5' to 3' end: Left side (first) ITR (inverted terminal repeat); promoter; Any one of the above nucleic acids encoding a fusion protein based on the FVIII-BDD and a heterologous signal peptide; polyadenylation signal; Right side (2nd) ITR.

[0032] The above structural elements of the expression cassette are operably linked to each other. As used herein, the term "operably linked" refers to the linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. The term "operably linked" means that the DNA sequences being linked are typically contiguous, and, where necessary to link two protein coding regions, contiguous and in reading frame.

[0033] In some embodiments, the expression cassette comprises a left (first) ITR having the nucleotide sequence of SEQ ID NO:14. In some embodiments, the expression cassette comprises an HLP promoter having the nucleotide sequence of SEQ ID NO:15.

[0034] In some embodiments, the expression cassette comprises a polyadenylation signal having the nucleotide sequence of SEQ ID NO:16. In some embodiments, the expression cassette comprises a right (second) ITR having the nucleotide sequence of SEQ ID NO:17.

[0035] In some embodiments, the expression cassette comprises the following elements from the 5' to 3' end: The left (first) ITR (inverted terminal repeat) having the nucleotide sequence of SEQ ID NO: 14; A promoter having the nucleotide sequence of SEQ ID NO: 15; Any one of the above nucleic acids encoding a fusion protein based on the FVIII-BDD and a heterologous signal peptide; a polyadenylation signal having the nucleotide sequence of SEQ ID NO: 16; The right (second) ITR having the nucleotide sequence of SEQ ID NO:17.

[0036] In one aspect, the invention relates to an expression vector comprising any one of the above-described nucleic acids encoding a fusion protein based on the FVIII-BDD and a heterologous signal peptide, or any one of the above-described expression cassettes.

[0037] In some aspects of the invention, vectors are plasmids, ie, circular double-stranded pieces of DNA into which additional DNA segments can be inserted. In some embodiments of the invention, the vector is a viral (expression) vector, in which additional DNA segments can be inserted into the viral genome.

[0038] In some aspects of the invention, the vector is capable of autonomous replication in a host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication site and episomal vectors). In further aspects of the invention, the vector (e.g., non-episomal vectors) can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genes. Furthermore, 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").

[0039] Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV, etc. DNA molecules can be inserted into vectors such that the transcriptional and translational control sequences in the vectors perform their intended function of regulating DNA transcription and translation. Expression vectors and expression control sequences can be selected to be compatible with the expression host cells used. DNA molecules can be introduced into expression vectors by standard methods (e.g., ligation of complementary restriction sites, or blunt-end ligation if no restriction sites are present).

[0040] In some aspects, the expression vector is a recombinant adeno-associated virus (AAV). In some embodiments, the AAV is selected from the group including the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, rAAV.rh8, rAAV.rhlO, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-l, AAV.hu37, rAAV.Anc80, rAAV.An c80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, A AV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 or AAV.HSC16.

[0041] In some aspects of the present invention, the vector or cassette can contain expression control sequences. As used in this description, the term "expression control sequence" refers to a polynucleotide sequence that is necessary to achieve the expression and processing of the inserted coding sequence. It will be understood by those skilled in the art that the design of the expression vector or cassette, including the selection of expression control sequences, may depend on factors such as the selection of the type of host cell to be transformed, the required level of protein expression, and the like. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion, if desired. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. Preferred expression control sequences for expression host cells in mammals include viral elements that ensure high levels of protein expression in mammalian cells, such as promoters and / or enhancers from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter / enhancer), simian virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), polyomavirus, and strong mammalian promoters such as the TTR promoter, the native immunoglobulin promoter, the actin promoter, and the HLP (hybrid liver-specific promoter). Expression control sequences encompass at least all components whose presence is important for expression and processing.

[0042] In addition to the genes and expression control sequences described above, the recombinant expression vectors of the invention can carry additional sequences, such as sequences that control replication of the vector in host cells (e.g., origins of replication) and selectable marker genes that facilitate selection of host cells into which the vector or cassette has been introduced.

[0043] In one aspect of the invention, an expression vector relates to a vector comprising one or more polynucleotide sequences of interest, a gene of interest, or a transgene flanked by parvovirus sequences or inverted terminal repeat (ITR) sequences.

[0044] None of the cassettes or vectors of the invention contain nucleotide sequences of the genes encoding the nonstructural (Rep) and structural (Cap) proteins of the adeno-associated virus. host cell In one aspect, the present invention relates to a host cell for producing a fusion protein based on FVIII-BDD and a heterologous signal peptide, comprising any one of the above-mentioned nucleic acids encoding a fusion protein based on FVIII-BDD and a heterologous signal peptide, or for producing any one of the above-mentioned expression vectors.

[0045] As used herein, the term "host cell" refers to a cell into which a recombinant expression vector or cassette according to the present invention has been introduced. The present invention relates to a host cell that can, for example, contain the vector according to the present invention. "Host cell" refers not only to a particular subject cell, but also to the progeny of such a cell. Such progeny may not actually be identical to the parent cell, since modifications due to either mutation or environmental influences may occur in successive generations; however, such cells are still included within the scope of the term "host cell" as used herein.

[0046] The expression vector or cassette according to the present invention can be used for transfection of mammalian cells, plant cells, bacteria or yeast host cells. Transfection can be carried out by any known technique for introducing polynucleotides into host cells. Methods for the introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, cationic polymer-nucleic acid complex transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells by viral (expression) vectors.

[0047] Mammalian cell lines used as hosts for transformation are well known in the art and include several immortalized cell lines available. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, FreeStyle293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, SK-HEP1, HUH7, Hep-RG and many other cell lines. Cell lines are selected by determining that the cell line has high expression levels and provides the required characteristics of the protein to be produced. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells. When the recombinant expression vector of the invention is introduced into a mammalian host cell, the fusion protein is produced by culturing the host cell for a period of time sufficient to express the fusion protein in the host cell or, more preferably, secrete the fusion protein into the culture medium in which the host cell is cultured. The fusion protein can be isolated from the culture medium using standard protein purification techniques. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include Escherichia and Streptomyces bacterial species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0048] The host cells do not refer to host cells generated using human embryos. The above host cells do not refer to host cells produced by altering the genetic integrity of human germline cells. Pharmaceutical Compositions In one aspect, the invention relates to a pharmaceutical composition for delivering the FVIII-BDD gene to a target cell, comprising any one of the above expression vectors or cassettes.

[0049] In some embodiments, a pharmaceutical composition for delivering the FVIII-BDD gene to a target cell comprises any one of the above expression vectors or cassettes in combination with one or more pharma- ceutically acceptable excipients.

[0050] The active agents in the compositions are present in effective amounts, for example, biologically effective amounts. "Pharmaceutical composition" means a composition comprising any one of the above expression vectors according to the present invention and at least one component selected from the group consisting of pharma- ceutically acceptable and pharmacologically compatible excipients, fillers, solvents, diluents, carriers, adjuvants, distribution agents, or delivery agents.

[0051] The pharmaceutical compositions of the present invention and methods for their preparation will be apparent to those skilled in the art. Pharmaceutical compositions should preferably be manufactured in accordance with GMP (Good Manufacturing Practice) requirements. In some embodiments of the pharmaceutical composition, the pharmaceutical composition may include a buffer composition, a tonicity agent (osmolyte or osmotic agent), a stabilizer, and / or a solubilizer.

[0052] "Pharmaceutically acceptable" means a substance that has no biological or other negative side effects, e.g., the substance can be administered to a subject without causing any undesirable biological effects. That is, such a pharmaceutical composition can be used, for example, in ex vivo transfection of cells or in vivo administration of any one of the above expression vectors of the present invention directly to a subject.

[0053] The term "excipient" is used herein to describe any other component of the present invention than the above-mentioned components. These are substances of inorganic or organic nature used in pharmaceutical production / manufacturing to impart the required physicochemical properties to the drug product.

[0054] The pharmaceutical compositions according to the present invention are stable compositions. A pharmaceutical composition is "stable" if the active agent retains its physical and / or chemical stability and / or biological activity during the stated shelf life at storage temperatures, e.g., 2-8°C. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. The shelf life is adjusted based on the results of stability testing under accelerated or natural aging conditions.

[0055] In some aspects, the pharmaceutical composition is a solution for intravenous administration. In some aspects, the pharmaceutical composition is a concentrate for preparation of a solution for intravenous administration. use In one aspect, the invention relates to the use of any one of the above expression cassettes or vectors or the above compositions for delivering the FVIII-BDD gene to a target cell.

[0056] In one aspect, the invention relates to the use of any one of the above expression cassettes or vectors or the above compositions to provide FVIII-BDD protein to a subject having hemophilia A and / or who does not have a functional copy of the FVIII gene.

[0057] The lack of a functional copy of the FVIII gene means an inactivating mutation or deletion in all copies of the FVIII gene in the genome, resulting in a loss or deficiency of function of the FVIII gene.

[0058] In one aspect, the present invention relates to a method for providing FVIII-BDD protein to a subject with hemophilia A, comprising the step of introducing a therapeutically effective amount of any one of the above expression vectors or compositions into cells of a subject in need thereof.

[0059] In one aspect, the invention relates to a method of delivering a FVIII-BDD gene to a target cell of a subject with hemophilia A, comprising the step of introducing into the cell of the subject any one of the above expression vectors or compositions.

[0060] A subject in need of delivery of the FVIII-BDD gene to a target cell or a subject in need of being provided with the FVIII-BDD protein means a subject with hemophilia A, or a subject with a deficiency in the coagulation factor FVIII, or a subject with an inactivating mutation or deletion in the FVIII gene that results in a loss or deficiency in the function of the FVIII gene.

[0061] In one aspect, the invention relates to the use of any one of the above expression vectors or the above compositions for treating hemophilia A in a subject having hemophilia A. In one aspect, the invention relates to a method for treating hemophilia A in a subject comprising administering to a subject having hemophilia A a therapeutically effective amount of any one of the above expression vectors or compositions.

[0062] In some aspects, hemophilia A is severe hemophilia A (less than 1% factor VIII activity) or moderate hemophilia A (1-5% factor VIII activity). Exemplary modes of administration include topical application, intranasal, inhalation, transmucosal, transdermal, enteral (e.g., oral, rectal), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular) administration, and direct tissue or organ injection.

[0063] In some aspects of the uses, the expression vector or any one of the compositions is administered to the subject as an intravenous infusion. Any one of the above expression vectors is administered to an organism in an effective amount. Any one of the above expression vectors is preferably administered to an organism in a biologically effective amount. The "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. When an expression vector is administered to a cell in vivo, the "biologically effective" amount of an expression vector is an amount that is sufficient to cause transduction of a target cell and expression of a nucleic acid sequence in the target cell.

[0064] The dosage of any one of the above expression vectors according to the invention will depend on the mode of administration of the particular vector and can be determined in a routine manner. Cells for administering any one of the above expression cassettes or vectors according to the invention can be any type of cell, including, but not limited to, epithelial cells (e.g., skin, respiratory and intestinal epithelial cells), liver cells, muscle cells, pancreatic cells (including islet cells), liver cells, spleen cells, fibroblasts, endothelial cells, etc.

[0065] Any one of the above expression cassettes or vectors according to the present invention is not used to modify the genetic integrity of human germline cells. In some embodiments of use, any one of the above expression vectors according to the present invention, as well as products based thereon, are used as a monotherapy.

[0066] In some embodiments of use, any one of the above expression vectors according to the present invention, as well as products based thereon, are used in combination with replacement therapy using clotting factor concentrates, desmopressin and / or fibrinolytic inhibitors.

[0067] In some embodiments of use, any one of the above expression vectors according to the invention, as well as products based thereon, are used in combination with a monoclonal antibody (eg, emicizumab).

[0068] In some embodiments of use, any one of the above expression vectors according to the present invention, as well as therapeutic agents based thereon, are used in combination with an RNA interference therapeutic agent (eg, Fitusiran).

[0069] In some embodiments of use, any one of the above expression vectors according to the invention, as well as products based thereon, are administered once to a subject. In some embodiments of use, any one of the above expression vectors according to the present invention, as well as products based thereon, are repeatedly administered to a subject. [Brief description of the drawings]

[0070] [Figure 1]1 is a graph showing the increased level of FVIII-BDD protein production into the culture medium after transfection with a nucleic acid encoding a fusion protein based on FVIII-BDD (B-domain deleted coagulation factor VIII) and one of the heterologous signal peptides, as compared to a nucleic acid encoding FVIII-BDD containing the wild-type FVIII signal peptide. 1 is the FVIII-BDD protein level after cell transfection with a nucleic acid encoding a fusion protein based on human FVIII-BDD and the naturally occurring FVIII signal peptide (SP-FVIII-FVIII-BDD) having the amino acid sequence of SEQ ID NO: 6. 2 is the FVIII-BDD protein level after cell transfection with a nucleic acid encoding a fusion protein based on FVIII-BDD and the FIX signal peptide (SP-FIX-FVIII-BDD) having the amino acid sequence of SEQ ID NO: 7. 3 is the FVIII-BDD protein level after cell transfection with a nucleic acid encoding a fusion protein based on human FVIII-BDD and lactalbumin signal peptide (SP-lactalbumin-FVIII-BDD) having the amino acid sequence of SEQ ID NO: 9. 4 is the FVIII-BDD protein level after cell transfection with a nucleic acid encoding a fusion protein based on human FVIII-BDD and immunoglobulin G kappa chain signal peptide (SP-IgGK-FVIII-BDD) having the amino acid sequence of SEQ ID NO: 8. [Diagram 2]2 is a graph showing the increase in the level of FVIII-BDD protein activity in the culture medium after transfection with a nucleic acid encoding a fusion protein based on FVIII-BDD (B-domain deleted coagulation factor VIII) and one of the heterologous signal peptides, as compared to a nucleic acid encoding FVIII-BDD containing the wild-type FVIII signal peptide. 1 is the FVIII-BDD activity level after cell transfection with a nucleic acid encoding a fusion protein based on human FVIII-BDD and a naturally occurring FVIII signal peptide (SP-FVIII-FVIII-BDD) having the amino acid sequence of SEQ ID NO: 6. 2 is the FVIII-BDD activity level after cell transfection with a nucleic acid encoding a fusion protein based on FVIII-BDD and a FIX signal peptide (SP-FIX-FVIII-BDD) having the amino acid sequence of SEQ ID NO: 7. 3 is the FVIII-BDD activity level after cell transfection with a nucleic acid encoding a fusion protein based on human FVIII-BDD and lactalbumin signal peptide (SP-lactalbumin-FVIII-BDD) having the amino acid sequence of SEQ ID NO: 9. 4 is the FVIII-BDD activity level after cell transfection with a nucleic acid encoding a fusion protein based on human FVIII-BDD and immunoglobulin G kappa chain signal peptide (SP-IgGK-FVIII-BDD) having the amino acid sequence of SEQ ID NO: 8. [Diagram 3]3 is a graph showing the increased level of FVIII-BDD protein production when nucleic acid is delivered in vitro in the form of a rAAV expression vector containing a nucleic acid encoding a fusion protein based on FVIII-BDD and one of the heterologous signal peptides, compared to a rAAV expression vector containing a nucleic acid encoding a FVIII-BDD containing a wild-type FVIII signal peptide. 1 is the FVIII-BDD protein level after cell transduction with an expression vector containing a nucleic acid encoding a fusion protein based on human FVIII-BDD and a naturally occurring FVIII signal peptide (SP-FVIII-FVIII-BDD) having the amino acid sequence of SEQ ID NO: 6. 2 is the FVIII-BDD protein level after cell transduction with an expression vector containing a nucleic acid encoding a fusion protein based on FVIII-BDD and a FIX signal peptide (SP-FIX-FVIII-BDD) having the amino acid sequence of SEQ ID NO: 7. 3 is the FVIII-BDD protein level after cell transduction with an expression vector containing a nucleic acid encoding a fusion protein based on human FVIII-BDD and lactalbumin signal peptide (SP-lactalbumin-FVIII-BDD) having the amino acid sequence of SEQ ID NO:9. [Figure 4]4 is a graph showing the increased level of FVIII-BDD protein activity when nucleic acid is delivered in vitro in the form of a rAAV expression vector containing a nucleic acid encoding a fusion protein based on FVIII-BDD and one of the heterologous signal peptides, compared to a rAAV expression vector containing a nucleic acid encoding a FVIII-BDD containing a wild-type FVIII signal peptide. 1 is the FVIII-BDD activity level after cell transduction with an expression vector containing a nucleic acid encoding a fusion protein based on human FVIII-BDD and a naturally occurring FVIII signal peptide (SP-FVIII-FVIII-BDD) having the amino acid sequence of SEQ ID NO: 6. 2 is the FVIII-BDD activity level after cell transduction with an expression vector containing a nucleic acid encoding a fusion protein based on FVIII-BDD and a FIX signal peptide (SP-FIX-FVIII-BDD) having the amino acid sequence of SEQ ID NO: 7. 3 is the FVIII-BDD activity level after cell transduction with an expression vector containing a nucleic acid encoding a fusion protein based on human FVIII-BDD and lactalbumin signal peptide (SP-lactalbumin-FVIII-BDD) having the amino acid sequence of SEQ ID NO:9. [Diagram 5]5 is a graph showing the increase in the level of FVIII-BDD protein when a nucleic acid encoding a fusion protein based on FVIII-BDD and one of the heterologous signal peptides is delivered in vivo to B6.129S-F8tm1Smoc(HemA) mice in the form of an rAAV expression vector. 1 is the FVIII-BDD protein level in the plasma of the animal after injection of a control solution (negative control) that does not contain AAV. 2 is the FVIII-BDD protein level in the plasma of the animal after injection of an expression vector containing a nucleic acid encoding a fusion protein based on FVIII-BDD and FIX signal peptide (SP-FIX-FVIII-BDD) having the amino acid sequence of SEQ ID NO: 7. 3 is the FVIII-BDD protein level in the plasma of the animal after injection of an expression vector containing a nucleic acid encoding a fusion protein based on human FVIII-BDD and lactalbumin signal peptide (SP-lactalbumin-FVIII-BDD) having the amino acid sequence of SEQ ID NO: 9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] EXAMPLES

[0072] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any manner.

[0073] All publications, patents, and patent applications cited herein are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the accompanying embodiments. Materials and General Methods Recombinant DNA Technology Standard methods were used to manipulate DNA 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 manufacturer's protocols. Briefly, plasmid DNA was generated for further manipulation in E. coli cells grown under selective antibiotic pressure so that the plasmid was not lost in the cell population. We isolated plasmid DNA from cells using a commercially available kit, measured the concentration, and used it for cloning by restriction endonuclease treatment or PCR amplification. DNA fragments were ligated together using ligase and transformed into bacterial cells for clone selection and further generation. All resulting gene constructs were confirmed by restriction patterns and complete Sanger sequencing. Gene synthesis The desired gene segments were prepared from oligonucleotides produced by chemical synthesis. Gene fragments of 300-1000 bp in length flanked by unique restriction sites were collected by refolding overlapping oligonucleotides, followed by PCR amplification from border primers. As a result, a mixture of fragments containing the desired. The fragments were cloned at the restriction sites into an intermediate vector, and the DNA sequences of the subcloned fragments were then confirmed by DNA sequencing. DNA sequencing DNA sequences were determined by Sanger sequencing. DNA and protein sequences were analyzed and sequence data were processed in SnapGene Viewer 4.2 or higher for sequence generation, mapping, analysis, annotation and illustration. Cultivation of cell cultures The experiments used the following cell lines: HEK293 (human embryonic kidney clone 293), HUH7 (human hepatocellular carcinoma cell line) and HepG2 (human hepatocellular carcinoma cell line). Suspension HEK293 cells used to generate AAV were cultured under standard conditions at 37 °C and 5% CO2 in complete culture medium without FBS and antibiotics. Adherent HUH7 and HepG2 cells used to test the efficacy of AAV products were cultured under standard conditions at 37 °C and 5% CO2 in complete DMEM medium supplemented with 10% FBS, antibiotics / antimycotics. HUH7 and HepG2 cells were passaged when they reached 80-90% confluence. TrypLE Select enzyme (10x) was used to disperse the cell monolayer. Cell viability was assessed using trypan blue staining and a disposable cell counting chamber using an automated Countess II counter. Transfection of cell cultures To evaluate the functionality of the new variants of the fusion protein after transfection, we used plasmids containing expression cassettes for the expression of different variants of the hFVIII-BDD transgene. The HepG2 cell line was cultured at 10,000 cells / cm. 2 The cells were pre-seeded into the wells of a 12-well plate at a density of 1000 μg / ml. After one day, the plasmids with the same copy number were introduced into the complexes using Lipofectamine3000. On the seventh day after transfection, the level and activity of FVIII-BDD protein in the culture medium were determined by ELISA and chromogenic assay. The study involving the evaluation of the level and activity of FVIII-BDD protein in the culture medium was carried out in six independent experiments. Intact HepG2 cells were used as a negative control. Assembly and purification of AAV-based expression vectors To assemble the AAV expression vector containing the codon-optimized mutant of the FVIII-BDD gene, we used HEK293 producer cells transfected with three plasmids as follows: 1) Plasmids containing AAV expression cassettes for expressing different variants of the hFVIII-BDD transgene; 2) A plasmid for expressing the AAV6 serotype Cap gene and the AAV2 serotype Rep gene, with alternative reading frames, each gene encoding several protein products; 3) A plasmid for expressing the adenovirus Ad2 genes required for AAV capsid assembly and packaging.

[0074] After 72 hours, the cells were lysed and the particles were purified and concentrated using filtration, chromatography and ultracentrifugation. The titers of the particles were determined by quantitative PCR using primers and samples specific for regions of the recombinant viral genome and expressed as copies of the viral genome per mL. Transduction of cell cultures HUH7 cell line was cultured at 10,000 cells / cm 2 The cells were pre-seeded into wells of a 12-well plate at a density of 100,000 vg / cell. After the cells attached to the adhesive substrate, the AAV preparation was transduced at an MOI of 500,000 vg / cell. Seven days after transduction, the levels and activity of FVIII-BDD protein in the culture medium were determined by ELISA and chromogenic assay. The studies involving the evaluation of the levels and activity of FVIII-BDD protein in the culture medium were performed in six independent experiments. Intact cells were used as a negative control. Determination of the amount of coagulation factor VIII-BDD protein by ELISA The content of blood coagulation factor VIII-BDD protein in the culture medium after HepG2 cell line transfection and HUH7 cell line transduction, and in the plasma after injection of animals (mice) with target candidates, was evaluated by the sandwich method of non-competitive enzyme-linked immunosorbent assay (ELISA). Briefly, samples diluted in dilution buffer were introduced into 96-well plate wells sensitized with a primary antibody specific for coagulation factor VIII-BDD. The same plate was loaded with standards, controls for plotting a calibration curve. The plate was incubated at a temperature of 37°C for 1 hour. Prior to the introduction of a solution of biotinylated antibody, streptavidin peroxidase conjugate and TMB, the plate wells were washed with a washing buffer. A solution containing a biotinylated detection antibody specific for factor VIII-BDD was introduced and the plate was incubated at a temperature of 37°C for 30 minutes. Subsequently, a streptavidin peroxidase conjugate solution was added to the resulting complex and the plate was incubated at a temperature of 37°C for 30 minutes. To visualize the enzymatic reaction, TMB solution was introduced. When the required degree of staining intensity was reached, a stop solution was added to all wells to stop the reaction. The optical density of the solution in the plate wells was then measured. The concentration of coagulation factor VIII-BDD in the test samples was determined by a calibration curve, taking into account the preliminary dilution of the samples. Determination of activity levels of coagulation factor VIII-BDD protein by ELISA The activity of coagulation factor VIII protein in the culture medium after transfection of HepG2 cells with target candidates and transduction of HUH7 cells was evaluated by a chromogenic assay. The assay is based on the fact that in the presence of calcium ions, phospholipids and factor IXa, factor X is converted to the activated form Xa, factor VIII functions as a cofactor in the reaction, and the rate of factor X activation is linearly related to the level of factor VIII. Briefly, culture medium samples diluted in dilution buffer, standards and controls for plotting a calibration curve were introduced into the wells of a 96-well plate. The plate was incubated at a temperature of 37° C. for 3 minutes. A factor reagent solution containing factor IXa, factor X, thrombin, CaCl2 and phospholipids was introduced into all wells of the plate. The plate was incubated at a temperature of 37° C. for 4 minutes. A solution of the chromogenic substrate S-2765+I-2581 was introduced into all wells of the plate. The plate was incubated at a temperature of 37° C. for 7 minutes. When the required degree of staining intensity was achieved, a 20% solution of acetic acid was added to all wells to stop the reaction.The optical density of the solution in the plate wells was then measured.The activity of coagulation factor VIII in the test samples was determined by a calibration curve, taking into account the preliminary dilution of the samples. In vivo studies on laboratory animals FVIII-deficient B6.129S-F8tm1Smoc(HemA) mice (male, 6-8 weeks old) were used for the experiments. The products were administered to the animals using a single intravenous injection into the tail vein. A buffer solution without AAV was administered to a negative control group of animals. Plasma samples were taken on the day of injection before administration of the products, followed by days 14 and 56 after introduction of the expression vector. Statistical Data Analysis Results are presented as mean ± standard deviation (SD), and experimental results were compared using one-way analysis of variance (ANOVA) followed by Dunnett's multiple pairwise comparisons and determined to be statistically significant.

[0075] Example 1 To increase the level of secretion of the protein FVIII-BDD (B-domain deleted coagulation factor VIII), the sequence of SEQ ID NO: 6 (SP-FVIII-BDD) was modified by replacing the sequence of the wild-type FVIII signal peptide by the signal peptide corresponding to the amino acid sequence defined in SEQ ID NO: 2 (SP-FIX) or SEQ ID NO: 3 (SP-IgGK) or SEQ ID NO: 4 (SP-lactalbumin), resulting in the generation of fusion proteins corresponding to the amino acid sequences of SEQ ID NO: 7 (SP-FIX-FVIII-BDD), SEQ ID NO: 8 (SP-IgGK-FVIII-BDD) and SEQ ID NO: 9 (SP-lactalbumin-FVIII-BDD).

[0076] The resulting nucleic acid encoding a fusion protein based on one of the FVIII-BDD and heterologous signal peptides, comprising an amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, was tested during in vitro cell transfection in an expression cassette consisting of a left (first) ITR (inverted terminal repeat) corresponding to the sequence of SEQ ID NO: 14, an HLP promoter (SEQ ID NO: 15), a gene of interest, a polyadenylation signal (SEQ ID NO: 16), a right (second) ITR (SEQ ID NO: 17), the gene of interest being one of the sequences of SEQ ID NO: 10 to 13. As a control, the inventors used a nucleic acid encoding a human FVIII-BDD protein (SP-FVIII-FVIII-BDD) comprising a naturally occurring FVIII-BDD signal peptide corresponding to the sequence of SEQ ID NO: 10.

[0077] The use of all nucleic acids SEQ ID NO: 11-13 encoding fusion proteins based on FVIII-BDD and one of the heterologous signal peptides, having sequences SEQ ID NO: 7-9, resulted in increased levels of FVIII-BDD protein production (Figure 1) and activity (Figure 2), when compared to the use of nucleic acid SEQ ID NO: 10 encoding FVIII-BDD protein (SP-FVIII-FVIII-BDD) containing the naturally occurring peptide of SEQ ID NO: 6. Furthermore, a surprising increase in the level of FVIII-BDD protein production in the culture medium resulting after transfection was shown by sequence SEQ ID NO: 11 (SP-FIX-FVIII-BDD) with a 4.3-fold increase, by sequence SEQ ID NO: 12 (SP-IgGK-FVIII-BDD) with a 1.3-fold increase, and by sequence SEQ ID NO: 13 (SP-lactalbumin-FVIII-BDD) with a 4.9-fold increase, when compared to the use of nucleic acid SEQ ID NO: 10 (SP-FVIII-FVIII-BDD). The inventors observed similar results in FVIII-BDD activity in the culture medium resulting after transfection: a surprising increase was observed for the nucleic acids of SEQ ID NO: 11 (SP-FIX-FVIII-BDD) (8.6-fold increase), SEQ ID NO: 12 (SP-IgGK-FVIII-BDD) (1.9-fold increase) and SEQ ID NO: 13 (SP-lactalbumin-FVIII-BDD) (7.2-fold increase) when compared to the nucleic acid of SEQ ID NO: 10 (SP-FVIII-FVIII-BDD). The correspondence observed between the FVIII-BDD protein production and activity results indicates that replacement of the naturally occurring FVIII-BDD signal peptide with a signal peptide corresponding to the amino acid sequence of SEQ ID NO: 2 (SP-FIX), or SEQ ID NO: 3 (SP-IgGK), or SEQ ID NO: 4 (SP-lactalbumin) results in a significant increase in FVIII-BDD protein production and activity, and does not affect FVIII-BDD protein functionality.

[0078] That is, the resulting nucleic acid encoding a fusion protein based on FVIII-BDD and one of the heterologous signal peptides, comprising an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, is capable of causing in vitro FVIII-BDD protein expression in a host cell and has a high potential for the production of recombinant FVIII-BDD protein in producer cells for therapy of hemophilia A.

[0079] Example 2 The inventors have generated rAAV expression vectors comprising the nucleic acids of SEQ ID NO: 11-13 encoding fusion proteins based on FVIII-BDD and heterologous signal peptides, comprising amino acid sequences selected from SEQ ID NO: 7-9. The expression vectors were validated by in vitro transduction of HUH7 cells. As a control, the inventors used a rAAV expression vector comprising the nucleic acid of SEQ ID NO: 10 encoding a human FVIII-BDD protein (SP-FVIII-FVIII-BDD) containing the naturally occurring FVIII signal peptide, corresponding to the sequence of SEQ ID NO: 6.

[0080] The use of expression vectors containing nucleic acids of SEQ ID NO: 11-13 resulted in increased levels of FVIII-BDD protein production (Figure 3) and activity (Figure 4) when compared to the use of an expression vector containing nucleic acid of SEQ ID NO: 10 (SP-FVIII-FVIII-BDD). Furthermore, a surprising increase in the level of FVIII-BDD protein production in the culture medium resulting after transduction was shown by the expression vector containing the sequence of SEQ ID NO: 11 (SP-FIX-FVIII-BDD) with a 2.7-fold increase, and the expression vector containing the sequence of SEQ ID NO: 13 (SP-lactalbumin-FVIII-BDD) with a 2.4-fold increase, when compared to the use of an expression vector containing nucleic acid of SEQ ID NO: 10 (SP-FVIII-FVIII-BDD). The inventors observed similar results in FVIII-BDD activity in the culture medium resulting after transduction. The inventors observed a surprising increase for expression vectors containing the nucleic acids of SEQ ID NO: 11 (SP-FIX-FVIII-BDD) (3.9-fold increase) and SEQ ID NO: 13 (SP-lactalbumin-FVIII-BDD) (3.5-fold increase) when compared to expression vectors containing the nucleic acid of SEQ ID NO: 10 (SP-FVIII-FVIII-BDD). The results are in agreement with the data generated by transfecting HepG2 cells.

[0081] That is, the developed rAAV expression vector encoding a fusion protein based on FVIII-BDD and one of the heterologous signal peptides, comprising an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, is capable of causing FVIII-BDD protein expression in vitro.

[0082] Example 3 To carry out in vivo studies of rAAV expression vectors containing a nucleic acid selected from SEQ ID NO: 11 or SEQ ID NO: 13 encoding a fusion protein based on one of the FVIII-BDD and heterologous signal peptides, containing an amino acid sequence selected from SEQ ID NO: 7 or SEQ ID NO: 9, the inventors used FVIII-deficient B6.129S-F8tm1Smoc(HemA) laboratory mice. The dose of rAAV product used in the studies was 6×10 13 vg / kg. A control solution without AAV was used as a negative control. The products were administered to the animals using a single intravenous hydrodynamic administration into the tail vein. Plasma sampling was performed on the day of injection before administration of the product, followed by 14 and 56 days after introduction of the product. The levels of coagulation factor VIII-BDD protein in the plasma samples were determined by ELISA as described above.

[0083] In vivo studies (FIG. 5) showed that the use of both rAAV expression vectors containing a nucleic acid selected from SEQ ID NO: 11 or SEQ ID NO: 13 showed a significant increase in the levels of factor VIII-BDD in the plasma of animals at days 14 and 56 (FIG. 5). When using an expression vector containing a nucleic acid of SEQ ID NO: 11, the inventors observed FVIII-BDD expression at 296 and 504 ng / mL in the plasma of animals at days 14 and 56, respectively, after injection. When using an expression vector containing a nucleic acid of SEQ ID NO: 13, the inventors observed FVIII-BDD expression at 270 and 381 ng / mL in the plasma of animals at days 14 and 56, respectively, after injection.

[0084] In other words, the developed rAAV expression vector comprising a nucleic acid selected from SEQ ID NO: 11 or SEQ ID NO: 13 encoding a fusion protein based on FVIII-BDD and one of the heterologous signal peptides, comprising an amino acid sequence selected from SEQ ID NO: 7 or SEQ ID NO: 9, is capable of causing FVIII-BDD protein expression in vivo and has high potential for gene therapy of hemophilia A.

Claims

1. Isolated nucleic acids encoding a fusion protein based on FVIII-BDD (B-domain deletion coagulation factor VIII) and a heterologous signal peptide, comprising an amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:

9.

2. The isolated nucleic acid according to claim 1, wherein the fusion protein based on FVIII-BDD and a heterologous signal peptide has the amino acid sequence of SEQ ID NO:

7.

3. The isolated nucleic acid according to claim 1, wherein the fusion protein based on FVIII-BDD and a heterologous signal peptide has the amino acid sequence of SEQ ID NO:

8.

4. The isolated nucleic acid according to claim 1, wherein the fusion protein based on FVIII-BDD and a heterologous signal peptide has the amino acid sequence of SEQ ID NO:

9.

5. The isolated nucleic acid according to claim 2, wherein the nucleotide sequence is sequence number 11.

6. The isolated nucleic acid according to claim 3, which is the nucleotide sequence of sequence number 12.

7. The isolated nucleic acid according to claim 4, wherein the nucleotide sequence is sequence number 13.

8. An expression cassette comprising the nucleic acid according to any one of claims 1 to 7.

9. The following elements are located in the direction from the 5' end to the 3' end: Left (first) ITR (inverted terminal repeat); promoter; The nucleic acid according to any one of claims 1 to 7; Polyadenylation signal; Right side (2nd) ITR An expression cassette according to claim 8, including the following:

10. An expression vector comprising a nucleic acid according to any one of claims 1 to 7, or an expression cassette containing the nucleic acid according to any one of claims 1 to 7.

11. The expression vector according to claim 10, wherein the vector is a recombinant adeno-associated virus (AAV).

12. The AAV has the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, rAAV. rh8, rAAV. rhlO, rAAV. rh20, rAAV. rh39, rAAV. Rh74, rAAV. RHM4-l, AAV. hu37, rAAV. Anc80, rAAV. Anc80L65, rAAV. 7m8, rAAV. PHP. An expression vector according to claim 11, selected from the group comprising 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.

13. A host cell for generating a fusion protein based on FVIII-BDD and a heterologous signal peptide, comprising the nucleic acid described in any one of claims 1 to 7.

14. A pharmaceutical composition for delivering the FVIII-BDD gene to target cells, comprising the expression vector according to claim 10 in combination with one or more pharmaceutically acceptable excipients.

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