Codon-optimized nucleic acid encoding FVIII-BDD

Abstract

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 encoding FVIII-BDD (B-domain deleted coagulation factor VIII) protein, an expression cassette and vector based thereon, a host cell for producing FVIII-BDD, and 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 codon-optimized nucleic acid encoding FVIII-BDD (B-domain deleted coagulation factor VIII) protein, an expression cassette and vector based thereon, a host cell for producing FVIII-BDD, and 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] One of the pressing research objectives in the area of ​​development of effective gene therapy is the codon optimization of the gene of interest in the vector to achieve maximum levels of production of the protein of interest, which in turn allows the use of relatively low doses of the vector to achieve a significant effect.

[0010] The authors of the present invention have surprisingly found that a codon-optimized nucleic acid encoding a FVIII-BDD (B-domain deleted coagulation factor VIII) protein having the amino acid sequence of SEQ ID NO: 11 and comprising a nucleotide sequence selected from the group of SEQ ID NO: 2 (mutant FVIII-BDDco-v1), SEQ ID NO: 3 (mutant FVIII-BDDco-v2), SEQ ID NO: 4 (mutant FVIII-BDDco-v3) or SEQ ID NO: 5 (mutant FVIII-BDDco-v4) surprisingly causes an increased level of FVIII-BDD gene expression with a fold increase, an increased level of coagulation factor VIII-BDD protein production as well as an increased level of coagulation factor VIII-BDD protein activity when compared to the wild-type gene encoding coagulation factor VIII-BDD (FVIII-BDD-wt). These variants of the codon-optimized nucleic acids according to the invention having a nucleotide sequence selected from the group of SEQ ID NO: 2 (mutant FVIII-BDDco-v1), SEQ ID NO: 3 (mutant FVIII-BDDco-v2), SEQ ID NO: 4 (mutant FVIII-BDDco-v3) or SEQ ID NO: 5 (mutant FVIII-BDDco-v4) are comprised in expression cassettes and vectors based thereon. 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] "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.

[0019] 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 codon-optimized nucleic acid encoding a FVIII-BDD (B-domain deleted coagulation factor VIII) protein having the amino acid sequence of SEQ ID NO:11 and comprising a nucleotide sequence selected from the group of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0020] 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.

[0021] One of the properties of the genetic code is degeneracy, i.e. the ability of different codons (trinucleotides) to code for the same amino acid. Such codons that are translated into the same amino acid are called synonymous codons. In natural sequences, one of the synonymous codons is randomly selected during evolution, but the frequency of use of the synonymous codons is different: each amino acid has a more favored codon and a less favored codon. Codon optimization is a widely known technique for amplifying the production of protein molecules, providing a rational mapping of one of the preferred synonymous codons for each amino acid in a protein sequence. While one of the general principles of codon optimization involves the use of the most frequent codons, other approaches such as harmonization (reproducing the distribution of codon usage) have been introduced since, but these do not always increase productivity. In addition to the codon frequency, the GC content of the sequence (ratio of guanine and cytosine to the total length of the sequence) can affect the production efficiency, and in particular, high GC content has been shown to be associated with increased mRNA levels in mammalian cells (Grzegorz Kudla ET AL. High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells, June 2006, Vol. 4, No. 6, e180, pp. 933-942). It is further noteworthy that stable secondary structure elements of mRNA, i.e., those with low free holding energy, can reduce the efficiency.

[0022] Different codon-optimized variants of the sequence of a gene of interest can lead to the following (when compared to the wild-type gene): a) the expression level of the gene of interest will be slightly increased; b) the expression level of the gene of interest will be significantly increased; c) expression levels of the gene of interest will remain at roughly the same level; d) The expression level of the gene of interest will be reduced.

[0023] The codon-optimized nucleic acid according to the present invention was generated by codon-optimizing a wild-type nucleic acid having the nucleotide sequence of SEQ ID NO:1. As a result of codon optimization of the wild-type nucleic acid encoding the FIX-BDD protein having the nucleotide sequence of SEQ ID NO:1, a number of codon-optimized nucleic acids were generated, which were further tested for levels of protein production compared to the control FVIII-BDD-wt (wild-type nucleic acid having SEQ ID NO:1).

[0024] All codon-optimized nucleic acids caused an increase in the level of FVIII-BDD protein production when compared to the wild-type nucleic acid; moreover, many codon-optimized nucleic acids caused a slight increase in the level of FVIII-BDD protein production when compared to the wild-type nucleic acid. Next, the codon-optimized nucleic acids according to the invention selected from the group of SEQ ID NO: 2 (mutant FVIII-BDDco-v1), SEQ ID NO: 3 (mutant FVIII-BDDco-v2), SEQ ID NO: 4 (mutant FVIII-BDDco-v3) or SEQ ID NO: 5 (mutant FVIII-BDDco-v4) surprisingly caused an increase in the level of FVIII-BDD gene expression with a fold increase, an increase in the level of coagulation factor VIII-BDD protein production, as well as an increase in the level of coagulation factor VIII-BDD protein activity when compared to the wild-type gene (FVIII-BDD-wt). Expression cassette.Expression vector In one aspect, the invention relates to an expression cassette comprising any one of the above codon-optimized nucleic acids encoding a FVIII-BDD protein.

[0025] 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.

[0026] 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.

[0027] 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 the 3' end: Left side (first) ITR (inverted terminal repeat); promoter; a nucleic acid encoding a signal peptide; Any one of the above codon-optimized nucleic acids encoding a FVIII-BDD protein; polyadenylation signal; Right side (2nd) ITR.

[0028] 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.

[0029] In some embodiments, the expression cassette comprises a left (first) ITR having the nucleotide sequence of SEQ ID NO:13. In some embodiments, the expression cassette comprises an HLP promoter having the nucleotide sequence of SEQ ID NO:14.

[0030] In some embodiments, the expression cassette comprises a polyadenylation signal having the nucleotide sequence of SEQ ID NO:15. In some embodiments, the expression cassette comprises a right (second) ITR having the nucleotide sequence of SEQ ID NO:16.

[0031] In some embodiments, the nucleic acid encoding the signal peptide has a nucleotide sequence selected from the group of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

[0032] In some embodiments, the codon-optimized nucleic acid encoding the FVIII-BDD protein including the signal peptide (FVIII-BDDco-v1 variant) has the nucleotide sequence of SEQ ID NO:7.

[0033] In some embodiments, the codon-optimized nucleic acid encoding the FVIII-BDD protein including the signal peptide (FVIII-BDDco-v2 variant) has the nucleotide sequence of SEQ ID NO:8.

[0034] In some embodiments, the codon-optimized nucleic acid encoding the FVIII-BDD protein including the signal peptide (FVIII-BDDco-v3 variant) has the nucleotide sequence of SEQ ID NO:9.

[0035] In some embodiments, the codon-optimized nucleic acid encoding the FVIII-BDD protein including the signal peptide (FVIII-BDDco-v4 variant) has the nucleotide sequence of SEQ ID NO:10.

[0036] In some embodiments, the expression cassette comprises the following elements from the 5' to the 3' end: The left (first) ITR (inverted terminal repeat) having the nucleotide sequence of SEQ ID NO: 13; A promoter having the nucleotide sequence of SEQ ID NO: 14; a nucleic acid encoding a signal peptide having a nucleotide sequence selected from the group of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21; Any one of the above codon-optimized nucleic acids encoding a FVIII-BDD protein; a polyadenylation signal having the nucleotide sequence of SEQ ID NO: 15; The right (second) ITR having the nucleotide sequence of SEQ ID NO:16.

[0037] In some embodiments, the expression cassette comprises the following elements from the 5' to the 3' end: The left (first) ITR (inverted terminal repeat) having the nucleotide sequence of SEQ ID NO: 13; A promoter having the nucleotide sequence of SEQ ID NO: 14; a codon-optimized nucleic acid encoding a FVIII-BDD protein comprising a signal peptide, having a nucleotide sequence selected from the group of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; a polyadenylation signal having the nucleotide sequence of SEQ ID NO: 15; The right (second) ITR having the nucleotide sequence of SEQ ID NO:16.

[0038] In one aspect, the present invention relates to an expression vector comprising any one of the above codon-optimized nucleic acids encoding a FVIII-BDD protein or any one of the above expression cassettes.

[0039] 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.

[0040] 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").

[0041] 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).

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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 FVIII-BDD protein, comprising any one of the above codon-optimized nucleic acids encoding a FVIII-BDD protein, or for producing any one of the above expression vectors.

[0047] 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.

[0048] 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.

[0049] 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 present invention is introduced into a mammalian host cell, the FVIII-BDD 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] "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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] In one aspect, the 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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 a monoclonal antibody (eg, emicizumab).

[0070] 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).

[0071] 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]

[0072] [Figure 1] 1 is a diagram of the plasmid pAAV-hFVIII-BDD, which contains a 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 further includes the following elements: AmpR is a β-lactamase gene that provides resistance to ampicillin; pUC origin is the pUC origin of replication in bacteria; ITR is an inverted terminal repeat; HLP promoter is a synthetic tissue-specific promoter; sPA is a synthetic polyadenylation signal sequence to increase mRNA stability. [Diagram 2] FIG. 2 is a graph showing increased levels of FVIII-BDD protein production in culture medium following transfection with a codon-optimized nucleic acid encoding a FVIII-BDD (B-domain deleted coagulation factor VIII) protein compared to a wild-type nucleic acid encoding the FVIII-BDD protein.

[0073] FVIII-BDD protein levels after cell transfection: 1--Using a wild-type nucleic acid encoding the FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:1.

[0074] 2--Using a codon-optimized nucleic acid encoding the FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:2. 3--Using a codon-optimized nucleic acid encoding the FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:3.

[0075] 4--Using a codon-optimized nucleic acid encoding the FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:4. 5--Using a codon-optimized nucleic acid encoding a FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:5. [Diagram 3] FIG. 3 is a graph showing increased levels of FVIII-BDD protein activity in culture medium following transfection with a codon-optimized nucleic acid encoding FVIII-BDD protein (B-domain deleted coagulation factor VIII) compared to a wild-type nucleic acid encoding the FVIII-BDD protein.

[0076] FVIII-BDD activity levels after cell transfection: 1--Using a wild-type nucleic acid encoding the FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:1.

[0077] 2--Using a codon-optimized nucleic acid encoding the FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:2. 3--Using a codon-optimized nucleic acid encoding the FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:3.

[0078] 4--Using a codon-optimized nucleic acid encoding the FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:4. 5--Using a codon-optimized nucleic acid encoding a FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:5. [Figure 4]FIG. 4 is a graph showing increased levels of FVIII-BDD protein production upon in vitro delivery of nucleic acid in the form of an AAV expression vector containing a codon-optimized nucleic acid encoding FVIII-BDD protein (B-domain deleted coagulation factor VIII) compared to an AAV expression vector containing a wild-type nucleic acid encoding the FVIII-BDD protein.

[0079] FVIII-BDD protein levels after cell transduction: 1 - control solution without AAV (negative control). 2- An AAV serotype 6 expression vector comprising a codon-optimized nucleic acid encoding the FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:2.

[0080] 3- An AAV serotype 6 expression vector comprising a codon-optimized nucleic acid encoding a FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:5. 4 - An AAV serotype 5 expression vector comprising a codon-optimized nucleic acid encoding a FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:2. [Diagram 5] FIG. 5 is a graph showing increased levels of FVIII-BDD protein activity upon in vitro delivery of nucleic acid in the form of an AAV expression vector comprising a codon-optimized nucleic acid encoding FVIII-BDD protein (B-domain deleted coagulation factor VIII) compared to an AAV expression vector comprising a wild-type nucleic acid encoding the FVIII-BDD protein.

[0081] FVIII-BDD activity levels after cell transduction: 1 - control solution without AAV (negative control). 2- An AAV serotype 6 expression vector comprising a codon-optimized nucleic acid encoding the FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:2.

[0082] 3- An AAV serotype 6 expression vector comprising a codon-optimized nucleic acid encoding a FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:5. 4 - An AAV serotype 5 expression vector comprising a codon-optimized nucleic acid encoding a FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:2. [Figure 6] FIG. 6 is a graph showing increased levels of FVIII-BDD protein upon in vivo delivery of a codon-optimized nucleic acid encoding the FVIII-BDD protein in the form of an AAV expression vector to B6.129S-F8tm1Smoc(HemA) mice.

[0083] FVIII-BDD protein levels in the plasma of animals after infusion: 1 - control solution without AAV (negative control). 2- An AAV serotype 5 expression vector comprising a codon-optimized nucleic acid encoding a FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:2.

[0084] 3- An AAV serotype 6 expression vector comprising a codon-optimized nucleic acid encoding a FVIII-BDD protein having the nucleotide sequence of SEQ ID NO:2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] EXAMPLES

[0086] 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.

[0087] 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), SK-HEP1 (human adenocarcinoma endothelial cell line) and HUH7 (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 SK-HEP1 cells used to test the efficacy of AAV products were cultured under standard conditions at 37 °C and 5% CO2 in complete EMEM medium supplemented with 10% FBS, antibiotics / antimycotics. Adherent HUH7 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. 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 novel codon-optimized nucleic acids encoding the FVIII-BDD protein, plasmids containing expression cassettes for expressing different variants of the hFVIII-BDD transgene were used in transfection. SK-HEP1 cells were cultured at 10,000 cells / cm. 2The cells were pre-seeded into wells of a 12-well plate at a density of 1000 μg / ml. After 1 day, the plasmid with the same copy number was introduced into the complex using Lipofectamine3000. On the 7th 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 performed in six independent experiments. Intact SK-HEP1 cells were used as a negative control. Assembly and purification of AAV-based expression vectors To assemble AAV expression vectors containing mutants 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 (Figure 1); 2) Plasmids for expressing the AAV6 or AAV5 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 Ad5 genes required for AAV capsid assembly and packaging.

[0088] After 72 hours, cells were lysed, and vectors were purified and concentrated using filtration, chromatography and ultracentrifugation. Viral vector titers were determined by quantitative PCR using primers and samples that were specific to regions of the recombinant viral genome, and expressed as the number of copies of viral genome per mL. Transduction of cell cultures HUH7 cell line was cultured at 10,000 cells / cm 2The cells were pre-seeded into wells of a 12-well plate at a density of 100000 vg / cell. After the cells attached to the adhesive substrate, the AAV preparation was transduced at an MOI of 500000 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 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 coagulation factor VIII-BDD protein levels in the culture medium after cell transfection with the target candidates were evaluated by a non-competitive enzyme-linked immunosorbent assay (ELISA) sandwich method. Briefly, the culture medium 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, a TMB solution was introduced. When the required level of staining intensity was reached, 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 sample was determined by a calibration curve, taking into account the preliminary dilution of the sample. 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 cells with target candidates 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 Experiments were performed on C57BL / 6 mice (male, 6-8 weeks old). 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 35 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.

[0089] Example 1: Modification of the hFVIII-BDD gene sequence using the developed codon optimization algorithm The genetic construct developed is a nucleotide sequence encoding an expression cassette containing the human B-domain deleted coagulation factor VIII gene (FVIII-BDD).

[0090] The resulting expression cassette contains all the elements required for both gene expression and assembly in the recombinant AAV genome (Figure 1): 1) ITRs at the ends of the sequence to be encapsidated into the expression vector; 2) elements for expressing the target gene (promoter, transgene, polyadenylation site); 3) A bacterial origin of replication and an antibiotic resistance gene for producing plasmid DNA in bacterial cells.

[0091] As wild-type nucleic acid, the inventors used a nucleic acid encoding a human B-domain deleted coagulation factor VIII protein and comprising the nucleotide sequence of SEQ ID NO: 1. The given nucleic acid is used in the expression cassette as a control.

[0092] Furthermore, to increase the efficiency of expression, the naturally occurring nucleotide sequence of the B-domain deleted coagulation factor VIII gene was modified using a codon optimization algorithm. Codon optimization of the nucleic acid corresponding to the sequence of SEQ ID NO:1 resulted in multiple codon-optimized nucleic acids of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, which were further tested for coagulation factor VIII-BDD protein expression and activity levels when compared to the control (nucleic acid of SEQ ID NO:1) in the expression cassette (Figure 1).

[0093] Example 2. Functional testing of codon-optimized mutants of the B-domain deleted FVIII gene sequence in vitro The resulting codon-optimized nucleic acid selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 encoding a FVIII-BDD protein was confirmed by transfecting SK-HEP1 cells in vitro as part of an expression cassette consisting of a left (first) ITR (inverted terminal repeat), a tissue-specific promoter, a transgene, a polyadenylation signal, a right (second) ITR, the transgene being one of the sequences SEQ ID NO:2 to 5. As a control, the transgene was a non-optimized nucleic acid encoding a human B-domain deleted FVIII protein corresponding to the sequence SEQ ID NO:1.

[0094] The use of nucleic acids encoding FVIII-BDD protein selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 all resulted in increased levels of FVIII-BDD protein production (Figure 2) and activity (Figure 3) when compared to the use of the non-optimized nucleic acid of SEQ ID NO:1.

[0095] Furthermore, the generated codon-optimized nucleic acid (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5) encoding a B-domain deleted coagulation factor VIII protein is capable of causing increased expression of FVIII-BDD protein in a host cell in vitro when compared to the use of a non-optimized nucleic acid of SEQ ID NO: 1. Thus, the codon-optimized nucleic acid according to the present invention has high potential for the generation of recombinant FVIII-BDD protein for the therapy of hemophilia A.

[0096] Example 3. Construction and functional testing of expression vectors carrying codon-optimized variants of the FVIII-BDD gene sequence in vitro To prove the efficiency of delivery of the codon-optimized nucleic acid developed with expression vectors in vitro, the inventors used plasmids encoding target gene constructs together with the rest of the necessary plasmids to generate expression vectors based on adeno-associated virus serotype 5 carrying the nucleotide sequence of SEQ ID NO:2 (hereinafter referred to as AAV5-FVIII-BDDco-v1) and based on adeno-associated virus serotype 6 carrying the nucleotide sequence of SEQ ID NO:2 (hereinafter referred to as AAV6-FVIII-BDDco-v1) or the nucleotide sequence of SEQ ID NO:5 (hereinafter referred to as AAV6-FVIII-BDDco-v4). The purified AAV5-FVIII-BDDco-v1, AAV6-FVIII-BDDco-v1 and AAV6-FVIII-BDDco-v4 products used for in vitro and in vivo studies were prepared using standard buffers and excipients that are safe and do not change AAV properties.

[0097] The AAV5-FVIII-BDDco-v1, AAV6-FVIII-BDDco-v1 and AAV6-FVIII-BDDco-v4 products were tested in vitro using adherent HUH7 cell lines (Figures 4 and 5). Cells of the cell lines were grown at 10,000 cells / cm. 2 The cells were plated into wells of a 12-well plate at a density of 100 μg / ml. 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. All samples were run in triplicate. Intact cells were used as a negative control.

[0098] As confirmed by culture medium analysis by ELISA (Figure 4) and activity analysis (Figure 5) of coagulation factor VIII-BDD protein, it was shown that the generated AAV5-FVIII-BDDco-v1, AAV6-FVIII-BDDco-v1 and AAV6-FVIII-BDDco-v4 expression vectors carrying codon-optimized variants of the coagulation factor VIII-BDD gene can effectively deliver the coagulation factor VIII-BDD transgene into HUH7 cells and provide for the production of the target 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 medium at 129 ng / mL and activity at 97%. Delivery of SEQ ID NO:2 using an AAV serotype 6 expression vector results in the production of FVIII-BDD protein in the culture medium at 104 ng / mL and activity at 91%. Delivery of SEQ ID NO:5 using an AAV serotype 6 expression vector results in the production of FVIII-BDD protein in the culture medium at 61 ng / mL and activity at 68%.

[0099] Example 4. Functional testing of AAV5-FVIII-BDDco-v1 and AAV6-FVIII-BDDco-v1 products in vivo To demonstrate the efficiency of delivery of the developed codon-optimized nucleic acids when delivered in an expression vector in vivo, the selected products AAV5-FVIII-BDDco-v1 and AAV6-FVIII-BDDco-v1 were administered to experimental C57BL / 6 mice. The dose of AAV product used in the study was 6×10 13 vg / mouse. 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 (day 0) before administration of the product, followed by days 35 and 56 after introduction of the product. The levels of coagulation factor VIII-BDD protein in the plasma samples were determined by ELISA as described above.

[0100] In vivo studies showed that a significant increase in the levels of factor VIII-BDD in the plasma of animals on days 35 and 56 was observed when using both products AAV5-FVIII-BDDco-v1 and AAV6-FVIII-BDDco-v1 containing the codon-optimized sequence of the coagulation factor VIII-BDD gene of SEQ ID NO:2 (Figure 6). The product AAV5-FVIII-BDDco-v1 containing the codon-optimized sequence of the coagulation factor VIII-BDD gene of SEQ ID NO:2 resulted in FVIII-BDD expression at 304 and 279 ng / mL in the plasma of animals on days 35 and 56, respectively, after infusion. The product AAV6-FVIII-BDDco-v1 containing the codon-optimized sequence of the coagulation factor VIII-BDD gene of SEQ ID NO:2 resulted in FVIII-BDD expression at 415 and 459 ng / mL in the plasma of animals on days 35 and 56, respectively, after infusion.

[0101] Therefore, the developed AAV5 and AAV6 expression vectors carrying a codon-optimized version of the B-domain deleted coagulation factor VIII gene (AAV5-FVIII-BDDco-v1 and AAV6-FVIII-BDDco-v1) are capable of causing expression of FVIII-BDD protein in vivo and have high potential for gene therapy of hemophilia A.

Claims

1. An isolated codon-optimized nucleic acid encoding the FVIII-BDD (B-domain deletion coagulation factor VIII) protein having the amino acid sequence of SEQ ID NO: 11, comprising 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 comprising the nucleic acid described in claim 1.

3. The following elements are located in the direction from the 5' end to the 3' end: Left (first) ITR (inverted terminal repeat); promoter; Nucleic acids that encode signal peptides; The nucleic acid according to claim 1; Polyadenylation signal; Right side (2nd) ITR An expression cassette according to claim 2, comprising:

4. An expression vector comprising the nucleic acid described in claim 1 or the expression cassette described in claim 2.

5. The expression vector according to claim 4, wherein the vector is a recombinant adeno-associated virus (AAV).

6. 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 5, 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.

7. A host cell for producing FVIII-BDD protein, comprising the nucleic acid described in claim 1.

8. A pharmaceutical composition for delivering the FVIII-BDD gene to target cells, comprising, in combination with one or more pharmaceutically acceptable excipients, the cassette according to claim 2, or an expression vector comprising the nucleic acid according to claim 1 or the expression cassette according to claim 2.

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