Linking peptide, blood coagulation factor VIII protein or variant thereof containing the linking peptide, and uses thereof

Abstract

The present invention relates to a linking peptide, a blood coagulation factor VIII protein or a variant thereof containing the linking peptide, and uses thereof, and belongs to the field of gene defect therapeutic drugs. The linking peptide can improve the expression level, activity and therapeutic effect of a blood coagulation factor VIII protein or a variant thereof using minicircle DNA as a carrier, and further has a good secondary administration therapeutic effect, and has advantages such as good safety and long-term expression in vivo.

Description

[Technical Field]

[0001] The present invention relates to the field of drugs for treating gene defects, and more particularly to a connecting peptide, a blood coagulation factor VIII protein or a variant thereof containing the connecting peptide, and uses thereof. [Background technology]

[0002] Hemophilia is an X-linked, single-gene genetic disorder characterized by a deficiency in a set of blood coagulation factors. Hemophilia A, characterized by a deficiency of blood coagulation factor VIII (FVIII), encoded by the F8 gene, is the most prevalent, accounting for 80-85% of patients. Currently, the standard treatment for hemophilia A is replacement therapy, namely, exogenous FVIII protein supplementation, including plasma-derived FVIII (pdFVIII) and recombinant FVIII (rFVIII). pdFVIII is an extract isolated from normal human plasma, which limits donor availability and poses the risk of bloodborne virus transmission. rFVIII is expressed and purified in vitro using mammalian cell lines, which can effectively reduce the risk of bloodborne virus infection without restricting the blood supply, but requires a high production process.

[0003] FVIII protein has a short half-life in the body (average 12 hours), requiring lifelong administration for patients on alternative therapy, and frequent injections (2-3 or even 4 times weekly intravenous injections), which increases costs and makes treatment inconvenient. Conventional methods for prolonging the half-life, such as Fc fusion (Drug Des Devel Ther 2014, 8:365-371) and PEG modification (Haemophilia 2019, 25:773-781), can extend its half-life to approximately 19 hours, but this extension is limited (only 50-60%) and does not significantly reduce the frequency of administration. Sanofi and Sobi have jointly developed a new, longer-lasting FVIII fusion protein (BIVV001; FVIIIFc-vWF-XTEN) that combines an antibody Fc fragment with the vWF factor D'D3 domain (D3 domain), further extending the half-life to 38-44 hours and reducing the dosing frequency to once a week (N Engl J Med. 2020, 383:1018-1027). While these long-acting modifications to protein molecules can slightly extend the half-life and improve patient experience to some extent, they cannot fundamentally change the "lifelong administration, repeated injection" treatment model.

[0004] Meanwhile, FVIII gene therapy is expected to provide a functional "cure" for hemophilia A patients by correcting the genetic defect at the genetic level. Currently, the industry generally considers AAV-hFVIII (targeted delivery of normal human FVIII genes to liver tissue is achieved using an adeno-associated virus vector) to be the "optimal" approach for hemophilia A gene therapy. However, existing liver-targeted AAV-FVIII gene therapies have clear flaws (Hemasphere 2021, 5:e540). i) AAV-hFVIII is only applicable to adult patients aged 18 years or older. Children's livers are in a stage of rapid growth and development, with active hepatocyte division, making this therapy unsuitable for use in such patients (the risk of gene integration is significantly increased, and unintegrated target genes are easily lost with cell division). ii) Because AAV is a common human virus and has a high prevalence in the human population, many patients have pre-existing AAV-neutralizing antibodies, making this treatment unavailable to these patients. iii) Administration cannot be repeated. AAV-hFVIII is not effective for life, and expression levels may fall below therapeutic levels after several years. In this case, re-administration is necessary to restore therapeutic levels. However, the AAV viral coat is highly immunogenic, and virus-specific immune responses are likely to occur after the first administration, making re-administration ineffective. iv) The large size of the hFVIII coding gene exceeds the capacity limit of the AAV vector, making packaging and production of AAV-hFVIII difficult. In addition, AAV therapy raises other safety concerns, such as the risk of random gene integration.

[0005] Expression of human factor VIII (hFVIII) is quite difficult, a major technical challenge facing both recombinant FVIII production (in vitro expression) and FVIII gene therapy (in vivo expression). The expressed amount of hFVIII is only 1 / 100-1 / 1000 of that of the normal protein (Hum Gene Ther 1993, 4:259-272; Blood 2004, 103:3412-3419). Therefore, efforts in related fields have focused on modifying the hFVIII molecule to increase its expression level. FVIII contains six structural domains, A1-A2-B-A3-C1-C2, of which the B domain is 908 aa long, accounting for approximately 40% of the total length (2332 aa). Deletion of the B domain does not affect blood coagulation function (PNAS 1986, 83:5939-5942), but significantly increases mRNA (17-fold) and protein product (30%) levels (Blood 2004, 103:3412-3419; Blood Coagul Fibrinolysis 1997, 8 Suppl 2:S3-14). Therefore, B domain deletion has become a widely adopted modification strategy. In fact, many B domain-deleted FVIII drugs (B domain deleted FVIII, BDD-FVIII) have been in large-scale clinical use for many years, such as Xyntha, produced by Pfizer. The first BDD-FVIII lacked the B domain almost entirely, leaving only the linker A1-A2 and A3-C1-C2 (called the SQ linker, with the sequence SFSQNPPVLKRHQR) consisting of the N-terminal four amino acids and the C-terminal ten amino acids (total 14 aa) of the B domain of native FVIII. For convenience, we refer to BDD-FVIII containing the SQ linker as BDD-FVIII-SQ. We have previously investigated the possibility of in vivo expression of therapeutic levels of BDD-FVIII-SQ using minicircle DNA vectors. However, experiments showed that the BDD-FVIII-SQ vector could only express weakly in vivo (less than 1% of normal levels) and did not achieve therapeutic efficacy. There are three therapeutic levels.i) Over 1% - initial therapeutic effect, with moderate relief of severe hemophilia; ii) Over 5% - significant therapeutic effect, with clear mild relief of moderate to severe hemophilia; iii) Over 50% - functional "cure" achieved, with complete recovery of normal blood clotting function.

[0006] The B region linker connects A1-A2 and A3-C1-C2, and its sequence has a significant impact on the expression level of BDD-FVIII protein. Miao et al. (Blood 2004, 103:3412-3419) reported that the expression level of BDD-FVIII protein was four times higher when the N-terminal 226 amino acids (226aa / N6) of the B region were retained than when the B region was almost completely deleted (wild-type SQ linker) (detected by ELISA). Based on this, McIntosh et al. (Blood 2013, 121:3335-3344; patent: WO 2013 / 186563) found that replacing 226aa / N6 with the 31-aa long v3 linker (sequence SFSQNATNVSNNSNTSNDSNVSPPVLKRHQR) further increased the expression level of the target protein (approximately 50%). In the present invention, BDD-FVIII containing a v3 linker is referred to as BDD-FVIII-v3. Currently, a phase I clinical trial (NCT03001830) of AAV gene therapy based on BDD-FVIII-v3 has been initiated. Novo Nordisk (Denmark) has developed another BDD-FVIII molecule containing a 21-aa linker (sequence SFSQNSRHPSQNPPVLKRHQR) that exhibits superior expression compared to BDD-FVIII-SQ (N8, Haemophilia 2010, 16:349-359; patent: WO2006103298). In the present invention, this molecule is referred to as BDD-FVIII-N8. However, BDD-FVIII-v3 and BDD-FVIII-N8 have problems such as low expression levels and poor therapeutic efficacy. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2013 / 186563 [Patent Document 2] International Publication No. 2006 / 103298 [Non-patent literature]

[0008] [Non-Patent Document 1] Drug Des Devel Ther 2014, 8:365-371 [Non-patent document 2] Haemophilia 2019, 25:773-781 [Non-patent document 3] N Engl J Med.2020,383:1018-1027 [Non-patent document 4] Hemasphere 2021,5:e540 [Non-Patent Document 5] Hum Gene Ther 1993,4:259-272 [Non-patent document 6] Blood 2004,103:3412-3419 [Non-Patent Document 7] Blood Coagul Fibrinolysis 1997,8 Suppl 2:S3-14 [Non-patent document 8] Blood 2013,121:3335-3344 [Non-Patent Document 9] Haemophilia 2010,16:349-359 Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, there is still an urgent need for drugs that have high expression levels, allow for secondary administration, are easy to prepare, have good therapeutic effects, and can be expressed in the body for a long time to treat diseases associated with blood coagulation factor VIII deficiency. [Means for solving the problem]

[0010] To solve the above problems, the present invention provides the following technical solutions. In a first aspect, a connecting peptide is provided. A recombinant blood coagulation factor VIII protein or a variant thereof using the connecting peptide of the present invention has advantages such as a high expression level, excellent secondary administration efficacy, simple preparation, and excellent therapeutic efficacy. A minicircle DNA of a recombinant blood coagulation factor VIII protein or a variant thereof using the connecting peptide of the present invention has advantages such as a high expression level, excellent secondary administration efficacy, simple preparation, and excellent therapeutic efficacy. It also overcomes the problems of AAV.hf8, such as production difficulties (packaging difficulties), limited application (cannot be used in children and cannot be used for secondary administration), and safety risks (carcinogenesis due to random integration). It thus has advantages such as simple production, wide application (can be used in children and has good therapeutic efficacy for secondary administration), good safety, and long-term expression in the body.

[0011] In a second aspect, there is provided a nucleotide sequence, said nucleotide sequence being capable of encoding a connecting peptide according to the first aspect.

[0012] In a third aspect, there is provided a use of a connecting peptide according to the first aspect or a nucleotide sequence according to the second aspect.

[0013] In a fourth aspect, there is provided a recombinant blood coagulation factor VIII protein or a variant thereof, which has advantages such as high expression level, high secondary administration efficacy, simple preparation, and high therapeutic efficacy.

[0014] In a fifth aspect, a nucleotide is provided.

[0015] In a sixth aspect, there is provided a recombinant gene vector.

[0016] In a seventh aspect, there is provided a parental plasmid for use in producing minicircle DNA.

[0017] In an eighth aspect, a method for producing minicircle DNA is provided.

[0018] In a ninth aspect, there is provided a minicircle DNA obtained by the production method described in the eighth aspect. The minicircle DNA has advantages such as high expression level, excellent secondary administration effect, simple preparation, and excellent therapeutic effect, and solves the problems of AAV.hf8 production difficulties (packaging difficulties), limited application range (cannot be used in children and secondary administration is not possible), and safety risks (carcinogenesis due to random integration), i.e., it has advantages such as simple production, wide application range (can be used in children and has good therapeutic effect in secondary administration), good safety, and long-term expression in the body.

[0019] In a tenth aspect, there is provided a host cell comprising a nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or variant thereof according to the fourth aspect, a nucleotide sequence according to the fifth aspect, a recombinant gene vector according to the sixth aspect, or a minicircle DNA according to the ninth aspect.

[0020] In an eleventh aspect, a pharmaceutical composition is provided.

[0021] In a twelfth aspect, there is provided use of the recombinant blood coagulation factor VIII protein or variant thereof according to the fourth aspect, the nucleotide sequence according to the fifth aspect, the recombinant gene vector according to the sixth aspect, the parent plasmid according to the seventh aspect, the minicircle DNA obtained by the production method according to the eighth aspect, the minicircle DNA according to the ninth aspect, the host cell according to the tenth aspect, or the pharmaceutical composition according to the eleventh aspect in the preparation of a medicament for treating a disease.

[0022] (Detailed Description of the Invention) To solve the above problems, the present invention provides the following technical solutions. In a first aspect, a connecting peptide is provided. A connecting peptide having an amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2, or an amino acid sequence having at least 80-99% identity to either of the nucleotide sequences, or at least a portion of either of the sequences. The recombinant blood coagulation factor VIII protein or variant thereof using the connecting peptide according to the first aspect of the present invention has advantages such as a high expression level, a good secondary administration effect, ease of production, and good therapeutic effect. The minicircle DNA of the recombinant blood coagulation factor VIII protein or variant thereof using the connecting peptide according to the first aspect of the present invention has advantages such as a high expression level, a good secondary administration effect, simple preparation, and good therapeutic effect. It solves the problems of AAV.hf8 production difficulties (packaging difficulties), limited application range (cannot be used in children and cannot be used in secondary administration), and safety risks (carcinogenesis due to random integration), and thus has advantages such as ease of production, wide application range (can be used in children and has good therapeutic effect in secondary administration), good safety, and long-term expression in the body.

[0023] In some embodiments, the nucleotide sequence encoding the connecting peptide having the amino acid sequence of SEQ ID NO. 1 (abbreviated as L1 linker in the present invention) can include SEQ ID NO. 3 or a codon-optimized sequence thereof.

[0024] In some embodiments, the nucleotide sequence encoding the connecting peptide having the amino acid sequence of SEQ ID NO. 2 (abbreviated as L2 linker in the present invention) can include SEQ ID NO. 4 or a codon-optimized sequence thereof.

[0025] In a second aspect, a nucleotide sequence is provided. A nucleotide sequence encoding a connecting peptide according to the first aspect.

[0026] In some embodiments, the nucleotide sequence comprises SEQ ID NO. 3 or SEQ ID NO. 4, or a codon-optimized version of either nucleotide sequence.

[0027] In some embodiments, the nucleotide sequence is SEQ ID NO. 3, or a codon-optimized version thereof can be used to encode a connecting peptide whose amino acid sequence is SEQ ID NO. 1.

[0028] In some embodiments, the nucleotide sequence can be SEQ ID NO. 4, or a codon-optimized version thereof, can be used to encode a connecting peptide whose amino acid sequence is SEQ ID NO. 2.

[0029] In a third aspect, there is provided a use of a connecting peptide according to the first aspect or a nucleotide sequence according to the second aspect. The use of the connecting peptide according to the first aspect or the nucleotide sequence according to the second aspect in the construction of a recombinant blood coagulation factor VIII protein or a variant thereof. The present invention relates to the use of the nucleotide sequence according to the second aspect in a nucleotide sequence for constructing a recombinant blood coagulation factor VIII protein or a variant thereof.

[0030] In a fourth aspect, there is provided a recombinant blood coagulation factor VIII protein or a variant thereof. The blood coagulation factor VIII protein or its variant has a connecting peptide selected from the connecting peptides of the first aspect, or a nucleotide sequence of the connecting peptide selected from the nucleotide sequences of the second aspect. The recombinant blood coagulation factor VIII protein or its variant provided in the fourth aspect of the present invention has advantages such as a high expression level, a high secondary administration effect, ease of preparation, and a high therapeutic effect.

[0031] In some embodiments, the recombinant blood coagulation factor VIII protein or a variant thereof has an amino acid sequence of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, or SEQ ID NO. 53, or an amino acid sequence having at least 80% to 99% identity to any of the amino acid sequences thereof, or at least a portion of any of the amino acid sequences thereof.

[0032] In some embodiments, the nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 5 may comprise SEQ ID NO. 7 or a codon-optimized sequence thereof.

[0033] In some embodiments, the nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 6 may comprise SEQ ID NO. 8 or a codon-optimized sequence thereof.

[0034] In some embodiments, the nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 21 may comprise SEQ ID NO. 22 or a codon-optimized sequence thereof.

[0035] In some embodiments, the nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 23 may comprise SEQ ID NO. 24 or a codon-optimized sequence thereof.

[0036] In some embodiments, the nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 43 may comprise SEQ ID NO. 38 or a codon-optimized sequence thereof.

[0037] In some embodiments, the nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 44 may comprise SEQ ID NO. 39 or a codon-optimized sequence thereof.

[0038] In some embodiments, the nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 50 may comprise SEQ ID NO. 56 or a codon-optimized sequence thereof.

[0039] In some embodiments, the nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 51 may comprise SEQ ID NO. 60 or a codon-optimized sequence thereof.

[0040] In some embodiments, the nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 52 may comprise SEQ ID NO. 58 or a codon-optimized sequence thereof.

[0041] In some embodiments, the nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 53 may comprise SEQ ID NO. 54 or a codon-optimized sequence thereof.

[0042] In a fifth aspect, a nucleotide is provided. A nucleotide sequence encoding a recombinant blood coagulation factor VIII protein or a variant thereof according to the fourth aspect. In some embodiments, the nucleotide sequence comprises SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.54, SEQ ID NO.56, SEQ ID NO.58 or SEQ ID NO.60, or a codon-optimized version of any of these nucleotide sequences.

[0043] In some embodiments, the nucleotide sequence SEQ ID NO. 7 can be used to encode the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 5.

[0044] In some embodiments, the nucleotide sequence SEQ ID NO. 8 can be used to encode the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 6.

[0045] In some embodiments, the nucleotide sequence SEQ ID NO. 22 can be used to encode the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 21.

[0046] In some embodiments, the nucleotide sequence SEQ ID NO. 24 can be used to encode the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 23.

[0047] In some embodiments, the nucleotide sequence SEQ ID NO. 38 can be used to encode a recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence SEQ ID NO. 43.

[0048] In some embodiments, the nucleotide sequence SEQ ID NO. 39 can be used to encode a recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence SEQ ID NO. 44.

[0049] In some embodiments, the nucleotide sequence SEQ ID NO. 54 can be used to encode the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 53.

[0050] In some embodiments, the nucleotide sequence SEQ ID NO. 56 can be used to encode the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 50.

[0051] In some embodiments, the nucleotide sequence SEQ ID NO. 58 can be used to encode the recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 52.

[0052] In some embodiments, the nucleotide sequence SEQ ID NO. 60 can be used to encode a recombinant blood coagulation factor VIII protein or a variant thereof having the amino acid sequence of SEQ ID NO. 51.

[0053] In a sixth aspect, there is provided a recombinant gene vector. A recombinant gene vector comprising a nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or a variant thereof according to the fourth aspect or the nucleotide sequence according to the fifth aspect.

[0054] In some embodiments, the recombinant gene vector may comprise a non-viral vector or a viral vector.

[0055] In some embodiments, the non-viral vector may be selected from standard plasmids or other circular expression cassettes.

[0056] In some embodiments, the viral vector may be selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0057] In some preferred embodiments, the non-viral vector may be selected from the group consisting of minicircle DNA vectors.

[0058] In a seventh aspect, there is provided a parental plasmid for use in producing minicircle DNA. The parental plasmid for producing the minicircle DNA comprises a plasmid vector comprising the nucleotide sequence according to the fifth aspect.

[0059] In some embodiments, a parent plasmid used for producing minicircle DNA is formed by inserting a promoter nucleotide sequence, an enhancer nucleotide sequence, a multicloning site nucleotide sequence, a polyA signal nucleotide sequence, and a target gene DNA fragment into a plasmid vector, wherein the target gene DNA fragment is located between restriction endonuclease enzyme cleavage sites of the multicloning site, and the target gene DNA fragment comprises the nucleotide sequence described in the fifth aspect.

[0060] In some embodiments, the promoter is a CMV promoter, the nucleotide sequence of which may be SEQ ID NO.29.

[0061] In some embodiments, the enhancer nucleotide sequence may be SEQ ID NO. 30.

[0062] In some embodiments, the multiple cloning site nucleotide sequence may be SEQ ID NO.31.

[0063] In some embodiments, the polyA signal may comprise a signal selected from the group consisting of a bovine growth hormone polyA signal, a human growth hormone polyA signal, or an SV40 polyA signal.

[0064] In some embodiments, the bovine growth hormone polyA signal nucleotide sequence may be SEQ ID NO.32.

[0065] In some embodiments, the target gene DNA fragment may further comprise a Kozak sequence and a human FVIII gene signal peptide coding sequence, which is advantageous for enhancing transcription of the target gene.

[0066] In some embodiments, the nucleotide sequence of the Kozak sequence may be SEQ ID NO. 63 (GCCACC).

[0067] In some embodiments, the human FVIII gene signal peptide coding sequence may be SEQ ID NO. 33 or SEQ ID NO. 34.

[0068] In some embodiments, the plasmid vector comprises one selected from the group consisting of pMC.BESPX plasmid or p2ΦC31 plasmid.

[0069] In some embodiments, the promoter nucleotide sequence, enhancer nucleotide sequence, multicloning site nucleotide sequence, and polyA signal nucleotide sequence are inserted into a plasmid via the attB and attP recombination sites of the plasmid.

[0070] In some embodiments, the target gene DNA fragment is inserted into the plasmid via the AgeI and EcoRV restriction endonuclease enzyme cleavage sites in the multiple cloning site.

[0071] In an eighth aspect, a method for producing minicircle DNA is provided. A method for producing minicircle DNA, comprising transforming the parental plasmid described in the seventh aspect into a host cell, and after induction, generating minicircle DNA and backbone DNA from the parental plasmid through site-specific recombination at a specific recombination site, and extracting the minicircle DNA using a plasmid DNA purification kit.

[0072] In some embodiments, the backbone DNA is linearized and then degraded within the host cell.

[0073] In some embodiments, the induction may include induction with L-arabinose.

[0074] In a ninth aspect, there is provided a minicircle DNA. The minicircle DNA according to the ninth aspect of the present invention, obtained by the manufacturing method described in the eighth aspect, has advantages such as high expression level, excellent secondary administration effect, simple preparation, and excellent therapeutic effect, and solves the problems of AAV.hf8's difficulty in production (difficulty in packaging), limited range of application (cannot be used in children and cannot be used for secondary administration), and safety risk (carcinogenesis due to random integration), i.e., it has advantages such as simple production, wide range of application (can be used in children and has good therapeutic effect in secondary administration), and good safety.

[0075] In some embodiments, the nucleotide sequence of the minicircle DNA may include one selected from the group consisting of SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.49, SEQ ID NO.55, SEQ ID NO.57, SEQ ID NO.59 or SEQ ID NO.61.

[0076] In a tenth aspect, a host cell is provided. A host cell comprising a nucleotide sequence encoding a recombinant blood coagulation factor VIII protein or a variant thereof according to the fourth aspect, a nucleotide sequence according to the fifth aspect, a recombinant gene vector according to the sixth aspect, or a minicircle DNA according to the ninth aspect.

[0077] In some embodiments, the host cell may comprise a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is selected from E. coli. In some embodiments, the host cell is selected from E. coli ZYCY10P3S2T.

[0078] In an eleventh aspect, a pharmaceutical composition is provided. A pharmaceutical composition comprising a recombinant blood coagulation factor VIII protein or a variant thereof according to the fourth aspect, a recombinant blood coagulation factor VIII protein or a variant thereof encoded by the nucleotide sequence according to the fifth aspect, a recombinant gene vector according to the sixth aspect, a minicircle DNA obtained by the production method according to the eighth aspect, or a minicircle DNA according to the ninth aspect, and a pharmaceutically acceptable adjuvant or carrier. The pharmaceutical composition according to the eleventh aspect of the present invention has advantages such as a high expression level, good secondary administration efficacy, ease of production, and good therapeutic efficacy. It overcomes the problems of AAV.hf8 production difficulties (packaging difficulties), limited application (cannot be used in children, cannot be used in secondary administration), and safety risks (carcinogenesis due to random integration), and thus has advantages such as ease of production, wide application (can be used in children, good therapeutic effect in secondary administration), good safety, and long-term expression in the body.

[0079] A twelfth aspect provides a use. Use of the recombinant blood coagulation factor VIII protein or variant thereof according to the fourth aspect, the nucleotide sequence according to the fifth aspect, the recombinant gene vector according to the sixth aspect, the parent plasmid according to the seventh aspect, the minicircle DNA obtained by the production method according to the eighth aspect, the minicircle DNA according to the ninth aspect, the host cell according to the tenth aspect, or the pharmaceutical composition according to the eleventh aspect in the production of a medicament for treating a disease. In some embodiments, the disease may be selected from inherited gene defect diseases. In some embodiments, the disease may be a disease caused by a blood coagulation factor deficiency. In some embodiments, the disease may be a disease caused by a deficiency of blood coagulation factor VIII. In some embodiments, the disease may be hemophilia. In some embodiments, the hemophilia is moderate hemophilia or severe hemophilia. [Effects of the Invention]

[0080] Certain embodiments of the present invention have at least one of the following beneficial technical effects compared to the prior art. (1) The recombinant blood coagulation factor VIII protein or its variants produced using the recombinant gene vector, minicircle DNA, pharmaceutical composition, or connecting peptide of the present invention have advantages such as high expression level, good secondary administration effect, easy production, good therapeutic effect, and long-term expression in the body. (2) It solves the problems of AAV.hf8's production difficulties (difficulty in packaging), limited application range (cannot be used in children, cannot be administered as a secondary dose), and safety risks (carcinogenesis due to random integration), and has the advantages of being easy to produce, having a wide range of applications (can be used in children, has good secondary administration effect), good safety, and long-term expression in the body.

[0081] Definition of Terms "Room temperature" means ambient temperature, which may be between 20°C and 30°C. In some embodiments, it is between 22°C and 28°C, in some embodiments, it is between 24°C and 26°C, and in some embodiments, it is 25°C.

[0082] In the description herein, a statement referring to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples" means that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, general expressions using the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, a person skilled in the art may combine different embodiments or examples described herein, as well as features of different embodiments or examples, as long as they are not mutually inconsistent.

[0083] The fusion proteins of the present invention are typically produced by biosynthesis. Based on the nucleotide sequences described herein, the encoding nucleic acids of the present invention can be produced by various methods conveniently known to those skilled in the art. These methods include, but are not limited to, PCR and artificial DNA synthesis. For specific methods, see J. Sambrook's "A Laboratory Guide to Molecular Cloning." In one embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by synthesizing a nucleotide sequence in fragments, followed by overlapping and extending the fragments.

[0084] The term "parental plasmid" refers to a plasmid containing an expression vector for a target gene and backbone DNA, which can generate the original plasmid of the minicircle DNA. After site-specific DNA recombination occurs in the plasmid, the minicircle DNA is generated.

[0085] The term "fusion protein" generally refers to a protein obtained by fusing two or more proteins or polypeptides. The two or more protein or polypeptide genes or nucleic acid molecules may be linked together to form a fusion gene or fusion nucleic acid molecule, which may then encode the fusion protein. Translation of the fusion gene produces a single polypeptide possessing the properties of at least one of the two or more proteins or polypeptides from which it was fused, or each of the two or more proteins or polypeptides. Recombinant fusion proteins are artificially created using recombinant DNA technology for biological research and therapy. A recombinant fusion protein is a protein produced by genetic engineering of a fusion gene. The present invention relates to recombinant fusion proteins, and the terms "fusion protein" and "recombinant fusion protein" are used interchangeably herein. Fusion proteins described herein typically contain at least two domains (A and C) and, optionally, a third component, a linker between the two domains. The production of recombinant fusion proteins is known in the art and typically involves removing a stop codon from a cDNA sequence encoding a first protein or polypeptide, followed by joining the cDNA sequence of a second protein in reading frame using ligation or overlap extension PCR. This DNA sequence is expressed by the cell into a single protein that can be engineered to contain the complete sequences of the two original proteins or polypeptides, or only portions of either.

[0086] As used herein, a "pharmaceutical composition" may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All of these methods include the step of bringing into association the active ingredient with the carrier, which constitutes one or more accessory ingredients. Generally, compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers, finely divided solid carriers, or both.

[0087] In this application, the term "recombinant gene vector" generally refers to a nucleic acid molecule capable of self-replicating in an appropriate host that transfers an inserted nucleic acid molecule into and / or between host cells. Examples of recombinant gene vectors include vectors primarily used to insert DNA or RNA into cells, recombinant gene vectors primarily used to replicate DNA or RNA, and recombinant gene vectors primarily used for transcriptional and / or translational expression of DNA or RNA. Recombinant gene vectors also include recombinant gene vectors having multiple functions. The recombinant gene vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into an appropriate host cell. Generally, a desired expression product is produced from the recombinant gene vector by culturing an appropriate host cell containing the recombinant gene vector.

[0088] In this application, the term "variant" can refer to any naturally occurring or engineered molecule that contains one or more nucleotide or amino acid mutations.

[0089] In this application, the term "nucleotide" refers to ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotide, and combinations thereof.

[0090] As used herein, "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. As used herein, the terms "transformed" or "transfected" refer to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with a DNA sequence of the present invention and used for the expression and / or secretion of a target protein.

[0091] As used herein, the term "identity," when used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). This formula was incorporated into the BLAST (Basic Local Alignment Search Tool) program of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Percent sequence identity can be determined using the most recent version of BLAST available as of the filing date of this application.

[0092] "At least 80-99% sequence identity" means at least 80%-99%, at least 81%-99%, at least 82%-99%, at least 83%-99%, at least 84%-99%, at least 85%-99%, at least 86%-99%, at least 87%-99%, at least 88%-99%, at least 89%-99%, at least 90%-99%, at least 91%-99%, at least 92%-99%, at least 93%-99%, at least 94%-99%, at least 95%-99%, at least 96%-99%, at least 97%-99%, at least 98%-99% or at least 99% sequence identity to a respective reference sequence.

[0093] In the present invention, "MC.BDD-FVIII-L1" or "MC.BDD-FVIII-L1 microring DNA" have the same meaning, and the meaning of other terms beginning with "MC.BDD" is inferred in this manner, for example, "MC.BDD-FVIII-L2" or "MC.BDD-FVIII-L2 microring DNA" have the same meaning. [Brief explanation of the drawings]

[0094] [Figure 1]FIG. 1 is a schematic diagram of the construction process of the minicircle DNA parent plasmid pMC.BDD-FVIII-SQ-CO2 in Example 1. [Figure 2] FIG. 1 is a schematic diagram of the structure of the minicircle DNA parent plasmid pMC.BDD-FVIII-L1 of Example 1. [Figure 3] FIG. 1 is a schematic diagram of the structure of the minicircle DNA parental plasmid pMC.BDD-FVIII-L2 of Example 1. [Figure 4] FIG. 1 is a schematic diagram of the structure of the minicircle DNA parental plasmid pMC.BDD-FVIII-L3 of Example 1. [Figure 5] FIG. 1 is a schematic diagram of the structure of the minicircle DNA parent plasmid pMC.BDD-FVIII-V3 of Example 1. [Figure 6] FIG. 1 is a schematic diagram of the structure of the minicircle DNA parent plasmid pMC.BDD-FVIII-N8 of Example 1. [Figure 7] 1 shows a Western blot analysis of MC.BDD-FVIII-SQ-CO2 in Example 4. Lane M is a protein marker. Lane 1 is a negative control sample (MC vector non-transfected blank control). Lane 2 is MC.BDD-FVIII-SQ-CO2 cell culture supernatant. Lane 3 is a positive control sample (hFVIII plasma extract, Guizhou Taibang Biological Products Co., Ltd.). [Figure 8] FIG. 11 is a statistical diagram showing the results of measuring the in vitro cellular expression level of the target protein by ELISA after transfection of MC.BDD-FVIII-SQ-WT, MC.BDD-FVIII-SQ-CO1, and MC.BDD-FVIII-SQ-CO2 into 293T cells in Example 5. [Figure 9] This is a statistical graph showing the therapeutic effect of MC.BDD-FVIII-SQ-CO2 on hemophilia A mice (F8-KO) in which the F8 gene was knocked out in Comparative Example 1. m1 / 2mm indicates a tail cut experiment performed one month after injection, with the mouse's tail diameter at 2 mm, and m2 / 2.5mm indicates a tail cut experiment performed two months after injection, with the mouse's tail diameter at 2.5 mm. [Figure 10]1 is a statistical diagram showing the results of in vitro cellular expression levels of target proteins after transfection of MC.BDD-FVIII-L1, MC.BDD-FVIII-L2 and other FVIII minicircle DNA vectors into 293T cells in Example 6. [Figure 11] 13 is a statistical diagram showing the expression levels of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 in normal mice in Example 8. [Figure 12] 13 is a statistical diagram of the expression levels of MC.BDD-FVIII-L1 in beagle dogs in Example 8. FIG. [Figure 13] 13 is a statistical diagram showing the expression levels of MC.BDD-FVIII-L1 in hemophilia A mice (F8-KO mice) in Example 9. FIG. [Figure 14] FIG. 11 is a statistical graph of bleeding volume in tail cut experiments in F8-KO mice in the blank control group (PBS), F8-KO mice in the MC.BDD-FVIII-L1 minicircle DNA treatment group (MC), and wild-type normal mice (WT) in Example 9. [Figure 15] FIG. 11 is a statistical graph of bleeding volume in tail cut experiments in F8-KO mice in the blank control group (PBS), F8-KO mice in the MC.BDD-FVIII-L2 minicircle DNA treatment group (MC), and wild-type normal mice (WT) in Example 9. [Figure 16] 10 is a statistical diagram showing the in vivo expression levels of MC.BDD-FVIII-CTP-Fc in Example 10. [Figure 17] 10 is a statistical diagram showing the results of measuring the in vitro cellular expression level of the target protein by ELISA after transfection of MC.BDD-FVIII-L1-X5 and MC.BDD-FVIII-L1 into 293T cells in Example 10. [Figure 18] FIG. 11 is a statistical diagram showing the results of expression levels of target proteins in plasma samples from in vivo experiments in mice using MC.BDD-FVIII-L1-X5 and MC.BDD-FVIII-L1 in Example 10. [Figure 19]11 is a statistical diagram showing the therapeutic effect of the second administration of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 in Example 11. FIG. [Figure 20] 10 is a graph showing the long-term expression of MC.BDD-FVIII-L1 minicircle DNA and MC.BDD-FVIII-L2 minicircle DNA in mice in Example 12. [Figure 21] FIG. 13 shows the results of pathological sections after injection of MC.BDD-FVIII-L1 minicircle DNA into beagle dogs in Example 13. DETAILED DESCRIPTION OF THE INVENTION

[0095] In order for those skilled in the art to better understand the technical configuration of the present invention, the following further discloses some non-limiting embodiments to describe the present invention in more detail. The reagents used in the present invention may be commercially available or may be prepared by the method described in the present invention. The range of plasma FVIII concentrations in normal humans is 100 to 200 ng / mL, with 200 ng / mL defined as 100% of the normal value (Reference: J Biol Chem 2001, 276: 46340-46346). In a specific embodiment, the amino acid and nucleotide sequences of each connecting peptide, FVIII protein, minicircle, etc. are as follows:

[0096] [Table 1] The coding sequences of BDD-FVIII with "WT" in the sequence name are coding sequences in which the B region is almost completely deleted (retaining the SQ linker) from the wild-type human FVIII coding sequence (SEQ ID NO: NM_000132). The coding sequences of BDD-FVIII with "CO1" or "CO2" in the sequence name are coding sequences in which the B region is almost completely deleted (retaining the SQ linker) from the wild-type human FVIII coding sequence (SEQ ID NO: NM_000132) after codon optimization. SEQ ID NO.63:GCCACC

[0097] Example 1: Construction of minicircle DNA parental plasmid (PP) Construct a minicircle DNA parental plasmid according to the following procedure. (1) The CMV promoter nucleotide sequence (SEQ ID NO. 29), CMV enhancer nucleotide sequence (SEQ ID NO. 30), multiple cloning site (MCS) nucleotide sequence (SEQ ID NO. 31), and bovine growth hormone polyadenylation signal (bpA) nucleotide sequence (SEQ ID NO. 32) were inserted between the attB and attP recombination sites of the minicircle DNA cloning vector (pMC.BESPX) to construct the pMC.CMV-MCS-bpA vector. (2) A DNA fragment of the target gene containing a Kozak sequence (SEQ ID NO. 63) that enhances transcription of the target gene, a human FVIII gene signal peptide coding sequence (SEQ ID NO. 33), and a BDD-FVIII coding sequence (pre-optimization) containing an SQ linker (SEQ ID NO. 35) was synthesized. The synthesized DNA fragment was cloned into the AgeI / EcoRV restriction sites of the pMC.CMV-MCS-bpA multiple cloning site to construct the wild-type BDD-FVIII minicircle DNA parent plasmid containing an SQ linker (pMC.BDD-FVIII-SQ-WT). (3) Referring to the steps (1) and (2) above, the BDD-FVIII coding sequence with SQ linker (before optimization) (SEQ ID NO. 35) was transformed into the BDD-FVIII optimized coding sequence with SQ linker-CO1 (SEQ ID NO. 45), the BDD-FVIII optimized coding sequence with SQ linker-CO2 (SEQ ID NO. 15), the BDD-FVIII optimized coding sequence with v3 linker (SEQ ID NO. 16), the BDD-FVIII optimized coding sequence with N8 linker (SEQ ID NO. 17), the BDD-FVIII optimized coding sequence with L1 linker (SEQ ID NO. 7), the BDD-FVIII optimized coding sequence with L2 linker (SEQ ID NO. 8), the BDD-FVIII optimized coding sequence with L3 linker (SEQ ID NO. 18), the BDD-FVIII coding sequence with v3 linker (SEQ ID NO. 36), and the N8 The BDD-FVIII coding sequence containing the L1 linker (SEQ ID NO. 37), the BDD-FVIII coding sequence containing the L1 linker (SEQ ID NO. 38), and the BDD-FVIII coding sequence containing the L2 linker (SEQ ID NO. 39) are replaced with the BDD-FVIII coding sequence containing the L1 linker (SEQ ID NO. 39). Construct different minicircle DNA parental plasmids, such as pMC.BDD-FVIII-SQ-CO1, pMC.BDD-FVIII-SQ-CO2, pMC.BDD-FVIII-v3, pMC.BDD-FVIII-N8, pMC.BDD-FVIII-L1, pMC.BDD-FVIII-L2, pMC.BDD-FVIII-L3, pMC.BDD-FVIII-v3-WT, pMC.BDD-FVIII-N8-WT, pMC.BDD-FVIII-L1-WT, and pMC.BDD-FVIII-L2-WT, respectively (the nucleotide sequences of each minicircle parental plasmid are in Table 1).

[0098] Example 2: Preparation of minicircle DNA Produce minicircle DNA according to the following procedure. (1) The parental plasmids (pMC.BDD-FVIII-SQ-WT, pMC.BDD-FVIII-SQ-CO1, pMC.BDD-FVIII-SQ-CO2, pMC.BDD-FVIII-v3, pMC.BDD-FVIII-N8, pMC.BDD-FVIII-L1, pMC.BDD-FVIII-L2, pMC.BDD-FVIII-L3, pMC.BDD-FVIII-L1-WT, and pMC.BDD-FVIII-L2-WT) were transformed into the genetically engineered Escherichia coli ZYCY10P3S2T strain, respectively. (2) Select positive monoclonal colonies, inoculate them into LB or TB medium, and culture them in a shaker at 37°C for 12 to 16 hours. (3) Induction is performed by adding L-arabinose (final concentration of L-arabinose: 0.2% by weight). The induction temperature is 30-32°C, and the induction time is 6-8 hours. Site-specific DNA recombination is induced in the parent plasmid, forming minicircle DNA and backbone DNA. The backbone DNA is linearized within the bacterial body and then degraded. (4) Using a plasmid DNA purification kit (QIAGEN EndoFree Plasmid Mega Kit, Qiagen, Germany), the corresponding minicircle DNAs were extracted (the nucleotide sequences of each minicircle DNA are shown in Table 1): MC.BDD-FVIII-SQ-WT, MC.BDD-FVIII-SQ-CO1, MC.BDD-FVIII-SQ-CO2, MC.BDD-FVIII-v3, MC.BDD-FVIII-N8, MC.BDD-FVIII-L1, MC.BDD-FVIII-L2, MC.BDD-FVIII-L3, MC.BDD-FVIII-L1-WT, and MC.BDD-FVIII-L2-WT.

[0099] Example 3: Cell transfection (1) 1 x 10 cells per well in a 6-well plate 6 Inoculate 293T cells at a cell inoculation density of 1000 x g. (2) The cells are cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 for 24 hours to obtain cells to be transfected. (3) Discard the serum-containing DMEM medium and replace it with serum-free expression medium (Expi293, Thermo Fisher Scientific), place in a 37°C incubator, and continue culturing for 4 hours. (4) Using a transfection reagent (X-tremeGENE HP DNA Transfection Reagent, Roche), the minicircle DNA is transferred to the cells to be transfected at a dose of 1.2 μg / well to obtain recombinant cells. (5) The recombinant cells are cultured for 72 hours, and the cell culture supernatant is collected and used for subsequent testing.

[0100] Example 4: Western blot detection (1) Sample preparation: Add reducing loading buffer to the collected cell culture supernatant, negative control sample (transfection reagent), and positive control sample (hFVIII plasma extract, Guizhou Taibang Biological Products Co., Ltd.). (2) Electrophoresis: Add an appropriate amount of loading buffer to the sample prepared in step (1), heat in boiling water for 3-5 minutes to denature the proteins, cool, then add the sample to the loading well of an SDS-PAGE gel and electrophorese at 80-100V for 1 hour. (3) Membrane transfer: Using a wet membrane transfer apparatus (Bio-Rad), the membrane is transferred at 300 mA for 1 hour to transfer proteins from the SDS-PAGE gel to a PVDF membrane. (4) Blocking: After the PVDF membrane is washed, blocking solution is added to block it. (5) Antibody incubation: Diluted HRP-labeled anti-human FVIII antibody (Affinity Biologicals) is added and incubated at room temperature for 1 hour. (6) Color development: Proteins are detected using ECL chemiluminescence reagents (Cell Signaling). Results: The detection results for MC.BDD-FVIII-SQ-CO2 are shown in FIG.

[0101] Example 5: Considerations of BDD-FVIII codon-optimized sequences The wild-type human FVIII coding sequence (SEQ ID NO: NM_000132) was obtained from the Genbank database, and the B region was almost completely deleted (retaining the SQ linker) to construct wild-type BDD-FVIII-SQ-WT (SEQ ID NO. 35). Wild-type BDD-FVIII was codon-optimized using two optimization algorithms to obtain two optimized BDD-FVIII sequences (BDD-FVIII-SQ-CO1 (SEQ ID NO. 45) and BDD-FVIII-SQ-CO2 (SEQ ID NO. 15)). MC.BDD-FVIII-SQ-WT, MC.BDD-FVIII-SQ-CO1, and MC.BDD-FVIII-SQ-CO2 were obtained by the method described in Example 1 (the nucleotide sequences of each minicircle are shown in Table 1). MC.BDD-FVIII-SQ-WT, MC.BDD-FVIII-SQ-CO1, and MC.BDD-FVIII-SQ-CO2 were transfected into 293T cells, and the cell culture supernatants were collected after 72 hours. The expression levels of the target proteins were measured by ELISA. Results are shown in Figure 8. Conclusion: The expression levels of the two optimized vectors were significantly improved, and the second optimized version (BDD-FVIII-CO2) showed an even greater improvement, exceeding 1-fold.

[0102] Comparative Example 1: In vivo expression and activity of MC.BDD-FVIII-SQ-CO2 MC.BDD-FVIII-SQ-CO2 plasma expression levels in wild-type C57 mice: Procedure: Wild-type C57 mice were intramuscularly injected with MC.BDD-FVIII-SQ-CO2 at a dose of 30 μg / mouse, and plasma expression levels were measured by ELISA. Results: Plasma expression levels were measured by ELISA and only weak expression (1-2 ng / mL) was observed. Therapeutic effect of MC.BDD-FVIII-SQ-CO2 on F8 gene knockout hemophilia A mice (F8-KO mice): Procedure: Six 12-week-old male F8-KO mice were randomly divided into two groups of three. Mice in the treatment group received an intramuscular injection of 30 μg / 50 μL (50 μL solution containing 30 μg of minicircle DNA) of minicircle DNA (MC.BDD-FVIII-SQ-CO2). Mice in the blank control group received an intramuscular injection of 50 μL of PBS. After intramuscular injection, a pulsed electric field (TERESA Biogene Transducer; parameters: 36V, 10 ms, 1 Hz) was applied. One month (m1) and two months (m2) after injection, tail cut experiments were performed twice, at 2 mm and 2.5 mm diameters, and the amount of bleeding was recorded. After cutting the tail, hemostasis was achieved with AgNO3 cautery. Results: As shown in Figure 9, after a period of observation, mice in the minicircle DNA (MC.BDD-FVIII-SQ-CO2)-injected group and untreated control mice (injected with an equal volume of PBS buffer solution without minicircle DNA) underwent tail cutting (2 mm tail diameter after 1 month, 2.5 mm tail diameter after 2 months) to compare the amount of bleeding. The results showed that the amount of bleeding in both groups was similar and not statistically different. This result indicates that MC.BDD-FVIII-SQ-CO2 treatment was not effective in improving the coagulation function of hemophilia A mice.

[0103] Example 6: Measurement of target protein expression levels by ELISA The cell culture supernatants of MC.BDD-FVIII-L1, MC.BDD-FVIII-L2, MC.BDD-FVIII-L3, MC.BDD-FVIII-v3, and MC.BDD-FVIII-N8 obtained in Example 3 were collected, and the expression levels of the target proteins were measured by ELISA. Results: As shown in Figure 10, the expression of target proteins by MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 was significantly higher than that of other FVIII minicircle DNA expression vectors, and the relative levels were as follows: (1) Expression level of target protein in MC.BDD-FVIII-L1 compared to MC.BDD-FVIII-N8 and MC.BDD-FVIII-v3: MC.BDD-FVIII-L1:MC.BDD-FVIII-N8=1.867 MC.BDD-FVIII-L1:MC.BDD-FVIII-v3=1.849. MC.BDD-FVIII-L1 is approximately 87% and 85% higher than MC.BDD-FVIII-N8 and MC.BDD-FVIII-v3, respectively. (2) Expression level of target protein in MC.BDD-FVIII-L2 compared to MC.BDD-FVIII-N8 and MC.BDD-FVIII-v3: MC.BDD-FVIII-L2:MC.BDD-FVIII-N8=1.924 MC.BDD-FVIII-L2:MC.BDD-FVIII-v3=1.906. MC.BDD-FVIII-L2 is approximately 92% and 91% higher than MC.BDD-FVIII-N8 and MC.BDD-FVIII-v3, respectively. (3) Expression level of target protein in MC.BDD-FVIII-L3 compared to MC.BDD-FVIII-N8 and MC.BDD-FVIII-v3: MC.BDD-FVIII-L3:MC.BDD-FVIII-N8=0.443 MC.BDD-FVIII-L3:MC.BDD-FVIII-v3=0.439. MC.BDD-FVIII-L3 corresponds to approximately 44% of MC.BDD-FVIII-N8 or MC.BDD-FVIII-v3. MC.BDD-FVIII-v3:MC.BDD-FVIII-N8=1.01, and there was almost no difference between MC.BDD-FVIII-v3 and MC.BDD-FVIII-N8. (4) Expression level of target protein in MC.BDD-FVIII-L1 relative to MC.BDD-FVIII-L2: MC.BDD-FVIII-L1:MC.BDD-FVIII-L2=0.97, and there is almost no difference between MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2. Conclusion: Factor VIII minicircle DNA vectors containing L1-linker or L2-linker linking peptides can express target proteins at higher levels than factor VIII minicircle DNA vectors containing other types of linking peptides.

[0104] Example 7: In vitro blood coagulation activity detection (one-stage APTT method) Procedure: Cell culture supernatants of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2, which expressed the highest levels of target protein, were collected and sent to the Institute of Blood Transfusion, Chinese Academy of Medical Sciences, for blood coagulation activity assay using the one-stage APTT method. The FVIII activity of normal pooled plasma (NPP) was defined as 100%. Results: The activity measurements of the expression products (72-h cell culture supernatant) of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 were 118.1% and 116%, respectively (the reference activity range for humans with normal blood coagulation function is 50%-150%), indicating that blood coagulation factor VIII containing L1-linker or L2-linker linked peptides has good blood coagulation activity.

[0105] Example 8: In vivo expression (1) In vivo expression in normal mice Procedure: Ten 6-8 week-old Balb / c mice were randomly divided into two groups (five mice per group): the MC.BDD-FVIII-L1 group (five mice) and the MC.BDD-FVIII-L2 group (five mice). Blood was collected one day before microring DNA injection, and plasma was separated and stored at -80°C. This served as a blank control sample (W0). Two groups of mice received intramuscular injections of MC.BDD-FVIII-L1 microring DNA and MC.BDD-FVIII-L2 microring DNA at a dose of 30 μg / 50 μL per mouse. A pulsed electric field (TERESA Biogene Transfection System; parameters: 36V, 10 ms, 1 Hz) was also applied. Blood was collected periodically after injection, and plasma was separated and stored at -80°C. The expression level of the target protein in the plasma samples was detected using a human FVIII ELISA kit (VisuLize Factor VIII PLUS Antigen ELISA Kit, Affinity Biologicals Inc.). The results are shown in Figure 11. There was no expression in the blank control sample. In the MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 groups, 4 weeks after injection, the in vivo expression levels of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 were similar, reaching 20 ng / mL or higher (equivalent to 10% or higher of the normal human plasma FVIII concentration (20 ng / mL)), exceeding the significant therapeutic level (>5%) required for mild and clear improvement of moderate or severe hemophilia. (2) In vivo expression in beagle dogs Procedure: Three adult beagle dogs weighing approximately 13 kg were blood-collected before minicircle DNA injection. Plasma was separated and stored at -80°C to serve as blank control samples (W0). On the day of blood collection, each dog was injected with 600 μg of MC.BDD-FVIII-L1 minicircle DNA and subjected to a pulsed electric field (TERESA Biogene Transfection Device; parameters: 36V, 10 ms, 1 Hz). Subsequently, blood samples were collected weekly, and plasma was separated and stored at -80°C. Expression levels of target proteins in plasma samples were measured using a human FVIII ELISA kit (VisuLize Factor VIII PLUS Antigen ELISA Kit, Affinity Biologicals Inc.) and converted to relative levels of FVIII in normal pooled plasma (NPP). The FVIII concentration in normal pooled plasma is approximately 200 ng / mL. Results are shown in Figure 12. There was no expression in the blank control sample. One week after MC.BDD-FVIII-L1 minicircle DNA injection, plasma hFVIII expression in one dog reached 5% or more of the normal human plasma FVIII concentration (10 ng / mL), while the values ​​in the other two dogs were lower. Two weeks after MC.BDD-FVIII-L1 minicircle DNA injection, expression levels gradually increased, and all exceeded 10% of the normal human plasma FVIII concentration (20 ng / mL).

[0106] Example 9: In vivo activity in hemophilia A mice (1) In vivo expression level of MC.BDD-FVIII-L1 in hemophilia A mice (F8-KO mice) Procedure: One day before minicircle DNA injection, blood was collected from five 6- to 8-week-old male F8-KO mice. Plasma was separated and stored at -80°C. This sample served as a blank control (W0). All mice received an intramuscular injection of minicircle DNA (MC.BDD-FVIII-L1 minicircle DNA) at a dose of 30 μg / 50 μL per mouse (50 μL of solution containing 30 μg of minicircle DNA). A pulsed electric field (TERESA Biogene Transfection System; parameters: 36V, 10 ms, 1 Hz) was applied. Six weeks later (W6), blood was collected, plasma was separated, and stored at -80°C. Expression levels of target proteins in plasma samples were measured using a human FVIII ELISA kit (VisuLize Factor VIII PLUS Antigen ELISA Kit, Affinity Biologicals Inc.). Results: The results are shown in Figure 13. There was no expression in the blank control sample. Six weeks after MC.BDD-FVIII-L1 minicircle DNA injection, plasma target protein levels measured by ELISA reached over 100 ng / mL, exceeding 50% of the normal plasma FVIII concentration in normal humans (100 ng / mL), suggesting that hemophilia can be effectively treated and normal coagulation function can be restored. (2) Treatment of hemophilia A mice (F8-KO mice) with MC.BDD-FVIII-L1 Procedure: Ten male F8-KO mice aged 6–8 weeks were randomly divided into two groups: a blank control group (PBS) and a minicircle DNA treatment group (MC treatment group, MC), each consisting of five mice. Mice in the minicircle DNA treatment group were intramuscularly injected with MC.BDD-FVIII-L1 minicircle DNA solution at a dose of 30 μg / 50 μL (50 μL of solution containing 30 μg of minicircle DNA). After injection, a pulsed electric field was applied (TERESA Biogene Transfection Device; parameters: 36V, 10 ms, 1 Hz). Mice in the blank control group were injected with an equal volume of PBS buffer in the same manner. Five other age-matched male C57 mice with normal coagulation function served as wild-type controls (WT). Four weeks after injection, a tail cut experiment was performed at a point 2 mm in diameter on the tail of each mouse, and the amount of bleeding within 15 minutes was recorded. Mice were sacrificed at the end of the experiment. Results: As shown in Figure 14, the bleeding volume of mice treated with MC.BDD-FVIII-L1 minicircle DNA (MC treatment group, MC) was significantly reduced compared to the blank control group (PBS), and the bleeding volume of mice treated with MC was comparable to that of wild-type normal mice (WT) (not statistically different, ns). This indicates that the blood coagulation function of hemophilia A mice was restored to normal after MC treatment. (3) Identification of the activity of MC.BDD-FVIII-L2 in hemophilia A mice (F8-KO mice) Procedure: Twenty male F8-KO mice, 9-10 weeks old, were randomly divided into two groups: a blank control group (PBS) consisting of five mice and a minicircle DNA injection group (MC treatment group, MC) consisting of 15 mice. Mice in the MC treatment group were injected with MC.BDD-FVIII-L2 minicircle DNA solution (3 μg of minicircle DNA dissolved in 1.8 mL of PBS buffer) at a dose of 3 μg / 1.8 mL per mouse (1.8 mL of solution containing 3 μg of minicircle DNA). The injection method was high-pressure tail vein injection (rapid injection of 1.8 mL of solution via the tail vein within 5-8 seconds). Mice in the blank control group were injected with an equal volume of PBS buffer (1.8 mL) in the same manner. Five other age-matched male C57 mice with normal coagulation function served as wild-type controls (WT). 24 hours after injection, a tail cut experiment was performed at a point 2 mm in diameter on the mouse tail, and the amount of bleeding within 15 minutes was recorded. After the experiment, the mice were sacrificed. One mouse in the blank control group died due to an abnormality in the high-pressure injection procedure, and the tail could not be clipped. Results: As shown in Figure 15, compared with the blank control group (PBS), the amount of bleeding in the MC.BDD-FVIII-L2 minicircle DNA-injected mice (MC treatment group, MC) was significantly reduced, and even less than that in untreated wild-type normal mice (WT), indicating that MC.BDD-FVIII-L2 has extremely high in vivo coagulation activity.

[0107] Example 10: Other modifications (1)Fc fusion Fc fusion is an important means of increasing the half-life of FVIII, and expressing molecules with longer half-lives in vivo may lead to the accumulation of more product in the body and achieve higher levels of expression. To test this hypothesis, we constructed a minicircle DNA expression vector (MC.BDD-FVIII-CTP-Fc, (the nucleotide sequence of the minicircle is shown in Table 1)) for the BDD-FVIII-Fc fusion protein (SEQ ID NO. 19). Procedure: Ten 6- to 8-week-old Balb / c mice were randomly divided into two groups, five in each group, with one group receiving minicircle DNA (MC.BDD-FVIII-CTP-Fc) injections and the other receiving a PBS blank control. Mice in the minicircle group received intramuscular injections of minicircle DNA (MC.BDD-FVIII-CTP-Fc) at a dose of 45 μg / 50 μL per mouse (50 μL of solution containing 45 μg of minicircle DNA, equimolar to 30 μg of MC.BDD-FVIII). Mice in the blank control group received intramuscular injections of 50 μL of PBS. After intramuscular injection, pulsed electric fields (TERESA Biogene Transfection System; parameters: 36V, 10 ms, 1 Hz) were applied. Blood samples were then collected periodically, and plasma was separated and stored at -80°C. The expression level of the target protein in the plasma samples was measured using a human FVIII ELISA kit (VisuLize Factor VIII PLUS Antigen ELISA Kit, Affinity Biologicals Inc.). The results are shown in Figure 16. There was no expression in the blank control sample. 11 to 15 weeks after injection of the MC.BDD-FVIII-CTP-Fc minicircle DNA, the BDD-FVIII-Fc fusion protein minicircle DNA did not further improve the in vivo expression level (only about 10 ng / mL, which is less than 5% of the normal level (10 ng / mL) of normal human plasma FVIII concentration). (2)X5 mutation Measuring in vitro cellular expression levels of a protein of interest by ELISA: Procedure: The X5 mutation (i.e., five key amino acids in the human FVIII A domain were mutated to the corresponding amino acids in porcine FVIII: I86V / A108S / G132K / M147T / L152P) was introduced into BDD-FVIII-L1 to obtain the BDD-FVIII-L1-X5 nucleotide sequence (SEQ ID NO. 22). The corresponding MC expression vector (the nucleotide sequence of each minicircle is shown in Table 1) was constructed with MC.BDD-FVIII-L1-X5 minicircle DNA. MC.BDD-FVIII-L1 and MC.BDD-FVIII-L1-X5 minicircle DNAs were transfected into 293T cells, respectively. After 72 hours, the cell culture supernatants were collected and the target protein levels were measured by ELISA. Results: As shown in Figure 17, the target protein level was increased after the introduction of X5 mutation compared to the MC.BDD-FVIII-L1 minicircle DNA. In vivo experiments in mice: Procedure: Ten 6- to 8-week-old Balb / c mice were randomly divided into two groups of five mice each. Each group received an intramuscular injection of minicircle DNA (MC.BDD-FVIII-L1 minicircle DNA or MC.BDD-FVIII-L1-X5 minicircle DNA) at a dose of 30 μg / 50 μL per mouse (50 μL solution containing 30 μg of minicircle DNA). After intramuscular injection, a pulsed electric field (TERESA Biogene Transfection System; parameters: 36V, 10 ms, 1 Hz) was applied. Four weeks after injection, blood was collected, plasma was separated, and the expression level of the target protein in the plasma samples was measured using a human FVIII ELISA kit (VisuLize Factor VIII PLUS Antigen ELISA Kit, Affinity Biologicals Inc.). Results: As shown in Figure 18, the level of the target protein was slightly increased after introducing the X5 mutation compared to the MC.BDD-FVIII-L1 minicircle DNA. (3) R1645H mutation Siner et al. (Blood 2013, 121:4396-4403) reported that the furin site (R) at the C-terminus of the SQ linker in the BDD-FVIII B region 1645 -H 1646 -Q 1647 -R 1648 (2014) reported that mutating RHQR resulted in a gene that was not recognized or cleaved by Furin protease, leading to increased expression. Because the furin site has a recognition pattern of RXXR (i.e., arginine at the beginning and end, and any amino acid at the middle two positions), theoretically, mutating the arginine at positions 1645 or 1648 to any amino acid other than arginine could prevent recognition and cleavage by Furin protease. This goal could also be achieved by deleting the furin site (including deleting all four amino acids of RHQR, or any one, two, or three of them). In the present invention, since a furin site called RHQR remains at the C-terminus of the L1 linker, the fourth arginine (Arg, R) from the bottom of the C-terminus of the L1 linker is mutated to histidine (His, H), i.e., the R1645H mutation, and the coding sequence of BDD-FVIII-L1 is BDD-FVIII-L1. R1645H (nucleotide sequence shown in Table 1) was obtained and the corresponding MC expression vector MC.BDD-FVIII-L1 R1645H (The nucleotide sequences of the minicircles are shown in Table 1) were constructed. (4) F309S mutation Swaroop et al. (J Biol Chem 1997, 272:24121-24124) reported that the F309S mutation (a mutation from phenylalanine to serine at position 309 in FVIII) enhances FVIII secretion. This resulted in the formation of BDD-FVIII-L1 and BDD-FVIII-L1. R1645H and BDD-FVIII-L2, the phenylalanine at position 309 was mutated to serine (Ser, S), resulting in BDD-FVIII F309S -L1, BDD-FVIII F309S -L1 R1645H and BDD-FVIIIF309S The coding sequences of MC.BDD-FVIII-L2 were obtained (the nucleotide sequences of each are shown in Table 1), and the corresponding MC expression vector MC.BDD-FVIII F309S -L1, MC.BDD-FVIII F309S -L1 R1645H and MC.BDD-FVIII F309S -L2 was constructed (the nucleotide sequence of each minicircle is shown in Table 1).

[0108] Example 11: Therapeutic effect of secondary administration Procedure: Ten 6- to 8-week-old Balb / c mice were randomly divided into two groups: five in the MC.BDD-FVIII-L1 group and five in the MC.BDD-FVIII-L2 group. Mice in the two groups received intramuscular injections of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 minicircle DNA at a dose of 30 μg / 50 μL (50 μL solution containing 30 μg of minicircle DNA each) and pulsed electric fields (TERESA Biogene Transfection Device; parameters: 36V, 10 ms, 1 Hz). At week 22 (W22), the mice were administered the same treatment again. After the first and second injections, blood samples were collected periodically, and plasma was separated and stored at -80°C. Expression levels of target proteins in plasma samples were measured using a human FVIII ELISA kit (VisuLize Factor VIII PLUS Antigen ELISA Kit, Affinity Biologicals Inc.). The FVIII levels were then converted to the relative levels of normal pooled plasma (NPP), which has a FVIII concentration of approximately 200 ng / mL. Results are shown in Figure 19. After minicircle DNA injection, the in vivo expression levels in both groups of mice were 20% (40 ng / mL) or more of the normal human plasma FVIII concentration at week 4 (W4). Although the expression levels decreased at week 12 (W12), they remained above 10% (20 ng / mL) of the normal human plasma FVIII concentration for a long period (W12-W20). After a booster injection at week 22 (W22), the expression levels at W23 (23 weeks after the first injection, i.e., 1 week after the second injection) rapidly recovered to levels close to those before the decrease. Secondary injections of the minicircle DNA provided by the present invention remained effective and did not induce a virus-specific immune response, demonstrating that secondary injections are possible and that repeated administration, as needed, can restore or enhance therapeutic efficacy.

[0109] Example 12: Long-term expression in vivo Procedure: Ten 6- to 8-week-old Balb / c mice were randomly divided into two groups: five in the MC.BDD-FVIII-L1 group and five in the MC.BDD-FVIII-L2 group. Blood was collected before minicircle DNA injection, and plasma was separated and stored at -80°C. This served as a blank control sample (W0). Mice in the two groups received intramuscular injections of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 minicircle DNA at a dose of 30 μg / 50 μL (50 μL solution containing 30 μg of minicircle DNA each) and pulsed electric fields (TERESA Biogene Transfection System; parameters: 36V, 10 ms, 1 Hz). The mice were re-administered in the same manner 22 weeks after injection. Blood samples were collected periodically after injection, and plasma was separated and stored at -80°C. The expression level of the target protein in the plasma samples was measured using a human FVIII ELISA kit (VisuLize Factor VIII PLUS Antigen ELISA Kit, Affinity Biologicals Inc.). Results: As shown in Figure 20, both MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 were expressed in mice for more than a year, and secondary injections of the minicircle DNA of the present invention were also effective for a long period of time and did not induce virus-specific immune responses.

[0110] Example 13: Safety study Procedure: One adult female beagle (number D0016) weighing 10-12 kg and one adult male beagle (number C957) weighing 10-12 kg were each injected with 1.2 mg of MC.BDD-FVIII-L1 minicircle DNA and subjected to a pulsed electric field (36 V, 10 ms, 1 Hz). Six weeks after injection, lung, liver, spleen, kidney, heart, brain, ovary or testis, muscle, and thymus tissues were collected, sliced, stained with HE staining, and subjected to pathological examination. The results are shown in Figure 21. Results: Six weeks after injection of MC.BDD-FVIII-L1 minicircle DNA, pathological examinations of the lungs, liver, spleen, kidneys, heart, brain, ovaries (female dog D0016), testes (male dog C957), muscle, and thymus tissues of the beagle dogs were all normal (Figure 21). No obvious toxicity was observed, indicating the safety of MC.BDD-FVIII-L1.

[0111] Although the method of the present invention has been described by way of preferred embodiments, it is obvious to those skilled in the art that modifications or appropriate modifications and combinations may be made to the method and application of the present invention within the content, spirit and scope of the present invention in order to realize and apply the technology of the present invention. Those skilled in the art can achieve this by referring to the contents of this specification and appropriately improving the process parameters. In particular, it should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention.

Claims

1. A connecting peptide comprising: A connecting peptide having the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO.

2.

2. A nucleic acid molecule comprising a nucleotide sequence encoding the connecting peptide of claim 1.

3. 3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence comprises SEQ ID NO. 3 or SEQ ID NO. 4, or a codon-optimized version of either nucleotide sequence.

4. 10. Use of the connecting peptide of claim 1 for constructing a recombinant blood coagulation factor VIII protein.

5. Use of the nucleic acid molecule according to claim 2 for constructing a nucleic acid molecule comprising the nucleotide sequence of a recombinant blood coagulation factor VIII protein.

6. A blood coagulation factor VIII protein, 2. A blood coagulation factor VIII protein, wherein the connecting peptide in the blood coagulation factor VIII protein is selected from the connecting peptides of claim 1.

7. 7. The blood coagulation factor VIII protein of claim 6, wherein the amino acid sequence is SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, or SEQ ID NO.

53.

8. A nucleic acid molecule comprising a nucleotide sequence encoding the blood coagulation factor VIII protein of claim 6, The nucleic acid molecule, wherein the nucleotide sequence comprises SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, or SEQ ID NO. 60, or a codon-optimized version of any of these nucleotide sequences.

9. A recombinant gene vector comprising: A recombinant gene vector comprising a nucleotide sequence encoding the blood coagulation factor VIII protein of claim 6.

10. The recombinant gene vector according to claim 9, characterized in that the recombinant gene vector comprises a non-viral vector or a viral vector.

11. The non-viral vector is selected from a standard plasmid or other circular expression cassette; Alternatively, the viral vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

12. The non-viral vector according to claim 10, characterized in that the non-viral vector is selected from minicircle DNA vectors.

13. A parent plasmid used to prepare minicircle DNA, A plasmid vector comprising the nucleic acid molecule of claim 8, Alternatively, a parent plasmid used for producing minicircle DNA is formed by inserting a promoter nucleotide sequence, an enhancer nucleotide sequence, a multicloning site nucleotide sequence, a polyA signal nucleotide sequence, and a target gene DNA fragment into a plasmid vector, wherein the target gene DNA fragment is located between the restriction endonuclease enzyme cleavage sites of the multicloning site, and the target gene DNA fragment comprises the nucleotide sequence described in claim 8.

14. A minicircle DNA, The nucleotide sequence of the minicircle DNA is selected from SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59 and SEQ ID NO.

61.

15. A host cell comprising: The recombinant gene vector according to claim 9, The host cell comprises a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

16. 1. A pharmaceutical composition comprising: A pharmaceutical composition comprising the recombinant gene vector of claim 9 and a pharmaceutically acceptable adjuvant or carrier.

17. 10. Use of the blood coagulation factor VIII protein according to claim 6 in the manufacture of a drug for treating a disease, comprising: The use, characterized in that the disease is selected from inherited gene deficiency diseases, or the disease is a disease caused by a blood coagulation factor deficiency.

18. 18. The use according to claim 17, wherein the disease is a disease caused by a deficiency of blood coagulation factor VIII.

19. 18. The use according to claim 17, wherein the disease is hemophilia.

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