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 gene defect therapeutic drugs, and specifically relates to a linking peptide, a blood coagulation factor VIII protein or a variant thereof containing the linking peptide, and their uses.

Background Art

[0002] Hemophilia is a group of blood coagulation disorders caused by a single gene defect on the X chromosome linked to a blood coagulation factor defect. Among them, hemophilia A is deficient in blood coagulation factor VIII (FVIII) encoded by the F8 gene, and the number of its patients is the largest, accounting for 80-85%. Currently, the standard treatment method for hemophilia A is replacement therapy, that is, to supplement exogenous FVIII protein, including plasma-derived FVIII (pdFVIII) and recombinant FVIII (rFVIII). pdFVIII is an extract separated from normal human plasma, with limited donor sources and a risk of transmission of blood-derived viruses. rFVIII is expressed and purified in vitro using mammalian cell lines, which can effectively reduce the risk of blood-derived virus infection without being restricted by blood supply, but has high requirements for the production process.

[0003] The in vivo half-life of FVIII protein is short (average 12 hours), and patients undergoing replacement therapy need to receive lifelong administration. They have to inject frequently (intravenously 2 - 3 times a week, or even 4 times), which is costly and inconvenient for treatment. By means of ordinary long-term modification methods such as Fc fusion (Drug Des Devel Ther 2014, 8:365 - 371) and PEG modification (Haemophilia 2019, 25:773 - 781), its half-life can be extended to about 19 hours, but the extent is limited (only 50 - 60% extension), and the dosing frequency cannot be significantly reduced. Sanofi and Sobi jointly developed a new type of long-lasting FVIII fusion protein (BIVV001; FVIIIFc-vWF-XTEN), and by fusing the antibody Fc fragment with the vWF factor D´D3 domain (D3 domain), the half-life was further extended to 38 - 44 hours, and the dosing frequency was reduced to once a week (N Engl J Med. 2020, 383:1018 - 1027). The modification for long-term action on these protein molecules can extend the half-life to some extent and improve the patient's dosing experience to a certain degree, but it cannot fundamentally change the treatment model of "lifelong administration and repeated injection".

[0004] On the other hand, FVIII gene therapy is expected to "cure" this disease functionally by correcting the genetic defect of patients with hemophilia A at the gene level. Currently, in the industry, AAV-hFVIII (targeted delivery of the normal human FVIII gene in liver tissue is achieved using an adeno-associated virus vector) is generally considered the "optimal" solution for hemophilia A gene therapy. However, there are obvious defects in the existing liver-targeted AAV-FVIII gene therapy (Hemasphere 2021, 5:e540). i) AAV-hFVIII is only applicable to adult patients over 18 years old. The livers of children are in the stage of rapid growth and development, with active hepatocyte division, and are not suitable for the use of this treatment method (the risk of gene integration is significantly increased, and the unintegrated target gene is likely to be lost during cell division). ii) AAV is a virus commonly found in humans, and due to its high infection rate within the human population, many patients pre-exist with AAV neutralizing antibodies, and these patient groups cannot use this treatment method either. iii) Repeated administration is not possible. AAV-hFVIII is not effective for life, and the expression level may decrease below the therapeutic level after several years. In this case, it is necessary to administer again to restore the therapeutic level. However, the AAV virus coat is highly immunogenic, and a virus-specific immune response is likely to occur after the first administration, rendering re-administration ineffective. iv) The hFVIII coding gene is large, exceeding the limitation of the AAV vector capacity, making the packaging and production of AAV-hFVIII difficult. In addition, there are other safety concerns such as random integration of genes in AAV therapy.

[0005] The expression of hFVIII (human blood coagulation factor VIII) is quite difficult and is a major common and crucial technical problem faced by the production of recombinant FVIII (in vitro expression) and FVIII gene therapy (in vivo expression). The expression level of hFVIII is only 1 / 100 to 1 / 1000 of that of a normal protein corresponding to its molecular weight (Hum Gene Ther 1993, 4:259 - 272; Blood 2004, 103:3412 - 3419). Therefore, modifying the hFVIII molecule to increase its expression level has become the focus of research in the related fields. FVIII contains a total of six structural domains, namely A1 - A2 - B - A3 - C1 - C2. Among them, the length of the B domain is 908 aa, accounting for approximately 40% of the full length (2332 aa). The deletion of the B domain does not affect the blood coagulation function (PNAS 1986, 83:5939 - 5942) and significantly increases the levels of mRNA (17 - fold) and protein product (30%) (Blood 2004, 103:3412 - 3419; Blood Coagul Fibrinolysis 1997, 8 Suppl 2:S3 - 14). Therefore, the deletion of the B domain has become a widely adopted modification method. In fact, many B domain - deleted FVIII drugs (B domain deleted FVIII, BDD - FVIII) have already been applied clinically on a large scale for many years. For example, Xyntha produced by Pfizer. The first BDD - FVIII almost completely deletes the B domain, leaving only the linker - linked A1 - A2 and A3 - C1 - C2 composed of the N - terminal 4 amino acids and C - terminal 10 amino acids (a total of 14 aa) of the B domain of natural FVIII. This linker is called the SQ linker, and its sequence is SFSQNPPVLKRHQR. For convenience, BDD - FVIII containing the SQ linker is denoted as BDD - FVIII - SQ. We have investigated the possibility of in vivo expressing therapeutic - level BDD - FVIII - SQ using a minicircle DNA vector. However, experiments have shown that the BDD - FVIII - SQ vector can only be weakly expressed in vivo (less than 1% of the normal level), and no therapeutic effect can be obtained. There are three levels of therapeutic levels.i) Exceeding 1%, having a preliminary therapeutic effect, and severe hemophilia can be moderately alleviated. ii) Exceeding 5%, having a significant therapeutic effect, and moderate to severe hemophilia is significantly alleviated to a mild degree. iii) Exceeding 50%, achieving functional "cure" and completely restoring normal blood coagulation function.

[0006] The B-domain linker connects A1 - A2 and A3 - C1 - C2, and its sequence has an important impact on the expression level of the BDD - FVIII protein. Miao et al. (Blood 2004, 103: 3412 - 3419) reported that retaining 226 amino acids (226aa / N6) at the N-terminus of the B-domain and having almost complete deletion of the B-domain (wild-type SQ linker) had an expression level 4 times higher (detected by ELISA). Based on this, McIntosh et al. (Blood 2013, 121: 3335 - 3344; Patent: WO 2013 / 186563) found that replacing the 31aa v3 linker (sequence SFSQNATNVSNNSNTSNDSNVSPPVLKRHQR) with 226aa / N6 could further increase the expression level of the target protein (by about 50%). In the present invention, BDD - FVIII containing the v3 linker is designated 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 in Denmark developed another BDD - FVIII molecule with better expression than BDD - FVIII - SQ and containing a 21aa linker (sequence SFSQNSRHPSQNPPVLKRHQR) (N8, Haemophilia 2010, 16: 349 - 359; Patent: WO2006103298). In the present invention, this molecule is designated as BDD - FVIII - N8. However, problems such as low expression levels and low therapeutic effects remain for BDD - FVIII - v3 and BDD - FVIII - N8.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

[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] [Problems to be Solved by the Invention]

[0009] Therefore, there is still an urgent need for a drug that can be highly expressed, can be administered secondarily, has a simple preparation, has a good therapeutic effect, and can be expressed in the body for a long time to treat diseases related to blood coagulation factor VIII deficiency. [Means for Solving the Problems]

[0010] In order to solve the above problems, the present invention provides the following technical solutions. In a first aspect, a linking peptide is provided. The recombinant blood coagulation factor VIII protein or its variant using the linking peptide according to the present invention has advantages such as high expression level, excellent secondary administration effect, simple preparation, and excellent therapeutic effect. The minicircle DNA of the recombinant blood coagulation factor VIII protein or its variant using the linking peptide according to the present invention has advantages such as high expression level, excellent secondary administration effect, simple preparation, and excellent therapeutic effect, and also solves the problems of difficult production (packaging difficulty) of AAV.hf8, limitation of application range (cannot be used in children and cannot be administered secondarily), and safety risk (cancer induction by random integration), that is, it has advantages such as simple production, wide application range (can be used in children and has good therapeutic effect for secondary administration), good safety, and long-term expression in the body.

[0011] In a second aspect, a nucleotide sequence is provided. The nucleotide sequence can encode the linking peptide described in the first aspect.

[0012] In a third aspect, uses of the linking peptide described in the first aspect or the nucleotide sequence described in the second aspect are provided.

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

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

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

[0016] In a seventh aspect, a parental plasmid used for the production of minicircle DNA is provided.

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

[0018] In a ninth aspect, minicircle DNA obtained by the production method described in the eighth aspect is provided. The minicircle DNA has advantages such as high expression levels, excellent secondary administration effects, simple preparation, and excellent therapeutic effects. It solves the problems of the difficulty in producing AAV.hf8 (packaging difficulty), the limitation of the application range (not applicable to children and cannot be administered secondarily), and the safety risk (cancer induction by random integration). That is, it has advantages such as simple production, a wide application range (applicable to children and has a good therapeutic effect for secondary administration), good safety, and long-term expression in the body.

[0019] In a tenth aspect, a host cell is provided that contains the nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or its variant described in the fourth aspect, the nucleotide sequence described in the fifth aspect, the recombinant gene vector described in the sixth aspect, or the minicircle DNA described in the ninth aspect.

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

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

[0022] (Detailed Description of the Invention) In order to solve the above problems, the present invention provides the following technical solutions. In a first aspect, a linking peptide is provided. A linking peptide, wherein the amino acid sequence is SEQ ID NO.1 or SEQ ID NO.2, or an amino acid sequence having at least 80-99% identity with any nucleotide sequence or at least a part of any sequence. The recombinant blood coagulation factor VIII protein or its variant using the linking peptide according to the first aspect of the present invention has advantages such as high expression level, good secondary administration effect, simple production, and good therapeutic effect. The minicircle DNA of the recombinant coagulation factor VIII protein or its variant using the linking peptide according to the first aspect of the present invention has advantages such as high expression level, excellent secondary administration effect, simple preparation, and excellent therapeutic effect, and solves the problems of difficult production of AAV.hf8 (difficult packaging), limited application range (cannot be used in children and cannot be administered secondarily), and safety risks (cancer induction by random integration), that is, it has advantages such as simple production, wide application range (can be used in children and has good therapeutic effect for secondary administration), good safety, and long-term expression in the body.

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

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

[0025] In the second aspect, a nucleotide sequence is provided. A nucleotide sequence encoding the linking peptide described in the first aspect.

[0026] In some examples, the nucleotide sequence includes SEQ ID NO.3 or SEQ ID NO.4, or the codon-optimized sequence of any of the nucleotide sequences thereof.

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

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

[0029] In a third aspect, provided is the use of the linker peptide described in the first aspect or the nucleotide sequence described in the second aspect. It is for use in constructing a recombinant blood coagulation factor VIII protein or a variant thereof with the linker peptide described in the first aspect or the nucleotide sequence described in the second aspect. It is for use in a nucleotide sequence for constructing a recombinant blood coagulation factor VIII protein or a variant thereof with the nucleotide sequence described in the second aspect.

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

[0031] In some embodiments, a recombinant blood coagulation factor VIII protein or a variant thereof, 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, or an amino acid sequence having at least 80% to 99% identity with any of the amino acid sequences or at least a part of any of the amino acid sequences.

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

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

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

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

[0036] In some embodiments, the nucleotide sequence encoding a recombinant blood coagulation factor VIII protein or a variant thereof having an amino acid sequence of SEQ ID NO.43 may include 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 can include 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 can include 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 can include 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 can include 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 can include SEQ ID NO. 54 or a codon-optimized sequence thereof.

[0042] In a fifth aspect, a nucleotide is provided. A nucleotide sequence encoding the 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 sequence of any of the nucleotide sequences thereof.

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

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

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

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

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

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

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

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

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

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

[0053] In a sixth aspect, a recombinant gene vector is provided. 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 a standard plasmid or other circular expression cassette.

[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, a parental plasmid used for the production of minicircle DNA is provided. The parental plasmid for minicircle DNA production includes a plasmid vector containing the nucleotide sequence described in the fifth aspect.

[0059] In some embodiments, a parental plasmid used for the production of minicircle DNA, wherein a promoter nucleotide sequence, an enhancer nucleotide sequence, a multiple cloning site nucleotide sequence, a polyA signal nucleotide sequence, and a target gene DNA fragment are inserted into a plasmid vector, the target gene DNA fragment is located between the restriction endonuclease cleavage sites of the multiple cloning site, and the target gene DNA fragment contains the nucleotide sequence described in the fifth aspect.

[0060] In some embodiments, the promoter is a CMV promoter, and its nucleotide sequence 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 multi-cloning site nucleotide sequence may be SEQ ID NO. 31.

[0063] In some embodiments, the polyA signal may include one selected from the group consisting of bovine growth hormone polyA signal, human growth hormone polyA signal, or 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 include a Kozak sequence and a human FVIII gene signal peptide coding sequence. The Kozak sequence is advantageous for enhancing the 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 includes one selected from the group consisting of pMC.BESPX plasmid or p2ΦC31 plasmid.

[0069] In some embodiments, the promoter nucleotide sequence, enhancer nucleotide sequence, multi-cloning site nucleotide sequence, and polyA signal nucleotide sequence are inserted into the 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 restriction endonuclease cleavage site of AgeI and EcoRV 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 by the site-specific recombination action of the parental plasmid at the site of the 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 in the host cell.

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

[0074] In a ninth aspect, minicircle DNA is provided. The minicircle DNA according to the ninth aspect of the present invention obtained by the production 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 difficult production (difficult packaging) of AAV.hf8, limited application range (cannot be used in children and cannot be administered secondarily), and safety risks (cancer induction by random integration), that is, it has advantages such as simple production, wide application range (can be used in children and has good therapeutic effect for secondary administration), and good safety.

[0075] In some embodiments, the nucleotide sequence of the minicircle DNA may include those 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 the recombinant blood coagulation factor VIII protein or a variant thereof according to the fourth aspect, the nucleotide sequence according to the fifth aspect, the recombinant gene vector according to the sixth aspect, or the minicircle DNA according to the ninth aspect.

[0077] In some embodiments, the host cell may include a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is selected from Escherichia coli. In some embodiments, the host cell is selected from Escherichia 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, or a minicircle DNA obtained by the production method according to the eighth aspect, or the 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 high expression level, good secondary administration effect, simple production, and good therapeutic effect. It solves the problems of difficult production (difficult packaging) of AAV.hf8, limited application range (not applicable to children and cannot be administered secondarily), and safety risks (cancer induction by random integration), that is, it has advantages such as simple production, wide application range (applicable to children and good therapeutic effect of secondary administration), good safety, and long-term expression in the body.

[0079] The twelfth aspect provides an application. Use in the manufacture of a drug for treating a disease of a recombinant blood coagulation factor VIII protein or a 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 parental 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 some embodiments, the disease may be selected from hereditary genetic defect diseases. In some embodiments, the disease may be a disease caused by a blood coagulation factor defect. 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.

Advantages of the Invention

[0080] Certain embodiments of the present invention have at least one of the following beneficial technical effects as compared with the prior art. (1) The recombinant gene vector, minicircle DNA, pharmaceutical composition according to the present invention, or the recombinant blood coagulation factor VIII protein or its variant using the linking peptide according to the present invention has advantages such as high expression level, good secondary administration effect, simple production, good therapeutic effect, and long-term expression in the body. (2) It solves the problems of difficult production (packaging difficulty) of AAV.hf8, limited application range (not applicable to children and cannot be administered secondarily), and safety risks (cancer induction by random integration), that is, it has advantages such as simple production, wide application range (applicable to children and good secondary administration effect), good safety, and long-term expression in the body.

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

[0082] In the description of this specification, a description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in relation to that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily target the same embodiment or example. Furthermore, the described specific features, structures, materials, or features may be combined in a suitable manner in any one or more embodiments or examples. Also, those skilled in the art may combine different embodiments or examples described in this specification, as well as the features of different embodiments or examples, as long as they do not conflict with each other.

[0083] The fusion protein of the present invention is usually produced by a biosynthesis method. According to the nucleotide sequence described in the present invention, the coding nucleic acid of the present invention can be produced by various methods that are conveniently known to those skilled in the art. These methods include, but are not limited to, for example, PCR, DNA artificial synthesis, etc. For specific methods, refer to "Molecular Cloning: A Laboratory Manual" by J. Sambrook. The coding nucleic acid sequence of the present invention can be constructed, as one embodiment of the present invention, by a method of fragmentarily synthesizing the nucleotide sequence and then overlapping and extending it.

[0084] The "parent plasmid" contains an expression cassette of the target gene and skeletal DNA, and the parent plasmid can generate the original plasmid of the minicircle DNA. After site-specific DNA recombination is performed with this plasmid, minicircle DNA is generated.

[0085] The term "fusion protein" generally means a protein obtained by fusing two or more proteins or polypeptides. The two or more protein or polypeptide genes or nucleic acid molecules may encode and be ligated to each other to form a fusion gene or a fusion nucleic acid molecule, and the fusion gene or the fusion nucleic acid molecule may encode the fusion protein. By translation of the fusion gene, at least one of the two or more proteins or polypeptides before fusion, or a single polypeptide having the respective properties, is produced. Recombinant fusion proteins are artificially created by recombinant DNA techniques used in biological research and therapy. A recombinant fusion protein is a protein produced by genetic engineering of a fusion gene. The present invention relates to a recombinant fusion protein, and the fusion protein and the recombinant fusion protein are used herein in the same meaning. The fusion protein described herein generally includes 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 generally involves removing the stop codon from the cDNA sequence encoding the first protein or polypeptide and then ligating the cDNA sequence of the second protein or joining it in the reading frame by overlap extension PCR. This DNA sequence is expressed by the cell as a single protein. The protein can be engineered to include the complete sequences of the two original proteins or polypeptides, or only a portion of either.

[0086] In the present application, a "pharmaceutical composition" can preferably be expressed in unit dosage form and can be prepared by any method well known in the pharmaceutical art. Any method includes the step of combining an active ingredient with a carrier constituting one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a fine solid carrier, or both.

[0087] In the present application, the "recombinant gene vector" generally means a nucleic acid molecule that can self-replicate in a suitable host for transferring the inserted nucleic acid molecule into and / or between host cells. Examples of the recombinant gene vector include vectors mainly used for inserting DNA or RNA into cells, recombinant gene vectors mainly used for replicating DNA or RNA, and recombinant gene vectors mainly used for the expression of transcription and / or translation of DNA or RNA. The recombinant gene vector also includes a recombinant gene vector having the above-mentioned multiple functions. The recombinant gene vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the recombinant gene vector, a desired expression product is generated from the recombinant gene vector.

[0088] In the present application, the term "variant" can mean any naturally occurring molecule or engineered molecule that contains mutations of one or more nucleotides or amino acids.

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

[0090] In the present specification, the "host cell" means a prokaryotic cell or a eukaryotic cell into which a recombinant expression vector can be introduced. In the present specification, the terms "transformed" or "transfected" mean introducing a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. A suitable host cell can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and / or secretion of the target protein.

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

[0093] In the present invention, both "MC.BDD-FVIII-L1" and "MC.BDD-FVIII-L1 microcircular DNA" represent the same meaning, and the meanings of other terms starting with "MC.BDD" can be analogized in this way, for example, "MC.BDD-FVIII-L2" or "MC.BDD-FVIII-L2 microcircular DNA" represent the same meaning.

Brief Description of the Drawings

[0094]

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Modes for Carrying Out the Invention

[0095] In order for those skilled in the art to better understand the technical configuration of the present invention, several non-limiting embodiments are further disclosed below to explain the present invention in more detail. The reagents used in the present invention may all be commercially available, or they can be prepared by the methods described in the present invention. The range of FVIII concentration in normal human plasma is 100 - 200 ng / mL, and 200 ng / mL is defined as 100% of the normal value (Reference: J Biol Chem 2001, 276: 46340 - 46346). The amino acid sequences and nucleotide sequences of each linker peptide, FVIII protein, mini-circle, etc. in the specific embodiments are as follows.

[0096]

Table 1

[0097] Example 1: Construction of minicircle DNA parental plasmid (PP) Construct a minicircle DNA parental plasmid according to the following procedure. (1) Insert the CMV promoter nucleotide sequence (SEQ ID NO.29), CMV enhancer nucleotide sequence (SEQ ID NO.30), multiple cloning site nucleotide sequence (MCS) (SEQ ID NO.31), and bovine growth hormone polyA signal nucleotide sequence (bpA) (SEQ ID NO.32) between the attB and attP recombination sites of the minicircle DNA cloning vector (pMC.BESPX) to construct the pMC.CMV-MCS-bpA vector. (2) Synthesize a target gene DNA fragment containing the Kozak sequence (SEQ ID NO.63) that enhances the transcription of the target gene, the human FVIII gene signal peptide coding sequence (SEQ ID NO.33), and the BDD-FVIII coding sequence (before optimization) containing the SQ linker (SEQ ID NO.35). Clone the synthesized DNA fragment between the AgeI / EcoRV restriction sites of the pMC.CMV-MCS-bpA multiple cloning site to construct the minicircle DNA parental plasmid of wild-type BDD-FVIII containing the SQ linker (pMC.BDD-FVIII-SQ-WT). (3) Referring to the operations in (1) and (2) above, the BDD-FVIII coding sequence (before optimization) (SEQ ID NO.35) containing the SQ linker is replaced with the BDD-FVIII optimized coding sequence - CO1 (SEQ ID NO.45) containing the SQ linker, the BDD-FVIII optimized coding sequence - CO2 (SEQ ID NO.15) containing the SQ linker, the BDD-FVIII optimized coding sequence (SEQ ID NO.16) containing the v3 linker, the BDD-FVIII optimized coding sequence (SEQ ID NO.17) containing the N8 linker, the BDD-FVIII optimized coding sequence (SEQ ID NO.7) containing the L1 linker, the BDD-FVIII optimized coding sequence (SEQ ID NO.8) containing the L2 linker, the BDD-FVIII optimized coding sequence (SEQ ID NO.18) containing the L3 linker, the BDD-FVIII coding sequence (SEQ ID NO.36) containing the v3 linker, the BDD-FVIII coding sequence (SEQ ID NO.37) containing the N8 linker, the BDD-FVIII coding sequence (SEQ ID NO.38) containing the L1 linker, and the BDD-FVIII coding sequence (SEQ ID NO.39) containing the L2 linker. Different mini-circle 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, pMC.BDD-FVIII-L2-WT (the nucleotide sequences of each micro-ring parental plasmid are shown in Table 1) are constructed.

[0098] Example 2: Production of Mini-Circle DNA Mini-circle DNA is produced 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, pMC.BDD-FVIII-L2-WT) were each transformed into genetically engineered Escherichia coli ZYCY10P3S2T. (2) Positive monoclonal colonies were selected, inoculated into LB medium or TB medium, and cultured on a shaker at 37 °C for 12 to 16 hours. (3) Induction was carried out by adding L - arabinose (final concentration of L - arabinose 0.2% by weight), the induction temperature was 30 °C to 32 °C, the induction time was 6 to 8 hours, site - specific DNA recombination of the parental plasmid was induced to form mini - circle DNA and backbone DNA, and the backbone DNA was linearized and then degraded in the bacteria. (4) The corresponding mini - circle DNA was extracted using a plasmid DNA purification kit (QIAGEN EndoFree Plasmid Mega Kit, Qiagen, Germany) (the nucleotide sequences of each mini - circle 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, MC.BDD-FVIII-L2-WT.

[0099] Example 3: Cell Transfection (1) 293T cells were inoculated in a 6 - well plate at an inoculation density of 1×10 6 cells per well. (2) The cells were cultured in DMEM medium containing 10% fetal bovine serum under the conditions of 37 °C and 5% CO2 for 24 hours to obtain the cells to be transfected. (3) Discard the DMEM medium containing serum, replace it with serum-free expression medium (Expi293, Thermo Fisher Scientific), and continue culturing in a 37 °C incubator for 4 hours. (4) Using a transfection reagent (X-tremeGENE HP DNA Transfection Reagent, Roche), transfer the mini-circle DNA to the target cells for transfection at a dose of 1.2 μg / well to obtain recombinant cells. (5) Culture the recombinant cells for 72 hours, collect the cell culture supernatant, and use it for subsequent inspections.

[0100] Example 4: Western blot detection (1) Sample preparation: To the collected cell culture supernatant, negative control sample (transfection reagent), and positive control sample (hFVIII plasma extract, "Guizhou Taibang Biological Products Co., Ltd."), add reducing loading buffer respectively. (2) Electrophoresis: After adding an appropriate amount of loading buffer to the samples prepared in step (1), heat them in boiling water for 3 - 5 minutes to denature the proteins. After cooling, add the samples to the loading wells of the SDS-PAGE gel and perform electrophoresis at 80 - 100 V for 1 hour. (3) Membrane transfer: Using a wet membrane transfer apparatus (Bio-Rad), transfer the proteins from the SDS-PAGE gel to the PVDF membrane at 300 mA for 1 hour. (4) Blocking: After the PVDF membrane is washed, add the blocking solution for blocking. (5) Antibody incubation: Add the diluted HRP-labeled anti-human FVIII antibody (Affinity Biologicals) and incubate at room temperature for 1 hour. (6) Color development: Detect the proteins using an ECL chemiluminescence reagent (Cell Signaling). Results: The detection results of MC.BDD-FVIII-SQ-CO2 are shown in Figure 7.

[0101] Example 5: Consideration of the BDD-FVIII codon optimization sequence 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). Codon optimization was performed on wild-type BDD-FVIII using two optimization algorithms, and two BDD-FVIII optimized sequences were obtained respectively (BDD-FVIII-SQ-CO1 (SEQ ID NO. 45) and BDD-FVIII-SQ-CO2 (SEQ ID NO. 15)). By the method described in Example 1, MC.BDD-FVIII-SQ-WT, MC.BDD-FVIII-SQ-CO1, and MC.BDD-FVIII-SQ-CO2 were obtained (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 collected, transfected into 293T cells respectively, and 72 hours later, the cell culture supernatant was collected, and the expression level of the target protein was measured by ELISA. Results: As shown in Figure 8. Conclusion: The expression levels of the two optimized vectors were significantly improved. Moreover, the improvement range of the second optimized version (BDD-FVIII-CO2) was even larger, exceeding 1-fold.

[0102] Comparative Example 1: Consideration of in vivo expression and activity of MC.BDD-FVIII-SQ-CO2 Plasma expression level of MC.BDD-FVIII-SQ-CO2 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 the plasma expression level was measured by ELISA. Results: When the plasma expression level was measured by ELISA, only weak expression (1 - 2 ng / mL) was observed. Therapeutic effect of MC.BDD-FVIII-SQ-CO2 on A-type hemophilia mice with F8 gene knockout (F8-KO mice): Operation: Six 12-week-old male F8-KO mice were randomly divided into two groups of three mice each. Mice in the treatment group were intramuscularly injected with 30 μg / 50 μL of mini-circle DNA (MC.BDD-FVIII-SQ-CO2) solution (50 μL of solution containing 30 μg of mini-circle DNA), and mice in the blank control group were intramuscularly injected with 50 μL of PBS. After intramuscular injection, a pulsed electric field (TERESA gene transfer device; parameters: 36 v, 10 ms, 1 Hz) was applied. One month (m1) and two months (m2) after injection, tail-cut experiments were performed twice at mouse tail diameters of 2 mm and 2.5 mm, respectively, and the blood loss was recorded. After cutting the tail, the wound was stopped bleeding with AgNO3 cauterization. Results: As shown in Figure 9, after a certain period of observation, for mice in the mini-circle DNA (MC.BDD-FVIII-SQ-CO2) injection group and untreated control group mice (without mini-circle DNA, only an equal volume of PBS buffer was injected), a mouse tail-cut experiment (tail diameter after cutting was 2 mm one month later and 2.5 mm two months later) was performed to compare the blood loss. As a result, the blood loss of mice in both groups was the same, and there was no statistical difference. 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 the expression level of the target protein 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 respectively, and the expression level of the target protein was measured by ELISA. Results: As shown in Figure 10, the expression of the target protein of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 was significantly higher than that of other FVIII mini-circle DNA expression vectors, and the relative levels were specifically as follows. (1) Values of the expression level of the target protein of MC.BDD-FVIII-L1 relative 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) Values of the expression levels of the target proteins of MC.BDD-FVIII-L2 with respect 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) Values of the expression levels of the target proteins of MC.BDD-FVIII-L3 with respect 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 is almost no difference between MC.BDD-FVIII-v3 and MC.BDD-FVIII-N8. (4) Values of the expression levels of the target proteins of MC.BDD-FVIII-L1 with respect 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: The minicircle DNA vector of blood coagulation factor VIII containing the L1-linker or L2-linker ligation peptide has a higher expression level of the target protein compared to the minicircle DNA vector of blood coagulation factor VIII containing other types of ligation peptides.

[0104] Example 7: Detection of in vitro blood coagulation activity (one-stage APTT method) Operation: The cell culture supernatants of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 with the highest expression levels of the target proteins obtained in Example 3 were collected, and the samples were sent to the "Institute of Blood Transfusion, Chinese Academy of Medical Sciences" to measure the blood coagulation activity using the one-stage APTT method. The FVIII activity of normal human pooled plasma (NPP) was defined as 100%. Results: The measured activity values of the expression products (72-hour cell culture supernatants) of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 were 118.1% and 116% respectively (the reference values for the activity range of humans with normal blood coagulation function are 50% - 150%), indicating that blood coagulation factor VIII containing the L1-linker or L2-linker ligation peptide has good blood coagulation activity.

[0105] Example 8: In vivo expression (1) In vivo expression in normal mice Operation: Ten 6-8-week-old Balb / c mice were randomly divided into two groups of 5 mice each, designated as the MC.BDD-FVIII-L1 group of 5 mice and the MC.BDD-FVIII-L2 group of 5 mice. Blood was collected 1 day before injecting the microcircular DNA, plasma was separated and stored at -80°C to serve as the blank control sample (W0). The two groups of mice were intramuscularly injected with MC.BDD-FVIII-L1 microcircular DNA and MC.BDD-FVIII-L2 microcircular DNA respectively according to a dose of 30 μg / 50 μL / mouse, and furthermore, a pulsed electric field (TERESA gene delivery device; parameters: 36 v, 10 ms, 1 Hz) was applied. Blood was collected regularly after injection, 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). Results: As shown in Figure 11. There was no expression in the blank control sample. In the MC.BDD-FVIII-L1 group and the MC.BDD-FVIII-L2 group, 4 weeks after injection, the in vivo expression levels of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 were similar and reached above 20 ng / mL (equivalent to 10% or more of the normal level of normal human plasma FVIII concentration (20 ng / mL)), exceeding the significant treatment level (>5%) that mildly and clearly improves moderate hemophilia or severe hemophilia. (2) In vivo expression in beagle dogs Operation: Three adult beagle dogs weighing approximately 13 kg were bled before injection with mini-circle DNA, and the 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 mini-circle DNA, and pulsed electric fields (TERESA gene introduction device; parameters: 36 v, 10 ms, 1 Hz) were applied. Thereafter, blood was collected weekly, the 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) and converted to the relative level of FVIII in normal human pooled plasma (NPP). The FVIII concentration in normal human pooled plasma is approximately 200 ng / mL. Results: As shown in Figure 12. There was no expression in the blank control samples. One week after injection with MC.BDD-FVIII-L1 mini-circle DNA, the hFVIII expression in the plasma of one dog reached 5% (10 ng / mL) or more of the normal level of FVIII concentration in normal human plasma, while the values of the other two dogs were low. Two weeks after injection with MC.BDD-FVIII-L1 mini-circle DNA, the expression level gradually increased and all exceeded 10% (20 ng / mL) of the normal level of FVIII concentration in normal human plasma.

[0106] Example 9: In vivo activity of hemophilia A mice (1) In vivo expression level of MC.BDD-FVIII-L1 in hemophilia A mice (F8-KO mice) Procedure: One day before the mini-circle DNA injection, blood was collected from 5 male F8-KO mice aged 6 - 8 weeks, plasma was separated and stored at -80 °C to serve as the blank control sample (W0). All mice were intramuscularly injected with mini-circle DNA (MC.BDD-FVIII-L1 mini-circle DNA) at a dosage of 30 μg / 50 μL / mouse (a 50 μL solution containing 30 μg of mini-circle DNA), and a pulsed electric field (TERESA in vivo gene transfection device; parameters: 36 v, 10 ms, 1 Hz) was applied. After 6 weeks (W6), blood was collected, 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: The results are shown in Figure 13. There was no expression in the blank control sample. Six weeks after the MC.BDD-FVIII-L1 mini-circle DNA injection, the plasma target protein level measured by ELISA reached over 100 ng / mL, exceeding 50% (100 ng / mL) of the normal level of plasma FVIII concentration in normal humans, suggesting that hemophilia can be functionally treated and normal coagulation function can be restored. (2) Treatment of hemophilia A mice (F8-KO mice) with MC.BDD-FVIII-L1 Procedure: 10 male F8-KO mice aged 6 - 8 weeks were randomly divided into two groups: a blank control group (PBS) and a mini-circle DNA treatment group (MC treatment group, MC), with 5 mice in each group. The mice in the mini-circle DNA treatment group were intramuscularly injected with the MC.BDD-FVIII-L1 mini-circle DNA solution at a dosage of 30 μg / 50 μL (a 50 μL solution containing 30 μg of mini-circle DNA), and a pulsed electric field was applied after the injection (TERESA in vivo gene transfection device; parameters: 36 v, 10 ms, 1 Hz). The mice in the blank control group were injected with an equal volume of PBS buffer in the same manner. Another 5 male C57 mice of the same age were used as wild-type controls (WT) with normal coagulation function. Four weeks after the injection, a tail cut experiment was performed at a location on the mouse's tail with a diameter of 2 mm, and the blood loss within 15 minutes was recorded. After the experiment, the mice were sacrificed. Results: As shown in Figure 14, the bleeding volume of mice in the MC.BDD-FVIII-L1 minicircle DNA treatment group (MC treatment group, MC) was significantly decreased compared with that in the blank control group (PBS), and the bleeding volume of mice in the MC treatment group was equivalent to that of wild-type normal mice (WT) (no statistical difference, ns). It was shown that the blood coagulation function of hemophilia A mice recovered normally after MC treatment. (3) Identification of the activity of MC.BDD-FVIII-L2 in hemophilia A mice (F8-KO mice) Procedure: Twenty 9- to 10-week-old male F8-KO mice were randomly divided into two groups, namely, 5 mice in the blank control group (PBS) and 15 mice in the minicircle DNA injection group (MC treatment group, MC). Mice in the MC treatment group were injected with an MC.BDD-FVIII-L2 minicircle DNA solution (dissolving 3 μg of minicircle DNA 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), and the injection method was high-pressure tail vein injection (rapidly injecting 1.8 mL of solution within 5 - 8 s via the tail vein). Mice in the blank control group were injected with an equal volume (1.8 mL) of PBS buffer in the same manner. Another 5 male C57 mice of the same age were used as wild-type controls (WT) with normal coagulation function. Twenty-four hours after injection, a tail cut experiment was performed at a site with a diameter of 2 mm on the tail of the mice, and the bleeding volume within 15 minutes was recorded. The mice were sacrificed after the experiment. One mouse in the blank control group died due to an abnormality in the high-pressure injection operation and the tail cut could not be performed. Results: As shown in Figure 15, compared with the blank control group (PBS), the bleeding volume of mice in the MC.BDD-FVIII-L2 minicircle DNA injection group (MC treatment group, MC) was significantly decreased and was also less than that of untreated wild-type normal mice (WT), indicating that the in vivo coagulation activity of MC.BDD-FVIII-L2 was extremely high.

[0107] Example 10: Other modifications (1) Fc fusion Fc fusion is an important means to increase the half-life of FVIII. By expressing molecules with a long half-life in the body, it is possible to accumulate more products in the body and achieve higher levels of expression. To verify this assumption, a mini-circle DNA expression vector of BDD-FVIII-Fc fusion protein (SEQ ID NO.19) (MC.BDD-FVIII-CTP-Fc, the nucleotide sequence of the mini-circle is shown in Table 1) was constructed. Operation: Ten 6- to 8-week-old Balb / c mice were randomly divided into two groups of 5 mice each, namely, a group of 5 mice injected with mini-circle DNA (MC.BDD-FVIII-CTP-Fc) and a group of 5 mice as a PBS blank control group (control). The mice in the mini-circle group were intramuscularly injected with mini-circle DNA (MC.BDD-FVIII-CTP-Fc) at a dose of 45 μg / 50 μL / mouse (a 50 μL solution containing 45 μg of mini-circle DNA, equimolar to 30 μg of MC.BDD-FVIII). The mice in the blank control group were intramuscularly injected with 50 μL of PBS each. After intramuscular injection, a pulsed electric field (TERESA in vivo gene transfection device; parameters: 36 v, 10 ms, 1 Hz) was applied. Then, blood was collected regularly, 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 16. There was no expression in the blank control samples. It was found that 11 to 15 weeks after injection of MC.BDD-FVIII-CTP-Fc mini-circle DNA, the mini-circle DNA of BDD-FVIII-Fc fusion protein did not further improve the in vivo expression level (only about 10 ng / mL, and some were less than 5% of the normal level (10 ng / mL) of normal human plasma FVIII concentration). (2) X5 mutation Measurement of the in vitro cell expression level of the target protein by ELISA: Operation: Introduce the X5 mutation (i.e., mutate the five important amino acids in the human FVIII A region to the corresponding amino acids in porcine FVIII. I86V / A108S / G132K / M147T / L152P) into BDD-FVIII-L1 to obtain the BDD-FVIII-L1-X5 nucleotide sequence (SEQ ID NO.22), and construct the corresponding MC expression vector (the nucleotide sequences of each minicircle are shown in Table 1) with MC.BDD-FVIII-L1-X5 minicircle DNA. Transfect MC.BDD-FVIII-L1 minicircle DNA and MC.BDD-FVIII-L1-X5 minicircle DNA into 293T cells respectively. After 72 hours, collect the cell culture supernatant and measure the level of the target protein by ELISA. Result: As shown in Figure 17, compared with MC.BDD-FVIII-L1 minicircle DNA, the level of the target protein increased after introducing the X5 mutation. In vivo experiment in mice: Operation: Randomly divide 10 six- to eight-week-old Balb / c mice into two groups of 5 each. Inject the minicircle DNA (MC.BDD-FVIII-L1 minicircle DNA or MC.BDD-FVIII-L1-X5 minicircle DNA) into the mice in each group at a dosage of 30 μg / 50 μL / mouse (a 50 μL solution containing 30 μg of minicircle DNA each), and apply a pulsed electric field (TERESA in vivo gene transfer device; parameters: 36 v, 10 ms, 1 Hz) after intramuscular injection. Blood is collected 4 weeks after injection, plasma is separated, and the expression level of the target protein in the plasma sample is measured using a human FVIII ELISA kit (VisuLize Factor VIII PLUS Antigen ELISA Kit, Affinity Biologicals Inc). Result: As shown in Figure 18, compared with MC.BDD-FVIII-L1 minicircle DNA, the level of the target protein increased slightly after introducing the X5 mutation. (3) R1645H mutation Siner et al. (Blood 2013, 121:4396-4403) reported that mutating the furin site (R 1645 -H 1646 -Q 1647 -R 1648 ) at the C-terminus of the SQ linker in the B domain of BDD-FVIII renders it unrecognizable and uncleavable by furin protease, and enables increased expression. Since the recognition pattern of the furin site is R-X-X-R (i.e., the first and last are arginine and the second position in the middle is any amino acid), theoretically, mutating the arginine at position 1645 or 1648 to any amino acid other than arginine can achieve the purpose of not being recognized and cleaved by furin protease. This purpose can also be achieved by deletion of the furin site (including deletion of all 4 amino acids of RHQR, or any 1, 2, or 3 of these amino acids). In the present invention, since a furin site of RHQR remains at the C-terminus of the L1 linker, the fourth arginine (Arg, R) from the bottom at 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 BDD-FVIII-L1 R1645H (the nucleotide sequence is shown in Table 1) is obtained, and the corresponding MC expression vector MC.BDD-FVIII-L1 R1645H (the nucleotide sequence of the minicircle is shown in Table 1) is constructed. (4) F309S Mutation Swaroop et al. (J Biol Chem 1997, 272:24121-24124) reported that the F309S mutation (mutation of phenylalanine at position 309 of FVIII to serine) promotes FVIII secretion. Accordingly, phenylalanine at position 309 of BDD-FVIII-L1, BDD-FVIII-L1 R1645H and BDD-FVIII-L2 is mutated to serine (Ser, S) to obtain BDD-FVIII F309S -L1, BDD-FVIII F309S -L1 R1645H and BDD-FVIIIF309S - The coding sequence of L2 was obtained (each nucleotide sequence is shown in Table 1). Then, 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 sequences of each minicircle are shown in Table 1).

[0108] Example 11: Therapeutic effect by secondary administration Procedure: Ten 6- to 8-week-old Balb / c mice were randomly divided into two groups of 5 mice each: the MC.BDD-FVIII-L1 group and the MC.BDD-FVIII-L2 group. The mice in both groups were intramuscularly injected with MC.BDD-FVIII-L1 minicircle DNA and MC.BDD-FVIII-L2 minicircle DNA, respectively, at a dose of 30 μg / 50 μL (a 50 μL solution injection containing 30 μg of minicircle DNA each), and a pulsed electric field was applied (TERESA in vivo gene transfer device; parameters: 36 v, 10 ms, 1 Hz). At 22 weeks (W22) after injection, the administration was repeated in the same manner. After the first and second injections, blood was collected regularly, 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). And it was converted to the relative level of normal human pooled plasma (NPP) FVIII. The normal human pooled plasma FVIII concentration is approximately 200 ng / mL. Results: Shown in Figure 19. After injection of the mini-circle DNA, the in vivo expression levels in both groups of mice were above 20% (40 ng / mL) of the normal level of normal human plasma FVIII concentration at week 4 (W4), and the expression levels decreased at week 12 (W12), but were able to maintain above 10% (20 ng / mL) of the normal level of normal human plasma FVIII concentration for a long period (weeks 12 - 20, W12 - W20). After the booster administration at week 22 (W22), the expression level at W23 (23 weeks after the first administration, i.e., 1 week after the second administration) rapidly recovered to a level close to that before the decrease. The secondary injection of the mini-circle DNA provided by the present invention is still effective, does not cause a virus-specific immune response, enables secondary injection, and has shown that the therapeutic effect can be restored or enhanced by a method of repeated administration as needed.

[0109] Example 12: Long-term in vivo expression Procedure: Ten 6 - 8-week-old Balb / c mice were randomly divided into two groups: 5 mice in the MC.BDD-FVIII-L1 group and 5 mice in the MC.BDD-FVIII-L2 group. Blood was collected before mini-circle DNA injection, plasma was separated and stored at -80 °C as a blank control sample (W0). The two groups of mice were intramuscularly injected with MC.BDD-FVIII-L1 mini-circle DNA and MC.BDD-FVIII-L2 mini-circle DNA, respectively, at a dose of 30 μg / 50 μL (injection of a 50 μL solution containing 30 μg of mini-circle DNA each), and a pulsed electric field was applied (TERESA gene transfer device; parameters: 36 v, 10 ms, 1 Hz). The same administration was carried out again at week 22 after injection. Blood was collected regularly after injection, 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 the mouse body for more than one year. The secondary injection of the minicircle DNA according to the present invention was also effective in the long term and did not induce a virus-specific immune response.

[0110] Example 13: Safety study Procedure: One adult female beagle weighing 10 - 12 kg (number D0016) and one adult male beagle weighing 10 - 12 kg (number C957) were each injected with 1.2 mg of MC.BDD-FVIII-L1 minicircle DNA, and a pulsed electric field (36 v, 10 ms, 1 Hz) was applied. At the 6th week after injection, lung, liver, spleen, kidney, heart, brain, ovary or testis, muscle and thymus tissues were collected, sliced, stained with HE, and subjected to pathological examination. The results are shown in Figure 21. Results: At 6 weeks after the injection of MC.BDD-FVIII-L1 minicircle DNA, the pathological examinations of the lung, liver, spleen, kidney, heart, brain, ovary (female dog D0016) or testis (male dog C957), muscle and thymus tissues of the beagle dogs were all normal (Figure 21). No obvious toxicity was observed, indicating that the safety of MC.BDD-FVIII-L1 is good.

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

Claims

1. A linking peptide, wherein the amino acid sequence is SEQ ID NO. 1 or SEQ ID NO. 2, or an amino acid sequence having at least 80-99% identity with any of the nucleotide sequences thereof or at least a part of any of the sequences thereof, and characterized by the linking peptide.

2. A nucleotide sequence, wherein the nucleotide sequence encodes the linking peptide according to Claim 1, preferably, the nucleotide sequence comprises SEQ ID NO. 3 or SEQ ID NO. 4, or a codon-optimized sequence of any of the nucleotide sequences thereof, and characterized by the nucleotide sequence.

3. Use of the linking peptide according to Claim 1 or the nucleotide sequence according to Claim 2 for constructing a recombinant blood coagulation factor VIII protein or a variant thereof, or use of the nucleotide sequence according to Claim 2 for constructing a recombinant blood coagulation factor VIII protein or a variant thereof.

4. A blood coagulation factor VIII protein or a variant thereof, wherein the linking peptide in the blood coagulation factor VIII protein or a variant thereof is selected from the linking peptides according to Claim 1, or the nucleotide sequence of the linking peptide in the blood coagulation factor VIII protein or a variant thereof is selected from the nucleotide sequences according to Claim 2, and characterized by the blood coagulation factor VIII protein or a variant thereof.

5. A recombinant blood coagulation factor VIII protein or a variant thereof, 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, or an amino acid sequence having at least 80%-99% identity with any of the amino acid sequences thereof or at least a part of any of the amino acid sequences thereof, and characterized by the recombinant blood coagulation factor VIII protein or a variant thereof.

6. A nucleotide sequence, wherein the nucleotide sequence encodes the blood coagulation factor VIII protein or a variant thereof according to Claim 4 or 5, Preferably, the nucleotide sequence comprises the nucleotide sequence of 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 sequence of any of the nucleotide sequences thereof. A nucleotide sequence characterized by that.

7. A recombinant gene vector, A nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or a variant thereof according to claim 4 or 5, or a nucleotide sequence according to claim 6, A recombinant gene vector characterized by that.

8. The recombinant gene vector includes a non-viral vector or a viral vector, The recombinant gene vector according to claim 7, characterized by that.

9. 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, The recombinant gene vector according to claim 8, characterized by that.

10. The non-viral vector is selected from a minicircle DNA vector, The recombinant gene vector according to claim 8, characterized by that.

11. A parental plasmid used for the production of minicircle DNA, Including a plasmid vector containing the nucleotide sequence according to claim 6, Alternatively, a promoter nucleotide sequence, an enhancer nucleotide sequence, a multiple cloning site nucleotide sequence, a polyA signal nucleotide sequence, and a target gene DNA fragment are inserted into a plasmid vector, and the target gene DNA fragment is located between the restriction endonuclease cleavage sites of the multiple cloning site, The parental plasmid used for the production of minicircle DNA, characterized in that the target gene DNA fragment contains the nucleotide sequence according to claim 6.

12. A method for producing minicircle DNA, The parental plasmid according to claim 8 is transformed into a host cell. After induction, the parental plasmid generates minicircle DNA and backbone DNA by site-specific recombination at the site of the specific recombination site, and the minicircle DNA is extracted using a plasmid DNA purification kit. A method for producing minicircle DNA, characterized by comprising the steps of:

13. Preferably, the nucleotide sequence of the minicircle DNA is 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. Minicircle DNA obtained by the production method according to claim 9, characterized in that it is selected from the group consisting of:

14. A host cell, Comprising the nucleotide sequence encoding the recombinant blood coagulation factor VIII protein or a variant thereof according to claim 4 or 5, the nucleotide sequence according to claim 6, the recombinant gene vector according to claim 7, or the minicircle DNA according to claim 10, Preferably, the host cell includes a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. A host cell, characterized by comprising:

15. A pharmaceutical composition, Comprising the recombinant blood coagulation factor VIII protein or a variant thereof according to claim 4 or 5, the recombinant blood coagulation factor VIII protein or a variant thereof encoded by the nucleotide sequence according to claim 6, the recombinant gene vector according to claim 7, or the minicircle DNA obtained by the production method according to claim 9 or the minicircle DNA according to claim 10, and a pharmaceutically acceptable adjuvant or carrier. A pharmaceutical composition, characterized by comprising:

16. Use in the manufacture of a drug for treating a disease of the recombinant blood coagulation factor VIII protein or a variant thereof according to claim 4 or 5, the nucleotide sequence according to claim 6, the recombinant gene vector according to claim 7, the parental plasmid according to claim 8, the minicircle DNA obtained by the production method according to claim 9, the minicircle DNA according to claim 10, the host cell according to claim 11, or the pharmaceutical composition according to claim 12, Preferably, the disease is selected from hereditary genetic deficiency diseases. Preferably, the disease is a disease caused by a blood coagulation factor defect, Preferably, the disease is a disease caused by a deficiency of blood coagulation factor VIII, Preferably, the disease is hemophilia, characterized by such use.

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