Connecting peptide, blood coagulation factor VIII protein containing connecting peptide or variant thereof and application of blood coagulation factor VIII protein or variant

Abstract

The invention relates to a connecting peptide, a blood coagulation factor VIII protein containing the connecting peptide or a variant thereof and application of the blood coagulation factor VIII protein and the variant, and belongs to the field of gene defect treatment medicines.

Description

Connecting peptide, coagulation factor VIII protein containing the connecting peptide or its variant and use thereof Technical Field

[0001] The present invention relates to the field of drugs for treating gene defects, and in particular to a connecting peptide, a coagulation factor VIII protein comprising the connecting peptide or a variant thereof, and uses thereof. Background Art

[0002] Hemophilia is a group of X-linked, single-gene genetic disorders of coagulation caused by deficiencies in coagulation factors. Hemophilia A, which is characterized by a deficiency in coagulation factor VIII (FVIII), encoded by the F8 gene, accounts for the largest number of patients, accounting for 80-85%. Currently, the standard treatment for hemophilia A is replacement therapy, which involves supplementing with exogenous FVIII protein, including plasma-derived FVIII (pdFVIII) and recombinant FVIII (rFVIII). pdFVIII is an extract isolated from normal human plasma, which has a limited donor source and carries the risk of blood-borne viral transmission. rFVIII is expressed and purified in vitro using mammalian cell lines. It is not restricted by blood supply and can effectively reduce the risk of blood-borne viral infection, but it has very high production process requirements.

[0003] FVIII protein has a short in vivo half-life (average 12 hours), requiring lifelong dosing and frequent injections (two to three, or even four, times per week) for replacement therapy patients. This is costly and inconvenient. Conventional approaches to prolonging the effect, such as Fc fusion (Drug Des Devel Ther 2014, 8:365-371) and PEGylation (Haemophilia 2019, 25:773-781), can extend its half-life to approximately 19 hours, but this is limited (only 50-60%) and does not significantly reduce dosing frequency. Sanofi and Sobi have collaborated to develop a novel, longer-acting FVIII fusion protein (BIVV001; FVIIIFc-vWF-XTEN), which further extends the half-life to 38-44 hours by fusing the antibody Fc region with the vWF factor D'D3 domain, reducing dosing frequency to once a week (N Engl J Med. 2020, 383:1018-1027). Although these long-term modifications to protein molecules can more or less extend the half-life and improve the patient's medication experience to a certain extent, they still cannot fundamentally change the treatment model of "lifelong medication, repeated injections."

[0004] On the contrary, FVIII gene therapy is expected to achieve a functional "cure" of the disease by correcting the genetic defects of hemophilia A patients at the genetic level. Currently, the industry generally believes that AAV-hFVIII (adeno-associated virus vector liver-targeted delivery of normal human FVIII gene) is the "best" solution for gene therapy of hemophilia A. However, existing liver-targeted AAV-FVIII gene therapy has obvious defects (Hemasphere 2021, 5:e540):

[0005] i) AAV-hFVIII is only suitable for adult patients aged 18 years and above. Children's livers are in a stage of rapid growth and development, and their liver cells are actively dividing, making this therapy unsuitable (the risk of gene integration is greatly increased; unintegrated target genes are easily lost during cell division).

[0006] ii) AAV is a common virus in the human body with a high infection rate in the population. Therefore, many patients have pre-existing AAV neutralizing antibodies, and these patient groups cannot use this therapy.

[0007] iii) Repeated administration is not possible. AAV-hFVIII is not lifelong, and expression levels may drop below therapeutic levels after several years, requiring repeat administration to restore therapeutic levels. However, the AAV viral capsid is highly immunogenic, and the initial administration can easily induce a virus-specific immune response, rendering repeat injections ineffective.

[0008] iv) The hFVIII gene is large, exceeding the capacity of AAV vectors, making AAV-hFVIII packaging and production difficult. Furthermore, AAV therapy poses other safety concerns, such as random gene integration.

[0009] The expression of hFVIII (human coagulation factor VIII) is quite difficult, a common and key technical challenge faced in both recombinant FVIII production (in vitro expression) and FVIII gene therapy (in vivo expression). The expression level of hFVIII is only 1 / 100th to 1 / 1000th of that of ordinary proteins of comparable molecular weight (Hum Gene Ther 1993, 4:259-272; Blood 2004, 103:3412-3419). Therefore, modifying the hFVIII molecule and improving its expression level have become the focus of research in this area. FVIII consists of six domains: A1-A2-B-A3-C1-C2. Domain B is 908 aa long, accounting for approximately 40% of the total length (2332 aa). Deletion of domain B does not affect its coagulation function (PNAS 1986, 83:5939-5942) and can significantly increase mRNA (17-fold) and protein product (30%) levels (Blood 2004, 103:3412-3419; Blood Coagul Fibrinolysis 1997, 8 Suppl 2:S3-14). Therefore, deleting domain B has become a widely adopted modification method; in fact, several B-domain deleted FVIII drugs (BDD-FVIII), such as Pfizer's Xyntha, have been in large-scale clinical use for many years. The earliest BDD-FVIII almost completely deleted the B region, retaining only a linker consisting of the N-terminal 4 amino acids and the C-terminal 10 amino acids (a total of 14 amino acids) of the native FVIII B region, connecting A1-A2 and A3-C1-C2 (this linker is called the SQ linker; the sequence is SFSQNPPVLKRHQR). For convenience, BDD-FVIII containing the SQ linker is denoted as BDD-FVIII-SQ. We have previously investigated the feasibility of expressing therapeutic levels of BDD-FVIII-SQ in vivo using minicircle DNA vectors; however, experiments revealed that the BDD-FVIII-SQ vector only expressed weakly in vivo (less than 1% of normal levels), failing to achieve a therapeutic effect. Therapeutic levels are categorized into three levels: i) above 1%, indicating a preliminary therapeutic effect, with severe hemophilia resolving to moderate severity; ii) above 5%, indicating a significant therapeutic effect, with moderate to severe hemophilia significantly resolving to mild severity; and iii) above 50%, indicating a functional "cure," with complete restoration of normal coagulation function.

[0010] The B-region linker connects A1-A2 and A3-C1-C2, and its sequence significantly influences BDD-FVIII protein expression. Miao et al. (Blood 2004, 103:3412-3419) reported that retaining the N-terminal 226 amino acids of the B-region (226aa / N6) resulted in a four-fold higher expression level (ELISA assay) than a near-complete deletion of the B-region (wild-type SQ linker). McIntosh et al. (Blood 2013, 121:3335-3344; Patent: WO 2013 / 186563) built on this finding by replacing the 226aa / N6 with a 31aa v3 linker (sequence: SFSQNATNVSNNSNTSNDSNVSPPVLKRHQR), finding that this further increased target protein expression by approximately 50%. In the present invention, BDD-FVIII containing the v3 linker is referred to as BDD-FVIII-v3. AAV gene therapy based on BDD-FVIII-v3 has entered Phase I clinical trials (NCT03001830). Novo Nordisk of Denmark has developed another BDD-FVIII molecule (N8, Haemophilia 2010, 16:349–359; patent: WO2006103298) that exhibits superior expression compared to BDD-FVIII-SQ and contains a 21aa linker (sequence: SFSQNSRHPSQNPPVLKRHQR). This molecule is referred to as BDD-FVIII-N8 in this paper. However, BDD-FVIII-v3 and BDD-FVIII-N8 still suffer from low expression levels and poor therapeutic efficacy.

[0011] Therefore, there is still an urgent need for a drug for treating diseases related to coagulation factor VIII deficiency that has a high expression level, can be administered twice, is simple to prepare, has a good therapeutic effect, and can be expressed in the body for a long time.

[0012] Summary of the Invention

[0013] SUMMARY OF THE INVENTION

[0014] To solve the above problems, the present invention provides the following technical solutions.

[0015] In the first aspect, a connecting peptide is provided. The recombinant coagulation factor VIII protein or its variant using the connecting peptide provided by the present invention has the advantages of high expression level, good secondary administration effect, simple preparation, good therapeutic effect, etc. The mini-circle DNA of the recombinant coagulation factor VIII protein or its variant using the connecting peptide provided by the present invention has the advantages of high expression level, good secondary administration effect, simple preparation, good therapeutic effect, etc., and also solves the problems of AAV.hf8 production difficulties (packaging difficulties), application scope limitations (cannot be used for children, cannot be secondary administered), and safety risks (random integration causes cancer), that is, it has the advantages of simple production, wide application range (can be used for children, good secondary administration effect), good safety, and can be expressed in vivo for a long time.

[0016] In a second aspect, a nucleotide sequence is provided, which can encode the connecting peptide described in the first aspect.

[0017] In a third aspect, a use of the connecting peptide described in the first aspect or the nucleotide sequence described in the second aspect is provided.

[0018] In a fourth aspect, a recombinant coagulation factor VIII protein or a variant thereof is provided. The recombinant coagulation factor VIII protein or a variant thereof has the advantages of high expression level, good secondary administration effect, simple preparation, and good therapeutic effect.

[0019] In a fifth aspect, a nucleotide is provided.

[0020] In a sixth aspect, a recombinant gene vector is provided.

[0021] In a seventh aspect, a parent plasmid for producing minicircle DNA is provided.

[0022] In an eighth aspect, a method for preparing minicircle DNA is provided.

[0023] In a ninth aspect, a minicircle DNA obtained by the preparation method according to the eighth aspect is provided. The minicircle DNA has the advantages of high expression level, 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 for children, cannot be secondary administration), and safety risks (random integration causes cancer). That is, it has the advantages of simple production, wide application range (can be used for children, good secondary administration effect), good safety, and long-term expression in vivo.

[0024] In the tenth aspect, a host cell is provided, which comprises the encoding nucleotide sequence of the recombinant 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.

[0025] In the eleventh aspect, a pharmaceutical composition is provided.

[0026] In the twelfth aspect, a method is provided for preparing a drug for treating a disease by providing the recombinant 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 parent plasmid described in the seventh aspect, the minicircle DNA obtained by the preparation 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.

[0027] Detailed Description of the Invention

[0028] To solve the above problems, the present invention provides the following technical solutions.

[0029] In a first aspect, a connecting peptide is provided.

[0030] A connecting peptide, whose 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 portion of any of the sequences thereof. The recombinant coagulation factor VIII protein or its variant using the connecting peptide provided in the first aspect of the present invention has the advantages of high expression level, good secondary administration effect, simple preparation, good therapeutic effect, etc. The minicircle DNA of the recombinant coagulation factor VIII protein or its variant using the connecting peptide provided in the first aspect of the present invention has the advantages of high expression level, good secondary administration effect, simple preparation, good therapeutic effect, etc., and also solves the problems of AAV.hf8 production difficulty (packaging difficulty), application range limitation (cannot be used for children, cannot be secondary administered), and safety risk (random integration causes cancer), that is, it has the advantages of simple production, wide application range (can be used for children, good secondary administration effect), good safety, and can be expressed in vivo for a long time.

[0031] In some embodiments, the nucleotide sequence encoding the linker peptide (referred to as L1 linker in the present invention) with the amino acid sequence of SEQ ID NO. 1 may include SEQ ID NO. 3 or a codon-optimized sequence thereof.

[0032] In some embodiments, the nucleotide sequence encoding the linker peptide with the amino acid sequence of SEQ ID NO. 2 (referred to as L2 linker in the present invention) may include SEQ ID NO. 4 or a codon-optimized sequence thereof.

[0033] In a second aspect, a nucleotide sequence is provided.

[0034] A nucleotide sequence encoding the connecting peptide according to the first aspect.

[0035] In some embodiments, the nucleotide sequence comprises: SEQ ID NO. 3 or SEQ ID NO. 4, or a codon-optimized sequence of either nucleotide sequence.

[0036] In some embodiments, the nucleotide sequence of SEQ ID NO. 3 or its codon-optimized sequence can be used to encode a connecting peptide of the amino acid sequence of SEQ ID NO. 1.

[0037] In some embodiments, the nucleotide sequence of SEQ ID NO. 4 or its codon-optimized sequence can be used to encode a connecting peptide of the amino acid sequence of SEQ ID NO. 2.

[0038] In a third aspect, a use of the connecting peptide described in the first aspect or the nucleotide sequence described in the second aspect is provided.

[0039] A use of the connecting peptide described in the first aspect or the nucleotide sequence described in the second aspect in constructing a recombinant coagulation factor VIII protein or a variant thereof.

[0040] A use of the nucleotide sequence of the second aspect in constructing the nucleotide sequence of a recombinant coagulation factor VIII protein or a variant thereof.

[0041] In a fourth aspect, a recombinant coagulation factor VIII protein or a variant thereof is provided.

[0042] A coagulation factor VIII protein or variant thereof, wherein the connecting peptide in the coagulation factor VIII protein or variant thereof is selected from the connecting peptide described in the first aspect, or the nucleotide sequence of the connecting peptide in the coagulation factor VIII protein or variant thereof is selected from the nucleotide sequence described in the second aspect. The recombinant coagulation factor VIII protein or variant thereof provided in the fourth aspect of the present invention has the advantages of high expression level, good secondary administration effect, simple preparation, and good therapeutic effect.

[0043] In some embodiments, a recombinant 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 that has at least 80%-99% identity with any of these amino acid sequences, or at least a portion of any of these amino acid sequences.

[0044] In some embodiments, the nucleotide sequence encoding the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO. 5 may include SEQ ID NO. 7 or a codon-optimized sequence thereof.

[0045] In some embodiments, the nucleotide sequence encoding the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO. 6 may include SEQ ID NO. 8 or a codon-optimized sequence thereof.

[0046] In some embodiments, the nucleotide sequence encoding the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO. 21 may include SEQ ID NO. 22 or a codon-optimized sequence thereof.

[0047] In some embodiments, the nucleotide sequence encoding the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO. 23 may include SEQ ID NO. 24 or a codon-optimized sequence thereof.

[0048] In some embodiments, the nucleotide sequence encoding the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO. 43 may include SEQ ID NO. 38 or a codon-optimized sequence thereof.

[0049] In some embodiments, the nucleotide sequence encoding the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO. 44 may include SEQ ID NO. 39 or a codon-optimized sequence thereof.

[0050] In some embodiments, the nucleotide sequence encoding the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO. 50 may include SEQ ID NO. 56 or a codon-optimized sequence thereof.

[0051] In some embodiments, the nucleotide sequence encoding the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO. 51 may include SEQ ID NO. 60 or a codon-optimized sequence thereof.

[0052] In some embodiments, the nucleotide sequence encoding the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO. 52 may include SEQ ID NO. 58 or a codon-optimized sequence thereof.

[0053] In some embodiments, the nucleotide sequence encoding the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO. 53 may include SEQ ID NO. 54 or a codon-optimized sequence thereof.

[0054] In a fifth aspect, a nucleotide is provided.

[0055] A nucleotide sequence encoding the recombinant coagulation factor VIII protein or a variant thereof as described in the fourth aspect.

[0056] 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 one of these nucleotide sequences.

[0057] In some embodiments, the nucleotide sequence of SEQ ID NO.7 can be used to encode the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO.5.

[0058] In some embodiments, the nucleotide sequence of SEQ ID NO. 8 can be used to encode a recombinant coagulation factor VIII protein or a variant thereof having an amino acid sequence of SEQ ID NO. 6.

[0059] In some embodiments, the nucleotide sequence of SEQ ID NO. 22 can be used to encode a recombinant coagulation factor VIII protein or a variant thereof having an amino acid sequence of SEQ ID NO. 21.

[0060] In some embodiments, the nucleotide sequence of SEQ ID NO. 24 can be used to encode the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO. 23.

[0061] In some embodiments, the nucleotide sequence of SEQ ID NO. 38 can be used to encode a recombinant coagulation factor VIII protein or a variant thereof having an amino acid sequence of SEQ ID NO. 43.

[0062] In some embodiments, the nucleotide sequence of SEQ ID NO. 39 can be used to encode the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO. 44.

[0063] In some embodiments, the nucleotide sequence of SEQ ID NO. 54 can be used to encode a recombinant coagulation factor VIII protein or a variant thereof having an amino acid sequence of SEQ ID NO. 53.

[0064] In some embodiments, the nucleotide sequence of SEQ ID NO. 56 can be used to encode a recombinant coagulation factor VIII protein or a variant thereof having an amino acid sequence of SEQ ID NO. 50.

[0065] In some embodiments, the nucleotide sequence of SEQ ID NO. 58 can be used to encode the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO. 52.

[0066] In some embodiments, the nucleotide sequence of SEQ ID NO. 60 can be used to encode the recombinant coagulation factor VIII protein or its variant having the amino acid sequence of SEQ ID NO. 51.

[0067] In a sixth aspect, a recombinant gene vector is provided.

[0068] A recombinant gene vector comprising the nucleotide sequence encoding the recombinant coagulation factor VIII protein or its variant described in the fourth aspect or the nucleotide sequence described in the fifth aspect.

[0069] In some embodiments, the recombinant gene vector may include a non-viral vector or a viral vector.

[0070] In some embodiments, the non-viral vector can be selected from standard plasmids or other circular expression cassettes.

[0071] In some embodiments, the viral vector may be selected from a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0072] In some preferred embodiments, the non-viral vector can be selected from minicircle DNA vectors.

[0073] In a seventh aspect, a parent plasmid for producing minicircle DNA is provided.

[0074] A parent plasmid for producing minicircle DNA, comprising a plasmid vector containing the nucleotide sequence described in the fifth aspect.

[0075] In some embodiments, a parent plasmid for producing minicircle DNA is formed by inserting 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 into a plasmid vector, wherein the target gene DNA fragment is located between the restriction endonuclease cutting sites of the multiple cloning site, and the target gene DNA fragment includes the nucleotide sequence described in the fifth aspect.

[0076] In some embodiments, the promoter may be a CMV promoter, and its nucleotide sequence may be SEQ ID NO.29.

[0077] In some embodiments, the enhancer nucleotide sequence may be SEQ ID NO.30.

[0078] In some embodiments, the multiple cloning site nucleotide sequence may be SEQ ID NO.31.

[0079] In some embodiments, the polyA signal may include a bovine growth hormone polyA signal, a human growth hormone polyA signal, or an SV40 polyA signal.

[0080] In some embodiments, the bovine growth hormone polyA signal nucleotide sequence may be SEQ ID NO.32.

[0081] 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 beneficial for enhancing transcription of the target gene.

[0082] In some embodiments, the nucleotide sequence of the Kozak sequence may be SEQ ID NO. 63 (GCCACC).

[0083] In some embodiments, the human FVIII gene signal peptide coding sequence may be SEQ ID NO. 33 or SEQ ID NO. 34.

[0084] In some embodiments, the plasmid vector comprises a plasmid selected from pMC.BESPX plasmid or p2ΦC31 plasmid.

[0085] In some embodiments, the promoter nucleotide sequence, enhancer nucleotide sequence, multiple cloning site nucleotide sequence and polyA signal nucleotide sequence are inserted into the plasmid through the attB and attP recombination sites of the plasmid.

[0086] In some embodiments, the target gene DNA fragment is inserted into the plasmid through the restriction endonuclease cutting sites of AgeI and EcoRV in the multiple cloning site.

[0087] In an eighth aspect, a method for preparing minicircle DNA is provided.

[0088] A method for preparing minicircle DNA, comprising: transforming the parent plasmid described in the seventh aspect into a host cell; after induction, the parent plasmid produces minicircle DNA and backbone DNA through site-specific recombination of specific recombination sites; and extracting the minicircle DNA using a plasmid DNA purification kit.

[0089] In some embodiments, the backbone DNA is linearized and subsequently degraded within the host cell.

[0090] In some embodiments, the induction can include induction with L-arabinose.

[0091] In the ninth aspect, a minicircular DNA is provided.

[0092] A minicircle DNA obtained according to the preparation method of the eighth aspect. The minicircle DNA provided in the ninth aspect of the present invention has the advantages of high expression level, 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 for children, cannot be used for secondary administration), and safety risks (random integration causes cancer). That is, it has the advantages of simple production, wide application range (can be used for children, good secondary administration effect), and good safety.

[0093] In some embodiments, the nucleotide sequence of the minicircle DNA may include 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.

[0094] In a tenth aspect, a host cell is provided.

[0095] A host cell comprising the nucleotide sequence encoding the recombinant 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.

[0096] 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.

[0097] In the eleventh aspect, a pharmaceutical composition is provided.

[0098] A pharmaceutical composition comprising the recombinant coagulation factor VIII protein or its variant described in the fourth aspect, the recombinant coagulation factor VIII protein or its variant encoded by the nucleotide sequence described in the fifth aspect, the recombinant gene vector described in the sixth aspect, or the minicircle DNA obtained by the preparation method described in the eighth aspect, or the minicircle DNA described in the ninth aspect, and a pharmaceutically acceptable excipient or carrier. The pharmaceutical composition provided in the eleventh aspect of the present invention has the advantages of high expression level, good secondary administration effect, simple preparation, good therapeutic effect, etc., and solves the problems of AAV.hf8 production difficulties (packaging difficulties), limited application range (cannot be used for children, cannot be secondary administered), and safety risks (random integration causes cancer). That is, it has the advantages of simple production, wide application range (can be used for children, good secondary administration effect), good safety, and long-term expression in the human body.

[0099] In the twelfth aspect, a use is provided.

[0100] Use of the recombinant 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 parent plasmid described in the seventh aspect, the minicircle DNA obtained by the preparation 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 a disease.

[0101] In some embodiments, the disease may be selected from a hereditary gene defect disease.

[0102] In some embodiments, the disease may be a disease caused by a coagulation factor deficiency.

[0103] In some embodiments, the disease may be a disease caused by a deficiency of coagulation factor VIII.

[0104] In some embodiments, the disease may be hemophilia.

[0105] In some embodiments, the hemophilia is moderate hemophilia or severe hemophilia. Beneficial effects

[0106] Compared with the prior art, an embodiment of the present invention has at least one of the following beneficial technical effects:

[0107] (1) The recombinant gene vector, minicircle DNA, pharmaceutical composition provided by the present invention, or the recombinant coagulation factor VIII protein or its variant using the connecting peptide provided by the present invention has the advantages of high expression level, good secondary administration effect, simple preparation, good therapeutic effect, and long-term expression in vivo.

[0108] (2) Solve the problems of AAV.hf8 production difficulties (packaging difficulties), application scope limitations (cannot be used for children, cannot be given a second time), and safety risks (random integration causes cancer), that is, it has the advantages of simple production, wide application range (can be used for children, second time administration has good efficacy), good safety, and can be expressed in the body for a long time.

[0109] Definition of terms:

[0110] The term "room temperature" refers to ambient temperature, which may be 20°C-30°C; in some embodiments, 22°C-28°C; in some embodiments, 24°C-26°C; and in some embodiments, 25°C.

[0111] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0112] The fusion proteins of the present invention are typically prepared by biosynthetic methods. Based on the nucleotide sequences described herein, those skilled in the art can readily produce the encoding nucleic acids of the present invention using various known methods. These methods include, but are not limited to, PCR and artificial DNA synthesis. For specific methods, see J. Sambrook, "Molecular Cloning: A Laboratory Manual." As one embodiment of the present invention, the encoding nucleic acid sequences of the present invention can be constructed by synthesizing nucleotide sequences in segments followed by overlap extension PCR.

[0113] The term "parent plasmid" refers to the original plasmid containing the target gene expression cassette and backbone DNA, which can produce minicircle DNA. After site-specific DNA recombination occurs in this plasmid, minicircle DNA is produced.

[0114] The term "fusion protein" generally refers to a protein obtained by fusing two or more proteins or polypeptides. The genes or nucleic acid molecules encoding the two or more proteins or polypeptides can be connected to each other to form a fusion gene or a fused nucleic acid molecule that encodes the fusion protein. Translation of the fusion gene produces a single polypeptide that has the properties of at least one, or even each, of the two or more proteins or polypeptides before fusion. Recombinant fusion proteins are artificially created by recombinant DNA technology for biological research or treatment. Recombinant fusion proteins are proteins created by genetic engineering of fusion genes. The present invention relates to recombinant fusion proteins, and the terms fusion protein and recombinant fusion protein are used herein with the same meaning. The fusion proteins described herein generally comprise at least two domains (A and C) and optionally comprise a third component, a linker between the two domains. The generation 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 attaching the cDNA sequence of the second protein in a frame-matched manner by ligation or overlap extension PCR. The DNA sequence is then expressed by the cell as a single protein. The protein can be engineered to include the entire sequence of both original proteins or polypeptides, or only a portion of either.

[0115] 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 pharmaceutical art. All methods include the step of bringing the active ingredient into association with a carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with a liquid carrier, a finely divided solid carrier, or both.

[0116] In this application, the term "recombinant gene vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The recombinant gene vector may include a vector primarily used for inserting DNA or RNA into a cell, a recombinant gene vector primarily used for replicating DNA or RNA, and a recombinant gene vector primarily used for expression of the transcription and / or translation of DNA or RNA. The recombinant gene vector also includes a recombinant gene vector with a variety of the above-mentioned functions. The recombinant gene vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the recombinant gene vector can produce a desired expression product by cultivating a suitable host cell that contains the recombinant gene vector.

[0117] In this application, the term "variant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0118] As used herein, the term "nucleotide" refers to ribonucleotides, deoxynucleotides or modified forms of either type of nucleotide, and combinations thereof.

[0119] As used herein, the term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. As used herein, the term "transformed" or "transfected" refers 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 the DNA sequences of the present invention and can be used for the expression and / or secretion of a target protein.

[0120] The term "identity" as used herein to describe an amino acid sequence or 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 is incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Sequence identity percentages can be determined using the most recent version of BLAST as of the filing date of this application.

[0121] The term "at least 80%-99% sequence identity" refers to 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 the respective reference sequence.

[0122] In the present invention, "MC.BDD-FVIII-L1" or "MC.BDD-FVIII-L1 minicircle DNA" have the same meaning, and the meanings of other terms beginning with "MC.BDD" are similar, such as "MC.BDD-FVIII-L2" or "MC.BDD-FVIII-L2 minicircle DNA" have the same meaning. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Figure 1 is a schematic diagram of the construction process of the minicircle DNA parent plasmid pMC.BDD-FVIII-SQ-CO2 in Example 1.

[0124] FIG2 is a schematic diagram of the structure of the minicircle DNA parent plasmid pMC.BDD-FVIII-L1 in Example 1.

[0125] FIG3 is a schematic diagram of the structure of the minicircle DNA parent plasmid pMC.BDD-FVIII-L2 in Example 1.

[0126] FIG4 is a schematic diagram of the structure of the minicircle DNA parent plasmid pMC.BDD-FVIII-L3 in Example 1.

[0127] FIG5 is a schematic diagram of the structure of the minicircle DNA parent plasmid pMC.BDD-FVIII-v3 in Example 1.

[0128] FIG6 is a schematic diagram of the structure of the minicircle DNA parent plasmid pMC.BDD-FVIII-N8 in Example 1.

[0129] Figure 7 is a Western blot detection diagram of MC.BDD-FVIII-SQ-CO2 in Example 4; wherein lane M is a protein marker; lane 1 is a negative control sample (blank control without transfection of MC vector); 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.).

[0130] Figure 8 is a statistical graph showing the results of in vitro cellular expression of the target protein after MC.BDD-FVIII-SQ-WT, MC.BDD-FVIII-SQ-CO1, and MC.BDD-FVIII-SQ-CO2 were transfected into 293T cells using ELISA in Example 5.

[0131] Figure 9 is a statistical graph of the therapeutic effect of MC.BDD-FVIII-SQ-CO2 on hemophilia A mice with F8 gene knockout (F8-KO) in Comparative Example 1, wherein m1 / 2mm indicates that the tail-cutting experiment was performed at a tail diameter of 2 mm one month after injection, and m2 / 2.5mm indicates that the tail-cutting experiment was performed at a tail diameter of 2.5 mm two months after injection.

[0132] Figure 10 is a statistical graph showing the in vitro cellular expression levels of the target protein after 293T cells were transfected with MC.BDD-FVIII-L1, MC.BDD-FVIII-L2 and other FVIII minicircle DNA vectors in Example 6.

[0133] FIG11 is a statistical diagram of the expression levels of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 in normal mice in Example 8.

[0134] FIG12 is a statistical diagram showing the expression level of MC.BDD-FVIII-L1 in beagle dogs in Example 8.

[0135] FIG13 is a statistical diagram of the expression level of MC.BDD-FVIII-L1 in hemophilia A mice (F8-KO mice) in Example 9.

[0136] Figure 14 is a statistical graph of the bleeding volume in the tail-cutting experiment of F8-KO mice in the blank control group (PBS), F8-KO mice in the MC.BDD-FVIII-L1 microcircle DNA treatment group (MC), and wild-type normal mice (WT) in Example 9.

[0137] Figure 15 is a statistical graph of the bleeding volume in the tail-cutting experiment of F8-KO mice in the blank control group (PBS), F8-KO mice in the MC.BDD-FVIII-L2 microcircle DNA treatment group (MC), and wild-type normal mice (WT) in Example 9.

[0138] FIG16 is a statistical diagram of the in vivo expression level of MC.BDD-FVIII-CTP-Fc in Example 10.

[0139] FIG17 is a statistical graph showing the results of in vitro cellular expression of the target protein after MC.BDD-FVIII-L1-X5 and MC.BDD-FVIII-L1 were transfected into 293T cells using ELISA in Example 10.

[0140] FIG18 is a statistical graph showing the expression levels of target proteins in plasma samples of MC.BDD-FVIII-L1-X5 and MC.BDD-FVIII-L1 in mice in vivo experiments in Example 10.

[0141] FIG19 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.

[0142] Figure 20 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.

[0143] FIG21 is a diagram showing the pathological section results of beagle dogs injected with MC.BDD-FVIII-L1 minicircle DNA in Example 13. DETAILED DESCRIPTION

[0144] In order to enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0145] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0146] The normal human plasma FVIII concentration ranges from 100 to 200 ng / mL, with 200 ng / mL being defined as 100% of the normal level (reference: J Biol Chem 2001, 276: 46340-46346).

[0147] The amino acid and nucleotide sequences of the connecting peptides, FVIII proteins, mini-rings, etc. in the specific embodiments are as follows:

[0148] Table 1: Amino acid and nucleotide sequences of various connecting peptides, FVIII proteins, mini-rings, etc. in specific examples

[0149] The sequence name with "WT" indicates that the coding sequence of BDD-FVIII is the coding sequence after almost completely deleting the B region (retaining the SQ linker) of the wild-type human FVIII coding sequence (sequence number: NM_000132); the sequence name with "CO1" or "CO2" indicates that the coding sequence of BDD-FVIII is the coding sequence obtained by codon optimization after almost completely deleting the B region (retaining the SQ linker) of the wild-type human FVIII coding sequence (sequence number: NM_000132).

[0150] SEQ ID NO.63: GCCACC

[0151] Example 1: Construction of minicircle DNA parental plasmid (PP)

[0152] Construct the minicircle DNA parent plasmid as follows:

[0153] (1) 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) were inserted between the attB and attP recombination sites of the minicircle DNA cloning vector (pMC.BESPX) to construct the vector pMC.CMV-MCS-bpA.

[0154] (2) Synthesize a target gene DNA fragment, including a Kozak sequence (SEQ ID NO. 63) that enhances target gene transcription, a human FVIII gene signal peptide coding sequence (SEQ ID NO. 33), and a BDD-FVIII coding sequence (before optimization) containing an SQ linker (SEQ ID NO. 35). The synthesized DNA fragment is then cloned into the pMC.CMV-MCS-bpA multiple cloning site between the AgeI / EcoRV restriction sites to construct a wild-type BDD-FVIII minicircle DNA parent plasmid containing an SQ linker (pMC.BDD-FVIII-SQ-WT).

[0155] (3) Referring to the operations of (1) and (2) above, the BDD-FVIII coding sequence containing SQ linker (before optimization) (SEQ ID NO.35) was replaced with the optimized coding sequence of BDD-FVIII containing SQ linker-CO1 (SEQ ID NO.45), the optimized coding sequence of BDD-FVIII containing SQ linker-CO2 (SEQ ID NO.15), the optimized coding sequence of BDD-FVIII containing v3 linker (SEQ ID NO.16), the optimized coding sequence of BDD-FVIII containing N8 linker (SEQ ID NO.17), the optimized coding sequence of BDD-FVIII containing L1 linker (SEQ ID NO.7), the optimized coding sequence of BDD-FVIII containing L2 linker (SEQ ID NO.8), the optimized coding sequence of BDD-FVIII containing L3 linker (SEQ ID NO.18), the optimized coding sequence of BDD-FVIII containing v3 linker (SEQ ID NO.36), the optimized coding sequence of BDD-FVIII containing N8 linker (SEQ ID NO.19), the optimized coding sequence of BDD-FVIII containing L1 linker (SEQ ID NO.7), the optimized coding sequence of BDD-FVIII containing L2 linker (SEQ ID NO.8), the optimized coding sequence of BDD-FVIII containing L3 linker (SEQ ID NO.19 ... ID NO.37), BDD-FVIII coding sequence containing L1 linker (SEQ ID NO.38), BDD-FVIII coding sequence containing L2 linker (SEQ ID NO.39), were constructed into different minicircle DNA parent 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 (the nucleotide sequences of each minicircle DNA parent plasmid are shown in Table 1).

[0156] Example 2: Minicircle DNA production

[0157] To produce minicircle DNA, follow these steps:

[0158] (1) The parent 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 respectively transformed into genetically engineered Escherichia coli ZYCY10P3S2T.

[0159] (2) Select positive monoclonal colonies, inoculate them into LB medium or TB medium, and culture them in a shaking incubator at 37°C for 12-16 hours.

[0160] (3) Add L-arabinose for induction (final L-arabinose concentration 0.2 wt%) at a temperature of 30°C-32°C for 6-8 hours to induce site-specific DNA recombination in the parental plasmid to form minicircular DNA and backbone DNA. The backbone DNA is linearized in the bacteria and then degraded.

[0161] (4) Use a plasmid DNA purification kit (QIAGEN EndoFree Plasmid Mega Kit, Qiagen, Germany) to extract the corresponding minicircle DNA (the nucleotide sequence of each minicircle DNA is 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.

[0162] Example 3: Cell transfection

[0163] (1) In a 6-well plate, the seeding density per well was 1x10 6 293T cells were inoculated into the cells;

[0164] (2) Culturing the cells for 24 hours in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 to obtain the cells to be transfected;

[0165] (3) Discard the serum-containing DMEM medium and replace it with serum-free expression medium (Expi293, Thermo Fisher Scientific), and continue culturing in a 37°C incubator for 4 h;

[0166] (4) Using a transfection reagent (X-tremeGENE HP DNA Transfection Reagent, Roche), the minicircle DNA was transferred into the cells to be transfected at a concentration of 1.2 μg / well to obtain recombinant cells;

[0167] (5) The recombinant cells were cultured for 72 hours, and the cell culture supernatant was collected for subsequent testing.

[0168] Example 4: Western blot detection

[0169] (1) Sample preparation: Add reducing loading buffer to the cell culture supernatant, negative control sample (transfection reagent), and positive control sample (hFVIII plasma extract; Guizhou Taibang Biological Products Co., Ltd.);

[0170] (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 protein, cool, load the sample into the SDS-PAGE gel well, and electrophoresed at 80-100V for 1 hour;

[0171] (3) Transfer: Use a wet transfer apparatus (Bio-Rad) at 300 mA for 1 h to transfer proteins from SDS-PAGE gel to PVDF membrane;

[0172] (4) Blocking: After washing the PVDF membrane, add blocking solution to block it;

[0173] (5) Antibody incubation: Add diluted HRP-labeled anti-human FVIII antibody (Affinity Biologicals) and incubate at room temperature for 1 hour;

[0174] (6) Color development: Detect protein using ECL chemiluminescence reagent (Cell Signaling).

[0175] Results: The MC.BDD-FVIII-SQ-CO2 test results are shown in Figure 7.

[0176] Example 5: Investigation of codon-optimized sequences of BDD-FVIII

[0177] 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 the wild-type BDD-FVIII-SQ-WT (SEQ ID NO. 35). The wild-type BDD-FVIII was codon optimized using two optimization algorithms to obtain two BDD-FVIII optimized sequences (BDD-FVIII-SQ-CO1 (SEQ ID NO. 45) and BDD-FVIII-SQ-CO2 (SEQ ID NO. 15), respectively. MC.BDD-FVIII-SQ-WT, MC.BDD-FVIII-SQ-CO1, and MC.BDD-FVIII-SQ-CO2 were prepared according to 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 supernatant was collected after 72 hours. The expression level of the target protein was detected by ELISA.

[0178] Results: As shown in Figure 8.

[0179] Conclusion: The expression levels of the two optimized vectors were significantly improved; and the second optimized version (BDD-FVIII-CO2) showed a greater improvement, more than 1-fold.

[0180] Comparative Example 1: In vivo expression and activity investigation of MC.BDD-FVIII-SQ-CO2

[0181] MC.BDD-FVIII-SQ-CO2 expression levels in the plasma of wild-type C57 mice:

[0182] Procedure: MC.BDD-FVIII-SQ-CO2 was injected intramuscularly into wild-type C57 mice at a dose of 30 μg / mouse, and the plasma expression level was determined by ELISA.

[0183] Results: The plasma expression level was determined by ELISA, and only weak expression (1-2 ng / mL) was found.

[0184] The therapeutic effect of MC.BDD-FVIII-SQ-CO2 on hemophilia A mice with F8 gene knockout (F8-KO mice):

[0185] Procedure: Six 12-week-old male F8-KO mice were randomly divided into two groups, with three mice in each group. Each mouse in the treatment group received an intramuscular injection of 30 μg / 50 μL of a microcircle DNA (MC.BDD-FVIII-SQ-CO2) solution (50 μL of solution containing 30 μg of microcircle DNA), while each mouse in the blank control group received an intramuscular injection of 50 μL of PBS. After intramuscular injection, a pulsed electric field (TERESA In Vivo Gene Transfer System; parameters: 36 V, 10 ms, 1 Hz) was applied. One month (m1) and two months (m2) after injection, tails were clipped twice at 2 mm and 2.5 mm diameters, respectively, and bleeding was recorded. After tail clipping, the wound was cauterized with AgNO3 to stop bleeding.

[0186] Results: As shown in Figure 9, after a period of time, the tails of the mice were cut (the diameter of the tail was 2 mm after 1 month; the diameter of the tail was 2.5 mm after 2 months) to compare the bleeding volume of the mice in the microcircular DNA (MC.BDD-FVIII-SQ-CO2) injection group and the untreated control group (no microcircular DNA, only injected with an equal volume of PBS buffer). It was found that the bleeding volume of the two groups was equivalent and there was no statistical difference, indicating that MC.BDD-FVIII-SQ-CO2 treatment failed to improve the coagulation function of hemophilia A mice.

[0187] Example 6: ELISA determination of target protein expression level

[0188] 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 taken and the expression levels of the target proteins were determined by ELISA.

[0189] Results: As shown in Figure 10, the target protein expression of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 was significantly higher than that of other FVIII minicircle DNA expression vectors. The relative levels are as follows:

[0190] (1) Ratio of target protein expression levels of MC.BDD-FVIII-L1, MC.BDD-FVIII-N8, and MC.BDD-FVIII-v3:

[0191] MC.BDD-FVIII-L1:MC.BDD-FVIII-N8=1.867;

[0192] MC.BDD-FVIII-L1:MC.BDD-FVIII-v3=1.849;

[0193] The protein content of MC.BDD-FVIII-L1 was approximately 87% and 85% higher than that of MC.BDD-FVIII-N8 and MC.BDD-FVIII-v3, respectively.

[0194] (2) Ratio of target protein expression levels of MC.BDD-FVIII-L2, MC.BDD-FVIII-N8, and MC.BDD-FVIII-v3:

[0195] MC.BDD-FVIII-L2:MC.BDD-FVIII-N8=1.924;

[0196] MC.BDD-FVIII-L2:MC.BDD-FVIII-v3=1.906;

[0197] MC.BDD-FVIII-L2 was approximately 92% and 91% higher than MC.BDD-FVIII-N8 and MC.BDD-FVIII-v3, respectively.

[0198] (3) Ratio of target protein expression levels of MC.BDD-FVIII-L3, MC.BDD-FVIII-N8, and MC.BDD-FVIII-v3:

[0199] MC.BDD-FVIII-L3:MC.BDD-FVIII-N8=0.443;

[0200] MC.BDD-FVIII-L3:MC.BDD-FVIII-v3=0.439;

[0201] MC.BDD-FVIII-L3 is approximately equivalent to 44% of MC.BDD-FVIII-N8 or MC.BDD-FVIII-v3.

[0202] 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.

[0203] (4) Ratio of target protein expression levels between MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2:

[0204] 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.

[0205] Conclusion: The minicircle DNA vectors of coagulation factor VIII containing L1-linker or L2-linker linker peptides have higher target protein expression levels than those containing other types of linker peptides.

[0206] Example 7: In vitro coagulation activity detection (one-stage APTT method)

[0207] Procedure: Cell culture supernatants from MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2, which showed the highest expression of the target protein as obtained in Example 3, were collected and sent to the Institute of Blood Transfusion Medicine, Chinese Academy of Medical Sciences. Coagulation activity was assayed using a one-stage APTT assay. FVIII activity in normal pooled plasma (NPP) was defined as 100%.

[0208] Results: The activity values ​​of the expression products of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 (72h cell culture supernatant) were 118.1% and 116%, respectively (the reference value of the activity range for humans with normal coagulation function is 50%-150%), indicating that coagulation factor VIII containing L1-linker or L2-linker connecting peptide has good coagulation activity.

[0209] Example 8: In vivo expression

[0210] (1) Expression in normal mice

[0211] Procedure: Ten 6-8 week old Balb / c mice were randomly divided into two groups of 5 mice each, 5 mice in the MC.BDD-FVIII-L1 group and 5 mice in the MC.BDD-FVIII-L2 group. Blood was collected one day before the injection of minicircle DNA, 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 minicircle DNA and MC.BDD-FVIII-L2 minicircle DNA at a dose of 30 μg / 50 μL / mouse, respectively, and then a pulsed electric field was applied (TERESA live gene introduction instrument; parameters: 36V, 10ms, 1Hz). After injection, blood was collected regularly, plasma was separated, and stored at -80°C. The expression level of the target protein in the plasma samples was detected using the human FVIII ELISA kit (VisuLize Factor VIII PLUS Antigen ELISA Kit, Affinity Biologicals Inc).

[0212] Results: As shown in Figure 11, while the blank control sample showed no expression, four weeks after injection, the in vivo expression levels of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 in the MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 groups were similar, reaching over 20 ng / mL (equivalent to over 10% of the normal plasma FVIII concentration in normal humans, exceeding the significant therapeutic level (>5%) that can significantly improve moderate or severe hemophilia to mild hemophilia.

[0213] (2) Expression in beagle dogs

[0214] Procedure: Three adult beagle dogs weighing approximately 13 kg were bled before injection with minicircle 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 then injected with 600 μg of MC.BDD-FVIII-L1 minicircle DNA, and then a pulsed electric field was applied (TERESA in vivo gene transduction instrument; parameters: 36V, 10ms, 1Hz). Thereafter, blood was collected weekly, 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) and converted to the relative level of FVIII in normal pooled plasma (NPP). The FVIII concentration in normal pooled plasma is approximately 200 ng / mL.

[0215] Results: As shown in Figure 12 , the blank control sample showed no expression. One week after injection with the MC.BDD-FVIII-L1 minicircle DNA, plasma hFVIII expression in one dog reached over 5% of the normal human plasma FVIII concentration (10 ng / mL), while levels were lower in the other two dogs. Two weeks after injection with the MC.BDD-FVIII-L1 minicircle DNA, expression levels gradually increased, exceeding 10% of the normal human plasma FVIII concentration (20 ng / mL) in all dogs.

[0216] Example 9: In vivo activity in hemophilia A mice

[0217] (1) In vivo expression level of MC.BDD-FVIII-L1 in hemophilia A mice (F8-KO mice)

[0218] Procedure: Blood was collected from 5 6-8 week old male F8-KO mice one day before microcircle DNA injection, plasma was separated, and stored at -80°C as a blank control sample (W0). All mice were intramuscularly injected with microcircle DNA (MC.BDD-FVIII-L1 microcircle DNA) at a dose of 30 μg / 50 μL / mouse (50 μL solution, containing 30 μg microcircle DNA), and then a pulsed electric field was applied (TERESA live gene introduction instrument; parameters: 36V, 10ms, 1Hz). 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 sample was detected using the human FVIII ELISA kit (VisuLize Factor VIII PLUS Antigen ELISA Kit, Affinity Biologicals Inc).

[0219] Results: The results are shown in Figure 13. No expression was observed in the blank control sample. Six weeks after injection of the MC.BDD-FVIII-L1 minicircle DNA, plasma target protein levels, as measured by ELISA, reached over 100 ng / mL. This represents 50% of the normal plasma FVIII concentration (100 ng / mL), indicating functional cure of hemophilia and restoration of normal coagulation function.

[0220] (2) Treatment of hemophilia A mice (F8-KO mice) with MC.BDD-FVIII-L1

[0221] Procedure: Ten 6-8-week-old male F8-KO mice were randomly divided into two groups, 5 mice each: a blank control (PBS) group and a microcircle DNA treatment group (MC treatment group, MC). Mice in the microcircle DNA treatment group received an intramuscular injection of MC.BDD-FVIII-L1 microcircle DNA solution at a dose of 30 μg / 50 μL (50 μL solution containing 30 μg microcircle DNA). After injection, a pulsed electric field (TERESA live gene delivery instrument; parameters: 36 V, 10 ms, 1 Hz) was applied. Mice in the blank control group received an equal volume of PBS buffer in the same manner. Five male C57 mice of the same age served as wild-type (WT) controls with normal coagulation function. Four weeks after injection, tail snips were performed at a 2 mm diameter, and bleeding volume was recorded over 15 minutes. Mice were sacrificed at the end of the experiment.

[0222] Results: As shown in Figure 14, compared with the blank control group (PBS), the amount of bleeding in the MC.BDD-FVIII-L1 microcircle DNA treated group (MC treated group, MC) was significantly reduced, while the amount of bleeding in the MC treated group was comparable to that in the wild-type normal mice (WT) (no statistical difference, ns); this indicated that after MC treatment, the coagulation function of hemophilia A mice had returned to normal.

[0223] (3) Identification of the activity of MC.BDD-FVIII-L2 in hemophilia A mice (F8-KO mice)

[0224] Procedure: Twenty 9-10-week-old male F8-KO mice were randomly divided into two groups: a blank control group (PBS) of 5 mice and a microcircle DNA injection group (MC treatment group, MC) of 15 mice. The MC treatment group received a 3 μg / 1.8 mL solution of MC.BDD-FVIII-L2 microcircle DNA (3 μg microcircle DNA dissolved in 1.8 mL PBS buffer) via high-pressure injection into the tail vein (1.8 mL of solution was rapidly injected over 5-8 seconds). The blank control group received an equal volume of 1.8 mL of PBS buffer via the same procedure. Five male C57 mice of the same age served as wild-type (WT) controls with normal coagulation function. Twenty-four hours after injection, tails were clipped at a 2 mm diameter, and bleeding was recorded over 15 minutes. The mice were then sacrificed. One mouse in the blank control group died due to an abnormality in the high-pressure injection procedure, preventing tail clipping.

[0225] Results: As shown in Figure 15, compared with the blank control group (PBS), the amount of bleeding in the MC.BDD-FVIII-L2 microcircle DNA injection group (MC treatment group, MC) was significantly reduced, even less than that of wild-type normal mice (WT) without any treatment, indicating that the coagulation activity of MC.BDD-FVIII-L2 in vivo is extremely high.

[0226] Example 10: Other modifications

[0227] (1) Fc fusion

[0228] Fc fusion is an important means of increasing the half-life of FVIII. In vivo expression of molecules with longer half-lives may lead to greater product accumulation and higher expression levels. To test this concept, 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).

[0229] Procedure: Ten 6-8-week-old Balb / c mice were randomly divided into two groups (5 mice each): a microcircle DNA (MC.BDD-FVIII-CTP-Fc) injection group (5 mice) and a PBS blank control group (5 mice). The microcircle DNA (MC.BDD-FVIII-CTP-Fc) was injected intramuscularly (IM) at a dose of 45 μg / 50 μL / mouse (50 μL per mouse, containing 45 μg of microcircle DNA, equivalent to 30 μg of MC.BDD-FVIII). The blank control group received an IM injection of 50 μL of PBS. After IM injection, a pulsed electric field (TERESA In Vivo Gene Transfer System; parameters: 36 V, 10 ms, 1 Hz) was applied. Blood was collected regularly, plasma was separated, and stored at -80°C. The expression level of the target protein in plasma samples was determined using a human FVIII ELISA kit (VisuLize Factor VIII PLUS Antigen ELISA Kit, Affinity Biologicals Inc.).

[0230] Results: As shown in Figure 16, the blank control sample showed no expression. Eleven to fifteen weeks after injection of the MC.BDD-FVIII-CTP-Fc minicircDNA, the minicircDNA of the BDD-FVIII-Fc fusion protein did not further increase in vivo expression levels (only approximately 10 ng / mL, less than 5% of the normal human plasma FVIII concentration (10 ng / mL)).

[0231] (2) X5 mutation

[0232] ELISA to determine the in vitro cell expression level of target protein:

[0233] Procedure: The X5 mutation (i.e., mutation of five key amino acids in the human FVIII A region to the corresponding amino acids in porcine FVIII: I86V / A108S / G132K / M147T / L152P) was introduced into BDD-FVIII-L1, resulting in the BDD-FVIII-L1-X5 nucleotide sequence (SEQ ID NO. 22). The corresponding MC expression vector (nucleotide sequences of each minicircle are shown in Table 1) was constructed: MC.BDD-FVIII-L1-X5 minicircle DNA. MC.BDD-FVIII-L1 minicircle DNA and MC.BDD-FVIII-L1-X5 minicircle DNA were transfected into 293T cells, and cell culture supernatants were collected 72 hours later for target protein levels assayed by ELISA.

[0234] Results: As shown in Figure 17, compared with MC.BDD-FVIII-L1 minicircle DNA, the level of target protein was increased after the introduction of X5 mutation.

[0235] In vivo experiments in mice:

[0236] Procedure: Ten 6-8-week-old Balb / c mice were randomly divided into two groups of five mice each. Each group received an intramuscular injection of 30 μg / 50 μL / mouse (50 μL per mouse, containing 30 μg of minicircle DNA) of minicircle DNA (MC.BDD-FVIII-L1 minicircle DNA or MC.BDD-FVIII-L1-X5 minicircle DNA). Following intramuscular injection, a pulsed electric field (TERESA In Vivo Gene Transfer System; parameters: 36 V, 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).

[0237] Results: As shown in Figure 18, compared with MC.BDD-FVIII-L1 minicircle DNA, the level of target protein was slightly increased after the introduction of X5 mutation.

[0238] (3) R1645H mutation

[0239] Siner et al. (Blood 2013, 121: 4396-4403) reported that the furin site (R 1645 -H 1646 -Q 1647 -R 1648 ), preventing it from being recognized and cleaved by the furin protease, thereby increasing expression. Since the recognition pattern of the furin site is RXXR (i.e., arginine at the first and last amino acids, with any two amino acids in between), theoretically, mutating the arginine at positions 1645 or 1648 to any amino acid other than arginine can prevent furin protease recognition and cleavage. This goal can also be achieved by deleting the furin site (including deleting all four amino acids in the RHQR sequence, or any one, two, or three of them).

[0240] In the present invention, the furin site RHQR is retained at the C-terminus of L1linker. Based on this, we mutated the fourth arginine (Arg, R) at the C-terminus of L1linker to histidine (His, H), i.e., R1645H mutation, to obtain the coding sequence of BDD-FVIII-L1. R1645H (The nucleotide sequence is shown in Table 1), and the corresponding MC expression vector MC.BDD-FVIII-L1 was constructed R1645H(The nucleotide sequence of the minicircle is shown in Table 1).

[0241] (4) F309S mutation

[0242] 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) can promote FVIII secretion. R1645H and BDD-FVIII-L2, the phenylalanine at position 309 was mutated to serine (Ser, S), respectively, to obtain BDD-FVIII F309S -L1, BDD-FVIII F309S -L1 R1645H and BDD-FVIII F309S -L2 coding sequence (each nucleotide sequence is shown in Table 1), and the corresponding MC expression vector MC.BDD-FVIII was constructed F309S -L1, MC.BDD-FVIII F309S -L1 R1645H and MC.BDD-FVIII F309S -L2 (the nucleotide sequences of each minicircle are shown in Table 1).

[0243] Example 11: Secondary administration treatment effect

[0244] Procedure: Ten 6-8-week-old Balb / c mice were randomly divided into two groups: MC.BDD-FVIII-L1 group (n=5) and MC.BDD-FVIII-L2 group (n=5). Each mouse in each group received an intramuscular injection of MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 minicircle DNA at a dose of 30 μg / 50 μL (50 μL per mouse, containing 30 μg of minicircle DNA). A pulsed electric field (TERESA In Vivo Gene Transfer System; parameters: 36 V, 10 ms, 1 Hz) was then applied. The mice were administered again in the same manner 22 weeks after injection (W22). 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 determined using a human FVIII ELISA kit (VisuLize Factor VIII PLUS Antigen ELISA Kit, Affinity Biologicals Inc). The FVIII concentration in normal pooled plasma (NPP) is approximately 200 ng / mL.

[0245] Results: As shown in Figure 19. After the injection of minicircle DNA, the 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). The expression level decreased at week 12 (W12), but was still able to maintain above 10% (20 ng / mL) of the normal level of normal human plasma FVIII concentration for a long time (weeks 12 to 20, W12-W20). After the booster administration at week 22 (W22), the expression level at W23 (week 23 after the first injection, i.e., 1 week after the second injection) quickly recovered to a level close to that before the decline. This shows that the second injection of minicircle DNA provided by the present invention is still effective and will not produce a virus-specific immune response. It can be injected a second time, and the therapeutic effect can be restored or enhanced by repeated administration as needed.

[0246] Example 12: Long-term expression in vivo

[0247] Procedure: Ten 6-8 week old Balb / c mice were randomly divided into two groups: MC.BDD-FVIII-L1 group (5 mice) and MC.BDD-FVIII-L2 group (5 mice). Before minicircle DNA injection, blood was collected, plasma was separated, and stored at -80°C to serve as a blank control sample (W0). Each mouse in both groups received an intramuscular injection of MC.BDD-FVIII-L1 minicircle DNA and MC.BDD-FVIII-L2 minicircle DNA at a dose of 30 μg / 50 μL (50 μL per mouse, containing 30 μg minicircle DNA). A pulsed electric field (TERESA live gene delivery instrument; parameters: 36 V, 10 ms, 1 Hz) was then applied. The same administration was repeated 22 weeks after injection. After injection, blood was collected regularly, plasma was separated, and stored at -80°C. The human FVIII ELISA kit (VisuLize Factor VIII PLUS Antigen ELISA Kit, Affinity Biologicals Inc) was used to detect the expression level of the target protein in plasma samples.

[0248] Results: As shown in Figure 20, MC.BDD-FVIII-L1 and MC.BDD-FVIII-L2 can be expressed in mice for more than one year, and the second injection of the minicircle DNA provided by the present invention is still effective in the long term and does not produce virus-specific immune response.

[0249] Example 13: Safety Study

[0250] Procedure: One adult female beagle dog (numbered D0016) weighing 10-12 kg and one adult male beagle dog (numbered 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 harvested, sectioned, and stained with hematoxylin and eosin for pathological examination. The results are shown in Figure 21.

[0251] Results: Six weeks after injection with MC.BDD-FVIII-L1 minicircle DNA, pathological examination of the beagle dogs' lungs, liver, spleen, kidneys, heart, brain, ovaries (female dog D0016) and testicles (male dog C957), muscle, and thymus tissues all appeared normal (Figure 21). This indicates that no significant toxicity was observed and that MC.BDD-FVIII-L1 is safe.

[0252] The methods of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art will be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and spirit of the present invention to implement and apply the technology of the present invention. Those skilled in the art may refer to the disclosure herein and appropriately modify the process parameters to achieve the desired effect. It is particularly important to note that all similar substitutions and modifications apparent to those skilled in the art are considered encompassed by the present invention.

Claims

1. 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% to 99% identity with either nucleotide sequence thereof, or at least a portion of either sequence.

2. A nucleotide sequence encoding the connecting 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 one of the nucleotide sequences.

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

4. A coagulation factor VIII protein or a variant thereof, wherein the connecting peptide in the coagulation factor VIII protein or the variant thereof is selected from the connecting peptide according to claim 1, or the nucleotide sequence of the connecting peptide in the coagulation factor VIII protein or the variant thereof is selected from the nucleotide sequence according to claim 2.

5. A coagulation factor VIII protein or a variant thereof, whose 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 that is at least 80%-99% identical to any of these amino acid sequences, or at least a portion of any of these amino acid sequences.

6. A nucleotide sequence encoding the coagulation factor VIII protein or a variant thereof according to any one of claims 4 to 5; preferably, 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 one of these nucleotide sequences.

7. A recombinant gene vector comprising a nucleotide sequence encoding the coagulation factor VIII protein or a variant thereof according to any one of claims 4 to 5 or the nucleotide sequence according to claim 6; 8. Preferably, the recombinant gene vector comprises a non-viral vector or a viral vector; and / or 9. The non-viral vector is selected from a standard plasmid or other circular expression cassette, or the viral vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector; 10. Preferably, the non-viral vector is selected from minicircle DNA vectors.

11. A parent plasmid for producing minicircle DNA, comprising a plasmid vector containing the nucleotide sequence of claim 6; or formed by inserting 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 into a plasmid vector, wherein the target gene DNA fragment is located between restriction endonuclease cleavage sites of the multiple cloning site, and the target gene DNA fragment comprises the nucleotide sequence of claim 6.

12. A method for preparing minicircle DNA, comprising: The parent plasmid according to claim 8 is transformed into a host cell. After induction, the parent plasmid produces minicircle DNA and backbone DNA through site-specific recombination of specific recombination sites, and the minicircle DNA is extracted using a plasmid DNA purification kit.

13. A minicircle DNA obtained according to the preparation method of claim 9; preferably, 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 or SEQ ID NO.

61.

14. A host cell comprising a nucleotide sequence encoding the recombinant coagulation factor VIII protein or a variant thereof according to any one of claims 4-5, 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 comprises a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

15. A pharmaceutical composition comprising the recombinant coagulation factor VIII protein or its variant according to any one of claims 4-5, the recombinant coagulation factor VIII protein or its variant encoded by the nucleotide sequence according to claim 6, the recombinant gene vector according to claim 7, or the minicircle DNA obtained by the preparation method according to claim 9, or the minicircle DNA according to claim 10, and a pharmaceutically acceptable excipient or carrier.

16. Use of the recombinant coagulation factor VIII protein or its variant according to any one of claims 4-5, the nucleotide sequence according to claim 6, the recombinant gene vector according to claim 7, the parent plasmid according to claim 8, the minicircle DNA obtained by the preparation 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 in the preparation of a medicament for treating a disease; preferably, the disease is selected from a hereditary gene defect disease; preferably, the disease is a disease caused by a coagulation factor deficiency; preferably, the disease is a disease caused by a deficiency of coagulation factor VIII; preferably, the disease is hemophilia.

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