Factor VIII mutant expression vectors with enhanced protein expression
阅读说明：本技术 具有增强的蛋白质表达的因子vⅲ突变体表达载体 (Factor VIII mutant expression vectors with enhanced protein expression ) 是由 胡成炫 朴秀振 于 2018-12-05 设计创作，主要内容包括：本公开涉及携带编码凝血因子VIII突变体的多核苷酸并增加蛋白质表达的表达载体,以及包含该表达载体的用于预防或治疗出血性疾病或出血的药物组合物。本公开的因子VIII突变体是通过在因子VIII中缺失一部分B结构域(残基784至1667)和一部分a3区域(残基1668至1671)而得到的,其结果是携带编码因子VIII突变体的多核苷酸的表达载体的蛋白质表达能力显著增加。(The present disclosure relates to an expression vector carrying a polynucleotide encoding a factor VIII mutant and increasing protein expression, and a pharmaceutical composition for preventing or treating a bleeding disease or bleeding comprising the expression vector. The factor VIII mutants of the present disclosure were obtained by deleting a portion of the B domain (residues 784 to 1667) and a portion of the a3 region (residues 1668 to 1671) in factor VIII, which resulted in a significant increase in protein expression capacity of expression vectors carrying polynucleotides encoding the factor VIII mutants.)
1. A factor VIII mutant expression vector comprising a single-chain polynucleotide encoding a factor VIII mutant, wherein amino acids Asp784 to Arg1671 are deleted from factor VIII represented by SEQ ID NO 1.
2. The factor VIII mutant expression vector according to claim 1, wherein the factor VIII mutant has an amino acid sequence represented by SEQ ID NO 3.
3. The factor VIII mutant expression vector of claim 1, wherein the vector is a vector selected from the group consisting of pCDNA3.1, pGP, and pEF.
4. The factor VIII mutant expression vector according to claim 1, which has increased protein expression compared to an expression vector comprising a polynucleotide encoding factor VIII represented by SEQ ID NO1, B-domain deleted factor VIII, or single chain factor VIII.
5. An expression system for expressing a factor VIII mutant comprising the expression vector of claim 1.
6. A pharmaceutical composition for preventing or treating a bleeding disease or hemorrhage, comprising the expression vector according to claim 1.
7. The pharmaceutical composition for preventing or treating a bleeding disease or hemorrhage according to claim 6, wherein the bleeding disease is hemophilia A or hemophilia B, hemophilia A being a hemophilia caused by or complicated by inhibitory antibodies against factor VIII or factor VIIIa.
8. A pharmaceutical composition for use in the prevention or treatment of a bleeding disease or hemorrhage according to claim 6, wherein the bleeding disease is selected from the group consisting of neonatal coagulopathy, severe liver disease, thrombocytopenia, congenital deficiency of factors V, VII, X or XI and von Willebrand disease with inhibitors against von Willebrand factor.
9. The pharmaceutical composition for preventing or treating a bleeding disease or hemorrhage according to claim 6, wherein the bleeding is caused by blood loss associated with high risk surgery, traumatic blood loss, bone marrow transplantation, or cerebral hemorrhage.
10. The pharmaceutical composition for preventing or treating bleeding disorders or hemorrhage according to claim 6, which is used for gene therapy.
Technical Field
The present disclosure relates to an expression vector comprising a polynucleotide encoding a coagulation factor VIII mutant and having increased protein expression, and a pharmaceutical composition for preventing or treating a bleeding disease or bleeding comprising the same.
Background
Hemophilia a (ha) is a serious genetic disease. Hemophilia is a disease associated with genes on the X chromosome, with hemophilia appearing in 1 of 5000 males. It is caused by a change in the plasma glycoprotein factor viii (fviii), an important component of the blood coagulation process. Factor VIII (hereinafter FVIII) encodes 2351 amino acids and has six domains (A1-A2-B-A3-C1-C2). Heterodimers are formed by a heavy chain comprising the a1, a2, and B domains and a light chain comprising the A3, C1, and C2 domains.
Prior to the 1980 s hemophiliacs received factor VIII extracted from the plasma of others for treatment. However, this method has serious problems such as viral infection. Since the 1980 s, to solve this problem, full-length factor VIII produced in CHO cells and the like by recombinant protein technology has been used. Later, it was known that B-domain deleted factor VIII (F8-BDD) could be produced at high protein productivity without activity difference, and such a double-chain F8-BDD-based recombinant protein (expressing heavy and light chains, respectively) had been developed and used. Due to its superior in vivo stability, the recently developed truncated single-chain F8-BDD recombinant protein was demonstrated to show a 2-to 4-fold increase in AUC compared to the double-chain F8-BDD.
Although the factor VIII recombinant protein shows excellent therapeutic effect, its concentration in the body should be always maintained at 5% or more than 5% in order to prevent internal bleeding in hemophilia patients. For this purpose, the protein should be administered 2 to 3 times per day or week. To overcome this drawback, recombinant virus-based gene therapeutics were developed in the early 2000 s. Recombinant virus-based gene therapy agents use B-domain deleted factor VIII (hereinafter referred to as "F8-BDD") having a size of less than 4.4kb, instead of full-length factor VIII having a size of 7kb or more than 7 kb. In particular, most therapeutic agents use a single-stranded F8-BDD gene rather than a double-stranded F8-BDD gene.
The inventors of the present disclosure have endeavored to develop a factor VIII gene therapeutic agent having further improved stability. To this end, a single-chain F8-BDD mutant exhibiting improved stability and increased protein expression in eukaryotic cells compared to the single-chain F8-BDD gene, and an expression vector expressing the mutant were developed. In addition, it has been determined that expression and stability can be further improved by using endogenous promoters such as elongation factor-1 α (EF-1 α) and codon-optimized (CO) single-stranded F8-BDD mutant, and the present disclosure has been completed.
[ reference to related Art ]
[ patent documents ]
(patent document 001) KR 10-1542752B.
Disclosed is a
Technical problem
The present disclosure relates to providing factor VIII mutant expression vectors with increased protein expression.
The present disclosure also relates to providing a pharmaceutical composition for preventing or treating a bleeding disease or hemorrhage, comprising an expression vector.
Technical scheme
The present disclosure provides factor VIII mutant expression vectors comprising a single-chain polynucleotide encoding a factor VIII mutant, wherein amino acids Asp784 to Arg1671 are deleted from factor VIII represented by SEQ ID NO 1.
In exemplary embodiments of the present disclosure, the factor VIII mutant may have an amino acid sequence represented by SEQ ID NO 3.
In exemplary embodiments of the present disclosure, the vector may be selected from pcdna3.1, pGP and pEF.
In exemplary embodiments of the present disclosure, the expression vector may have increased protein expression compared to an expression vector comprising a polynucleotide encoding factor VIII represented by SEQ ID NO1, B-domain deleted factor VIII, or single chain factor VIII.
In addition, the present disclosure provides expression systems for expressing factor VIII mutants, including expression vectors.
In addition, the present disclosure provides a pharmaceutical composition for preventing or treating a bleeding disease or hemorrhage, comprising the expression vector.
In exemplary embodiments of the present disclosure, the bleeding disorder may be hemophilia a or hemophilia B, hemophilia a being a hemophilia caused by or complicated by inhibitory antibodies to factor VIII or factor VIIIa.
In exemplary embodiments of the present disclosure, the bleeding disorder may be selected from neonatal coagulopathy, severe liver disease, thrombocytopenia, congenital deficiency of factors V, VII, X or XI, and von willebrand disease with inhibitors against von willebrand factor.
In exemplary embodiments of the present disclosure, the bleeding may be caused by blood loss associated with high risk surgery, traumatic blood loss, bone marrow transplantation, or cerebral hemorrhage.
In exemplary embodiments of the present disclosure, the pharmaceutical composition may be used for gene therapy.
Advantageous effects
Expression vectors comprising polynucleotides encoding the factor VIII mutants of the present disclosure deleted a portion of the B domain of factor VIII (residues 784 to 1667) and a portion of the a3 region (residues 1668 to 1671), with significant increase in protein expression.
Drawings
Figure 1 schematically shows a process of designing a factor VIII mutant according to an exemplary embodiment of the present disclosure.
FIG. 2 shows the enzyme cleavage map of the pCDNA3.1 vector.
FIG. 3 shows the enzyme cleavage map of the pGP vector.
FIG. 4 shows the enzyme cleavage map of the pEF vector.
FIG. 5 shows the results of measuring protein expression levels using a sample obtained from cells transfected with the pCDNA3.1 plasmid of preparation example 1.
FIG. 6 shows the results of measuring coagulation activity using a sample obtained from the cell transfected with the pCDNA3.1 plasmid of preparation example 1.
FIG. 7 shows the results of measuring protein expression levels using samples obtained from cells transfected with pGP plasmids of preparation example 2.
FIG. 8 shows the results of measuring protein expression levels using samples obtained from cells transfected with pEP plasmid of preparation example 3.
Best mode for carrying out the invention
Hereinafter, the present disclosure is described in detail.
In one aspect, the present disclosure provides a factor VIII mutant expression vector comprising a single-chain polynucleotide encoding a factor VIII mutant, wherein amino acids Asp784 to Arg1671 are deleted from factor VIII represented by SEQ ID NO 1.
Factor VIII mutants
In the present specification, the terms "coagulation factor VIII", "FVIII" and "F8" are used interchangeably. Mature human factor VIII consists of 2351 amino acids (including the signal peptide) and has the following domains:
signal peptide: (ii) residues 1 to 19 of the amino acid sequence,
a1: (ii)
a2: residues 392 to 729 of the amino acid sequence,
b: the residues 760 to 1667 of the amino acid sequence,
a3: residues 1709 to 2038 of the total number of,
c1: residues 2039 to 2191 and
c2: residues 2192 to 2351.
In addition, there are three acidic domains a1(356 to 391), a2(730 to 759), and a3(1668 to 1708). The acidic domain a3 is known to be involved in the binding of the factor VIII molecule to von willebrand factor (vWF), which plays an important role in blood coagulation. During secretion, factor VIII is cleaved between the B domain and the a3 acidic domain, producing a heterodimeric polypeptide. The factor VIII heterodimer consists of a variable size light chain (including A3, C1, and C2) and heavy chain (including a1, a2, and B). Due to limited proteolysis within the B domain, the heavy chains are heterologous. In the case of heterodimeric B domain deleted factor VIII, the "heavy chain" includes a1 and a2, but lacks part or all of the B domain.
The amino acid sequence of mature wild-type human factor VIII is shown in SEQ ID NO 1. Reference to an amino acid position in a particular sequence refers to the position of the corresponding amino acid in the wild-type FVIII protein and does not exclude mutations (deletions, insertions and/or substitutions) at other positions in the sequence. The DNA sequence encoding SEQ ID NO1 is SEQ ID NO 2.
"factor VIII" includes not only wild-type factor VIII but also derivatives of wild-type factor VIII having the procoagulant activity of wild-type factor VIII. The derivative may have deletions, insertions and/or additions compared to the amino acid sequence of wild-type factor VIII. Preferred derivatives are FVIII molecules which have been deleted for all or part of the B domain. The amino acid positions indicated throughout this specification always refer to the positions of the individual amino acids in mature full length wild type factor VIII (including signal peptide cleavage).
In the present specification, the term "mutant" includes conservative or non-conservative substitutions, insertions or deletions of amino acid sequences, nucleic acid sequences, and the like. This change does not substantially alter the active site or active domain that confers the biological activity of FVIII.
A factor VIII mutant according to the present disclosure is a mutant in which amino acids Asp784 to Arg1671 are deleted from factor VIII represented by SEQ ID NO 1. The mutant is a single chain factor VIII mutant. Factor VIII mutants are mutants in which a portion of the B domain (residues 784 to 1667) and a portion of the a3 region (residues 1668 to 1671) are deleted, which have enhanced protein expression and improved clotting activity and stability compared to factor VIII, B domain deleted factor VIII and single chain factor VIII. The factor VIII mutant has an amino acid sequence represented by SEQ ID NO 3.
"single chain factor VIII" refers to a factor VIII molecule that exists as a single polypeptide chain during secretion by a cell expressing the factor VIII molecule without being cleaved into two chains (e.g., a heavy chain and a light chain).
Polynucleotide
The disclosure also relates to polynucleotides encoding factor VIII mutants having the amino acid sequence represented by SEQ ID NO 3.
In the present specification, the term "polynucleotide" refers to any polyribonucleotide or polydeoxyribonucleotide which may be unmodified RNA or DNA, or modified RNA or DNA. The polynucleotides of the present disclosure may be single stranded DNA or RNA. The term "polynucleotide" as used in this specification includes DNA or RNA containing modified bases and/or unusual bases such as inosine. Obviously, various modifications can be made to DNA or RNA that provide a known useful purpose. The term "polynucleotide" as used in this specification also includes chemically, enzymatically or metabolically modified polynucleotides.
It will be appreciated by those skilled in the art that due to the degeneracy of the genetic code, factor VIII mutants may be encoded by several polynucleotides. That is, it is understood that the polynucleotide sequence encoding a factor VIII mutant that can be used in the present disclosure also includes nucleotide sequences having substantial identity to the amino acid sequence represented by SEQ ID NO 3.
In particular, the polynucleotides of the present disclosure may be isolated polynucleotides. An "isolated" polynucleotide refers to a polynucleotide that is substantially free of other nucleic acid sequences, such as chromosomal and extrachromosomal DNA and RNA, but is not so limited. The isolated polynucleotide may be purified from the host cell. Isolated polynucleotides can be obtained using conventional nucleic acid purification methods known to those skilled in the art. The term also includes recombinant polynucleotides and chemically synthesized polynucleotides.
Expression system for expressing factor VIII mutants
Expression vector
In another aspect, the present disclosure provides an expression vector comprising a single-chain polynucleotide encoding a factor VIII mutant (hereinafter "factor VIII mutant expression vector").
In the present specification, the term "expression" refers to the production of a factor VIII mutant in a cell.
In the present specification, the term "expression vector" refers to a vector capable of expressing the gene VIII mutant in a suitable host cell, and refers to a gene construct containing essential regulatory elements to which the gene insert is operably linked in a manner to be expressed.
In the present specification, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a polynucleotide encoding a factor VIII mutant that performs the overall function. For example, a promoter may be operably linked to a polynucleotide encoding a factor VIII mutant to affect expression of the polynucleotide. Operable linkage to a recombinant vector can be achieved using genetic recombination techniques well known in the art. For site-specific DNA cleavage and ligation, enzymes generally known in the art can be used.
Although not limited thereto, the expression vector of the present disclosure is constructed using a plasmid, a vector or a viral vector. Suitable expression vectors can include regulatory elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, and can be prepared in a variety of ways depending on the intended use. The promoter of the vector may be constitutive or inducible.
Specifically, the expression vector may be prepared by using a vector selected from the group consisting of pcdna3.1, pGP and pEF. In particular, the factor VIII mutant expression vectors have significantly increased protein expression compared to polynucleotides encoding factor VIII represented by SEQ ID NO1, B-domain deleted factor VIII, or single chain factor VIII. In particular, it was demonstrated that protein expression and stability can be further improved by using endogenous promoters such as the EF1a promoter and the like and by codon-optimizing (CO) single-stranded F8-BDD mutants.
In order to produce factor VIII mutants at high levels in host cells, appropriate regulatory elements must be present in the recombinant expression vector, which can be propagated in various expression systems according to methods known to those skilled in the art, as well as the assembled modified cDNA. Effective transcriptional control elements may be derived from viruses that have animal cells as the natural host or from chromosomal DNA of animal cells. In particular, promoter-enhancer combinations derived from the long terminal repeat of
Expression of factor VIII mutants
The expression vector may be transfected into a suitable host cell and may therefore result in the expression of the factor VIII mutant of the present disclosure and the production of a functional protein.
In another aspect, the present disclosure provides a host cell comprising the polynucleotide or expression vector.
The host cells of the disclosure can be used in methods of producing factor VIII mutants of the disclosure. The method can comprise the following steps: (a) a step of culturing the host cell under conditions in which the factor VIII mutant can be expressed; and (b) recovering the factor VIII mutant from the host cell or the culture medium.
In particular, for expression of factor VIII mutants, nucleotides such as cDNA are incorporated into the genome of a suitable host cell. In particular, the host cell should be an animal cell of vertebrate origin to ensure proper folding, disulfide bond formation, asparagine-linked glycosylation and other post-translational modifications, and to ensure secretion into the culture medium. Examples of other post-translational modifications include O-sulfation of tyrosine and proteolytic processing of nascent polypeptide chains. Examples of cells that can be used include monkey COS cells, mouse L cells, mouse C127 cells, hamster BHK-21 cells, human embryonic kidney 293 cells, and hamster CHO cells.
Recombinant expression vectors encoding the corresponding cDNAs can be introduced into animal cells in different ways. For example, recombinant expression vectors can be constructed from virus-based vectors. Examples include baculovirus-, vaccinia-and adenovirus-based vectors, in particular bovine papilloma virus.
The transcription unit encoding the corresponding DNA may also be introduced into animal cells together with another recombinant gene which may serve as a dominant selectable marker in the cell to facilitate isolation of a particular cell clone of recombinant DNA integrated into the genome. Examples of dominant selectable marker genes include Tn5 aminoglycoside phosphotransferase conferring resistance to geneticin (G418), hygromycin phosphotransferase conferring resistance to hygromycin, and puromycin acetyltransferase conferring resistance to puromycin. Recombinant expression vectors encoding such selectable markers may be present on the same vector as the cDNA encoding the desired protein, or may be encoded on separate vectors that are simultaneously introduced and integrated into the genome of the host cell, which typically results in a strong physical linkage between the different transcription units.
Other types of selectable marker genes that can be used in conjunction with cDNA for a desired protein are based on various transcription units encoding dihydrofolate reductase (DHFR). Introduction of this type of gene into cells lacking endogenous DHFR activity, typically CHO cells (DUKX-B11, DG-44), will enable the cells to grow in medium lacking nucleosides. For example, the medium may be Ham's F12 without hypoxanthine, thymidine, and glycine. The DHFR gene can be introduced into CHO cells along with the factor VIII cDNA transcription unit and ligated on the same vector or on different vectors to generate DHFR positive cells producing the recombinant protein.
If the cells proliferate in the presence of the cytotoxic DHFR inhibitor, methotrexate, new cells are generated that are resistant to methotrexate. These cells can produce recombinant proteins more rapidly due to the increased number of transcription units linked to DHFR. When these cells proliferate while increasing the concentration of methotrexate (from 1nM to 10000nM), new cells producing the desired protein can be obtained very rapidly.
Cells producing the desired protein can be grown in suspension culture or on a large scale on various solid supports. Examples of carriers include microcarriers based on a dextran or collagen matrix, or solid carriers in the form of hollow fibres or various ceramic materials. When propagated by suspension culture or on microcarriers, cell culture can be performed by bath culture or perfusion culture, thereby continuously producing conditioned medium for a longer period of time. Therefore, according to the present invention, the above-mentioned cells are very suitable for developing an industrial process for producing a desired recombinant mutant protein.
Pharmaceutical composition
In addition, the present disclosure provides a pharmaceutical composition for preventing or treating a bleeding disease or hemorrhage, comprising the expression vector as described above.
The bleeding disorder may be hemophilia a or hemophilia B, hemophilia a being a hemophilia caused by or complicated by inhibitory antibodies to factor VIII or factor VIIIa. In addition, the bleeding disorder may be selected from neonatal coagulopathy, severe liver disease, thrombocytopenia, congenital deficiencies of factors V, VII, X or XI and von willebrand disease with inhibitors against von willebrand factor.
Also, bleeding may be caused by blood loss associated with high risk surgery, traumatic blood loss, bone marrow transplantation, or cerebral hemorrhage.
The pharmaceutical compositions of the present disclosure may be used for gene therapy.
Purification of factor VIII mutants
Factor VIII mutants secreted from the above cells and accumulated in the culture of the above cells can be concentrated and purified by various biochemical and chromatographic methods, including methods utilizing differences in size, charge, hydrophobicity, solubility, specific affinity, etc., of the desired protein and other substances in the cell culture medium.
Specifically, the factor VIII mutants of the present disclosure can be purified to a purity of 80% or greater than 80%, more specifically 95% or greater than 95%. In particular, it is desirable that the mutant is in a pharmaceutically pure state, with a purity higher than 99.9% with respect to contaminating macromolecules, in particular other proteins and nucleic acids, and free from infectious substances and pyrogens. Specifically, isolated or purified mutants of the present disclosure are substantially free of other unrelated polypeptides.
As an example of such purification, after adsorption of the factor VIII mutant on a solid support, washing and desorption are carried out, and the protein may then be further purified by various chromatographic techniques based on its properties. The order of purification steps is selected according to the capacity or selectivity of the steps, the stability of the support or other aspects. Preferred purification steps include, for example, but are not limited to, ion exchange chromatography, immunoaffinity chromatography, affinity chromatography, hydrophobic interaction chromatography, dye chromatography, and size exclusion chromatography steps.
To minimize the theoretical risk of viral contamination, additional steps may be included in the method to effectively inactivate or eliminate the virus. Such steps are, for example, liquid or solid state heat treatment, solvent and/or surfactant treatment, light irradiation in the visible spectrum or UV spectrum, gamma irradiation or nanofiltration.
The modified polynucleotides (e.g., DNA) of the present disclosure may also be incorporated into transfer vectors for human gene therapy.
The various aspects described in this specification may be combined with each other. Hereinafter, the present disclosure will be described in more detail by way of examples. A description of certain aspects of the present disclosure will be set forth in connection with the appended drawings.
Preparation
The insertion proteins described in the present disclosure may be formulated into pharmaceutical formulations for therapeutic use. The purified protein is dissolved in a common physiologically acceptable buffered aqueous solution, and pharmaceutical excipients may optionally be added thereto to provide a pharmaceutical formulation.
Pharmaceutical carriers and Excipients, as well as suitable Pharmaceutical formulations, are well known in the art (e.g., "Pharmaceutical Formulation of Peptides and Proteins", Frokjaer et al, Taylor and Francis (2000) or "Handbook of Pharmaceutical Excipients", 3 rd edition, Kibbe et al, Pharmaceutical Press (2000)). In particular, pharmaceutical compositions comprising the mutants of the present disclosure may be formulated in lyophilized form or in a stable liquid form. The mutants of the present disclosure can be lyophilized by a variety of methods known in the art. The lyophilized formulation may be reconstituted prior to use by the addition of one or more pharmaceutically acceptable diluents such as sterile water for injection or sterile saline.
The formulation of the composition is delivered to the subject by any pharmaceutically suitable mode of administration. A variety of delivery systems are known and can be used to administer the compositions by any convenient route. Typically, the compositions of the present disclosure are administered systemically. In the case of systemic administration, the insertion proteins of the present disclosure are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, intracerebral, intrapulmonary, intranasal, or transdermal) or enteral (e.g., oral, vaginal, or rectal) delivery. The most preferred routes of administration are intravenous and subcutaneous. These formulations may be administered continuously by infusion or bolus injection. Some formulations include sustained release systems.
The intercalating proteins of the present disclosure can be administered to a patient in a therapeutically effective amount that does not reach a dose that causes unacceptable side effects, and is sufficient to produce the desired effect, while preventing or reducing the severity or development of the disorder or symptom to be treated. The exact dosage will depend on a variety of factors including the signs, formulation and method of administration, and each indication will be determined by preclinical and clinical trials.
The pharmaceutical compositions of the present disclosure may be administered alone or in combination with other therapeutic agents. Such agents may be contained in the same formulation. An example of such a preparation is von willebrand factor.
Method of treatment
The disclosure also relates to methods of treating a subject having hemophilia a, hemophilia B, or a bleeding disorder, such as acquired hemophilia. The method of treatment may comprise the step of administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an expression vector of the present disclosure. Alternatively, it may comprise the step of administering to the subject an effective amount of a host cell of the present disclosure.
According to exemplary embodiments of the present disclosure, the factor VIII mutant of the present disclosure may be administered in an amount of 10ng to 100mg, and the polynucleotide encoding the protein may be administered in an amount of 1 μ g to 100 mg. When the factor VIII mutant or a polynucleotide encoding the factor VIII mutant is administered more than once, the dose per administration may be the same or different.
The present disclosure will be described in detail below by way of examples. However, the following examples are for illustrative purposes only, and the scope of the present disclosure is not limited by the examples.
Examples
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