KR100969268B1 - Recombinant expression vector system for variants of coagulation factor VIII and von Willebrand factor - Google Patents

Abstract

The present invention relates to the expression system of the eighth coagulation factor and von Willebrand factor variant, more specifically, the mutant von Willebrand that significantly increases the stabilization and activation efficiency of the eighth coagulation factor, even though the size of the exon is greatly reduced The present invention relates to a vector system capable of simultaneously expressing a factor and an eighth coagulation factor useful for treating hemophilia.
According to the present invention, by using the reduced sized von Willebrand factor, the viral vector can be efficiently expressed with the eighth coagulation factor and the activity of the eighth coagulation factor can be significantly increased. In addition, the viral vector of the present invention can be efficiently used for gene therapy for the treatment of hemophilia.

Description

Recombinant expression vector system for variants of coagulation factor VIII and von Willebrand factor

The present invention relates to an expression system of the 8th blood coagulation factor and the von Willebrand factor variant, and more particularly, the mutant von Willebrand, which significantly increases the stabilization and activation efficiency of the 8th blood coagulation factor even though the exon is deleted and the size is greatly reduced. The present invention relates to a vector system capable of simultaneously expressing the factor and the 8th blood coagulation factor useful for the treatment of hemophilia.

Hemophilia A is a congenital blood clotting disease related to X-chromosome, which causes a disorder of blood clotting when bleeding due to a deficiency of the 8th coagulation factor. Symptoms include frequent bleeding in muscles, bones, stomach or urinary tract, and swelling or pain associated therewith. Currently, treatment is performed by supplementing a coagulation factor from the outside, but this has to be administered throughout the life, resulting in many difficulties in terms of economy as well as the hassle of daily life. In addition, there is a problem to be careful about secondary infection during administration.

The 8th blood coagulation factor is a 180Kb gene located in the X chromosome and is a glycoprotein with several domains of A1-A2-B-A3-C1-C2, and its synthesis is mostly performed in the liver. Until now, many studies have been attempted to transmit the 8th blood coagulation cognition, but the size of the 8th blood coagulation factor gene is not expressed or secreted. Followed. Among the structures of the 8th blood coagulation factor, the B domain is composed of one large exon, and has a structure that is highly glycosylated in asparagin, serin, and threonine residues. According to recent functional studies, this is not an essential domain for procoagulant activity, and thus it has been found that removal of this part has no problem in the formation of functional molecules of the 8th coagulation factor. When the B-domain-removed blood coagulation factor 8 (BDD-FVIII) was expressed in cells, the unstable coagulation factor 8 mRNA structure and the reaction of ER chaperones were overcome, so that a large number of mRNA levels could be secured. In BDD-FVIII, when 226 amino acids were left toward the N-terminus and 6 consensus sites of N-linked glycosylaton were placed, the secretion of coagulation factor 8 was remarkably increased.

When hemophilia A is approached genetically, the target cells are bone marrow cells, and stem cells or progenitor cells are most suitable among bone marrow cells. A lentivirus-based vector is used as a gene transfer medium, and when the cell is infected, the vector is expressed by intercalating the gene material into the chromosome of the infected cell, thus enabling stable and continuous expression. In the case of other types of viruses, such as the existing Moloney murine leukemia virus, there is a problem in targeting and infecting cells such as stem cells or progenitor cells because they are only infected with differentiating cells. In addition, in the case of adenovirus, it is possible to express a large amount of protein, but it is not possible to continuously express it because it is continuously diluted according to the differentiation of cells.

Therefore, the necessity of a safe and continuous delivery method of the 8th blood coagulation factor is emphasized, and method development is therefore required. Lentiviral vector is capable of infecting not only dividing cells but also almost all non-dividing cells, and is inserted into the chromosome of cells after infection, allowing stable expression for a long time. Therefore, if the 8th blood coagulation factor is developed through a lentivirus-based vector, it is very useful in gene therapy.

The von Willebrand factor (vWF) is an important factor for the smooth activation of the 8th blood coagulation factor in the blood coagulation effect during bleeding in the body. vWF is a blood glycoprotein that binds to the 8th blood coagulation factor and prevents the 8th blood coagulation factor from being decomposed in the blood. In addition, when there is a wound on the blood endothelial cells, it binds to collagen or binds to platelets and plays a large role in blood coagulation. This vWF consists of a domain of D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2, of which the D'-D3 portion binds to the 8th blood coagulation factor. The vWF is a 250 kDa protein, and the gene size is about 9 kb. Therefore, it is impossible to insert vWF into the lentiviral vector to help the role of the 8th blood coagulation factor. Accordingly, by studying the minimum part necessary for the role of the 8th blood coagulation factor in the domain structure of vWF, it was found that vWF up to exon 32 of the gene functions most efficiently. A lentivirus-based vector was prepared by adding the 8th blood coagulation factor followed by the internal ribosomal entry site (IRES) and von Willebrand factor. The invention of a viral vector that expresses the 8th blood coagulation factor and von Willebrand factor when infected with cells by expressing its vector and proteins essential for the generation of lentivirus, gag-pol, env, tat, and rev, respectively, was completed. This has never been attempted before, and it is highly valued for its usefulness in gene therapy and hemophilia research in the future.

Accordingly, an object of the present invention is to provide a mutant von Willebrand factor that significantly increases the stabilization and activation efficiency of the 8th blood coagulation factor even though the exon is deleted and the size is greatly reduced.

In addition, an object of the present invention is to provide a vector that continuously and stably expresses the 8th blood coagulation factor and von Willebrand factor, respectively, in cells.

The ultimate object of the present invention is to succeed in gene therapy in hemophilia A due to the expression of these factors, in that it expresses not only the 8th coagulation factor, but also the von Willebrand factor essential for its function. It is distinct from other existing inventions.

The above object of the present invention is to generate a VSV-G pseudotyped lentivirus expressing the 8th blood coagulation factor and the von Willebrand factor using a lentivirus-based vector, and then infect the cells to obtain the 8th blood coagulation factor in various types of cells. Is to effectively express and measure its activity. This was finally confirmed by quantifying and measuring the degree to which the 8th blood coagulation factor activated the 10th blood coagulation factor together with the ninth coagulation factor activated.

According to one aspect of the present invention, the present invention provides a mutant von Willebrand factor (vWF23) having the amino acid sequence of SEQ ID NO: 2 in which exon 24-46 in the von Willebrand factor is deleted.

According to another aspect of the present invention, the present invention provides a mutant von Willy Brandt factor (vWF23) gene having a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 2. Preferably, the gene is characterized by having the nucleotide sequence of SEQ ID NO: 1.

According to another aspect of the present invention, the present invention provides a mutant von Willebrand factor (vWF28) having the amino acid sequence of SEQ ID NO: 4 in which exons 29-46 in the von Willebrand factor are deleted.

According to another aspect of the present invention, the present invention provides a mutant von Willy Brand factor (vWF28) gene having a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 4. Preferably, the gene is characterized by having a nucleotide sequence of SEQ ID NO: 3.

According to another aspect of the present invention, the present invention provides a mutant von Willebrand factor (vWF32) having the amino acid sequence of SEQ ID NO: 6 in which exons 33-46 in the von Willebrand factor are deleted.

According to another aspect of the present invention, the present invention provides a mutant von Willy Brandt factor (vWF32) gene having a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 6. Preferably, the gene is characterized by having the nucleotide sequence of SEQ ID NO: 5.

According to another aspect of the present invention, the present invention provides an animal cell expression vector comprising a gene encoding the mutant von Willy Brandt factor (vWF23, vWF28 or vWF32) according to the present invention.

In the present invention, the animal cell expression vector includes any non-viral (plasmid or liposome) or viral vector that can be expressed by delivering the gene in an animal cell, but preferably retrovirus, lentivirus, adeno It may be a viral vector such as a virus or an adeno-associated virus, more preferably a lentiviral vector. In FIG. 12 of the present invention, pvEx23, pvEx28 and pvEx32, which are lentiviral vectors expressing vWF23, vWF28 or vWF32, are disclosed.

In the present invention, the animal cell expression vector further includes a gene encoding a human blood coagulation factor VIII (Factor VIII) in which the B domain is deleted in addition to the gene encoding the mutant von Willy Brand factor (vWF23, vWF28 or vWF32). Characterized in that. In this case, two components of the active ingredient for hemophilia treatment can be expressed in one vector at the same time.

In the present invention, preferably, the human blood coagulation factor VIII from which the B domain is deleted is characterized in that it has an amino line sequence of SEQ ID NO: 8, and the gene has a nucleotide sequence of SEQ ID NO: 7 It is characterized by that.

In the present invention, an animal cell expression vector capable of simultaneously expressing a mutant von Willybrand factor (vWF23, vWF28 or vWF32) and a human blood coagulation factor VIII (Factor VIII) in which the B domain is deleted is any non-viral (plasmid) Or liposome) or a viral vector is also included, preferably a retrovirus, a lentivirus, an adenovirus, a viral vector such as an adenovirus, and more preferably a lentiviral vector. 13 of the present invention discloses a pvBDD.FVIII.ires.vWex32 lentiviral vector, a bicistronic expression system in which the two genes are linked by an internal ribosome entry site (IRS).

According to another aspect of the present invention, the present invention is packaged by transfecting a packaging cell with a lentiviral vector capable of expressing the mutant von Willy Brandt factor of the present invention or the human coagulation factor 8 in which the B domain is deleted. Provides lentiviral particles.

In the present invention, the packaging cells may be any packaging cells such as 293T cells and HT1080 cells capable of packaging the lentiviral vector of the present invention to produce lentiviral particles, but preferably 293T cells.

In the present invention, it is characterized in that the lentiviral vector of the present invention is coat-transfected with pGag-pol, pRev, pTat, and pVSV-G to the packaging cells to make the lentiviral particles. In the examples of the present invention, a split gene expression system was used to produce a safer virus. In other words, only gag-pol, tat, rev, and VSV-G, which are essential factors for viral production, were expressed, but they were all transported to other vectors to lower the probability of recombination.

According to another aspect of the present invention, the present invention is a pharmaceutical for the treatment and prevention of hemophilia using the animal cell expression vector of the present invention or the mutant von Willy Brandt factor expressed therefrom and the human blood coagulation factor 8 deleted from the B domain Provide the appropriate composition.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for treating and preventing hemophilia containing the lentiviral particles according to the present invention as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The present inventors have developed a system that simultaneously expresses the 8th blood coagulation factor and the mutant von Willebrand factor in a lentiviral-based vector. Specifically, the present invention consists of the steps of confirming the expression and activity of the 8th blood coagulation factor in a lentiviral-based system and the steps of identifying the essential domain of von Willebrand factor for the activity of the 8th coagulation factor and expressing them together. .

The eighth blood coagulation factor of the present invention is a form in which the B domain is removed from its domain to further increase the secretion of the eighth blood coagulation factor (BDD-FVIII). Among the structures of the 8th blood coagulation factor, the B domain is composed of one large exon, and has a structure that is highly glycosylated in asparagin, serin, and threonine residues. According to recent functional studies, this is not an essential domain for procoagulant activity, and thus it has been found that removal of this part has no problem in the formation of functional molecules of the 8th coagulation factor. When the B-domain-removed blood coagulation factor 8 (BDD-FVIII) was expressed in cells, the unstable coagulation factor 8 mRNA structure and the reaction of ER chaperones were overcome, so that a large number of mRNA levels could be secured.

In BDD-FVIII, when 226 amino acids were left toward the N-terminus and 6 consensus sites of N-linked glycosylaton were placed, the secretion of coagulation factor 8 was remarkably increased. In addition, almost all non-dividing cells can be infected, and stable expression for a long time is possible by being inserted into the chromosome of the cell after infection. Therefore, if the 8th blood coagulation factor is developed through a lentivirus-based vector, it is very useful in gene therapy.

The mutant von Willebrand factor of the present invention removes some of the entire exons so that only the D1-D2-D'-D3 domain (vWF23), the D1-D2-D'-D3-A1 domain (vWF28), or D1 -D2-D'-D3-A1-A2 domain only (vWF32) is included. This domain has a binding site to the 8th blood coagulation factor, the 8th blood coagulation factor and the binding site of platelets to GP1b, or the 8th blood coagulation factor, the binding site of platelets to GP1b and collagen.

In the present invention, the expression of von Willebrand factor is required to maximize the activity of the 8th blood coagulation factor. This is to protect against factors that inactivate the 8th blood coagulation factor such as thrombin in vivo, and also to help the stable structure of the 8th blood coagulation factor. However, when the 8th coagulation factor is expressed externally together with the full-lengh von Willebrand factor, they co-localize in the cell, resulting in the result that the von Willebrand factor inhibits the secretion of the 8th coagulation factor. Therefore, the function of the eighth blood coagulation factor is maximized, and a mutant von Willebrand factor in the form of leaving only a portion that does not inhibit secretion is preferable.

Other mammalian expression vectors may be used as carriers for the expression of the 8th blood coagulation factor and von Willebrand factor of the present invention, but lentiviral-based vectors are used for cells that are not easy to transfer genes, such as stem cells and hematopoietic progenitor cells. It is more advantageous to use. However, in the case of a lentiviral-based vector, there is a limitation on the size of the expressed gene. The size of the B-domain deleted blood coagulation factor 8 (BDD.FVIII) is 4.4 kb, and the size of the von Willebrand factor up to the A2 domain is 5.6 kb. Therefore, these genes can be expressed without significant loss of viral titer, and in order to increase viral titer, the envelope protein of lentivirus can be produced by pseudotyping with VSV-G, and then the virus can be concentrated.

The animal cell expression vector of the present invention may include a promoter derived from eukaryotic or prokaryotic cells capable of inducing the transcription of foreign genes in the animal cell, and the promoter region may also include a regulatory element for increasing and inhibiting transcription. have. Suitable promoters include the cytomegalovirus class early promoter (pCMV), the mouse sarcoma virus long terminal repeat promoter (pRSV) and the SP6, T3 or T7 promoter. An enhancer upstream of the promoter or a terminator sequence downstream of the coding region may be optionally included in the vector of the present invention to facilitate expression. In addition, the vector of the present invention may contain an additional nucleic acid sequence such as a polyadenylate sequence, a localized sequence, or a signal sequence sufficient for the cell to efficiently and effectively process the protein expressed by the nucleic acid of the vector. Examples of preferred polyadenylate sequences are SV40 early site polyadenylate sites (CV Hall et al., J. Molec. App. Genet. 2, 101 (1983)) and SV40 late site polyadenylate sites (S. Carswell and JC Alwine, Mol. Cell Biol. 9, 4248 (1989)). Such additional sequences are inserted into the vector and are operably linked to the promoter sequence if transcription is desired, or to the initiation and processing sequences if additional translation and processing are desired. The insertion sequence can be placed at any position in the vector. The term “operably linked” is used to describe the linkage between a gene sequence and a promoter or other regulatory or processing sequence. As a result, transcription of the gene sequence is related by operative linkage with the promoter sequence, translation of the gene sequence is related by operably linking with the translational control sequence, and post-transcription processing of the gene sequence is operatively linked with the processing sequence. It is related by becoming.

Standard techniques for constructing the vector of the present invention are well known to those skilled in the art and can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, NY, 1989). Various strategies are useful for joining segments of DNA, the choice of which depends on the nature of the ends of the DNA segments and is easily made by skilled technicians.

Examples of lentiviruses usable in the present invention include HIV-1 and HIV-2, SIV, FIV, BLV, EIAV, CEV, and visna viruses. Of these, HIV and SIV are preferred for use in gene therapy. Human immunodeficiency virus type 1 (HIV-1) belongs to a small lineage within retroviruses, lentiviruses, and, like other members of these lineages, HIV can infect dormant cells. This makes lentiviruses an attractive vector for gene therapy.

HIV-1-derived vectors are most often used as gene carriers due to their ability to infect dividing and non-dividing cells with their cytoplasmic and nuclear entry proteins (Kohn, 2001). This ability is often thought to be due to the various properties of the vectors, which generate nuclear localization signals in a variety of multiple virion proteins and triple-stranded'DNA flaps' in the reverse-transcribed genome. It includes a central polypurine tract (Zennou et al., 2000). As a result of these properties, bioengineered HIV-1 can infect hematopoietic progenitor cells very effectively at a fairly low MOI (Park and Choi, 2004). The first consideration with regard to the use of lentiviral vectors as a tool for gene therapy is that the transfer vector is derived from HIV-1. However, all viral components required for viral replication were removed from the viral vector used in previous studies (Hofmann et al., 1999), and the transfer vector finally contained less than 5% of the HIV-1 genome (Kohn, 2001). . Another barrier to using lentiviral vectors is that they are limited to the size of the transferred genes. The vWF contains 52 exons with a cDNA size of approximately 9 Kb, which exceeds the size limit of most lentiviral vectors. In the present invention, the present inventors successfully introduced vWF cDNA into a lentiviral vector (Figs. 1 and 2). In the preparation and preparation of the lentivirus, the present inventors replaced the env of HIV-1 with the VSV-G protein. VSV-G mediates viral invasion into cells through membrane fusion rather than through specific cell surface receptor proteins, resulting in a remarkably widening of the host range (Hofmann et al., 1999). More importantly, VSV-G confers structural stability during ultracentrifugation, enabling concentration of viruses with high titers with little loss of infectivity (Burns et al., 1993; Hofmann et al., 1999). ). By utilizing these properties of VSV-G, the present inventors successfully prepared and concentrated vEx52, resulting in a transduction efficiency 6 times or more higher with only 1/100 of the volume of the lentiviral supernatant (FIG. 3). These results were analyzed by FACS and clearly observed under fluorescence: a significantly greater amount of eGFP was observed in cells transduced with vEx52 enriched than vEx52 which was not enriched (FIG. 4 ). A recent study by De Meyer et al. has shown the incorporation of long vWF cDNA into a lentiviral vector, and hematogenous endothelial obtained from dogs with VWD type 3 to develop gene therapy for von Willybrand disease type 3 (VWD type 3). Transduction of cells (blood-outgrowth endothelial cells, BOECs) was included (De Meyer et al., 2006). However, since concentrating the virus of low titer requires additional time and a difficult process, it cannot prove ideal for practical application in the treatment of hemophilia A. Therefore, the present inventors attempted to reduce the size of the vWF cDNA inserted into the lentiviral vector. We removed the domains of vWF, leaving only a minimal site for the interaction between vWF and FVIII. Mature vWF is composed of the D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2 domains. FVIII binds to the D'-D3 domain, and the A1 domain binds to platelet glycoprotein Ib, heparin and collagen. This facilitates the aggregation of platelets and aids in adhesion to the site of damage to blood vessels. The vWF gene is located on chromosome 12 and includes 52 exons having 178,000 bases. The present inventors removed exon 24-46 to prepare pRex23 and pvEx23, leaving only the site that binds to FVIII. FVIII binds to vWF within 272 amino acid residues located at the amino terminus of vWF (Sadler, 1998). In addition, the present inventors prepared pRex28 and pvEx28, which had exons 29-46 removed, leaving a platelet binding site in addition to the FVIII binding site. The platelet binding site on vWF is located in the A1 domain (Sadler, 1998). When pvEx23, pvEx28 and pvEx52 were packed in lentivirus, the viral preparations obtained from pvEx23 and pvEx28 were significantly more than those obtained from pvEx52. In general, the viral titer of unconcentrated vEx52 is 2×10 ⁴ to 4×10 ⁴ particles/ml (see FIG. 3), whereas the titers of vEx23 and vEx28 are ^{between 1×10 5} and 3×10 ⁵ particles/ml. Was in (Fig. 5). In Figures 3 and 5, the transduction efficiency of the three viruses can be compared with a graph (histogram). When 500 μl of vEx23, vEx28, and vEx52 were used to transduce Jurkat cells, 35.02%, 26.30% and 4.64% of cells were respectively positive for eGFP. Therefore, the present inventors were able to improve viral titer and transduction efficiency by removing domains in vWF, which are hardly needed for interaction with FVIII, and thus reduce the packing size. When functional FVIII was measured in the supernatant by transfecting pRex23, pRex28 and pRex52 into 293T cells, pRex23 and pRex28 showed lower FVIII activity than that observed with pRex52, a full-length vWF. However, when using the viral system, the supernatant obtained from cells transduced with vEx28 showed higher secreted BDD.FVIII activity than that obtained from vEx52 (FIG. 6 ). This may be because the large size of the full-length vWF limits the packing and expression efficiency of vWF. However, the inventors could not determine whether the expression of FVIII was altered by vWF, whether vEx28 increased the secreted level of FVIII expressed in the supernatant, and whether this effect was probably due to the protection of the BDD.FVIII structure. This is consistent with the observation that more FVIII activity was detected in cells in the absence of vWF (Kaufman et al., 1998). Another indicator that vWF stabilizes FVIII is the fact that FVIII decomposes rapidly in the absence of vWF (Over et al., 1978), while FVIII is eliminated more slowly in the presence of vWF (Tuddenham et al., 1982). . Taking a closer look at the properties of vWF and FVIII, both proteins may have been designed to provide powerful genetic tools in repairing FVIII-deficient cells.

Formulations of the pharmaceutical composition of the present invention include those suitable for parenteral (eg, intradermal, subcutaneous, intramuscular, intravenous, intraarterial), oral or inhalation administration. Alternatively, the pharmaceutical formulation of the present invention may be suitable for intramucosal administration (eg, nasal administration) in a subject. The formulation can be simply prepared in a disposable dosage form and can be prepared by any method known in the art.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, severity of disease symptoms, food, administration time, route of administration, excretion rate and response sensitivity. In general, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment. In general, the pharmaceutical compositions of the present invention once about 10 ³ per dose is suitable to be administered in an amount of protein of 10 ⁷ viral particles, or 0.001-100 ㎎ / ㎏.

1 is a schematic diagram of packaging constructs including a mutant vWF gene as an embodiment of the present invention.
2 shows the integration and transcription of vWF from transduced COS-1 cells as an embodiment of the present invention.
Figure 3 shows the concentration (concentration) of vWF-expressing HIV-1 as an embodiment of the present invention.
4 shows transduction by pseudotyped HIV-1 expressing vWF as an embodiment of the present invention.
5 is a schematic diagram of a deletion structure of vWF as an embodiment of the present invention.
6 shows the result of detecting the activity of secreted functional FVIII as an embodiment of the present invention.
7 is a photograph of the result of an expression test of eGFP in order to confirm the expression of the 8th blood coagulation factor as an embodiment of the present invention.
8 is a result of measuring the activity of the 8th blood coagulation factor by each mutant vWF as an embodiment of the present invention by a Chromogenic Assay.
9 is a diagram illustrating a manufacturing process of a Lentivirus vector including the gene of the 8th blood coagulation factor in which the B-domain is deleted as an embodiment of the present invention.
10 is a diagram showing a manufacturing process of a Lentivirus vector including a gene of vWF (von Willebrand Factor) as an embodiment of the present invention.
FIG. 11 is a diagram showing the manufacturing process of pRex23, pRex28, and pRex32, which are vectors containing vWF variants, as an embodiment of the present invention.
12 is a diagram showing the manufacturing process of pvEx23, pvEx28, and pvEx32, which are Lentivirus vectors including genes of vWF variants, as an embodiment of the present invention.
13 shows the manufacturing process of pvBDD.FVIII.vWEx32, a Lentivirus vector including BDD.FVIII and vWF variant genes as an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

Example 1: vector preparation

-GAACCGAAGCTGGTACCT-3

및 5

-GACAGGAGGGGCATTAAATTGCTTTTGCCT-3

를 이용하고, 하향 3‘-영역을 프라이머쌍 5

-TTTAATGCCCCACCAGTCTTGAAACGCCAT-3

및 5

-ATGCTCGCCAATAAGGCATTCCA-3

을 이용하여 PCR 증폭하였고, 다음 이들 산물을 열-변성(denaturation)과 재결합(renaturation)을 거쳐 원하는 결실을 얻었다. 그 산물을 KpnI 및 BglI 로 분해하고 원 pRF8 플라스미드의 KpnI - BglI 내로 서브클로닝하여 pREP7 (Invitrogen, USA)의 RSV 3

LTR 제어하에 B-도메인 결실된(BDD) FVIII cDNA가 삽입된 pREP7-BDD.FVIII를 제조하였다 (참조: The Journal of Gene Medicine, Volume 6, Issue 7 , Pages 760 - 768). 본 발명자는 상기 논문의 저자인 Subrata Banerjee에게서 pREP7-BDD.FVII를 제공받아 사용하였다. 또한, 전장 vWF cDNA (ATCC #59126)를 상기와 마찬가지로 링커를 이용하여 pREP7 vector (Invitrogen, USA)의 NotI 부위에 클로닝하여 pRex52 플라스미드를 얻었다. Full-length FVIII cDNA (ATCC Accession No. 40086) was cloned into the NotI site of the modified pREP7 vector (Invitrogen, USA) using a linker to obtain plasmid pRF8, and then the B-domain was raised to remove most of the B-domain. 5'-region primer pair 5

-GAACCGAAGCTGGTACCT-3

And 5

-GACAGGAGGGGCATTAAATTGCTTTTGCCT-3

And the downward 3'-region primer pair 5

-TTTAATGCCCCACCAGTCTTGAAACGCCAT-3

And 5

-ATGCTCGCCAATAAGGCATTCCA-3

PCR amplification was performed using, and then these products were subjected to heat-denaturation and recombination to obtain a desired deletion. The product was digested with KpnI and BglI and subcloned into KpnI - BglI of the original pRF8 plasmid, and RSV 3 of pREP7 (Invitrogen, USA)

PREP7-BDD.FVIII into which the B-domain deleted (BDD) FVIII cDNA was inserted under LTR control was prepared (see The Journal of Gene Medicine, Volume 6, Issue 7, Pages 760-768). The present inventor received and used pREP7-BDD.FVII from Subrata Banerjee, the author of the paper. In addition, the full length vWF cDNA (ATCC #59126) was cloned into the NotI site of the pREP7 vector (Invitrogen, USA) using a linker in the same manner as above to obtain a pRex52 plasmid.

The Lentivirus backbone (transfer vector) used in the experiment was Journal of Virology, December 1999, p. 10020-10028, Vol. 73, No. The HIV-1 vector pHIvec2.GFP received by Joseph Sodroski, the author of 12, was used (see above). The pHIvec2.GFP was prepared by removing the env and vpu sequences from the v653 rtatpC virus, maintaining the Rev-responsive element, and inserting the eGFP gene (Clontech) behind the IRFS. The vector map of pHIvec2.GFP is the same as that in FIG. 1 from which the vWF gene has been removed.

To make a Lentivirus vector containing the gene of the 8th coagulation factor in which the B-domain is deleted, cDNA of BDD.FVIII (B-Domain-Deleted Coagulation Factor VIII) was obtained from pREP7-BDD.FVIII using NotI, and This was put into the lentivirus backbone using the same enzyme to generate pvBDD.FVIII. The manufacturing process is shown in FIG. 9. Specifically, pRep7-BDD.FVIII and lentivirus backbone were digested with NotI, and the BDD.FVIII fragment from pREP7-BDD.FVIII was ligate to the NotI site of the lentivirus backbone.

To make a Lentivirus vector containing the gene of vWF (von Willebrand Factor), the pRex52 and lentivirus backbone were digested with NotI, and the vWF fragment from pRex52 was ligate to the NotI site of the lentivirus backbone, thereby preparing pvEx52. The manufacturing process is shown in FIG. 10. A detailed map of pvEx52, which is the result of FIG. 10, is disclosed in FIG. 1. The vWF gene is located between the long terminal repeats (LTR) and is fused to IRES-eGFP in a viral vector.

The vWF variants were created using PCR (polymerase chain reaction) in pRex52. Each PCR was reacted with each dNTP 25 mM, each phosphorlyated primers 10 μM, 2 mM Mg ²⁺ , and a DNA template, and amplified with PfuUltraTMII Fusion HS DNA Polymerase. The total volume of PCR was 50 µl and was carried out under the following conditions. At 95℃ for 5 minutes, at 95℃ for 30 seconds, at 52℃ for 30 seconds, and at 72℃ for 30 seconds per 1 Kb, the reaction was repeated 18 times, and at 72℃ for 10 minutes. The sequence of the forward primer for making vWF variants was 5-CGT GATGAGACGCTCCAG-3, the sequence of the reverse primers was 5-TTTTCTGGTGTCAGCACACTG-3 for pRex23, 5-AGGTGCAGGGGAGAGGGT-3 for pRex28, and 5-AGAGCACAGTTTGTGGAG-3 for pRex32. After performing PCR, the amplified product was isolated using a PCR removal kit (Qiagen), and reacted at 15° C. for about 24 hours using a ligase (Takara) to prepare pRex23, pRex28, and pRex32. The manufacturing process is shown in FIG. 11. Ligated mixture was transformed into TOP10.

To make a Lentivirus vector containing the genes of the vWF variants, the pRex23, pRex28 or pRex32 and the lentivirus backbone were digested with NotI, and the vWF fragment from pRex23, pRex28 or pRex32 was ligate to the NotI site of the lentivirus backbone, and pvEx23, pvEx28 and pvEx32 was prepared. The manufacturing process is shown in FIG. 12.

In pvBDD.FVIII, ires-eGFP is attached behind BDD.FVIII, where only the eGFP portion was removed by PCR, and then the vWF variant of pRex32 was added to this site to create pvBDD.FVIII.vWEx32. The manufacturing process is shown in FIG. 13. For transformation, TOP10 was used. In order to express vWF32 together following BDD.FVIII, an IRES (internal ribosomal entry site) sequence was inserted after BDD.FVIII, followed by vWF32. As a result, two proteins can be simultaneously expressed under one promoter, and vWF32 expressed later plays a role in helping the activity and function of BDD.FVIII.

Example 2: Generation of viruses

Vesicular stomatitis G protein (VSV-G) pseudotyped HIV-1 is a method of quinquepartite plasmid transient transfection of 293T (see Park and Choi, 2004 Mol. Cells 17, 297-303) by gag-pol, tat, rev, VSV-G. And by transfection with a transfer vector. The 293T was split into 100 mm plates at 4.5×10 ⁶ 24 hours before transfection, and then 4 hours before transfection, the supernatant was replaced with a culture medium containing 10% FCS and 25 mM HEPES. For transfection, 10 μg of Gag and Pol packaging plasmid, 2 μg of VSV-G plasmid, 1 μg of Tat plasmid, 1 μg of Rev plasmid, and 10 μg of transfer vector were added. Add these DNAs to 62 µl of 2.5 M CaCl ₂ and adjust the volume to 500 µl with water, and then vortex and add 500 µl of 2x HBS (281 mM NaCl, 100 mM hepes, 1.5 mM Na ₂ HPO ₄ pH 7.12 to this mixture). ) Was added and allowed to stand at room temperature for 30 minutes, and then sprinkled on 293T cells. 16 hours after transfection, the supernatant was changed to RPMI in 10 mM HEPES buffer, and the virus generated after 48 hours and released as a supernatant was harvested using a 0.45 μm filter.

Example 3: viral titration

NIH3T3 cells 3 x 10 ⁵ cells were put into 60 cm ² dishes, and after 20 hours, several volumes of virus were added to the cells. At this time, the total volume was 2 ml of culture media, and polybrene was added at a concentration of 2 μg/ml. After 6 hours, the virus was removed, and the cells were washed with DMEM containing 2% FCS to completely remove the virus, and then the cells were placed in an incubator. After 2 days, the cells were removed using 0.25% trypsin, washed with 1x PBS, and fixed with 3.7% formaldehyde. ^{The percentage of eGFP +} emitted from infected cells was read using FACScan (Becton Dickinson Immunocytometry Systems) and CellQuest program (Becton Dickinson), and the virus titer was calculated using the following formula. (2 × ^{number of cells × eGFP +} percent of number of cells) ÷ amount of virus.

Example 4: Concentration of virus ( Concentration )

The generated virus was placed in a polyallomer tube and centrifuged at 4° C. for 1.5 hours at 50,000 xg in a SW28 rotor. After dissolving the pellets in a small volume of medium, the tube was covered with parafilm and left at 4°C for 24 hours. For long-term storage, concentrated virus was placed at -80°C. Figure 3 shows the concentration (concentration) of vWF-expressing HIV-1 as an embodiment of the present invention. 500 μl unconcentrated (center) and 5 μl concentrated (right) vWF-expressing lentiviral supernatant were used to transduce Jurkat cells. The fraction of eGFP+ cells in the transduced cells was determined by flow cytometry.

Example 5: Transduction of cells ( Transduction )

After counting the cells on a hemocytometer, the desired number of cells was spread on a 24-well plate or a 6-well plate. The virus supernatant was added to the cells and added to the desired MOI. At this time, the total volume was adjusted to the medium to the desired volume, and at this time, polybrene was added at a concentration of 2 µg/ml. Infection was carried out at 37° C. for 6 hours in the presence of _{5% CO 2.} After infection, the cells were washed with medium. 5 is a schematic diagram of a deletion structure of vWF as an embodiment of the present invention. A. From pRex52 and lentiviral vectors, respectively, pRex23 and pvEx23 were constructed by removing exons 24-46, and pRex28 and pvEx28 were prepared by removing exons 29-46. B. vEx23 and vEx28 were produced from pvEx23 and pvEx28, respectively, and 500 μl of virus supernatant was used to transduce Jurkat cells. The proportion of transduced cells was analyzed by FACS.

Example 6: DNA Wow RNA Separation

Genomic DNA was separated with 500 μl lysis buffer (0.1 M Tris HCl, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, and 100 μg/ ml protease K). This was precipitated with isopropanol and washed with 70% Ethanol. RNA was obtained with Trizol (Invitrogen), and cDNA was synthesized using ImPromII (Promega). PCR was amplified with Pfu (Solgent) using 1x reaction buffer, 25 mM each dNTP, 10 μM each primer, 2 mM Mg ^{2 +} , and a DNA template to make a total volume of 50 μl. 2 shows the integration and transcription of vWF from transduced COS-1 cells as an embodiment of the present invention. COS-1 cells were transduced with lentivirus expressing vWF. A. The introduction of the transduced vWF gene was detected by PCR for 421-bp vWF and 227-bp LTR in genomic DNA of transduced COS-1 cells. B. cDNA was prepared from transduced cells and amplified with primer pairs specific for vWF, LTR, and GAPDH with 421-, 227- and 187-bp, respectively. vEx52: transduced with vEx52, eGFP: transduced with eGFP-expressing lentivirus.

Example 7: Plasmid transfection ( Transfection )

DNA to be transfected (pRex23, pRex28, and pRex32) was put in 50 µl of 150 mM NaCl based on a 12-well pate, vortexed, and then spin down. PEI of 3 times the volume of DNA was added to this mixture, then vortexed and spin down again. After standing at room temperature for 10 minutes, it was carefully dropped onto the cells.

Example 8: Cell immunostaining method ( Immunocytochemistry )

Transduced cells were grown on glycogen-coated coverslipsas in 6-well tissue culture plates. The cells were fixed with cooled 100% methanol, washed with TBS [50mM Tris-HCl (pH7.4), 150 mM NaCl], quenched in 0.1% sodium borohydride in fresh TBS for 5 minutes, and then 3 with TBS for 5 minutes. Washed twice. Cells were blocked with blocking buffer (10% horse serum, 10% bovine serum albumin, 0.02% NaN ₃ in 1x PBS) for 60 minutes and washed with TBS for 5 minutes. The primary vWF antibody (Abcam) was diluted with 1% BSA in TBS and incubated overnight with cells at 4°C. After washing the cells three times with TBS for 5 minutes, they were labeled with a secondary goat-anti-mouse IgG TRITC antibody (Santa Cruz) in 1% BSA in TBS for 30 minutes at room temperature covered with aluminum foil. Then, they were washed three times with TBS for 5 minutes and mounted on slides using a ProLong antifade kit (Cell Signaling).

4 shows transduction by pseudotyped HIV-1 expressing vWF as an embodiment of the present invention. A. 293T cells were transfected with a plasmid containing viral components for viral production, including a transfer vector containing vWF-IRES-eGFP. The eGFP obtained from the transfer vector was visualized under a fluorescence microscope at 10× magnification. B. Jurkat cells were visualized with an optical microscope and a fluorescence microscope. C. Jurkat cells were transduced with 500 μl of unenriched vWF-expressing pseudotype HIV-1 and visualized under a microscope. D. Jurkat cells were transduced with 5 μl of 160-fold concentrated vWF-expressing pseudotype HIV-1, and visualized in white and fluorescence. E. COS-1 cells were transduced with vWF-pseudotype HIV-1 at 0.5 MOI. eGFP was visualized under a fluorescence microscope (left). vWF was visualized by staining VWF with antibody and TRITC (center). The detected vWF was derived from the transferred gene, not from COS-1 cells (right).

BDD.FVIII was made excluding the B domain from the sequence of the 8th blood coagulation factor and inserted into a lentiviral vector. For Lentivirus expression, lenvirius expressing BDD.FVIII was produced by expressing BDD.FVIII in 293T cells together with gag, pol, VSV-G, tat, and rev. When the cells were infected with this, BDD.FVIII was expressed in the cells, and its expression was finally confirmed by measuring the activity of the 8th coagulation factor. Virus generation and infection were indirectly confirmed by expression of eGFP in the vector (FIG. 7). 7 is a photograph of the result of an expression test of eGFP in order to confirm the expression of the 8th blood coagulation factor as an embodiment of the present invention (corresponding to FIG. 4A). Virus generation and infection were indirectly confirmed by expression of eGFP.

Example 9: Factor VIII Activity measurement experiment

Activated FVIII activity (FVIII:C) was quantified with Coatest VIII:C/4 kit (DiaPharm). Phospholipids, 100 mg/l ciprofloxacin, and 5 times the volume of factor IXa and factor X were mixed, and 50 μl of this was added to 96-well microtiter plates. 25 µl of cell culture supernatant was added thereto, and incubated at 37C for 5 minutes. After adding 25 µl of 0.025 mol/L CaCl2 and incubating at 37C for 10 minutes, 50 µl of S-2765 and I-2581 were added and reacted again at 37C for 10 minutes. The reaction was stopped with 20% acetic acid and the activity of factor 8 was measured at 405 nm. A standard curve was created and quantified using a known concentration of recombinant human FVIII.

6 shows the result of detecting the activity of secreted functional FVIII as an embodiment of the present invention. A. 293T cells were transfected with pREP7-BDD.FVIII with pRex23, pRex28 or pRex52. The supernatant of the transduced cells was collected and quantitatively analyzed for FVIII activity. B. K562 cells were transduced with vEx23, vEx28 or vEx52 with HIV-1-BDD.FVIII at 1.5 MOI. The supernatant of the transduced cells was collected and screened for FVIII activity. Data are expressed as the mean±standard error (means±SE) of at least 3 independent experiments. C. RT-PCR was performed with RNAs obtained from transduced cells. The B-domain-deleted FVIII showed 1.1 Kb of preparation. vEx23: transduced with vEx23, vEx28: transduced with vEx28, vEx52: transduced with vEx52.

Example 10: PMA With or without processing Factor VIII Activity measurement experiment

The domain of the von Willebrand factor was removed from the exon 23, exon 28, and exon 32 to the C-terminus and named pRex23, pRex28, pRex32, and the full length vWF is pRex52. This was when pREP7-BF was transfected with pRex32 as a result of finding the von Willebrand factor vector with the highest activity of the 8th coagulation factor secreted when expressed with pREP7-BF carrying BDD.FVIII. (Fig. 8). 8 is a result of measuring the activity of the 8th blood coagulation factor by each mutant vWF as an embodiment of the present invention by a Chromogenic Assay. After transfecting pREP7, pREP7-BF and pRex23, pREP7-BF and pRex28, pREP7-BF and pRex32, and pREP7-BF and pRex52 into HeLa cells, the functional activity of FVIII secreted as supernatant was measured. 8A is a measurement of the activity of the 8th blood coagulation factor in a normal state (no wound). All of them showed an increase in activity compared to the basal level, with 32 being the largest. Fig. 8b shows that PMA (phorbol ester) was treated to artificially create a wounded state, all of which induced FVIII secretion by at least three times and showed a significant difference compared to the basal level. Although it is not overexpressed normally (a low level is usually preferable for reasons of antibody formation, etc.), the result of large induction of FVIII secretion in a wounded state has very important significance in the clinical practice of hemophilia.

Experiment result 1. Lentivirus produce

In order to prepare pvEx52 (LTRs), 8.8 Kb von Willebrand factor cDNA was cloned between two long terminal repeats (LTRs) of HIV-1-derived lentivirus (Fig. 1). ). The vWF cDNA was isolated from vW-8 (ATCC #59126) using Not I and then cloned into a lentiviral vector. By fusing the vWF gene with IRES-eGFP, the enhanced green fluorescence protein as an indirect indicator of virus production after transfection into 293T cells along with other viral genes required for packaging viral particles. , eGFP) enabled the use of (Parolin et al., 1996; Yee et al., 1994). The packing vector, including gag and pol under the control of the CMV promoter, individually expressed tat and rev of HIV-1 under the control of the CMV promoter. The present inventors used vesicular stomatitis virus G protein (VSV-G) instead of HIV-1 env (Hofmann et al., 1999).

Experimental result 2. HIV -One False type VSV -G( VSV -G pseudotyped HIV -1) transduction

COS-1 cells were transduced with vEx52 with intact vWF, or with HIV-1-eGFP lentivirus expressing eGFP. Genomic DNA was isolated from the transduced COS-1, and PCR was performed using a primer pair specific for human vWF gene and HIV-1 LTR. vWF could only be amplified in vEx52-transduced cells, whereas LTR could be amplified in both vEx52 and HIV-1-eGFP-transduced cells (Fig. 2A). And RNA was prepared from the transduced cells, the cDNA was synthesized, and PCR was performed for vWF and LTR (FIG. 2B). The result of RT-PCR was consistent with the result of PCR amplification of genomic DNA. vWF was amplified only in vEx52-transduced cells, and LTR was detected in both vEx52 and HIV-1-eGFP transduced cells.

Experimental Results 3. Transduced From lymphoblasts vWF And the expression of vEx52 Concentration of

Jurkat cells were cultured at 2×10 ⁵ and transduced with 500 μl of unconcentrated vEx52 virus. FACS analysis shows that 5.55% of cells are positive for GFP expression (FIG. 3 ). After the virus suspension was concentrated to 160 times by centrifugation at 50,000×g, 5 μl of the concentrated virus was used to transduce the same number of Jurkat cells to obtain a yield of 29.51% eGFP+ (FIG. 3). By concentrating the vEx52 virus, the titer was increased from 2.8×10 ⁴ particles/ml to 2.3×10 ⁷ particles/ml. Transfection of the vector induces lentiviral production, which was indirectly confirmed by the expression of gGFP (Fig. 4A). And transduction and expression of both unenriched or enriched vEx52 were visualized by fluorescence from eGFP expression (BD in FIG. 4). In order to demonstrate vWF expression from vEx52, COS-1 cells were transduced with vEx52 at MOI 0.5 and labeled with human vWF antibody, followed by TRITC staining (E in FIG. 4). And in order to confirm whether the transduced constructs were maintained for an extended period of time, 1×10 ⁵ Jurkat cells were transduced at an MOI of 0.5. After 4 days, 38.27% of cells were positive for eGFP by FACS analysis. In the analysis at 9, 15, 35, 50 and 90 days after transduction, 33.01%, 11.99%, 11.32%, 6.13%, and 5.56% of cells were eGFP+, respectively (data not shown).

Experimental Results 4. Domain-removed vWF Composition of

pRex23 and pRex28 were prepared from pREP7-vWF (fully provided by Dr. Subrata Banerjee) by removing exons 24-46 and 29-46. pvEx23 and pvEx28 were prepared from pvEx52 by the same method (Fig. 5A). The sequences were forward primers 5′-CGTGATGAGACGCTCCAG-3′ and Ex23PR reverse primers 5′-TTTTCTGGTGTCAGCACACTG-3′ for pRex23 and pvEx23, and Ex28PR reverse primer 5′-CAGGTGCAGGGGAGAGG-3′ for pRex28 and pvEx28. It was removed by PCR. Thereafter, pvEx23 and pvEx28 were used to prepare HIV-1 pseudotype VSV-G together with the packing vector, and titrated in Jurkat cells. When 500 μl of viral supernatants of vEx23 and vEx28 were used for transduction, respectively, 35.02% and 26.30% of cells were analyzed as positive for eGFP (FIG. 5B).

Experimental Results 5. Secreted FVIII Functional activity of

To test the effect of domain-depleted vWF on secretion of FVIII, 293T cells were transfected with pREP7-BDD.FVIII with one pRex23, pRex28, or pRex52. 48 hours after transfection, the supernatant was collected and the functional activity of FVIII was screened by chromogenic assays. From transduction with pRex23, pRex28 and pRex52, FVIII activity levels of 28.89±18.86, 107.22±30.64 and 199.44±58.93, respectively, were obtained (Fig. 6). Therefore, the functional activity of secreted FVIII decreased as part of the vWF domain was removed. Next, the present inventors evaluated the effect when the domain-deleted vWF as a component of the vWF lentivirus was transduced. K562 cells were transduced at MOI 1.5 with vBDD.FVIII, a BDD-FVIII expressing HIV-1 with vEx23, vEx28 or vEx52. Expression of the transduced FVIII was confirmed from RT-PCR performed with RNA obtained from the transduced cells (Fig. 6). The FVIII activity in the supernatant of the transduced cells was 28.33± 5.50 for vEx52-transduced cells, and 33.89± 3.93 and 53.33± 9.43 for vEx23- and vEx28-transduced cells, respectively (Fig. 6). These data suggest that the deleted form of vWF, vEx28, is the most efficient at promoting secretion of FVIII via interaction of its minimal essential domains with FVIII. These data indicate that vEx28, a deleted form of vWF, is most effective in inducing the secretion of FVIII through the interaction of FVIII with the minimal essential domains of vWF.

As described above, according to the present invention, by using the mutant von Willebrand factor reduced in size, it is possible to efficiently express the 8th coagulation factor together with the 8th coagulation factor in a viral vector and significantly increase the activity of the 8th coagulation factor. In addition, the viral vector of the present invention can be effectively used for gene therapy for hemophilia treatment. In addition, the simultaneous expression of the 8th coagulation factor and von Willebrand factor of the present invention is very useful in clinical applications such as gene therapy for hemophilia A.

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Claims (13)

Mutant von Willybrand factor (vWF23) having the amino acid sequence of SEQ ID NO: 2, which has deleted exon 24-46 in von Willebrand factor. A mutant von Willibrand factor (vWF23) gene having a nucleotide sequence encoding a protein having an amino acid sequence of SEQ ID NO: 2. The mutant von Willibrand factor (vWF23) gene according to claim 2, wherein the gene has a nucleotide sequence of SEQ ID NO: 1. An animal cell expression vector comprising a gene encoding the mutant von Willibrand factor of claim 1. 5. The animal cell expression vector of claim 4, wherein the animal cell expression vector is a lentiviral vector. 5. The animal cell expression vector according to claim 4, further comprising a gene encoding human blood coagulation factor (Factor VIII) deleted from the B domain. 7. The animal cell expression vector of claim 6, wherein the human coagulation factor 8 having the B domain deleted has an amino acid sequence represented by SEQ ID NO: 8. The animal cell expression vector according to claim 6, which is a pvBDD.FVIII.ires.vWex32 lentivirus vector having a cleavage map of FIG. A lentiviral particle packaged by transfecting the lentiviral vector of claim 5 or 8 into a packaging cell. 10. The lentivirus particle of claim 9, wherein said packaging cell is a 293T cell. The lentiviral particle of claim 9, wherein the lentiviral vector is co-fected with pGag-pol, pRev, pTat, and pVSV-G. A pharmaceutical composition for treating and preventing hemophilia comprising the animal cell expression vector of claim 4 or 6 or a mutant von Willibrand factor expressed therefrom and a human blood coagulation factor 8 deleted from the B domain as an active ingredient. A pharmaceutical composition for treating and preventing hemophilia, comprising the lentiviral particles according to claim 9 as an active ingredient.

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