WO2005095623A1

WO2005095623A1 - Method of introducing gene in target cell

Info

Publication number: WO2005095623A1
Application number: PCT/JP2005/006182
Authority: WO
Inventors: Shuji Hayashi; Makoto Inoue; Mamoru Hasegawa
Original assignee: Dnavec Research Inc.
Priority date: 2004-03-31
Filing date: 2005-03-30
Publication date: 2005-10-13
Also published as: JP2007228802A

Abstract

There is provided a method characterized by including the steps of: (1) effecting binding of a ligand to be received by a cell surface receptor of target cell to a surface protein of vector; and (2) performing cell infection with the resultant vector under at least one condition selected from among low temperature, low oxygen and short time.

Description

Gene transfer method to target cells

Technical field

The present invention relates to a method for introducing a gene under adverse conditions such as low-temperature, low-oxygen or short-term infection.

Background art

[0002] Gene transfer is a basic technique for genetic manipulation of cells and animals. Technology for gene transfer has been developed and improved in order to examine the function of a certain gene or gene product, or to use a gene whose gene function has been identified or to prevent or treat a disease. For humans, treatment of diseases by gene transfer is being studied as gene therapy.

[0003] Gene transfer methods include a physicochemical method such as a calcium phosphate method, an electoral poration method, and a method using a gene gun, and a biological method such as a transfer method using a virus vector. Gene transfer by a biological method is generally performed by contacting a cell with a gene at room temperature under oxygen for a relatively long time. But in the clinic, low temperature, low oxygen, short contact! / ヽぅ In some cases, gene transfer under special circumstances is required. For example, in genetic manipulation of the transplanted organ vasculature, the transplanted organ is removed from an organ donor, and a gene such as an immunosuppressive gene is introduced during storage of the organ under low temperature and low oxygen for a short time so as not to cause organ damage. There is a need. By transplanting this organ into patients, organ transplantation is possible without using immunosuppressants.

[0004] For example, among the viral vectors, the adenovirus vector is capable of very efficiently introducing genes into both dividing and non-dividing cells, and is also capable of preparing a virus solution having a very high concentration. For example, it satisfies the conditions for in vivo gene transfer into living organisms. The adenowill vector is one of the existing gene transfer vectors that is excellent in gene transfer efficiency into the vascular system (Lemarchand P et al., Proc Natl Acad Sci US A. 89 (14) , 6482—6486 (1992)). This is a typical example of the above-mentioned adverse conditions of low temperature, low oxygen, and short-term infection. Examination of the efficiency of gene transfer into transplanted organs revealed that the gene transfer efficiency and the expression level at low temperatures were significantly lower than at room temperature. Therefore, there is a need for a method for efficiently introducing a gene under such adverse conditions.

Non-patent literature l: Lemarchand P et al., Proc Natl Acad Sci U S A. 89 (14), 6482-6486 (1992)

Non-Patent Document 2: Watkins SJ et al., Gene Ther. 4, 1004-1012 (1997)

Non-Patent Document 3: Haisma HJ. Et al., Cancer Gene Ther. 7, 901-904 (2000)

Non-patent document 4: Dmitriev I. et al., J. Viol. 74, 6875-6884 (2000) Non-patent document 5: Wesseling JG. Et al., Gene Ther. 8, 969-976 (200 1)

Non-Patent Document 6: Nieklin SA. Et al., Circulation 102, 231-237 (2000)

Non-Patent Document 7: Nettelbeck DM. Et al., Mol. Ther. 3, 882—891 (2 001)

Disclosure of the invention

Problems to be solved by the invention

An object of the present invention is to provide a method for introducing a gene into a target cell using a vector into which the gene has been incorporated.

The method of the present invention comprises:

A method for introducing a gene into a target cell using a vector incorporating the gene is described below.

1) binding the ligand for the cell surface receptor of the target cell to the surface protein of the vector, and

2) Infect the cells with the vector under at least one condition of low temperature, low oxygen and short time.

It is characterized by including. [0006] The present invention also provides a conjugate of a ligand for a cell surface receptor of a target cell with a vector surface protein, and the use of the conjugate in the above-described gene transfer method of the present invention.

Means for solving the problem

[0007] The inventors of the present invention have made intensive studies to solve the above-described problems, and as a result, a receptor for vascular endothelial growth factor (VEGF) is highly expressed in the vascular system. With this in mind, the present invention has been conceived. Specifically, the VEGF protein was bound to the fiber knob protein via an antibody against the fiber knob of the adenovirus vector, and the vector was modified so that the vector could be bound and introduced into the cell via the VEGF receptor. As a result, even under special adverse conditions such as low temperature, low oxygen, and Z or short time, gene transfer via the VEGF receptor functions effectively, and both gene expression efficiency and expression level can be improved.

Brief Description of Drawings

FIG. 1 is a view showing the effect of mixing knobscFv-VEGF protein on gene transfer under low temperature.

FIG. 2 is a view showing FACS data. The vertical axis indicates the number of cells, and the horizontal axis indicates GFP intensity. NC: Negative control (no infection); C (l): (Yes infections, KnobscFv- mixing teeth of VEGF protein); C (1) V ( 20) ( Yes infections, KnobscFv- incorporation of VEGF protein 20 / zeta ₈ Ζπι1). N: nega GFP +: indicates GFP positive.

BEST MODE FOR CARRYING OUT THE INVENTION

[0009] A method for coupling and targeting a protein ligand to an antibody against a fiber knob of an adenovirus vector has already been reported for VEGF protein (Watkins SJ et al., Gene Therapy 4, 1004-1012 (1997 )). The method of the present invention is an improvement of the method for the purpose of gene transfer under adverse conditions such as low temperatures. Specifically, the present invention makes it possible to use VEGF protein even under adverse conditions such as low temperature, and in particular, to enable gene transfer to the vascular endothelium of a transplanted organ.

[0010] Therefore, the method of the present invention uses a vector into which a gene has been incorporated to transfer the gene to a target cell. How to introduce a child

It is characterized by including.

[0011] In one aspect of the method of the present invention,

1) mixing a substance that recognizes the surface protein of the vector, the conjugate of the ligand for the cell surface receptor of the target cell and the vector, and binding the ligand for the cell surface receptor of the target cell to the surface protein of the vector; And

Including.

[0012] The vector of the present invention

The vector in the present invention is not particularly limited as long as it has a function of expressing a desired gene in a cell by a biological method and producing a desired protein. The present invention utilizes a conjugate of a vector surface protein and a ligand for the cell surface receptor of the target cell. Therefore, vectors that can utilize surface proteins, for example, viral vectors, phage vectors, plasmids, ribosomes, and the like are applicable in the present invention.

[0013] In one embodiment of the present invention, the binding of the ligand to the cell surface receptor of the target cell to the vector surface protein is carried out by first, a substance that recognizes the surface protein of the vector, preferably an antibody, and the target cell A conjugate of a ligand to the cell surface receptor of the present invention may be prepared, and such a conjugate may be mixed with a vector. The ligand for the cell surface receptor binds to the surface protein of the vector via a substance that recognizes the surface protein of the vector. Alternatively, the vector surface protein and a ligand for the cell surface receptor of the target cell may be expressed as a fusion protein on the particle surface of the vector without using a substance that recognizes the surface protein. Such a fusion protein Vectors capable of expressing the same are also included in the scope of the present invention because they can be specifically targeted to cell surface receptors of target cells. Alternatively, a fusion protein of a vector surface protein and a ligand for a cell surface receptor of a target cell is prepared, and the fusion protein is chemically covalently bound to the vector.

[0014] The vector of the present invention is preferably a paramicrovirus vector including an adenovirus vector, an adeno-associated virus (AAV) vector, a retrowinores vector, a henoreus innores vector, a Sendai winores vector. Orthomyxovirus vectors containing influenza virus, and adenovirus vector Z adeno-associated virus hybrid vector are selected as well as virus vectors that can be selected as a group. Preferably, it is an adenovirus vector.

[0015] Gene transfer methods and general methods for gene expression using a viral vector are described, for example, in Muramatsu 'Yamamoto, "Experimental Medicine Separate Volume, New Revision, New Genetic Engineering, Non-book" Revised Edition (Yodosha) , 1999, p. 202-215; Or, Kazunori Nakajima and Yoshihiro Kitamura, eds., “Experimental Medical Science Supplement: A Gene Transfer and Expression Analysis Protocol That Must Be Successful” (Yodosha), September 2003, p. 79—130 The ability to perform according to S can.

[0016] The method of the present invention can also be used with non-viral vectors such as ribosomes. For example, non-viral vectors such as ribosomes, phage vectors and plasmids can be used. General methods for gene transfer and gene expression using ribosomes are described, for example, in the manual for ribosome experiments in life sciences, edited by Hiroshi Terada and Tetsuro Yoshimura, Springer-I 'Fairerak Tokyo (1992), and Miller AD, Methods Mol. Med References such as 90, 107-137, (2004), etc. Phage vectors One example includes a lambda phage vector and an M13 vector.

[0017] In the present invention, the type of the gene incorporated into the vector and introduced into the target cell is not particularly limited, and any gene can be used. Genes involved directly or indirectly in disease prevention and Z or cure, tissue and organ maintenance and Z or repair, and the like. As the disease, clinical application is possible, for example, by introducing a gene into a new blood vessel of a cancer in addition to a transplanted organ, or introducing a gene into an ischemic cardiovascular disease such as myocardial infarction. Alternatively, genes encoding factors involved in immunosuppression can be used Noh. As used herein, “a gene involved in immunosuppression refers to a protein or peptide having an activity of weakening, circumventing, or positively inducing an immune response. Alternatively, a specific cell, for example, a cancer cell For example, genes encoding lysine, herpes virus thymidine kinase (HSV-tk), yeast cytidine deaminase, and the like can be used.

As one embodiment of the gene that can be used in the present invention, for example, vascular endothelial growth factor (VEGF), Tie-1, Tie-2, CD34 (PECAM), E-selectin, VE-cadherin, von Willebrand (von Willebrand) factor, CD146, CD36, thrombomodulin, indegrin (V (3, angiopoietin, keratinocyte growth factor (KGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF)) The nucleic acid sequence information of such a gene is available from a gene database (for example, NCBI), so that those skilled in the art can use the obtained genetic information to obtain information on the nucleic acid sequence. , Expression vectors and the like.

[0019] In the present invention, the gene incorporated into the vector may be a "marker gene". As used herein, the term “marker” refers to a gene encoding a protein suitable for immunohistological staining or direct or indirect immunofluorescent staining of a tissue or a cell. These marker genes are expressed in transformed cells and can be detected by a known detection method depending on the type of the marker gene. Therefore, the localization of the transformed cells in the tissue can be observed using a fluorescence microscope by direct or indirect immunofluorescence staining. It is also possible to detect and observe using known flow cytometry (FCM) and FACS (flororescenc activated cell sorter).

[0020] The size (length) of a gene that can be incorporated into a vector is appropriately selected depending on the type of the vector. For example, in the case of a viral vector, a gene having a length of 6bp-5kbp can be inserted. For example, in the case of an adenovirus vector, it is preferably 18 bp-2 kbp. In the case of a phage vector, a gene with a length of 6 bp to 40 kbp can be inserted. In the case of ribosomes, it is possible to integrate a gene with a length of 6bp-20kbp.

[0021] The conjugate of the present invention The present invention includes a step of binding a ligand for a cell surface receptor of a target cell to a vector surface protein before or simultaneously with infection of the target cell with the vector.

[0022] In the present specification, the "surface protein of a vector" is present on the surface of a vector, and is preferably recognized by a substance that specifically recognizes the surface protein (for example, a specific antibody). A possible protein. Appropriate vector-surface proteins can be used as appropriate depending on the type of vector. For example, in the case of adenovirus, fiber proteins such as “knob”, hexon, and penton base can be used as surface proteins. Knob proteins are fibrous proteins present on the surface of adenovirus and are known to initiate infection with adenovirus with high binding affinity for adenovirus receptors.

[0023] Other virus As surface proteins of the vector, adeno-associated virus (AAV) vector is a forcepsid protein (VP1, VP2, VP3), retrovirus vector is envelope protein (Env), Virus vectors are gB and gC present in the envelope, Sendai virus vectors are fusion protein (F), HN protein (HN), and adenovirus vector Z adeno-associated virus hybrid vector is adenovirus “knob”. Hexon, penton base, etc. are available.

[0024] In the case of a ribosome vector, a ribosome surface protein, for example, HVJ-ribosome constructed by utilizing the membrane fusion ability of Sendai virus, in the case of Sendai virus fusion protein (F) and HN protein (HN ) Is available.

[0025] The "ligand for the cell surface receptor of the target cell" can be appropriately selected depending on the type of the target cell into which the gene is to be introduced. For example, it is known that receptors for vascular endothelial growth factor (VEGF) are highly expressed in the vascular system. Therefore, VEGF can be used in the present invention to target a vector containing the gene to cells of the vascular system. Tie-1, Tie-2, CD34 (PECAM), E-selectin, VE-cadherin, von Willebrand factor, CD146, CD36, thrombomodulin, indegulin (V (3 , Angiopoietin, epidermal growth factor (EGF) and fibroblast growth factor (FGF) also target vascular cells. Keratinocyte growth factor (KGF) targets the epithelium, especially the lung airways.

[0026] In one embodiment of the present invention, the binding of the ligand to the cell surface receptor of the target cell to the surface protein of the vector is performed by, first, a substance that recognizes the surface protein of the vector, preferably an antibody, and the target cell A conjugate of a ligand to a cell surface receptor of the present invention may be prepared, and such a conjugate may be mixed with a vector. The ligand for the cell surface receptor binds to the vector surface protein via a substance that recognizes the vector surface protein.

[0027] Therefore, the method of the present invention comprises, in one aspect thereof,

Including.

[0028] In the present invention, the substance that recognizes the surface protein of the vector is not particularly limited as long as it can specifically recognize the surface protein of the vector. It includes the extracellular domain of the receptor on the cell side recognized by the antibody and the vector. Preferably, it is an antibody.

[0029] In the present specification, the "antibody" is preferably a monoclonal antibody that can recognize the "surface protein of the vector". Antibodies specific for the vector surface protein can be generated using known methods. For example, it can be obtained by preparing a phage display library from the spleen of an immunized animal (for example, mouse) immunized with the vector surface protein and screening for an antibody specific to the antigen. "Antibody" refers to an intact antibody, including: polyclonal antibodies (see, eg, Ant ibodies: A Laboratory Manual, Harlow and Lane (eds.), Old spring Harbor Laboratory Press (1988)), and Monoclonal antibodies (see Monoclonal al Antibodies: A New Dimension in Biological Analysis, Plenum Press, Kennet, McKearn and Bechtol (ed.) (1980)). [0030] "Antibody" refers to an antibody fragment produced by recombinant DNA methods or enzymatic or chemical cleavage of an intact antibody, such as F (ab), F (ab,), F (ab,), Fv, Fc , And single-chain anti-

2

It also represents the body. The term "antibody" also refers to a bispecific or bifunctional antibody that is an artificial hybrid antibody having two different heavy Z light chain pairs and two different binding sites. Bispecific antibodies can be prepared by a variety of methods, including hybridoma fusion or Fab 'fragment conjugation (Songsivilai et al., 1990, Clin. Exp. Immunol., 79: 315-321; Kostelny et al.). , 1992, J. Immunol., 148: 1547-1553;).

[0031] "Antibody" also refers to a chimeric antibody, ie, an antibody in which a human antibody immunoglobulin constant domain is bound to one or more non-human antibody immunoglobulin variable domains, or a fragment thereof. Antibodies include "humanized antibodies", "minibodies", and transgenic animals capable of producing human antibodies, including transgenic animals that contain a certain percentage of human antibody-producing genes but do not produce endogenous antibodies. Includes antibodies produced by

[0032] In general, the variable regions of the heavy and light chains of an antibody contain an antigen-binding site, and the DNAs of both regions are linked by a linker, and expressed in a host cell to form a single-chain having an antigen-binding ability. It is known that FV protein (sFv) can be obtained. Thus, for example, in the present invention, a gene encoding both the heavy chain and light chain variable regions of an antibody is expressed in a host cell such as Escherichia coli, whereby an antibody protein capable of binding to a cell surface protein of a vector is obtained. Can be obtained in large quantities. This is cheaper than maintaining monoclonal antibodies obtained by culturing in a medium requiring serum, and application to enzyme immunoassay can be expected. In one embodiment of the present invention, the antibody is preferably a single-chain Fv protein (sFv).

[0033] The conjugate of the substance that recognizes the surface protein of the vector and the ligand for the cell surface receptor of the target cell can be prepared using a known method. Preferably, when the substance is a protein (preferably an antibody), the protein of the present invention is fused by expressing the gene encoding the substance and the gene encoding the ligand in the same reading frame (in flame). It may be expressed as a protein. The fusion protein can be prepared by any genetic engineering method, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Brother Edition 3, Cold Spring Harbor Laboratory, New York It can be carried out in accordance with the protein expression method described in Ichido 'Spring', Ichibar (2001).

[0034] The gene encoding an antibody against the surface protein of the vector (or a fragment thereof) can be obtained, for example, from an antibody-producing hybridoma based on the amino acid sequence of the antibody protein. First, mRNA is prepared from these hybridomas by a known method, and based on the mRNA, a single-stranded cDNA is synthesized using reverse transcriptase. Based on the amino acid sequence or base sequence of the antibody, PCR, hybridization, and the like are performed. By using the method and the like, the gene of the present invention can be selectively obtained. Such a method is well known, and those skilled in the art can easily isolate the gene of the present invention based on the disclosure in the present specification.

[0035] Alternatively, a cDNA library of cells producing the desired antibody is constructed, and cDNAs encoding the highly conserved immunoglobulin heavy and light chain constant regions are used as probes to prepare the cDNA library. The rally may be screened to isolate the light and heavy chain cDNAs of the desired antibody. As described above, genes encoding the variable regions of both the heavy and light chains of the antibody may be used to express the sFv antibody.

[0036] Information on the nucleic acid sequence of a gene for encoding a ligand for a cell surface receptor is available from a gene database (for example, NCBI). Therefore, those skilled in the art can use the obtained genetic information to integrate the gene into an expression vector or the like. For example, the VEGF gene has been registered with NCBI as AB021221, AF486837.

Alternatively, a substance that recognizes a surface protein of a vector and a ligand protein for the cell surface receptor of the target cell may be bound by a linker or the like. The linkers include, for example, dimethylsuccinate imidate dihydrochloride (DMS), suberic acid di-N-hydroxysuccinimide ester (DSS), tartaric acid N-hydroxydisuccinimide ester (DST), and p-phenyl-bismaleimide (pPDM) , Methyl-4-mercaptobutyrimidate hydrochloride (MBI), methyl-4 azidobenzimidate hydrochloride (ABI) and the like. The binding of a substance (preferably a protein) to the surface protein of the vector using the linker and the ligand protein to the cell surface receptor of the target cell can be performed, for example, by Seismic Chemistry Experiment Course 2 Chemistry of Proteins It can be carried out according to the method described in p.604-618 (1987).

[0038] The mixing ratio of the vector with the conjugate is, for example, 1 x 10 ⁸ pfuZml of the vector, the conjugate is preferably 2.5 g to 20 gZml, more preferably 10 g to 20 gZml. is there.

[0039] The present invention is characterized by specifically promoting the infection of a vector with a target cell by utilizing a surface protein on the vector and a ligand bound to the cell surface receptor of the target cell. Things. Therefore, the vector surface protein and the ligand for the cell surface receptor of the target cell may be expressed as a fusion protein on the particle surface of the vector by a genetic engineering technique without using a substance that recognizes the surface protein. Specifically, the protein of the present invention is expressed as a fusion protein by expressing the gene encoding the vector surface protein and the ligand gene for the cell surface receptor of the target cell in the same reading frame (in flame). It is possible to do so. The fusion protein can be prepared by any genetic engineering techniques, for example, as described in Sambrook et al., Molecular Science: A Laboratory Manual, Brother Edition 3, Cold spring Harb or Laboratory, Cold 'Spring' Nover, NY (2001). It can be carried out according to the protein expression method. For example, in the case of a viral vector, the gene encoding the fusion protein may be integrated into the viral genome using a known technique. In the case of a ribosome vector, the fusion protein can be expressed on the surface of the ribosome vector using an appropriate expression vector, for example, a plasmid.

Alternatively, a conjugate of the vector surface protein and the ligand for the cell surface receptor of the target cell may be separately prepared, and the conjugate may be chemically covalently bonded to the vector surface. Conjugates of the vector surface protein and the ligand for the cell surface receptor of the target cell are preferably prepared as a fusion protein. Alternatively, as described above, the conjugate of the substance that recognizes the surface protein of the vector and the ligand protein for the cell surface receptor of the target cell may form the vector surface protein and the ligand for the cell surface receptor of the target cell. You may couple | bond with a linker etc. Those skilled in the art can appropriately use a method for binding a conjugate, preferably a fusion protein, to the surface of a vector depending on the type of the vector. An embodiment in which a conjugate is separately prepared is useful, for example, when a plasmid vector is applied as the vector of the present invention.

[0041] The present invention includes a step 1) of binding a ligand for a cell surface receptor of a target cell to a vector surface protein before or simultaneously with the infection of the vector with a target cell. The surface protein on the vector binds to the ligand for the cell surface receptor, while the ligand for the cell surface receptor of the target cell binds to the cell surface receptor, thereby specifically promoting the infection of the vector to the target cell. Is performed. In Examples 2 and 3 described later in the present specification, 0-20 μg / ml of knobFv-VEGF protein was mixed with 1 × 10 ⁸ pfuZml of adenovirus, and as the amount of knobFv-VEGF protein increased, In addition, the percentage of gene-expressing cells and MCS were also high (Figure 1).

[0042] The method for guiding a person to send a message early according to the present invention

Gene transfer by a biological method is generally carried out by bringing cells into contact with a gene at room temperature under oxygen for a relatively long time in order to achieve efficient gene transfer. On the other hand, clinical practice requires gene transfer under special adverse conditions of low temperature, low oxygen, and short contact time. Nevertheless, a method for efficiently performing gene transfer under such so-called adverse conditions has not been provided despite the necessity.

[0043] The gene transfer method of the present invention includes, in order to solve the above problem, infecting the cell with the vector under at least one condition of low temperature, low oxygen and short time in step 2). .

[0044] In the above step 1), infection of the vector with the target cell is specifically promoted by binding a ligand for the cell surface receptor of the target cell to the surface protein of the vector. The use of such conjugates has enabled, for the first time, highly efficient gene transfer under at least one adverse condition at low temperature, low oxygen and short time.

[0045] In the present invention, the term "low temperature" refers to a temperature lower than normal temperature (less than normal temperature), preferably 10 ° C or lower, more preferably 7 ° C or lower, and most preferably 4 ° C or lower. However, gene transfer is not It is preferable that the lower limit temperature is at least o ° c or more in consideration of the state of the target cell, the fibrous tissue and the like. Cold conditions can be achieved, for example, by performing the infection in an ice slush tray or in an ice nos (ice bath).

[0046] In the present invention, "under hypoxia" means a condition in which blood is blocked and oxygen is not substantially delivered to tissues. Although not limited, for example, hypoxic conditions after 15 minutes or more after blood cutoff, more preferably hypoxic conditions after 1 hour or more, 3 hours or more, or 24 hours or more after blood cutoff It is. In the harshest conditions, it is desirable that gene transfer by vector infection under hypoxic conditions 72 hours or more after blood blockage is possible.

[0047] In the present invention, the term "short time" generally means a time shorter than a time for infecting a target cell with a vector in a gene transfer method using a vector. For example, in the case of a viral vector such as Adenovirus vector, the vector is generally infected for 1 hour to 24 hours. Alternatively, in the case of non-viral vectors such as ribosomes, the infection time is about 1 to 24 hours. In the present invention, the infection time of the vector to the target cell is preferably 1 hour or less, more preferably 30 minutes or less, and most preferably 15 minutes or less.

[0048] Using the method of the present invention, a desired gene can be introduced into a target cell with high efficiency. The target cell is not particularly limited as long as it can be transformed by a biological method using the vector. It may be a cultured cell in vitro, or a cell in an in vivo or in vivo tissue for transplantation or in a living tissue! ヽ. As the type of cells, those which present a cell surface receptor which can be used particularly on the cell surface, for example, vascular endothelial cells, epidermal cells, dermal cells, epithelial cells, keratinocytes and the like are preferable.

[0049] The method of the present invention can also be clinically applied, for example, to gene transfer to transplanted organs and other neovascular vessels of cancer and gene transfer to ischemic cardiovascular diseases such as myocardial infarction.

[0050] All the prior art documents cited in the present specification are incorporated herein by reference.

Example [0051] Hereinafter, the ability to specifically explain the present invention by way of examples is not intended to limit the technical scope of the present invention. Those skilled in the art can easily modify and change the present invention based on the description in the present specification, and they are included in the technical scope of the present invention.

Example 1 Preparation of fusion protein of VEGF and fiber knob protein

First, VEGF gene (NCBI databank, AB021221, AF486837) (SEQ ID NO: 1) the pcDNA3 1 (Invitrogen, Carlsbad, CA ) Sabukuro the EcoRI site of -. The ring with plasmid _(P cDNA3 1ZVEGF.) Was prepared. Primer 1 (5′—gcggccg cttccatgaactt—3 ′) (Tori selfie 2) and Primer 2 (5, —gcacactcgaggctgatcag—3) designed to add the NotlZXhoI restriction enzyme site to the type I plasmid ') (SEQ ID NO: 3) was used for PCR. A VEGF gene (NotlZXhoI) fragment was prepared.

On the other hand, the knob scFv-EGF gene (Watkins SJ et al., Gene Therapy 4, 1004-1012 (1997)) provided by the University of Bristol (Hawkins RE) was cut out with Ncol ZEcoRI. This fragment was inserted into the EcoRl restriction enzyme site of pcDNA3.1 ZHis A (Invitrogen) by blunt-end ligation to construct pcDNA3.1 / HisA / knobs cFv-EGF. This pcDNA3.1 / HisA / knobscFv—EGF was cut with NotlZX hoi to cut out the EGF gene (NotlZXhoI fragment). Instead, the VEGF gene (NotlZXhoI) fragment prepared above was inserted by ligation to prepare pcDNA3.1 / His AZknobscFv-VEGF.

Using the thus prepared plasmid, a gene was introduced into COS7 by the lipofectamine method (Invitrogen).

(0418 sulfate: 018-0—: 61 ^ Rockville, MD). After lysing the selected cells, knobscFv-VEGF protein was purified using a purification system of Ni-NTA Purification system with anti-Xpress ™ antibody (Invitrogen) according to the attached protocol. Finally, the cells were treated with enterokinase to cleave the His and X-press tags.

Example 2 Preparation of Adenovirus Vector for Targeting VEGF Receptor

The knobscFv-VEGF protein prepared in Example 1 was mixed with an adenovirus vector incorporating the GFP gene (adexGFP: RIKEN BioResource Center (RDB No 1727)). It was prepared by summing. Specifically, adexGFP of lxl0 ⁸ pfuZml, mixed at various concentrations KnobFv- VEGF protein (0- 20 g / ml), and incubated Chillon in 1 hour incubator until use in subsequent experiments (until the next day) in a refrigerator saved.

Example 3 Gene Transfer Test into Vascular Endothelial Cells Under Low Temperature Conditions

Experiments were performed to infect human or porcine vascular endothelial cells for 1 hour under cold conditions. First, porcine vascular endothelial cells were prepared as follows. Porcine aortic force The vascular endothelium was mechanically removed, and after 5 passages, it was confirmed that it was a vascular endothelial cell by the incorporation of Dil acetylated LDL (Di-Ac-LDL: Biogenesis L TD). .

[0057] The obtained vascular endothelial cells were confluent in a 6-well plate and infected with MOI 10. Specifically, in the case of low-temperature infection, a 6-well plate was placed in an ice slush tray and infected for 1 hour. Then, the medium was changed to a D-MEM medium containing 10% FCS, and cultured in an incubator (37 ° C) for 24 hours. In the case of room temperature infection, the cells were infected in an incubator (37 ° C) for 1 hour, and then the medium was changed to D-MEM containing 10% FCS, and cultured in an incubator ( ₃₇ ° _C) for 24 hours.

[0058] 24 hours after the infection, the cells were collected, and the gene expression was examined by FACS (flororescenc activated cell sorter) analysis. Gene expression at infection time of 1 hour at normal temperature and low temperature, and GFP gene expression status by adexGFP mixed with various concentrations (0-20 g / ml) of knobscFv-VEGF protein, the percentage of expressing cells and GFP The intensity was represented by mean channel shift (MCS), which reflects the change in the mean value (Fig. 1).

FIG. 1 shows the results. In FIG. 1, it was determined that the expression of the GFP gene was increased by mixing knobFv-VEGF protein with respect to the extremely low expression efficiency (C (l)) at low temperature. MCS values are expressed as relative values. As shown in FIG. 1, as the mixing amount of the knobscFv-VEGF protein was increased, the GFP gene expression was increased in a dose-dependent manner. In other words, the presence of knobscFv-V EGF protein bound to adexGFP proves that gene transfer via the VEGF receptor functions effectively at low temperatures and under special conditions, and improves both gene expression efficiency and expression level. It was done.

FIG. 2 shows FACS data. For C (1) V (20), control NC (infected It is evident that the number of cells showing high and GFP intensity is higher than that of (none) and C (l) (infected, no knobscFv-VEGF protein admixture).

Industrial applicability

[0061] The use of the method of the present invention makes it possible for the first time to perform gene transfer with high efficiency even under adverse conditions such as low temperature, low oxygen, and short time. For example, in Examples described later in this specification, infection was performed at a low temperature of 0 ° C. for 1 hour. When 0-20 μgZml of knobFv-VEGF protein was mixed with lxl0 ⁸ pfuZml of adenovirus, the higher the amount of knobFv-VEGF protein, the higher the percentage of gene-expressing cells and MCS (Fig. 1). ). Specifically, knobFv-VEGF protein at 10 / z gZml showed approximately the same ratio of gene-expressing cells and MCS as in the case of infection at room temperature for 1 hour. When 20 gZml of the conjugate was mixed, the transfection efficiency was increased by about 20% (about 68% and also about 87%) and the MCS was increased by about 4.7 times as compared to the case of infection at room temperature for 1 hour (10. 1 to 47.8). When compared at a low temperature of 0 ° C, the gene transfer efficiency is about 2.7 times (about 32% to about 87%) and the MSC is about 22 times (2%) compared to the case without knobFv-VEGF protein. . 2 power also increased by 47. 8).

[0062] The method of the present invention preferably achieves the same gene transfer efficiency as that in the case where the conjugate is not mixed without specific adverse conditions by mixing the conjugate even under specific adverse conditions. Things.

[0063] The present invention also relates to a conjugate of a ligand for a cell surface receptor of a target cell and a vector surface protein, and the use of the conjugate in the above-described gene transfer method of the present invention.

Claims

The scope of the claims

[1] A method for introducing a gene into target cells using a vector incorporating the gene

The above method, comprising:

[2] The vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a retrovirus vector, a herpes virus vector, a paramyxovirus vector including a Sendai virus vector, an orthomyxovirus vector including an influenza virus, and The adenovirus vector z adeno-associated virus hybrid vector force is a group force selected from the group consisting of a virus vector selected from the group consisting of ribosome, phage vector, and plasmid. Method.

[3] The method according to claim 1 or 2, wherein the vector is an adenovirus vector.

[4] Ligands to cell surface receptors on target cells include vascular endothelial growth factor (VEGF), Tie-1, Tie-2, CD34 (PECAM), E-selectin, VE-cadherin, von Willebrand factor, CD146, CD36, thrombomodulin, indegrin (V (3, angiopoietin, keratinocyte growth factor (KGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF)). Item 1. The method according to any one of Items 1 and 3 above.

[5] The method according to any one of claims 1 to 4, wherein the low temperature is 10 ° C or lower.

[6] The method according to any one of claims 1 to 4, wherein hypoxia is a condition in which blood is blocked and oxygen is not substantially delivered to a tissue.

[7] The method according to any one of claims 1 to 4, wherein the short time is one hour or less.

[8] The target cell according to any one of claims 1 to 7, wherein the target cell is a vascular endothelial cell, an epidermal cell, a dermal cell, an epithelial cell, or a keratinocyte in a cultured or transplanted tissue or in a living tissue. The method described in the section.

[9] Use of the conjugate of a ligand for a cell surface receptor of a target cell and a vector surface protein in the gene transfer method according to any one of claims 1 to 8.

[10] A conjugate of a ligand for a cell surface receptor of a target cell and a vector surface protein for use in the method for gene transfer according to any one of claims 1 to 8.