WO2008068945A1 - Gene transfer aid comprising peptide capable of migrating into cell as active ingredient, and gene transfer method utilizing the gene transfer aid - Google Patents

Abstract

[PROBLEMS] The object is to enable the gene transfer in a part of cancer cells, blood cells or the like in which the expression of both of CAR and an integrin is poor or lost. are expressed poorly [MEANS FOR SOLVING PROBLEMS] A gene transfer aid comprising a peptide capable of migrating into a cell as an active ingredient, characterized in that the peptide has a NHS (N-hydroxysuccinimidyl) group bound thereto; and a gene transfer method characterized in that the gene transfer aid is bound covalently to the surface of a coat protein of a virus to be used as a gene transfer vector.

Description

 Specification

 Gene transfer aid containing intracellular translocation peptide as active ingredient and gene transfer method using the gene transfer aid

 Technical field

 [0001] The present invention relates to a gene introduction auxiliary agent containing an intracellular translocating peptide as an active ingredient and a gene introduction method using the gene introduction auxiliary agent. More specifically, by covalently binding to the coat protein of the virus used as a gene transfer vector, both CAR and integrin are poorly expressed! / Or defective! /, Some cancers The present invention also relates to a “gene transfer aid using an intracellular translocation peptide as an active ingredient” and “a gene transfer method using the gene transfer aid”, which enable gene transfer even in cells and blood cells. Background art

 [0002] The regulatory mechanism of gene expression in cells of mammals and the like, and the functional analysis of proteins, which are gene products, are the central themes of current pharmacology and biology. Technology to introduce is indispensable. Currently, gene transfer methods are mainly divided into methods using non-viral vectors such as ribosomes and methods using viral vectors, and methods using viral vectors from the viewpoint of gene transfer / expression efficiency.

 Among viral vectors, the gene transfer method using an adenovirus vector (hereinafter referred to as “Ad”) shows high gene transfer and expression efficiency, which is very important for conducting basic research at the cell and animal level. It is known to be an important tool.

[0003] Currently, Ad widely used in gene therapy research and basic research has been developed based on type 2 or type 5 Ad, and the gene transfer mechanism uses the infection mechanism of Ad as it is. Ad has a regular icosahedron structure consisting of 252 capsomers on a virus particle with a diameter of about 80 nm, of which 12 at the apex are called pentons with protrusions, and the other 240 are hexons. be called. Penton consists of a Penton base and fiber, and the fiber is further divided into a tail, shaft and knob.

The entry of Ad into the target cell is caused by CAR (coxacki e-virus adenovirus receptor), followed by a two-step process where the Penton'-based RGD (Arg-Gly-Asp) motif binds to the α-integrin on the cell surface. . Ad that reaches the endosome undergoes a structural change in the force psid protein under acidic conditions, destroys the endosome and moves into the cell. Thereafter, Ad is transported to the vicinity of the nucleus by retrograde transport along the microtubule, and binds to the nuclear pore complex to deliver the genome into which the target gene is incorporated into the nucleus.

 Based on the mechanism described above, adenoviral vectors (Ad) can efficiently introduce genes into a wide variety of cells and tissues, regardless of whether they are dividing or stationary. In addition, since it can be expressed by administration to individual animals, it has been actively developed as a vector for gene therapy. However, for cell types with poor or defective Ad infection receptor (CAR) expression, such as malignant tumor cells and blood cell lineage cells, even using force ¾Ad is not an important target for gene therapy. Gene expression cannot be obtained.

 For some cancer cells and hematopoietic cells with poor expression of Ad infection receptor (CAR), the gene transfer efficiency is low. To overcome the problems of Ad, RGD (Arg — Improved Ad (hereinafter referred to as “AdRGD”) with α-integrin orientation has been developed by presenting a Gly— Asp peptide.

 AdRGD is a cell that has been difficult to transduce with conventional Ad.

V, cells, etc.) can be expressed very efficiently (Non-patent Documents 1 and 2).

 AdRGD is equipped with an expression cassette for the gene of interest in the E1-deficient region, as in the case of conventional Ad. In addition, an oligonucleotide corresponding to the RGD sequence having affinity for α-integrin is added to the region encoding fiber knob. Due to the modification of this fiber region, AdRGD has acquired a-integrin orientation, and the expression of CAR, which was difficult to introduce genes with conventional Ad, is poor or missing! /, The gene can be efficiently introduced into the cells to be treated.

In fact, the present inventors applied AdRGD to (1) optimization of cancer gene therapy using a site force-in gene or a suicide gene (Non-Patent Documents 2 to 4), (2) a tree by gene modification. Optimization of cellular immunotherapy based on enhancement of cellular functions (Non-patent documents 1, 5), (3) Construction of pharmacokinetics control method of immune cells by introduction of chemokine 'chemokine receptor gene (non-patent Reference 5) is underway, and a new DDS (Drug Delivery System) strategy that could not be achieved by conventional vector systems is being introduced to advanced medicine.

 However, in some cancer cells and blood cells that are poorly or deficient in both CAR and integrin expression, AdRGD still does not provide sufficient gene expression (Fig. 1). For this reason, development of vectors capable of gene transfer independently of CAR and integrin is required.

[0005] In some cancer cells and blood cells that are poorly or deficient in both CAR and integrin expression as described above, AdRGD is still sufficient for gene transfer. Was difficult.

 In addition, even in the cell types where CAR and integrin expression is observed, it is necessary to administer high doses in order for conventional virus vectors (Ad, AdRGD, etc.) to exhibit sufficient gene transfer activity. For example, there is a problem of side effects when applied to gene therapy. Therefore, new vectors with strong gene transfer activity that can be clinically applied at low doses have been required!

 Furthermore, from the viewpoint of basic research, for the purpose of gene transfer into cells with low receptor expression, development of vectors capable of gene transfer independent of CAR and integrin, and vectors capable of efficient gene transfer into all cells Development is required

[0006] Non-patent literature l: Okada, N. et al .: Cancer Res., 61: 7913-7919, 2001

 Non-Patent Document 2: Okada, N. et al .: Cancer Lett., 177: 57-63, 2002

 Non-Patent Document 3: Okada, N. et al .: Gene Ther., 10: 700-705, 2003

 Non-Patent Document 4: Okada, N. et al .: Cancer Gene Ther., 12: 608-616, 2005

 Non-Patent Document 5: Okada, N. et al .: Gene Ther., 12: 129-139, 2005

 Disclosure of the invention

 Problems to be solved by the invention

[0007] In view of the above-mentioned problems of the prior art, the present invention has poor expression of both CAR and integrin! / Or is defective! / In some cancer cells, blood cells, etc. However, gene transfer is possible. It is an object to provide a new gene transfer vector having the following requirements (1) to (3).

 (1) The expression of both CAR and integrin is poor! / ヽ or or! / Is deficient! / Can also be introduced into some cancer cells such as blood cells. (Expansion of applicable area).

 (2) As a gene therapy vector, it should be clinically applicable at a low dose (that is, side effects are reduced and gene transfer activity is strong).

 (3) As a gene function analysis tool in basic research, it should be possible to efficiently introduce genes into any cell.

 Means for solving the problem

[0008] As a result of diligent research, the present inventors have used a translocation peptide such as a Tat peptide bound to an NHS (N-hydroxysuccinimidyl) group as a gene transfer aid and used as a gene transfer vector. Covalently linked to the outer shell protein of the virus, the application range is expanded to hematopoietic cancers where gene transfer was difficult (with conventional gene transfer vectors), and regardless of the cell type. The present inventors have found that gene transfer activity is very strong (regardless of the presence or absence of receptor expression) and have completed the present invention.

 [0009] That is, the invention according to claim 1 is a gene transfer aid containing an intracellular translocation peptide as an active ingredient, wherein NHS (N-hydroxysuccinimidyl) group is bound to the intracellular translocation peptide. To a gene transfer aid.

 The invention according to claim 2 relates to the gene transfer auxiliary agent according to claim 1, wherein the intracellular translocation peptide is a Tat peptide.

 The invention according to claim 3 is a gene transfer auxiliary agent comprising a cell translocation peptide represented by any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 as an active ingredient, wherein the cell The present invention relates to a gene transfer aid characterized by binding an NHS group to an internalization peptide.

The invention according to claim 4 is selected from the group consisting of amino acid sequences in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence of the intracellular translocation peptide. 4. The gene transfer auxiliary agent according to claim 3, wherein the force is one. The invention according to claim 5 relates to the gene transfer aid according to any one of claims 1 to 4, wherein the gene transfer aid is covalently bound to the surface of a viral outer protein used as a gene transfer vector.

 The invention according to claim 6 relates to the gene transfer auxiliary agent according to claim 5, wherein the virus used as the gene transfer vector is adenovirus.

 The invention according to claim 7 is such that the covalent bond between the gene introduction auxiliary agent and the virus used as the gene introduction vector is 10 to 50 ° C., 100 to 1000 rpm, 5 to 60 minutes. The gene transfer auxiliary agent according to claim 5 or 6, which is carried out below.

 The invention according to claim 8 relates to the gene transfer auxiliary agent according to any one of claims 1 to 7, wherein the cell type to be transferred is an adherent cell or a floating cell.

 The invention according to claim 9 is the cell type according to any one of claims 1 to 8, wherein the cell type to be gene-transferred is a cell type in which expression of both CAR and integrin is poor or defective. It relates to a gene transfer aid.

 The invention according to claim 10 relates to the gene transfer auxiliary agent according to any one of claims 1 to 9, which is in the form of a freeze-dried powder.

 The invention according to claim 11 relates to the gene transfer auxiliary agent according to any one of claims 1 to 10 used for gene therapy.

 In the invention according to claim 12, the gene introduction auxiliary agent according to any one of claims 1 to 11 is bonded to the surface of the outer shell protein of the virus used as the gene introduction vector in the stage before gene introduction. A virus vector for gene transfer characterized in that

Yes

 In the invention according to claim 13, the gene introduction auxiliary agent according to any one of claims 1 to 11 is covalently bonded to the surface of the outer shell protein of the virus used as the gene introduction vector in the stage before gene introduction. The present invention relates to a gene introduction method characterized by the above.

The invention's effect

Coordinated to the surface of the outer shell protein of a virus used as a gene transfer vector, the “gene transfer aid containing an NHS (N-hydroxysuccinimidyl) group-bound intracellular translocation peptide as an active ingredient”. Virus vector characterized by The conventional gene transfer vectors (Ad, AdRGD, etc.) are difficult to transfer genes, but some cancer cells and blood cells (cells with poor or defective expression of both CAR and integrin) Species) can also exert a sufficient gene transfer effect (“expansion of application range”). At the same time, even cell types that can be transferred with conventional gene transfer vectors can exhibit much higher gene transfer activity than conventional cells (see “Improvement of gene transfer efficiency”). ").

 In other words, it enables gene transfer (CAR, integrin-independent gene transfer) to cells with low receptor expression, thus expanding the range of application of viral vectors (such as Ad) in cancer gene therapy, and at the same time, In the species (regardless of the presence or absence of receptor expression), the in vivo (in vivo) dose reduction can be achieved (that is, it can be clinically applied at a lower dose, thus contributing to reduction of side effects).

 In addition, since gene transfer can be performed efficiently for any cell, it can be applied in basic research as a gene transfer tool.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0011] In the present invention, an NHS group bound to an intracellular translocation peptide such as a Tat peptide is used as a gene introduction auxiliary agent (in the stage before gene introduction) to the outer shell protein of a virus vector. Application to some cancer cells and hematopoietic cells (cell types in which both CAR and integrin are poorly expressed or lacked! /) That have been difficult to introduce genes by covalent bonding. At the same time it is expanded, it is based on the knowledge that gene transfer activity is very strong (even in cell types that could be transferred with conventional viral vectors).

 Therefore, the virus vector in which the gene introduction auxiliary agent according to the present invention is covalently bound to the surface of the outer shell protein is an effective drug delivery system (Drug Delivery System) with improved gene introduction efficiency in vivo. DDS).

 Hereinafter, embodiments of the present invention will be described. The terms used in the present specification shall have the meanings usually used in the art unless otherwise specified, and the expression in the singular unless otherwise specified. It also includes the plural concept.

[0012] First, "intracellular translocation peptide (which is an essential component of the gene transfer adjuvant according to the present invention ( PTD) ”will be described. The intracellular translocation peptide is a peptide consisting of 10 to 20 amino acids containing a lot of basic amino acids, and is also called a membrane-permeable peptide (MPP). The details of the intracellular translocation mechanism are unknown, and it has been suggested that translocation into cells via sugar chains such as heparan sulfate existing on the surface of almost all cells. Typical intracellular translocation peptides include HIV-1 derived Tat peptides, etc. [Table 1] lists the intracellular translocation peptides preferably used in the present invention.

Yes

 [0013] [Table 1]

 Typical examples of PTD

 Peptide origin sequence

 Tat HiV-1 GRKK RQR RPPQ

 Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Pro Pro Gin

Antennapedia Drosophi! A QIKIWFQN KWKK

 Arg Gin He Lys iie Trp Phe Gin Asn Arg Arg Met Lys Trp Lys Lys

Rev HIV-1 TQ A RNRRRRW ERQR

 Thr Gin Arg Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg Gin Arg

VP22 HSV NAKT HERRRKLAIE

 Asn Ala Lys Thr Arg Arg His Glu Arg Arg Arg Lys Leu Ala lie Glu Arg,, H: Basic amino acid

 [0014] As shown in [Table 1], the "intracellular translocation peptide" that is an essential component of the gene transfer aid that is effective in the present invention includes Tat peptide (SEQ ID NO: 1), Antennapedia (SEQ ID NO: 2). , Rev (SEQ ID NO: 3), VP22 (SEQ ID NO: 4), etc. Power to enumerate Tat peptide in order to exert the effects of the present invention described above (expansion of application range, improvement of gene transfer efficiency, etc.) It is best to use The Tat peptide is derived from the transcriptional activator of HIV and has a peptide combination IJ “Gly—Arg—Lys—Lys—Arg—Arg—Gin Arg Arg Arg Pro Pro Gln” that contains many basic amino acids. Among the internalization peptides, it has a particularly high intracellular internalization activity and is also used as a carrier for introduction into cells into proteins, ribosomes and the like.

As long as it has an intracellular translocation function (membrane permeation function), the representative ones shown in [Table 1] In addition to the intracellular translocation peptide, these amino acid sequences (SEQ ID NOs: 1 to 4) may have amino acid sequences in which one or more amino acids are deleted, substituted, inserted, or added. It is an active ingredient for gene transfer aids.

[0015] The present invention relates to a gene transfer vector comprising an NHS (N-hydroxysuccinimidyl) group bound to an intracellular translocation peptide typified by the Tat peptide as an auxiliary for "gene transfer". By covalently binding to the outer protein of the virus used as a gene, the application range is expanded to some cancer cells and blood cells that have been difficult to transduce, and at the same time, “gene transfer efficiency” And “gene transfer activity” itself is (further) enhanced.

 In the present specification, “gene transfer” means that a target polynucleotide sequence (nucleic acid) is introduced into a cell. In addition, gene transfer may be to exert the action of the nucleic acid in the cell into which the nucleic acid has been introduced. For example, by introducing a nucleic acid containing an expression unit (promoter and a gene linked downstream thereof), the gene encoded by the expression unit is expressed (transcribed, translated, or both) in the cell. As used herein, “gene transfer efficiency” refers to the efficiency of nucleic acid introduction into a cell! /, For example, the proportion of transfected cells in the total cell, The amount of uptake or the expression level of the transgene in the whole cell population. When the gene is expressed in cells, it is preferably at the level of transgene expression in the entire cell population. The expression level is measured, for example, 24 hours after transfection. The expression level of the transgene in the cell population is determined by preparing a cell sample and measuring the expression level of the nucleic acid per cell extract equivalent to 1 mg of total cell protein. The expression level can be determined by the mRNA level, protein level, or activity level of the protein. In this specification, the term “gene transfer activity” refers to the activity of “gene transfer” by a vector. The function of the introduced gene (for example, in the case of an expression vector, expression of the encoded protein and And / or the activity of the protein).

[0016] As shown in the examples described later, the present inventors have used adenowines vectors, in which an “NHS (N-hydroxysuccinimidyl) group” is bound to a Tat peptide as an auxiliary for gene transfer. Gene transfer vector covalently linked to the outer shell protein (hereinafter “Tat It has been clarified that it has about 10 to 500 times stronger gene expression activity compared to conventional Ad regardless of CAR negative and positive cells. It came to do.

 The method of attaching the “NHS group” to the above-mentioned intracellular translocation peptide is not particularly limited as long as it is an experimental method within the range that can be easily performed by those skilled in the art.

 The gene transfer aid of the present invention is more effective by binding an “NHS group” to an intracellular translocation peptide as an active ingredient (specifically, when the intracellular translocation peptide is a Tat peptide, its lysine group). It imparts strong intracellular translocation activity (to the bound intracellular translocation peptide), and can exert strong gene expression activity regardless of the presence or absence of receptors (CAR, integrin, etc.).

 [0017] The above-described "gene introduction adjuvant containing an NHS group-bound intracellular translocation peptide as an active ingredient" according to the present invention can be changed or modified as appropriate, and these also substantially. It belongs to the technical scope of the present invention. For example, chemical linkers that bind to intracellular translocation peptides include dicyclohexylcarbodiimide (DCC), maleimidobenzoyl N-hydroxysuccinimide (MBS), N-ethynoxycarbonyl 2-ruethyloxyl in addition to the NHS group. 1,2-Dihydroquinoline (EEDQ), N-isobutyoxymonocarbonyl 2-isobutyloxy 1,2-dihydroquinoline (IIDQ) can be listed as IJ. However, it is desirable to use an “NHS group” when exhibiting the effects of the present invention described above (expansion of application range, improvement of gene transfer efficiency, etc.)!

 [0018] The gene transfer adjuvant of the present invention having an NHS group bound to an intracellular translocation peptide such as a Tat peptide is a "virus (inactivated)" used as a gene transfer vector. By covalently binding to the surface of the outer shell protein, sufficient gene transfer is possible even for some cancer cells and blood cells that have been difficult to transfer using conventional viral vectors (Ad, AdRGD, etc.). The introduction effect can be demonstrated.

As used herein, “virus” refers to an infectious microstructure that has either DNA or RNA as its genome and that grows only in infected cells. Viruses include: retrovirus family, togavirus family, coronavirus family, flaviviridae, paramyxoviridae, orthomyxoviridae, bunyaviridae, rhabdoviridae, box virus family, The power S includes viruses belonging to a family selected from the group consisting of herpesviridae, baculoviridae and hepadnaviridae, and the virus used more preferably in the present invention is “adenovirus”. Adenoviruses can efficiently transfect a wide variety of cells and tissues, regardless of whether they are in mitotic phase or stationary phase, and also have a transiently high gene transduction efficiency and expression efficiency as a gene transduction vector for mammals. Therefore, in order to achieve the purpose of the present invention (expansion of application range / improvement of gene transfer efficiency, etc.) (and as a viral vector modified by the gene transfer aid of the present invention) .

 As used herein, “inactivation” refers to inactivation of the genome when referred to a virus (eg, adenovirus). Inactivated viruses are replication deficient. Inactivation is accomplished by a variety of means normally available to those skilled in the art.

[0019] The "covalent bond" between the gene transfer aid of the present invention and the virus used as the gene transfer vector is usually an experimental method within the range that can be easily performed by those skilled in the art. Although not limited, in order to produce a complex (for example, “Tat peptide-modified Ad”) between the gene transfer aid and the virus vector, which is efficient, 10 to 50 ° C., 100 to 100 ° C. Under the condition of 1000 rpm, 5 minutes to 60 minutes (covalent binding force is desirable. Here, since the NHS group is bound to the intracellular translocation peptide in the gene transfer aid of the present invention, (NHS This makes it easier to covalently bind to viral vectors (see Figure 3).

 The ability of the gene transfer adjuvant of the present invention S, whether or not it is covalently bound to the surface of the outer protein of a virus used as a gene transfer vector is usually determined by an experimental method within the range that can be easily performed by those skilled in the art. Specifically, from the viewpoint of molecular weight, "confirmation of binding by SDS-PAGE" (see Fig. 4), and intracellular translocation peptides such as Tat peptide stray to "+"! / Therefore, confirm the presence or absence of binding (indirectly) by “surface charge (mV)” etc.

[0020] By applying the gene transfer adjuvant of the present invention to the surface of the outer shell protein of the virus vector (positively) before gene transfer, the application range of the virus vector for gene transfer is expanded, Its gene transfer efficiency is also improved (see Figures 9 and 10). That is, as a vector for gene transfer, compared to a vector in which a cell transfer peptide and a virus vector are simply mixed (as described above), the gene transfer aid of the present invention is bound to a virus vector (Tat peptide modified Ad Etc.) is used to enhance gene transfer activity (efficiency) and the like. This indicates that the peptide bound to the virus surface is involved in the enhancement of gene expression.

[0021] By using the gene introduction auxiliary agent of the present invention, the "gene introduction efficiency (activity)" is statistically significant (for example, the significance level) compared to the case where the transfusion is performed without the gene introduction auxiliary agent. p <0. 05) (see Figures 5-8 and 10). That is, (before the gene transfer), the gene transfer virus vector in which the gene transfer aid of the present invention is covalently bound to the surface of the outer protein of the virus has improved gene transfer efficiency (activity) in vivo. An effective drug delivery system (DDS). Measurement of gene transfer efficiency (activity) is not particularly limited as long as it is an experimental method that can be easily performed by those skilled in the art. Measurement by luciferase activity (luciferase activity) [RLU (relative lig ht unit) / well]. The increase in gene transfer efficiency by the gene transfer adjuvant of the present invention can be confirmed (in terms of luciferase activity [RLU / well]) by about 10 to 500 times compared to the case without the gene transfer adjuvant. That is, the gene introduction virus vector in which the gene introduction aid of the present invention is bound to the surface of the outer shell protein of a virus (such as adenovirus) used as a gene introduction vector in the stage before gene introduction. Since the gene transfer efficiency increases by about 10 to 500 times, for example, when it is clinically applied as a gene therapy vector, it can be clinically applied at a low dose, so that side effects are reduced and it is very beneficial.

[0022] The gene transfer adjuvant of the present invention and the cell type to which the viral vector for gene transfer is combined with the adjuvant are not particularly limited. That is, it can be any type of cells such as small intestine, nasal mucosa, skin tissue, subcutaneous tissue, bone tissue, cartilage tissue, etc. Cells of all species such as microorganisms, fish, reptiles, birds and insects can be used. Cell culture is not particularly limited, and can be performed according to known culture conditions using a known liquid medium according to the type of each cell. The cell introduction target of the gene transfer adjuvant of the present invention and the gene transfer virus vector to which the adjuvant is bound is preferably exemplified by cell types such as “adhesive cells” and “floating cells”. (From the examples described later).

 [0023] As a cell type to be a gene transfer target of the viral vector combined with the gene transfer auxiliary agent of the present invention, commonly used cultured cells such as A549 cells (human alveolar epithelial cancer cells), B16BL6 Cells, CHO cells, EL4 cells (mouse thymus-derived T cells), HE K293T cells, HT1080 cells (human fibrosarcoma cells), HeLa cells (human cervical cancer cells), KG-la cells (human myeloid leukemia cells), The ability to list NIH3T3 cells is not particularly limited.

 However, since none of the receptor CAR / integrin is expressed, some cancer cells and blood cells that have been difficult to introduce with existing gene transfer virus vectors (Ad, AdRGD, etc.) Even in (see FIGS. 1 and 2), gene transfer is possible (can also exhibit sufficient gene expression activity) with the gene transfer virus vector to which the gene transfer aid of the present invention is bound. For this reason, “cell types that are poorly expressed or defective in both CAR and integrin! /” (Some cancer cells, blood cells, etc.) ” LV is a cell type that is the target of gene transfer. Specific examples of cell types in which expression of both CAR and integrin cannot be confirmed include “KG-la cells”.

 [0024] Furthermore, the viral vector to which the gene transfer aid of the present invention is bound is a cell type that can be transferred even by a conventional viral vector, ie, a cell type that can sufficiently confirm the expression of both CAR and integrin. (For example, A549 cells, HT1080 cells, EL4 cells, HeLa cells, etc.) and “cell types in which CAR expression cannot be confirmed and integrin expression can be sufficiently confirmed” (for example, B16BL6 cells). However, the gene transfer activity can be significantly higher than that of conventional vectors (improvement of gene transfer efficiency). Therefore, when the viral vector to which the gene transfer aid of the present invention is bound is used as a gene therapy vector, it can be clinically applied even at a low dose (that is, side effects are reduced).

[0025] The gene introduction adjuvant of the present invention and the gene introduction virus combined with the adjuvant! The vector can also be preferably used in the form of “lyophilized powder”.

 The lyophilized powder can be obtained by lyophilizing the gene transfer adjuvant of the present invention and the virus vector for gene transfer combined with the adjuvant, and lyophilization can be carried out using a known method. For example, after freezing in liquid nitrogen, it can be performed with a freeze-dryer (manufactured by Fin Aqua). The lyophilized gene transfer adjuvant is enclosed in a vial and preferably stored at low temperature until use. The gene transfer adjuvant of the present invention and the gene transfer virus vector to which the adjuvant is bound can be regenerated with water at the time of use.

[0026] As described above, “the gene transfer adjuvant containing the NHS group-bound intracellular translocation peptide as an active ingredient” has been specially described, but changes and modifications can be made, and these are within the technical scope of the present invention. It belongs to. In addition, the gene transfer aid of the present invention can be applied to all currently used viral vectors including adenovirus vectors.

 [0027] The gene introduction method using the gene introduction auxiliary agent of the present invention (hereinafter referred to as "the present method") is applied to the surface of the outer protein of a virus used as a gene introduction vector in the stage before gene introduction. Compared with conventional Ad that expresses gene-dependent infectivity 'gene in a CAR-dependent manner, this method is characterized by using a viral vector covalently bound to the above-mentioned gene transfer aid. This is a gene transfer method based on a new idea that improves the efficiency of gene transfer by transferring Ad into cells efficiently and rapidly using its intracellular transfer activity. In other words, it is a system that can efficiently introduce and express even many CAR-negative cells in order to establish a CAR-independent infection 'gene transfer pathway.

[0028] Examples of genes to be treated in this method include enzymes, hormones, lymphokines, receptors, growth factors, regulatory proteins, polypeptides that affect the immune system, immune regulatory factors, antibodies, and the like. Examples include, but are not limited to, encoding genes. Specifically, these genes include, for example, human growth hormone, insulin, interleukin 2, tumor necrosis factor, nerve growth factor (NGF), epidermal growth factor, tissue plasminogen activator 1 (TPA), factor VIII : C, canorecitonin, thymidine kinase, interferon, granulocyte macrophage (GMCSF), erythropoietin (EPO), hepatocyte growth factor Examples include, but are not limited to, genes coding for offspring (HGF). These genes may be present in the form of nucleic acids or polypeptides in the viral vector to which the gene transfer aid of the present invention is bound.

[0029] The gene transfer adjuvant of the present invention and the virus vector to which the adjuvant is bound may be prepared by using any sterile biocompatible pharmaceutical carrier (saline, buffered saline, dextrose and water). Including, but not limited to). Any of these molecules can be administered to a patient alone or in combination with other drugs in a pharmaceutical composition mixed with suitable excipients, adjuvants, and / or pharmaceutically acceptable carriers. obtain. In certain embodiments of the invention, the pharmaceutically acceptable carrier is pharmaceutically inert.

 [0030] Administration of the gene transfer adjuvant of the present invention and the viral vector for gene transfer combined with the adjuvant can be achieved orally or parenterally. Parenteral delivery methods include topical, intraarterial (eg, via the carotid artery), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, or intraperitoneal. This method may be any route as long as it reaches the treatment site.

 [0031] Examples are shown below, but the present invention is not limited thereto.

 Example

 [0032] (1) Preparation of Tat peptide-modified Ad

 As a gene transfer aid that is useful for the present invention, a Tat peptide with an NHS group bound thereto (hereinafter referred to as “Tat-NHS”) was prepared and stored in a frozen state.

 The Ad dilution was added to the frozen Tat-NHS solution to dissolve Tat-NHS. After that, mixing with a vortex mixer and incubating under conditions of 37 ° C, 300 rpm, 30 min, the adenoviral vector for gene transfer (hereinafter referred to as “Tat-Ad”) to which “Tat-NHS” is bound. (Refer to [Fig. 3]).

 Specific examples (experimental examples;! To 3) of “Tat-Ad” production are shown below.

[Experiment 1]

 i) Tat-NHS

Amino acid sequence: Ac-GRKKRRERRRPPQG-GC— NHS Molecular weight: about 2000

2. 5 X 10— ⁶mol/tube Concentration (amount):

2. 5 X 10— ⁶ mol / tube

= 1.5 X 10 ¹ molecules / tube

ii) Reaction conditions

 Lysine residues present in the outer shell protein of Ad [7500 / vp (virus particle)]: “Tat—N HS” = 1: 1000

The number of Ad particles required is 2X10 ^U vp / tube

iii) "Ad", "Tat- NHS" suspension (composition)

• Ad (7X10 ¹² vp / ml): 28.6 / iL

• PBS: 121.4 _U L

 • Tat-NHS: 50

iv) "Tat-Ad" production

 Using “Tat-NHS” in i) in a frozen state, adjust the suspension with the composition shown in iii) under “Reaction conditions” in ii), and mix with a vortex mixer. Next, “Tat-Ad” was obtained by incubation under conditions of 37 ° C., 300 rpm, and 30 min.

(Experiment 2)

i) Tat-NHS

 Amino acid sequence: Ac-GRKKRRERRRPPQG-GC— NHS

 Molecular weight: about 2000

1. 5X10¹⁸m olecules/ tube Concentration (amount):

1. 5X10 ¹⁸ m olecules / tube

ii) Reaction conditions

Lysine residue present in Ad outer shell protein (7500 / vp): “Tat—NHS” = 1: 200 Necessary number of Ad particles is 1 X 10 ¹² vp / tube

iii) "Ad", "Tat- NHS" suspension (composition)

• Ad (2.3X10 ¹² vp / ml): 435

• PBS: 515 • Tat-NHS: 50

→ l X 10 ¹² vp / lmL

iv) "Tat-Ad" production

 Using “Tat-NHS” in i) in a frozen state, adjust the suspension with the composition shown in iii) under “Reaction conditions” in ii), and mix with a vortex mixer. Next, “Tat-Ad” was obtained by incubation under conditions of 37 ° C., 300 rpm, and 30 min.

(Experiment 3)

i) Tat-NHS

 Amino acid sequence: Ac-GRKKRRERRRPPQG-GC— NHS

 Molecular weight: about 2000

Concentration (amount): 2.5 mg / 100 _i L / tube = 1.25 X 10— ⁶ mol / tube = 7.5 X 10 molecules / tube

ii) Reaction conditions

Lysine residue (7500 / vp) in the outer shell protein of Ad: “Tat—NHS” = 1: 1000 Necessary Ad particle count is 1 X 10 ^U vp / tube

iii) "Ad", "Tat- NHS" suspension (composition)

• Ad (2.5 X 10 ¹² vp / ml): 40 / i L

 • PBS: 860

 • Tat -NHS: lOO ^ L

→ l X 10 ^U vp / lmL

iv) "Tat-Ad" production

 Use `` Tat-NHS '' in i) in a frozen state, mix with vortex mixer under `` Reaction conditions '' in ii), incubate at 37 ° C, 300 rpm, 45 min. Tat—A d ”.

V) Other

In addition, except that the reaction time in incubation was changed from 45 min to 45 min! /, The force S and NHS group reaction that produced “Tat-Ad” in the same manner as in Examples 1 and 2. Considering the nature, it is considered that there is almost no impact from the change. [0036] (2) Binding confirmation by SDS-PAGE

 Whether Tat-NHS was covalently bound to the surface of Ad's outer shell protein was confirmed by SDS-PAGE from the viewpoint of molecular weight (kDa).

Specifically, 2 × 101 ⁽ Vp (virus particle) “Ad” and “Tat-8” were concentrated using an evaporator. Then, loading buffer + 2ME was mixed at 95: 5, and the suspension was mixed. 15 µL was added to the solution (Ad, Tat peptide-modified Ad), and after pipetting, incubated at 96 ° C for 5 min and applied to the gel (PAG mini 1/20) Electrophoresis at 20 mA, staining with CBB staining solution for 2 hours, and decoloring with CBB decoloring solution for 2 hours, and then capturing the gel image with a scanner. The sample of Example 2] was used with “FULL RANGE RAINBOW (Amersham Bioscience # RPN800)” as the “marker”.

 The results are shown in [Figure 4]. As apparent from the gel image in [Fig. 4], the molecular weight of hexon increased, suggesting Tat-NHS binding to the Ad surface. In other words, the Tat peptide-modified Ad was larger than the normal Ad by the molecular weight (kDa) corresponding to “Tat-NHS”. As a result, in “Tat-Ad” prepared in (1) above, it was considered that Tat-NHS was covalently bound to the surface of the outer shell protein of Ad.

 [0037] (3) Binding confirmation by surface charge (mV) [Zeta potential measurement]

 “Tat peptide”, which is rich in basic amino acids, has a positive charge (+) while it has an “Ad” force S (—) charge. If Tat-NHS binds (to Ad's outer protein surface), the surface charge should shift from (-) to (+). Therefore, it was confirmed by surface charge (mV) whether or not Tat-NHS was covalently bound to the surface of the outer protein of Ad. Specifically, the sample “Ding & 1-8 (1)?” Was diluted with BS and the surface charge (mV) was measured with Zetasizer 3000HS (Malvern Instrument Ltd.). The sample of [Experimental Example 2] was used, and the results are shown in [Table 2] because the surface charge is shifted to (+), that is, “Tat-Ad” compared to Ad Is charged to (+), suggesting that Tat-NHS is covalently bound to the surface of Ad's outer shell protein.

[0038] [Table 2] Mean (mV) Width (mV)

 Z eta potential

 Ad -18,7 2.7

 Tat-Ad 2.3 1.6

[0039] The usefulness of "Tat-Ad" was evaluated and examined in the following experiments from the viewpoints of "targets for gene transfer (/ application area)" and "gene expression efficiency (/ gene expression activity)". It was. In the above evaluation / examination, the standard adenovirus vector (Ad) and the improved Ad (AdRGD) imparted with integrin-directed superiority were used as targets for comparison with Tat-Ad. In addition, the cell types used in the above evaluation / examination were forces that are adherent cells and floating cells. Specifically, the cultured cells listed in [Table 3] below were used.

[0040] [Table 3]

Cuftui medium (¾ss rrE hod 549 human alveolar male trypsin

* i 1 1 d J t line bell MEM + 1 (M FBS EDT A / PBS 摄 作 摄 S

¾La human 隱 trypB n

B16BL6 Mouse memory, << MEM + 73% Fffi EDTA Pffi

KG-1a HI RP Garden 640 + 10¾ PS + 2 E dilute

 孚 逷 繡 龐

EL4 mouse PM1640 + 1 (¾ FBS ^ 2 E dilute

[0041] (4) Usefulness evaluation of “Tat— Ad” for gene expression efficiency (B16BL6 cells)

 In B16BL6 cells (cell types in which CAR expression cannot be confirmed and integrin expression can be confirmed)! /, The gene expression efficiency (activity) of “Tat-Ad” can be changed to luciferase erase assay. 7 measurements.

 In addition, we also examined whether the gene expression efficiency (activity) of Tat-Ad is seen in a dose-dependent manner. Specifically, luciferase assay was performed according to the following procedure.

[0042] [Procedure] luciferase assay in B16BL6 cells

1) B16BL6 cells were seeded at 10 ⁴ cells / well in 500 medium in a 48-well flat bottom plate.

 2) Incubated for 24 hours.

3) Aspirate medium (see Table 3 above for medium for each cell type) 400 μL medium was added.

4) Various vectors diluted to 3 X 10 ⁷ vp / ml, 1 X loVp / ml, 3 X loVp / ml, 1 X 10 ⁹ vp / ml (“Tat-Ad” uses the sample from Experiment 3 above) 100 L / well (ie, 300 vp / cell, lOOOvp / cell, 3000 vp / cell, lOOOOvp / cell).

 5) Incubated for 24 hours.

 6) The medium was aspirated and washed twice with PBS.

 7) 100 μL / well of Cell Culture Lysis Reagent 5x (promega # E1531) diluted with MiliQ water was added.

 8) 10 L of the cell lysate was taken and placed in a lumino tube.

 9) After 10 L of reaction substrate (Promega # E1501 Luciferase Assay System) was vortexed lightly, [RLU (relative light unit) / well] was measured with a luminometer.

For samples with RLU exceeding 10 ⁷ , the cell lysis reagent was diluted 100-fold with cell lysis reagent, 100 L of substrate was added to 10 L of the diluted solution, and similarly measured with a luminometer. Correction was made by multiplying the measured value by 100.

 The results are shown in FIG. Fig. 5 shows the effectiveness of Tat-Ad in terms of gene expression efficiency. Luciferase activity (RLU / well) in B16BL6 cells is measured between "Ad", "AdR GD" and "Tat-Ad". It is the graph compared by.

 The horizontal axis in Fig. 5 shows the number of virus particles (cells / cell) infected, and the vertical axis shows the luciferase activity value [RLU / well] measured with a luminometer. In the number (vp / cell) area, “Tat-Ad” clearly showed a gene transfer activity much higher than “Ad” and “AdRGD”. In particular, in B16BL6 cells in which CAR expression could not be confirmed, “Tat-8 (1)” showed 400 times as much luciferase activity as “8 (1”), ie, 400 times as much gene transfer activity. Compared with “AdRGD”, which is capable of gene transfer even in cell types where CAR expression cannot be confirmed, the gene transfer activity was significantly higher.

From the results in FIG. 5, it was also confirmed that the gene expression efficiency (activity) of Tat-Ad is seen in a dose-dependent manner (similar to Ad and AdRGD). Based on the above, it was proved that Tat-Ad exhibits high gene transfer activity against B16BL6 cells with low CAR expression as compared to conventional virus vectors.

[0044] (5) Usefulness evaluation of “Tat— Ad” for gene expression efficiency (adherent cells)

 Various types of adherent cells: HeLa cells (human cervical cancer cells), A549 cells (human alveolar carcinoma cells), and HT1080 cells (human fibrosarcoma cells) (all of which can be confirmed to express CAR and integrin) ) And the gene expression efficiency (activity) of “Tat-Ad” was examined by Lucifera Zeatsu.

 Specifically, luciferase assay was performed according to the following procedure.

[0045] [Procedure] lucif erase assay in adherent cells

1) Various cells were seeded at 10 ⁴ cells / well in 500 medium on a 48-well flat bottom plate.

 2) Incubated for 24 hours.

 3) Medium (see Table 3 above for medium for each cell type) was aspirated and 400 μL of medium was newly added.

4) Various vectors diluted to 10 ⁸ or 10 ⁹ vp / ml (“Tat-Ad” was the sample from Experimental Example 1) were added at 100 μL / well.

• 10 ⁷ or 10 ⁸ vp / well

. 10 ³ or 10 ⁴ vp / cell

 5) Incubated for 24 hours.

 6) The medium was aspirated and washed twice with PBS.

 7) 100 μL / well of Cell Culture Lysis Reagent 5x (promega # E1531) diluted with MiliQ water was added.

 8) 10 L of the cell lysate was taken and placed in a lumino tube.

 9) Carry 10 L of reaction substrate (Promega # E1501 Lucif erase Assay System), mix gently by vortexing, and measure RLU (relative light unit) / well with a luminometer.

For samples with RLU exceeding 10 ⁷ , the cell lysis reagent was diluted 100-fold with cell lysis reagent, 100 L of substrate was added to 10 L of the diluted solution, and similarly measured with a luminometer. Measurement It was corrected by multiplying the value by 100.

[0046] The results are shown in FIG. Figure 6 shows the luciferase activity (RLU / well) in various adherent cells (HeLa cells, A549 cells, HT1080 cells) that should evaluate the usefulness of Tat-Ad in terms of gene expression efficiency (activity). This is a graph comparing “Ad”, “AdRGD” and “Tat-Ad”.

 HeLa cells, A549 cells, and HT1080 cells are cell types that can confirm the expression of CAR and integrin, so there is a difference in gene transfer activity between `` 8 (1) '' and `` 8 (11¾ ^) ''. There was a significant difference in the gene transfer activity between “Tat-Ad” and “Ad” and “AdRGD” and “Tat-Ad” (p <0.01).

 It is also shown that “Tat-Ad” has 10 times the luciferase activity of “Ad” in HeLa cells, 30 times in A549 cells and 40 times in HT1080 cells, ie, gene transfer activity. It was.

 From the above, it was proved that Tat-Ad exhibits high gene transfer activity against various adherent cells (HeLa cells, A549 cells, HT1080 cells) as compared with conventional virus vectors. At the same time, Tat-Ad has been shown to be a vector that can achieve a broader range of application of Ad in cancer gene therapy and reduced in vivo dose (ie, reduced side effects).

 [0047] Next, Fig. 7 shows the results of examination of the gene expression efficiency (activity) of "Tat-Ad" in A549 cells, HT1080 cells, and B16BL6 cells as adherent cells by luciferase assay. The experimental procedure is as described above (in each cell).

Fig. 7 shows the luciferase activity (RLU / well) in various adhesion cells (A549 cells, HT1080 cells, B16BL6 cells) that should evaluate the usefulness of Tat-Ad in terms of gene expression efficiency. It is the graph compared between "AdRGD" and "Tat-Ad". A 549 cells and HT1080 cells are both cell types in which the expression of CAR and integrin can be confirmed. However, between "Ad" and "AdRGD" and "Tat-Ad", the gene transfer activity is significant. There was a difference (Ρ <0 · 01). “Tat-Ad” was also shown to have a luciferase activity 30 times higher than that of “Eight luciferase activity (ie, gene transfer activity)” in Α549 cells and 40 times higher in HT1080 cells. On the other hand, in B16BL6 cells, CAR expression cannot be confirmed (integrin expression can be confirmed). Therefore, there was a significant difference between “Ad” WAdRGD and S, “Tat-Ad”. Was able to confirm the gene transfer activity more than “AdRGD” and 500 times the luciferase activity of “Ad”.

 [0048] (6) Usefulness evaluation of “Tat— Ad” for gene expression efficiency (Floating cells)

 In EL cells (mouse thymus-derived T cells) and KG-la cells (human myeloid leukemia cells), the gene expression efficiency (activity) of "Tat-Ad" was measured with Luciferase Atsey (RLU / well) investigated. Note that EL cells are cell types in which the expression of both CAR and integrin can be confirmed, and KG-la cells are cell types in which the expression of both CAR and integrin cannot be confirmed. Specifically, luciferase assay was performed according to the following procedure.

 [0049] [Procedure] lucif erase assay in suspension cells

1) Various cells were seeded on a 48-well flat bottom plate at 10 ⁴ cells / well in 400 medium (see Table 3 above for the medium for each cell type).

 2) Incubated for 24 hours.

3) Various vectors diluted to 10 ⁹ vp / ml (“Tat-Ad” was the sample of Experimental Example 1) were added at 100 L / well.

• 10 vp / well · 10 ⁴ vp / cell

 4) Incubated for 24 hours.

 5) The cell suspension was taken in a 100 HL luminotube.

 6) Bright-Glo (Promega # E2610) was 100: L.

 7) Left at room temperature for several minutes.

 8) After lightly mixing by vortex, RLU (relative light unit) / well was measured with a luminometer.

 [0050] The results are shown in FIG. Fig. 8 shows the luciferase activity (RLU / well) in various floating cells (EL cells, KG-la cells) that should evaluate the usefulness of Tat-Ad in terms of gene expression efficiency (activity). , “AdRGD” and “Tat-Ad”

Since EL cells are cell types that can confirm the expression of CAR and integrin, `` Eight (1) '' and `` Eight (1 Although there was no significant difference in luciferase activity (gene transfer activity) from RGD, “Tat-Ad” had significantly higher luciferase activity than “Ad” and “AdRGD”. The luciferase activity 5 times that of “Ad” was confirmed.

 On the other hand, since KG-la cells are cell types in which neither CAR nor integrin expression can be confirmed, “Ad” and “AdRGD” cannot exhibit sufficient gene transfer activity, but “Tat-Ad” Even in KG-la cells, it was possible to show sufficient gene transfer activity, and it was confirmed to have 10 times the luciferase activity of “Ad”.

 From the above, Tat-Ad is a cell type that has been difficult to transduce with conventional viral vectors (cells with poor expression of both CAR and integrin! / Or! It has been shown that a sufficient gene transfer effect can be exhibited in the species. At the same time, cell types that can be transferred with conventional wineless vectors are also different from conventional ones.

It was also shown that the gene transfer activity can be remarkably high.

[0051] (7) Comparison of gene expression activity (efficiency) by covalently binding “Tat—N HS” to the surface of the viral coat protein used as a gene transfer vector (B16BL6 cells)

In order to evaluate / examine the effect of gene expression activity (efficiency) by covalently binding “Tat—NHS” to the surface of the outer shell protein of the viral vector before gene transfer, we used B16BL6 cells. “Tat—Eight (1)” and “Ding & 1 peptide mixed Ad (non-NHS group-attached Tat peptide and Ad adsorbed non-specifically)” (Luciferase Atasei) (See Fig. 9).

 As “Tat-Ad”, the sample of [Experimental Example 3] is used, and the experimental procedure (luciferase, etc.) and the sample “Tat-Ad” are as described above. In addition, “Ad” and “AdRGD” were also used as comparison targets of gene expression activity.

[0052] FIG. 10 shows the results of the study using luciferase atsey.

 Figures 9 and 10 show the efficiency of gene expression (activity) in luciferase in B16BL6 cells to evaluate the usefulness of covalently binding to the surface of the adenovirus coat protein (positively) before gene introduction. It is the graph compared with "Ad", "AdRGD", "Tat pep tide mixed Ad", and "Tat-Ad" by Atsey.

As is clear from Fig. 10, "Ding & 8 (1) is Lucifera of" Ding & 1 peptide mixed Ad ". Remarkably higher strength than the activity.

 This indicates that the gene transfer activity is remarkably increased as compared with the case where “Tat-Ad” is simply mixed with the virus used in the vector (Ad in this case). In other words, by co-binding “Tat-NHS” to the surface of the outer shell protein of the virus used as a vector before gene transfer, the conventional virus vector, or simply the intracellular translocation peptide and virus vector can be combined. It was shown that the gene transfer activity was remarkably enhanced as compared with the case of just mixing.

 From the above, it was proved that “Tat-NHS” chemically bound to the surface of the virus used as a vector was involved in the enhancement of gene expression.

Brief Description of Drawings

[Fig. L] Diagram showing Ad and AdRGD invasion mode in relation to receptors

Yes

 [Fig. 2] A diagram showing the intracellular entry mode of Ad, AdRGD and Tat-modified Ad in relation to receptors.

 FIG. 3 shows a specific example of Tat-modified Ad production.

 [Fig. 4] Confirmation of binding between Ad and Tat-NHS by SDS-PAGE.

 FIG. 5 is a graph comparing gene expression efficiency in B16BL6 cells with “Ad, AdRGD and Tat-modified Ad”.

 FIG. 6 is a graph comparing gene expression activity between “Ad, AdRGD and Tat-modified Ad” in various adherent cells (HeLa cells, A549 cells, HT1080 cells).

 FIG. 7 is a graph comparing gene expression activity between “Ad, AdRGD and Tat-modified Ad” in various adherent cells (A549 cells, HT1080 cells, B16BL6 cells).

 [Fig.8] Gene expression efficiency in various floating cells (EL cells, KG-la cells)

It is the graph compared between "RGD and Tat modification Ad".

 FIG. 9 is a schematic diagram showing “Tat peptide-mixed Ad” and “Tat-modified Ad”.

 [Fig. 10] Gene expression activity is expressed as “Ad, AdRGD, Tat peptide-mixed Ad and Tat modification.

It is the graph compared with Adj.

Claims

 The scope of the claims  [I] A gene introduction adjuvant comprising an intracellular translocation peptide as an active ingredient, wherein an NHS (N-hydroxysuccinimidyl) group is bound to the intracellular translocation peptide.  [2] The gene transfer adjuvant according to claim 1, wherein the intracellular translocation peptide is a Tat peptide.  [3] A gene transfer aid containing an intracellular translocation peptide represented by any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 as an active ingredient, wherein NHS is added to the intracellular translocation peptide. A gene transfer adjuvant characterized by linking a group.  [4] In the amino acid sequence of the intracellular translocation peptide, any one force selected from the group consisting of amino acid sequences in which one or more amino acids are deleted, substituted, inserted or added The gene transfer adjuvant according to claim 3.  [5] The gene transfer adjuvant according to any one of [1] to [4], wherein the gene transfer aid is covalently bound to the surface of a viral outer shell protein used as a gene transfer vector.  6. The gene transfer adjuvant according to claim 5, wherein the virus used as the gene transfer vector is an adenovirus.  [7] The covalent bond between the gene transfer aid and the virus used as the gene transfer vector is performed under conditions of 10 to 50 ° C, 100 to 1000 rpm, 5 to 60 minutes. The gene transfer adjuvant according to claim 5 or 6. [8] The gene transfer adjuvant according to any one of claims 1 to 7, wherein the cell type to be transferred is an adherent cell or a floating cell. [9] The gene transfer adjuvant according to any one of [1] to [8], wherein the cell type to be transferred is a cell type in which expression of both CAR and integrin is poor or deficient. [10] The gene transfer adjuvant according to any one of [1] to [9], which is in the form of a lyophilized powder.  [I I] The gene transfer adjuvant according to any one of claims 1 to 10, which is used for gene therapy. [12] A viral vector for gene transfer characterized in that, in the stage before gene transfer, the gene transfer auxiliary agent S according to any one of claims 1 to 11 is covalently bound to the outer protein surface. . 12. A gene introduction method characterized in that, in the stage before gene introduction, the gene introduction auxiliary agent according to any one of claims 1 to 11 is covalently bound to the surface of the outer protein of a virus used as a gene introduction vector. .