WO2008068982A1 - Adjuvant for gene transfer comprising cell migration peptide as the active ingredient and gene transfer method using the adjuvant for gene transfer - Google Patents

Abstract

[PROBLEMS] To enable gene transfer even in some cancer cells, hemocytes and so on wherein CAR and integrin are both little or not expressed. [MEANS FOR SOLVING PROBLEMS] An adjuvant for gene transfer which comprises a cell migration peptide as the active ingredient, characterized in that a chemical linker has been attached to the cell migration peptide; and a gene transfer method characterized by comprising covalently bonding the adjuvant for gene transfer as described above to the surface of the coat protein of a virus which is to be used as a gene transfer vector. As the above-described chemical linker, it is preferable to use MHS (6-maleimidohexanoic acid N-hydroxysuccinimide ester) having an NHS (N-hydroxysuccinimidyl) group, etc.

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 earnest research, the present inventors have used a virus in which a chemical linker is bound to an intracellular translocation peptide such as a Tat peptide as a gene transfer aid and used as a gene transfer vector. By covalently binding to the outer shell protein, the range of application can be expanded to hematopoietic cancer, etc. where gene transfer has been difficult (with conventional gene transfer vectors), and regardless of cell type (receptor expression The present inventors have found that the gene transfer activity is very strong (with or without) and 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, characterized in that a chemical linker is bound to the intracellular translocation peptide. It relates to an introduction aid.

 The invention according to claim 2 relates to the gene transfer auxiliary agent according to claim 1, which has the NHK (N-hydroxysuccinimidyl) group.

 The invention according to claim 3 relates to the gene transfer auxiliary agent according to claim 2, which is MHS (6-maleimidohexanoic acid N-hydroxysuccinimide ester) having the NHS group.

 The invention according to claim 4 relates to the gene transfer auxiliary agent according to claim 2, wherein the intracellular translocation peptide is a Tat peptide.

The invention according to claim 5 relates to the gene introduction auxiliary agent according to claim 2, wherein the intracellular translocation peptide is a peptide consisting of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5. . The invention according to claim 6 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. The gene transfer adjuvant according to claim 5, which is one force. The invention according to claim 7 relates to the gene transfer aid according to any one of claims 1 to 6, 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 8 relates to the gene transfer auxiliary agent according to claim 7, wherein the virus used as the gene transfer vector is adenovirus.

 The invention according to claim 9 is the condition 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 adjuvant according to claim 7 or 8, which is performed under the following conditions.

 The invention according to claim 10 relates to the gene transfer auxiliary agent according to any one of claims 1 to 9, wherein the cell type to be transferred is an adherent cell or a floating cell.

 The invention according to claim 11 relates to the gene transfer auxiliary agent according to any one of claims 1 to 10, wherein the cell type to be transfected is a cell type in which expression of both CAR and integrin is poor or deficient. .

 The invention according to claim 12 relates to the gene transfer auxiliary agent according to any one of claims 1 to 11, which is in the form of a freeze-dried powder.

 The invention according to claim 13 relates to the gene transfer auxiliary agent according to any one of claims 1 to 12, which is used for gene therapy.

 In the invention according to claim 14, in the stage before gene introduction, the gene introduction auxiliary agent according to any one of claims 1 to 13 is bound to the surface of the outer shell protein of a virus used as a gene introduction vector. A virus vector for gene transfer characterized in that

Yes

 In the invention according to claim 15, the gene introduction auxiliary agent according to any one of claims 1 to 13 is covalently bound to the surface of the outer protein of a virus used as a gene introduction vector in a stage before gene introduction. The present invention relates to a gene introduction method characterized by the above.

The invention's effect [0010] The "gene transfer aid comprising an intracellular translocation peptide bound with a chemical linker as an active ingredient" according to the present invention is covalently bound to the surface of the outer shell protein of a virus used as a gene transfer vector. Is a viral vector characterized by poor expression of some cancer cells and hematopoietic cells (both CAR and integrin) that were difficult to transduce with conventional gene transfer vectors (Ad, AdRGD, etc.)? In addition, sufficient gene transfer effects can be exerted even on cell types that are deficient ("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 (“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] The present invention relates to an agent for introducing a chemical linker bound to an intracellular translocation peptide such as a Tat peptide as a gene transfer aid (in the stage before gene transfer). By covalently binding to proteins, the application range is expanded to some cancer cells and blood cells that have been difficult to introduce genes (cell types in which expression of both CAR and integrin is poor or defective). At the same time, it is based on the knowledge that gene transfer activity is very strong (even in cell types that could be transferred with conventional virus 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, but terms used in the present specification will be described. Unless otherwise stated, the meanings commonly used in the field are intended, and the expression of the singular includes the concept of the plural unless specifically stated otherwise.

[0012] First, "intracellular translocation peptide (PTD)" which is an essential component of the gene transfer adjuvant according to the present invention 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.

[0013] [Table 1]

Typical examples of PTD

 Peptide origin sequence

 Tat HIV-1 GRKKRRQ RPPQ

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

 Antennapedia Drosop ila QIKIWFQNRRMK KK

 Arg Gin lie Lys lie Trp Phe Gin Asn Arg Arg Met Lys Trp Lys Lys

 Rev HIV-1 TQRARRNRRRRWRERQR

 Thr Gin Arg Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Giu Arg Gin Arg

 VP22 HSV NAKTRRHERRRKLAIE

 Asn Ala Lys Thr Arg Arg His Glu Arg Arg Arg Lys Leu Ala lie Glu Arg

Ho —

 Arg Arg Arg Arg Arg Arg Arg Arg

 R, K, 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), R8 peptide (SEQ ID NO: 5), etc., the ability to enumerate S, the effects of the present invention described above (expansion of application range, gene transfer efficiency It is best to use the Tat peptide in order to achieve improvement. The Tat peptide is derived from a transcriptional activator of HIV and is a peptide combination containing many basic amino acids. IJ Gly—Arg—Lys—Lys—Arg—Arg—Gin—Arg—Arg—Arg—Pro—Pro—Gln 』. Power,, Among intracellular translocation peptides, it has a particularly high intracellular translocation activity and is also used as a carrier for introduction into cells into proteins, ribosomes and the like.

 R8 peptide is also a homopolymer of arginine, also called Octa arginine peptide, which is similar to STat peptide in efficiency and uptake mechanism, and has the same effect in Ad infection, so it can be a candidate for replacing Tat peptide. .

 In addition to the typical intracellular translocation peptides shown in [Table 1], one or more of these amino acid sequences (SEQ ID NOs: 1 to 5) can be used as long as they have an intracellular translocation function (membrane permeation function). Is an active ingredient of the gene transfer aid according to the present invention, even if it has an amino acid sequence deleted, substituted, inserted or added.

 The present invention relates to the outer shell of a virus used as a gene transfer vector, using a chemical linker linked to the intracellular translocation peptide represented by the above-described Tat peptide as an auxiliary for “gene transfer”. By covalently binding to proteins, the application range is expanded to some cancer cells and blood cells that have been difficult to transduce, and at the same time, “gene transduction efficiency” and “gene transduction activity” itself (more More)

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” using a vector. In other words, the function of the introduced gene (for example, in the case of an expression vector, expression of the encoded protein and / or activity of the protein) is detected as an index.

[0016] As shown in the examples below, the present inventors have also described that "the compound having an NHS (N-hydroxysuccinimidyl) group (chemical linker)" as the chemical linker in the Tat peptide, particularly "NH ≥> A gene transfer vector (hereinafter referred to as “Tat peptide”) covalently linked to the outer shell protein of an adenovirus vector using 2 MHc3 (6-maleimidohexanoic acid N-hydroxysuccmimide ester) as a gene transfer aid. ”Modified Ad” (sometimes referred to as “modified Ad”), but it is clear that it exhibits about 10 to 500 times stronger gene expression activity compared to conventional Ad regardless of CAR negative and positive cells, and the present invention was completed. It came to be.

 The method for binding the “chemical linker having an 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. An example of a method for binding MHS, which is a chemical linker having an NHS group, to Tat will be described later in Examples.

 The gene transfer auxiliary agent of the present invention has a chemical substance having an NHS group in the intracellular translocation peptide as an active ingredient (specifically, when the intracellular translocation peptide is a Tat peptide, a cysteine group inserted into the C terminus thereof). By binding the “linker”, it gives a stronger intracellular translocation activity (to the coupled intracellular translocation peptide) and is strong regardless of the presence of receptors (CAR, integrins, etc.). Gene expression activity can be exhibited.

[0017] The above-described "gene transfer adjuvant containing an intracellular translocation peptide bound with a chemical linker as an active ingredient" according to the present invention can be changed or modified as appropriate. In particular, it belongs to the technical scope of the present invention. For example, as chemical linkers that bind to intracellular translocation peptides, in addition to MHS (6-maleimidohexanoic acid N hydroxysuccinimide ester), dicyclohexyl carpositimide (DCC), maleimide benzoyl N hydroxy List succinimide (MBS), N-ethyloxycarbonyl —2-ethyloxy-1,2-dihydroquinoline (EEDQ), N-isobutyloxy-1,2-isobutyloxy 1,2-dihydroquinoline (IIDQ) can do. However, the effects of the present invention described above (expansion of application range, improvement of gene transfer efficiency, etc.) are exhibited. It is desirable to use “chemical linkers with NHS groups (MHS, etc.)”

Yes

 [0018] The gene transfer adjuvant of the present invention comprising a chemical linker, particularly a chemical linker having an NHS group (such as MHS) bound to an intracellular translocation peptide such as a Tat peptide as an active ingredient is a gene transfer vector. Some cancers that were difficult to transduce with conventional viral vectors (such as Ad and AdRGD) by covalently binding to the surface of the outer protein of the “virus (inactivated)” used A sufficient gene transfer effect can be exerted on cells and blood cells.

 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 viridae, herpesviridae, baculoviridae and The ability to include viruses belonging to a family selected from the group consisting of the family Hepadnaviridae S, and the virus more preferably used 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 bonding force is desirable. Here In the gene transfer aid of the present invention, MHS having an NHS group is bound to the intracellular translocation peptide! /, So it is easy to covalently bind to a viral vector (due to the reactivity of the NHS group). (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, in the stage before gene introduction, The gene transfer virus vector in which the gene transfer 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 transfer vector has a gene transfer efficiency of about 10 to 500 times. For example, when applied clinically as a gene therapy vector, since it can be clinically applied at a low dose, 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.) ” From the point of view) LV, the 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 virus vector for gene introduction combined with the adjuvant 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.

Yes

 [0026] As described above, it is possible to make a force change or deformation specially described for "a gene transfer aid containing a chemical linker, particularly a chemical linker having an NHS group (such as MHS) bound to an intracellular transit peptide" as an active ingredient. These belong to the technical scope of the present invention. In addition, the gene transfer aid of the present invention can be applied to any Winores betater that is currently widely used, 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. , Using a viral vector covalently linked to the above gene transfer aid Compared with the conventional Ad that expresses the gene's ability to infect 'dependently in a CAR-dependent manner, this method makes use of the intracellular translocation activity of the intracellular translocation peptide. It is a gene transfer method based on a new idea to increase gene transfer efficiency by making a rapid transition. 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 (TPA), factor VIII : Genes encoding C, canorecitonin, thymidine kinase, interferon, granulocyte macrophage (GMCSF), erythropoietin (EPO), hepatocyte growth factor (HGF), and the like, but are not limited thereto. 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 1

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

 As a gene transfer aid that is useful in the present invention, a chemical linker having an NHS group (specific example in this example is MHS (6-maleimidohexanoic acid N hydroxysuccinimide ester) is bound to the Tat peptide. (Referred to as “Tat-NHS”) (see [Fig. 3]) and stored in a frozen state.

 The Ad dilution was added to the frozen Tat-NHS solution to dissolve Tat-NHS. After that, by mixing with a vortex mixer and incubating under conditions of 37 ° C, 300rpm, 30min, the adenoviral vector for gene transfer (hereinafter referred to as "Tat Ad") bound to "Tat_NHS" It was fabricated (see [Fig. 4]).

 Specific examples (experimental examples;! To 5) of “Tat-NHS” and “Tat Ad” are shown below.

[Experiment 1]

 i) Tat-NHS

 Purified peptide containing the amino acid sequence of Tat peptide (Ac—GRKKRRQRRRPPQ—GC-NH hydrochloride) (15.6 a mol) 30 mg¾PBS (pH 7.2) 1.2 mL was dissolved. That

 2

 The lysate was mixed with 4.6 mg of MHS (15.6 μmol) dissolved in 12 L of dimethyl sulfoxide (DMS) and stirred for 30 minutes. The product (Ac—GRKKRRQRRRPPQGC—M HS) was immediately stored frozen at 80 ° C. and used in subsequent experiments.

[Experiment 2]

 i) Tat-NHS

 Amino acid sequence: Ac-GRKKRRQRRRPPQ— GC— MHS

 Molecular weight: about 2000

Concentration (amount): 5mg / 50 _i L / tube = 2.5 X 10— ⁶ mol / tube

 = 1.5 x 10 molecules / tube

 ii) Reaction conditions

Lysine residues present in the outer shell protein of Ad [7500 / vp (virus particle)]: “Tat— N HS” = 1: 1000 Necessary number of Ad particles is 2X10 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 3)

i) Tat-NHS

 Amino acid sequence: Ac-GRKKRRQRRRPPQ— GC— MHS

 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

→ lX10 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 4]

 i) Tat -NHS

 Amino Acid Sequence: Ac-GRKKRRQRRRPPQ— GC— MHS

 Molecular weight: about 2000

7.5X 10 molecules/ tube Concentration (amount):

7.5X 10 molecules / tube

 ii) Reaction conditions

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

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

• Ad (2.5X10 ¹² vp / ml): 40 / iL

 • PBS: 860

 • Tat -NHS: lOO ^ L

→ lX10 ^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.

[Experiment 5]

 i) Tat-NHS

 Amino acid sequence: Ac-GRKKRRQRRRPPQ— GC— MHS

 Molecular weight: about 2000

3.125 X 10— ⁷〜5 X 10— ol /tube =1.875X10¹⁷〜3X10¹⁸molecules/tube ii)反応条件 Concentration (amount):

3.125 X 10— ^{7 to} 5 X 10— ol / tube = 1.875X10 ^{17 to} 3X10 ¹⁸ molecules / tube ii) Reaction conditions

 Lysine residues present in the outer protein of Ad [7500 / vp (virus particle)]: Tat— NH S = l: 12.5, 1:25, 1: 50,1: 100, 1: 250, 1: 500, 1: 1000, 1: 2000

React 2 x 10 ^u vp / ml Ad solution with Tat-NHS.

iii) "Ad", "Tat- NHS" suspension (composition 1:25)

• Ad (2.7X10 ¹² vp / ml): 59.3 L

 • PBS: 740.7 L

 • Tat—NHS (2.511¾ / 50): 2 L

→ 2X10 ^U vp / ml

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, incubate under conditions of 37 ° C, 300rpm, 30min. Eight types with different Tat modification rates (lysine residues: Tat—NHS = 1: 12.5, 1:25, 1:50, 1: 100, 1: 250, 1: 500, 1: 1000, 1: 2000).

 (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, 2X101 ⁽ Vp (virus particle) “Ad” and “Tat-8” were concentrated using an evaporator. After that, loading buffer + 2ME was mixed at 95: 5, and the suspension ( 15 μL was added to 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, “Tat-Ad” is the above [Experimental Example 3 The “marker” was “FULL RANGE RAINBOW (Amersham Bioscience # RPN800)”.

The results are shown in [Figure 5]. As is apparent from the gel image in [Fig. 5], the molecular weight of hexon increased, suggesting Tat-NHS binding to the Ad surface. That is, Tat peptide repair The decorated 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.

 [0039] (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.

 [0040] [Table 2]

 [0041] The usefulness of “Tat—Ad” was evaluated from the viewpoint of “Gene transfer target (/ application area)” and “Gene expression efficiency (/ Gene expression activity)”. It was. In the above evaluation / examination, as a comparison target of Tat-Ad, normal adenovirus vector (Ad), improved Ad with integrin directivity (AdRGD) and Tat peptide without NHS group were added. A vector (Tat peptide mixed ad) adsorbed non-specifically by mixing Ad was used.

 The cell types used for the evaluation / examination were force S, which are adherent cells and suspension cells, specifically, cultured cells listed in [Table 3] below.

[0042] [Table 3] Passage cyst name origin Culture medium

 method

 A549 Human alveolar carcinoma DME + G¾ FBS trypsin HT1080 G Fibrosarcoma MEM + 10¾ FBS EDTA / P8S

He a human cervical cancer MEM + 10¾ FBS trypsin

 Adherent cells B 1 6BL6 Mouse Melanoma MEM + 7.5% FBS EDTA / PBS A 264J Mouse Macphage DMEM + 10% FBS EDTA / P8S CT26 Mouse Colon cancer RP I1640 + 10% FBS trypsin

KG-1a human acute myeloid leukemia cells RPMI1640 + W% FBS + 2ME ailute

 E 4 mice Lymphoma PMI1640 ÷ 10% FBS + 2M £ dilute Suspension cells U937 Human globular gland lymphoma RP I1640 + 10% FBS dilute

[0043] (4) Usefulness evaluation of “Tat-Ad” for gene transfer efficiency (B16BL6 cells)

 Use the EGF P (Ennanced green fluorescent protein) regressor to determine the efficiency of gene transfer (activity) of “Tat-Ad” or “Ad” in B16BL6 cells.

 Specifically, EGFP gene introduction activity was observed according to the following procedure.

[0044] [Procedure] EGFP gene transfer in B 16BL6 cells

To B16BL6 cells (10 ⁴ cells / well), “Tat-Ad” or “Ad” of lOOOOvp / cell loaded with EGFP gene was introduced. After 24 hours of incubation, the introduction of the EGFP gene into these cells was observed using a fluorescence microscope (BZ-8000).

 As “Tat-Ad”, “Tat-Ad” having one modification rate (1:25) among the samples obtained in Experimental Example 5 was used.

[0045] The results are shown in FIG. Fig. 6 is a graph comparing the observation of GFP fluorescence by EGFP gene transfer in B16BL6 cells with "Eight (1)" and "Tat-Adj" for evaluating the usefulness of Tat-Ad for gene transfer efficiency.

 From Fig. 6, it was confirmed that GFP fluorescence was not observed at all for Ad unmodified with Tat, whereas a large amount of fluorescence was observed with Tat-Ad, confirming high EGFP gene transfer.

 [0046] (5) 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. I examined Yotsuka.

In addition, whether or not the gene expression efficiency (activity) of Tat—Ad is seen in a dose-dependent manner. Even examined. Specifically, luciferase assay was performed according to the following procedure.

[0047] [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) 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 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 4 above) 100 HL / well (ie, 300 vp / cell, lOOOOvp / 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. Figure 7 shows the effectiveness of Tat-Ad in terms of gene expression efficiency. The 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. 7 shows the number of virus particles / 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, CAR expression In B16BL6 cells, “Tat-8 (1)” showed 400-fold luciferase activity compared to “8 (1”), that is, 400-fold gene transfer activity. It was not! /, And even when compared to “AdRGD”, which is capable of gene transfer even for cell types, the gene transfer activity was much higher! /.

 From the results shown in FIG. 7, it was also confirmed that the gene expression efficiency (activity) of Tat-Ad was observed 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.

[0049] (6) Assessing the relationship between gene expression efficiency and Tat-Ad modification rate (B16BL6 cells)

 In B16BL6 cells, the gene expression efficiency (activity) of “Tat-Ad” with different Tat modification rates was examined by luciferase assay. That is, the relationship between the Tat modification rate and the gene expression activity was examined.

[0050] In addition, as "Tat-Ad", the eight types of modification ratios obtained in Experimental Example 5 (1: 12.5, 1: 2 5, 1:50, 1: 100, 1: 250, 1: 500, 1: 1000, 1: 2000). The experimental procedure (luciferase, etc.) and the sample “Tat-Ad” are as described above. “Ad” was also used as a comparison target of gene expression activity. However, the amount of various vectors (“Tat-Ad” and “Ad”) was only lOOOvp / cell.

[0051] The results are shown in FIG. Figure 8 shows the luciferase activity (RLU / well) in B16BL6 cells that evaluate the relationship between the gene expression efficiency and the Tat modification rate of “Tat—Ad”. It is the graph compared between "Ad".

The horizontal axis in FIG. 8 represents each sample, ie, “Ad” and “Tat-Ad” having eight different modification rates, and the vertical axis represents the luciferase activity value [RLU / well] (average soil SD) measured with a luminometer. ( _n = 4)).

Figure 8 shows that Tat-Ad produced with a modification ratio of 1:25 (lysine residue: Tat-NHS) showed the highest gene transfer activity, and a Tat-Ad vector with a high modification ratio (1: 100 to 1: 2000). In this case, the gene transfer activity was lost. The detailed cause of this disappearance is unknown. It was speculated that one of the steps was hindered by the gene-transduction mechanism inherent to Ad by excessive Tat peptide modification! Based on the above, it was confirmed that Ad modified with Tat with an NHS group as the active group has high gene transfer efficiency, and the gene transfer efficiency is greatly influenced by the Tat modification rate.

[0052] (7) 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.

[0053] [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 an RLU exceeding 10 ⁷ , dilute the cell lysate 100 times with cell lysis reagent. Then, 100 L of the 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.

[0054] The results are shown in FIG. Figure 9 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).

 [0055] Next, Fig. 10 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). Figure 10 shows the luciferase activity (RL U / well) in various types of adherent cells (A549 cells, HT1080 cells, B16BL6 cells) for which the usefulness of Tat-Ad is evaluated for gene expression efficiency. It is the graph compared between "AdRGD" and "Tat-Ad".

A549 cells and HT1080 cells are both cell types that can confirm the expression of CAR and integrin. The strengths between Ad, AdRGD, and Tat-Ad are significant in their gene transfer activity. There was a difference (Ρ <0 · 01). “Tat-Ad” is 30 times the luciferase activity (ie, gene transfer activity) of “Ad” in Α549 cells and 40 times in HT1080 cells. It has also been shown to have luciferase activity.

 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”.

[0056] (8) Assessing the relationship between gene expression efficiency and Tat modification rate of “Tat—Ad” (adherent cells)

 As various types of adherent cells, RAW264.7 cells (macrophage cells in which CAR expression cannot be confirmed and integrin expression can be confirmed) and CT26 cells (carcinoma in which CAR expression can be confirmed at low levels and integrin expression can be confirmed) Cell)), “Tat-Adj gene expression efficiency (activity) with different Tat modification rates was examined by luciferase assay.

[0057] As "Tat-Ad", three types of modification rates (1: 12 · 5, 1:25, 1:50) of the samples obtained in the above experimental example 5 were used. Eight (1) was used.

 The experimental procedure (luciferase, etc.) and the sample “Tat-Ad” are as described above. “Ad” was also used as a comparison target of gene expression activity. However, the amount of various vectors (“Tat-Ad” and “Ad”) was only lOOOOvp / cell.

[0058] The results are shown in FIG. Figure 11 shows the relationship between gene expression efficiency and Tat-modification rate of “Tat-Ad”. Luciferase activity (RLU / 7) in various adherent cells (RAW264. 7 cells and CT2 6 cells) with different CAR expression levels. is a graph comparing “Ad” and “Tat-Ad” having different Tat modification rates.

The horizontal axis in FIG. 11 represents each sample, that is, “Ad” and three types of “Tat-Ad” with different modification rates, and the vertical axis represents the luciferase activity value [RLU / well] (average soil) measured with a noremeter. SD ( _n = 4)).

 Figure 11 shows that even in RAW264. 7 cells and CT2 6 cells where the expression of CAR is not confirmed or low, “Tat—Ad” made with a modification ratio of 1:25 (lysine residue: Tat—NHS) is the most. The gene transfer activity was high and several hundred times higher than that of “Ad”.

Based on the above, Ad modified with Tat to which an MHS group is added as an active group has a higher gene transfer activity against various types of adherent cells (RAW264.7 cells and CT26 cells) than conventional virus vectors. Proved to show. At the same time, MHS group grant Tat—Ad It has been shown that this is a vector that can achieve a wide range of application of Ad in child therapy and dose reduction (ie, side effect reduction) in vivo.

 [0059] (9) 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.

 [0060] [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.

 [0061] The results are shown in FIG. Fig. 12 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 in which the expression of CAR and integrin can be confirmed, there was no significant difference in luciferase activity (gene transfer activity) between “Eight (1)” and “Eight (1 RGD)” However, “Tat—Ad” is much higher than “Ad” and “AdRGD”. As a result, 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.

[0062] (10) Assessing the relationship between the gene expression efficiency and the Tat modification rate of “Tat—Ad” (floating cells)

 In U937 cells (tissue spherical lymphoma cells in which CAR expression cannot be confirmed and integrin expression can be confirmed) in suspension cells, the expression efficiency (activity) of “Tat-Ad” with different Tat modification rates can be changed to luciferase assembly. And examined.

[0063] As "Tat-Ad", three types of modification ratios (1: 12 · 5, 1:25, 1:50) of the samples obtained in Experimental Example 5 above were used. Eight (1) was used.

 The experimental procedure (luciferase, etc.) and the sample “Tat-Ad” are as described above. “Ad” was also used as a comparison target of gene expression activity.

[0064] The results are shown in FIG. Figure 13 shows the luciferase activity (RLU / well) in U937 cells, which are the floating cells to evaluate the relationship between the gene expression efficiency and the Tat modification rate of “Tat-Ad”. It is the graph compared between different "Tat-Ad". In FIG. 13, the horizontal axis represents each sample, ie, “Ad” and “Tat-Ad” with three different modification rates, and the vertical axis represents the luciferase activity value [RLU / well] (average soil) measured with a noreminometer. SD ( _n = 4)).

 Figure 13 shows that in U937 cells where CAR expression cannot be confirmed, “Tat-Ad” produced at a modification ratio of 1: 12.5 (lysine residue: Tat—NHS) is about 10 times higher than “Ad”. Showed activity.

Based on the above, Ad modified with Tat with NHS group as the active group is It has been proved that the gene transfer effect can be sufficiently exerted on floating cells (U937 cells) compared to conventional virus vectors.

[0065] (11) 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. 14).

 As “Tat-Ad”, the sample of [Experimental Example 4] 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.

[0066] FIG. 15 shows the results of the study by luciferase atsey.

 Figures 14 and 15 show the luciferase in B16BL6 cells to evaluate the usefulness of the gene expression efficiency (activity) by covalently binding to the surface of adenovirus outer shell protein (actively) before gene introduction. It is the graph compared between "Ad", "AdRGD", "Tat ptide mixed Ad", and "Tat-Ad" by Atsey.

 As is clear from FIG. 15, “Ding & 8 (1)” was significantly higher than the luciferase activity of “Ding & 1 peptide mixed Ad”.

 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.

[0067] (12) On the surface of the outer protein of the virus used as a gene transfer vector, “Tat— Comparison of gene expression activity by changing the modification rate of NHS and covalently binding (B16BL 6 cells)

 Evaluate 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. investigated. That is, in B16BL6 cells, “Ding & 8” (1) and “Ding & 1 peptide mixed Ad” with different Tat modification rates (or mixing ratios) were compared by luciferase assembly (see FIG. 14). .

 As “Tat-Ad”, two types of modification ratios (1: 12 · 5, 1: 25) of the samples obtained in Experimental Example 5 above are used. “Tat peptide mixed Ad” is a lysine residue (7500 / vp) in the outer shell protein of Tat that does not have MHS added: Tat = l: 25, 1: 100, 1: 1000 Ad that was calorized with the amount of peptide was 37. C, incubated for 40 min.

 The experimental procedure (luciferase, etc.) and the sample “Tat-Ad” are as described above. “Ad” was also used as a comparison target of gene expression activity.

[0068] FIG. 16 shows the results of the study by luciferase assay.

 Figure 16 shows the gene expression efficiency (activity) obtained by covalently binding “Tat—NHS” to the surface of adenovirus outer shell protein (positively) at different Tat modification rates before gene transfer. The effect of gene expression should be evaluated. By luciferase analysis in B16BL6 cells, comparison was made between “Ad”, “Tat-Ad” and “Tat peptide mixed Ad” with different Tat modification rate (or mixing rate), respectively. It is a graph.

 As is clear from Fig. 16, “Tat—Ad” is more than the luciferase activity of “Tat peptide mixed Ad” with three Tat mixing ratios at any Tat modification rate (1: 12 · 5, 1:25). Remarkably high.

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

[0069] (13) Examination of Tat—Ad's ability to avoid neutralizing antibodies

Whether Ad has a surface covered by Tat modification! Was examined.

 Specifically, anti-Ad serum was prepared according to the following procedure, and the gene expression activity of “Tat-Ad” in the presence of serum was examined by luciferase assay.

[0070] Anti-Ad Serum Production Method

BALB / c mice were given 5 X 10 ^{1 (} Vp “Ad” (unmodified Ad) intravenously, and 2 weeks later, the same amount was intravenously administered. Two weeks after the second administration, mouse serum Was collected and stored frozen at 80 ° C.

 [0071] Luciferase activity in A549 cells in the presence or absence of anti-Ad serum

 In A549 cells, the gene expression efficiency (activity) of “Ad” and “Tat-Ad” in the presence of anti-Ad serum diluted 3200 times and 12800 times and in the absence of anti-Ad serum was examined by luciferase assay.

 As “Tat-Ad”, “Tat Ad” having one modification rate (1:25) of the samples obtained in Experimental Example 5 was used.

 The experimental procedure (luciferase, etc.) and the sample “Tat Ad” are as described above. “Ad” was also used as a comparison target of gene expression activity. The amount of each vector (“Tat-Ad” and “Ad”) was only lOOOOvp / cell.

[0072] The results are shown in FIG. Figure 17 shows the luciferase activity (RLU / well) in A549 cells in the presence and absence of anti-Ad sera to evaluate the usefulness of Tat Ad for gene expression efficiency. It is the graph compared between "Tat-Ad".

 The horizontal axis in Fig. 17 shows the presence or absence of anti-Ad serum (by dilution concentration), and the vertical axis shows the luciferase activity ratio (%, activity in the absence of anti-Ad serum as 100% measured with a luminometer. Show). From these results, “Ad” showed a markedly decreased gene expression activity in the presence of anti-Ad serum (neutralizing antibody), whereas “Tat Ad” showed a relatively high activity.

 From the above, it was revealed that Tat Ad has the ability to avoid neutralizing antibodies.

[0073] Next, Ad modified with an R8 peptide (hereinafter referred to as "R8-Ad") as an intracellular transit peptide (PTD) other than the Tat peptide will be described.

Example 2 [0074] (1) Preparation of R8 peptide-modified Ad

 As a gene transfer aid that is useful for the present invention, an MHS having NHS group (6-maleimidohexanoic acid N-hydroxysuccinimide ester) bound to R8 peptide in the same manner as Tat-NHS ( (Hereinafter referred to as “R8—NHS”) and stored in a frozen state.

 Using the frozen R8-NHS solution, a “R8_NH Sj-bound adenoviral vector (hereinafter referred to as“ R8-Ad ”) was prepared in the same manner as“ Tat-Ad ”.

 “R8—Ad” was prepared by lysine residue [7500 / vp]: “R8—N HS” = 1: 25 present in the outer shell protein of Ad.

[0075] (2) Usefulness evaluation of “R8—Ad” for gene expression efficiency (B16BL6 cells)

 In B16BL6 cells (a cell type in which CAR expression cannot be confirmed and integrin expression can be confirmed)! I reviewed it.

 The experimental procedure (luciferase, etc.) and the sample “R8-Adj are as described above.“ Tat-Ad ”and“ Ad ”were also used as comparison targets for gene expression activity. However, the amount of each vector (“R8—Ad”, “Tat—Ad” and “Ad”) was set to ΙΟΟΟΟνρ / cell only.

 The results are shown in FIG. Fig. 18 shows the luciferase activity (RLU / well) in B16BL6 cells, which evaluates the usefulness of R8-Ad in terms of gene expression efficiency. Ad, Ta1-8 (1) and ¾8-8 ( It is a graph compared between "1".

 The horizontal axis in FIG. 18 represents each sample, that is, “Ad”, “Tat-Ad” and “R8-Ad”, and the vertical axis represents the luciferase activity value [RLU / well] (average soil SD ( n = 4)).

FIG. 18 shows that although R8-Ad has a gene transfer activity that is slightly inferior to that of Tat-Ad, it has a luciferase activity that is more than 10 times that of, ie, a gene transfer activity. As with Tat-Ad, it was revealed that gene transfer into cells with low CAR expression was possible. 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 is a flow diagram showing a process of binding MHS having an NHS group to Tat and modifying Ad with Tat.

 FIG. 4 shows a specific example of Tat-modified Ad production.

 [Fig. 5] Confirmation of binding between Ad and Tat-NHS by SDS-PAGE.

 [Fig.6] GFP fluorescence by EGFP gene transfer in B16BL6 cells, Ad and Tat—

It is the figure comparatively observed by Ad.

 FIG. 7 is a graph comparing gene expression efficiency in B16BL6 cells with “Ad, AdRGD and Tat modified Ad”.

 FIG. 8 is a graph comparing gene expression efficiency in B16BL6 cells between Ad and Tat—Ad with different Tat modification rates.

 FIG. 9 is a graph comparing gene expression activity in various adherent cells (HeLa cells, A549 cells, HT1080 cells) with “Ad, AdRGD, and Tat-modified Ad”.

 FIG. 10 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. 11 is a graph comparing gene expression efficiency between Ad and Tat-Ad with different Tat modification rates in various adherent cells (RAW264.7 cells, CT26 cells HeLa cells) with different CAR expression levels.

 FIG. 12 is a graph comparing gene expression efficiency in various floating cells (EL cells, KG-la cells) with “Ad, Ad RGD, and Tat-modified Ad”.

 FIG. 13 is a graph comparing gene expression efficiency in U937 cells, which are floating cells, between Ad and Tat-Ad with different Tat modification rates.

 FIG. 14 is a schematic diagram showing “Tat peptide-mixed Ad” and “Tat-modified Ad”.

[Figure 15] Gene expression activity is expressed as “Ad, AdRGD, Tat peptide -mixed Ad and Tat modification. It is the graph compared with Adj.

 FIG. 16 is a graph comparing gene expression efficiency in B16BL6 cells between Ad, Tat-Ad and Tat peptide mixed Ad with different Tat modification rates (or mixing rates).

FIG. 17 is a graph comparing the gene expression efficiency in A549 cells in the presence and absence of anti-Ad serum between Ad and Tat-Ad.

 FIG. 18 is a graph comparing the gene expression efficiency in B16BL6 cells among Ad, Tat-Ad and R8-Ad.

Claims

The scope of the claims  [I] A gene transfer aid comprising a cell translocation peptide as an active ingredient, wherein a chemical linker is bound to the cell transfer peptide.  [2] The gene transfer adjuvant according to claim 1, wherein the chemical linker has an NHS (N-hydroxysuccinimidyl) group. [3] MHS (6-maleimidohexanoic acid N-hydroxysuccinimide ester) having the NHS group as a chemical linker. [4] The gene transfer adjuvant according to claim 2, wherein the intracellular translocation peptide is a Tat peptide.  [5] The gene transfer adjuvant according to claim 2, wherein the intracellular translocation peptide is a peptide consisting of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5. [6] In the amino acid sequence of the intracellular translocation peptide, any 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 5. [7] The gene transfer adjuvant according to any one of [1] to [6], wherein the gene transfer aid is covalently bound to the surface of a viral coat protein used as a gene transfer vector. [8] The gene transfer adjuvant according to claim 7, wherein the virus used as the gene transfer vector is an adenovirus.  [9] The covalent bond between the gene introduction auxiliary agent and the virus used as the gene introduction 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 7 or 8.  [10] The gene transfer adjuvant according to any one of [1] to [9], wherein the cell type to be transferred is an adherent cell or a floating cell.  [I I] The gene transfer adjuvant according to any one of claims 1 to 10, wherein the cell type to be transferred is a cell type in which expression of both CAR and integrin is poor or deficient.  12. The gene transfer adjuvant according to any one of claims 1 to 11, which is in the form of a lyophilized powder.  [13] The gene transfer adjuvant according to any one of claims 1 to 12, which is used for gene therapy. [14] The gene introduction auxiliary agent according to any one of claims 1 to 13, in the stage before gene introduction A virus vector for gene transfer, characterized in that it is covalently bonded to the surface of the outer shell protein. A gene characterized in that the gene introduction auxiliary agent according to any one of claims 1 to 13 is covalently bound to the surface of the outer protein of a virus used as a gene introduction vector at a stage before gene introduction. Introduction method.