JP2010041919A - Gene transfer adjuvant containing intracellular migration peptide as active ingredient and gene transfer method using the gene transfer adjuvant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable transfer of a gene even in a part of cancer cells, blood cells, and the like in which the expression of CAR and integrin are both poor or deleted. <P>SOLUTION: Provided are the gene transfer adjuvant containing an intracellular migration peptide as an active ingredient, in which NHS (N-hydroxysuccinimidyl) group are attached to the intracellular migration peptide. The gene transfer method comprises covalent-bonding the gene transfer adjuvant to the surface of the coat protein of a virus used as a gene transfer vector. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a gene transfer aid containing an intracellular translocating peptide as an active ingredient and a gene transfer method using the gene transfer aid. More specifically, even in some cancer cells and blood cells that are poorly or deficient in expression of both CAR and integrin by covalently binding to the viral coat protein used as a gene transfer vector. Furthermore, the present invention relates to a “gene introduction auxiliary agent using an intracellular translocation peptide as an active ingredient” and “a gene introduction method using the gene introduction auxiliary agent” that enable gene introduction.

The regulation of gene expression in mammalian cells and the functional analysis of proteins, which are gene products, are the central themes of current pharmacology and biology. In order to analyze them, target genes are introduced into animal cells. Technology is essential. At present, gene transfer methods are roughly classified into methods using non-viral vectors such as liposomes and methods using viral vectors, and methods using viral vectors dominate from the viewpoint of gene transfer / expression efficiency.
Among viral vectors, the gene transfer method using an adenovirus vector (hereinafter referred to as “Ad”) exhibits high gene transfer / expression efficiency, and is therefore very important for basic research at the cell and animal level. It is known to be a powerful tool.

Currently, Ad widely used in gene therapy research and basic research has been developed on the basis of type 2 or type 5 Ad, and its gene transfer mechanism uses the infection mechanism of Ad as it is. Ad has a regular icosahedron structure consisting of 252 capsomeres on a virus particle having a diameter of about 80 nm, of which 12 at the apex are called pentons having a protruding structure, and the other 240 are called hexons. . Penton consists of a penton base and a fiber, which is further divided into a tail, shaft and knob.
Entry into a target cell of Ad are fiber knob binds to CAR (coxackievirus adenovirus receptor) are infectious receptor, followed by penton base RGD (Arg-Gly-Asp) motif on the cell surface alpha _v -It occurs by endocytosis through a two-step process of binding to integrins. Ad that reaches the endosome undergoes a structural change of the capsid 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 microtubules, 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, the adenoviral vector (Ad) can efficiently introduce a gene into a wide variety of cells and tissues regardless of the mitotic or stationary phase. In addition, since it can be expressed by administration to individual animals, it has been actively developed as a gene therapy vector.
However, in cell types with poor or defective expression of Ad infection receptor (CAR) such as malignant tumor cells and blood cell lineage cells, it is an important target for gene therapy, but even with Ad No expression can be obtained.

In order to overcome the problem of Ad that the gene transfer efficiency is low for some cancer cells and blood cells that have poor expression of Ad infection receptor (CAR), RGD (Arg) is added to the fiber tip of Ad. An improved Ad imparted with α _v -integrin directivity (hereinafter referred to as “AdRGD”) has been developed by presenting a (Gly-Asp) peptide.
AdRGD can express a gene very efficiently even in cells (such as cells with poor CAR expression) in which gene transfer is difficult with conventional Ad (Non-patent Documents 1 and 2).
In the same way as conventional Ad, AdRGD is equipped with an expression cassette for the target gene in the E1 deficient region, and in the region encoding fiber knob, an oligonucleotide corresponding to the RGD sequence having affinity for α _v -integrin is present. Due to the modification of this fiber region, AdRGD has acquired α _v -integrin-directed properties, and the expression of CAR, which has been difficult to transduce with conventional Ad, is difficult to express. However, the gene can be introduced efficiently.
In fact, the present inventors applied AdRGD to (1) optimization of cancer gene therapy using cytokine genes and suicide genes (Non-Patent Documents 2 to 4), (2) dendritic cell function by gene modification Optimization of cellular immunotherapy based on the enhancement of non-patent documents (Non-patent Documents 1 and 5), (3) Construction of immune cell pharmacokinetics control method by introducing chemokine / chemokine receptor gene (Non-patent Document 5) A new DDS (Drug Delivery System) strategy that could not be achieved by a conventional vector system has been introduced to advanced medicine.
However, in some cancer cells and blood cells that are poorly or deficient in expression of both CAR and integrin, sufficient gene expression cannot be obtained even with AdRGD. 1). Therefore, development of a vector capable of gene transfer independent of CAR and integrin is required.

As described above, in some cancer cells and blood cells that are poorly or deficient in expression of both CAR and integrin, it is still difficult to introduce a gene sufficiently even with AdRGD. It was.
Further, even in cell types in which CAR and integrin expression is observed, in order for conventional virus vectors (Ad, AdRGD, etc.) to exhibit sufficient gene transfer activity, it is necessary to administer a high dose, for example, There are side effects when applied to gene therapy. Therefore, a new vector having strong gene transfer activity that can be clinically applied at a low dose has been demanded.
Furthermore, from the viewpoint of basic research, with the aim of gene transfer into cells with low receptor expression, the development of vectors capable of gene transfer independent of CAR and integrin, and vectors that can efficiently transfer genes into any cell Development is required.

Okada, N. et al .: Cancer Res., 61: 7913-7919, 2001 Okada, N. et al .: Cancer Lett., 177: 57-63, 2002 Okada, N. et al .: Gene Ther., 10: 700-705, 2003 Okada, N. et al .: Cancer Gene Ther., 12: 608-616, 2005 Okada, N. et al .: Gene Ther., 12: 129-139, 2005

In view of the above-mentioned problems of the prior art, the present invention enables gene transfer even in some cancer cells or blood cells that are poorly or deficient in expression of both CAR and integrin.
An object is to provide a new gene transfer vector having the following requirements (1) to (3).
(1) Gene transfer is possible even in some cancer cells or blood cells that are poorly or deficient in expression of both CAR and integrin (expansion of application range).
(2) As a gene therapy vector, it can be clinically applied 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.

  As a result of diligent research, the inventors of the present invention have used, as a gene transfer aid, a gene transfer vector obtained by binding an NHS (N-hydroxysuccinimidyl) group to an intracellular translocation peptide such as a Tat peptide. By covalently binding to the outer shell protein, the range of application can be expanded to blood cell lineage cancer, etc. where gene transfer was difficult (with conventional gene transfer vectors), and regardless of cell type (receptor expression or not Regardless of this, the present inventors have found that gene transfer activity is very strong and have completed the present invention.

That is, the invention according to claim 1 is a gene transfer aid containing an intracellular translocating peptide as an active ingredient, wherein an NHS (N-hydroxysuccinimidyl) group is bound to the intracellular translocating peptide. It relates to an introduction 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 aid comprising as an active ingredient an intracellular translocation peptide represented by any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4, and the intracellular translocation The present invention relates to a gene introduction auxiliary agent characterized by having an NHS group bonded to a peptide.
The invention according to claim 4 is any one 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. It is related with the gene transfer adjuvant of Claim 3 which is one.
The invention according to claim 5 relates to the gene transfer auxiliary agent according to any one of claims 1 to 4, wherein the gene transfer aid is covalently bound to the surface of a viral coat 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 an adenovirus.
In the invention according to claim 7, the covalent bond between the gene introduction auxiliary agent and the virus used as the gene introduction vector is under conditions of 10 to 50 ° C., 100 to 1000 rpm, and 5 minutes to 60 minutes. The gene transfer adjuvant according to claim 5 or 6, which is performed.
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 relates to the gene transfer auxiliary agent according to any one of claims 1 to 8, 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 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 introduction auxiliary agent according to any one of claims 1 to 10, which is used for gene therapy.
In the invention according to claim 12, in the stage before gene introduction, the gene introduction auxiliary agent according to any one of claims 1 to 11 is bound to the surface of the outer shell protein of a virus used as a gene introduction vector. The present invention relates to a virus vector for gene transfer.
According to a thirteenth aspect of the present invention, the gene introduction auxiliary agent according to any one of the first to eleventh aspects 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 “gene transfer auxiliary agent comprising an intracellular translocation peptide linked with an NHS (N-hydroxysuccinimidyl) group” as an active ingredient according to the present invention is covalently bonded to the surface of the outer shell protein of a virus used as a gene transfer vector. The viral vector is characterized in that the expression of some cancer cells and blood cells (both CAR and integrin), in which gene transfer is difficult with conventional gene transfer vectors (Ad, AdRGD, etc.), or A sufficient gene transfer effect can be exerted even on deficient cell types) (“expansion of application range”). At the same time, even in cell types that can be transferred with conventional gene transfer vectors, the gene transfer activity can be significantly higher than that of conventional cells ("improvement of gene transfer efficiency"). ).
That is, gene transfer (CAR, integrin-independent gene transfer) to cells with low receptor expression is possible, so that the range of application of viral vectors (such as Ad) in cancer gene therapy can be expanded and all cell types A gene transfer vector can be achieved in vivo (with or without receptor expression) in vivo (in other words, it can be clinically applied at a lower dose, thus reducing side effects).
In addition, since gene transfer can be efficiently performed on all cells, it can be applied as a gene transfer tool in basic research.

In the present invention, by binding a NHS group to a cell translocation peptide such as a Tat peptide as a gene transfer aid, it is covalently bonded to the outer shell protein of the viral vector (in the stage before gene transfer). At the same time, the application range has been 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 deficient). Based on the finding that gene transfer activity is very strong (even in cell types that could be transferred with a viral vector).
Therefore, the viral vector in which the gene transfer aid according to the present invention is covalently bonded to the outer shell protein surface is considered to be an effective drug delivery system (DDS) with improved gene transfer efficiency in vivo. It is done.
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 of the singular unless otherwise specified. It also includes the plural concept.

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

As shown in [Table 1], the “intracellular peptide” as an essential component of the gene transfer adjuvant according to 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) and the like can be listed, but in order to exert the effects of the present invention described above (expansion of application range, improvement of gene transfer efficiency, etc.), it is preferable to use a Tat peptide. Is optimal. The Tat peptide is derived from the transcriptional activator of HIV and has a peptide sequence “Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln” containing a large amount of basic amino acids. However, among intracellular translocation peptides, it has particularly high intracellular translocation activity and is also used as a carrier for introduction into cells into proteins, liposomes and the like.
As long as it has an intracellular translocation function (membrane permeation function), in addition to the typical intracellular translocation peptides shown in [Table 1], one or more of these amino sequences (SEQ ID NOs: 1 to 4) Even if it has an amino acid sequence deleted, substituted, inserted or added, it is an active ingredient of the gene transfer auxiliary according to the present invention.

The present invention is a virus that is used as a gene transfer vector, with an "NHS (N-hydroxysuccinimidyl) group" linked to an intracellular translocation peptide typified by the Tat peptide described above as an auxiliary for "gene transfer" By covalently binding to the outer shell protein, the application range is expanded to some cancer cells and blood cells that have been difficult to introduce genes, and at the same time, “gene transfer efficiency” and “gene transfer activity” itself Is also (further) enhanced.
As used herein, “gene transfer” means introducing a target polynucleotide sequence (nucleic acid) into a cell. In addition, gene transfer may be to exhibit the action of a nucleic acid in a 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 introduction of a nucleic acid into a cell. For example, the ratio of transfected cells in the whole cell, the amount of nucleic acid incorporated in the cell population, or the total cell population It is the expression level of the transgene. When the gene is expressed in a cell, it is preferably the expression level of the transgene in the whole 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 mRNA level, protein level, or activity level of the protein.
As used herein, “gene transfer activity” refers to the activity of “gene transfer” by a vector, and functions of the introduced gene (for example, in the case of an expression vector, expression of the encoded protein and / or activity of the protein). Etc.) are detected as indicators.

As shown in Examples described later, the present inventors used an Nt (N-hydroxysuccinimidyl) group bound to a Tat peptide as an auxiliary agent for gene transfer to the outer shell protein of an adenovirus vector. Covalently linked gene transfer vector (hereinafter sometimes referred to as “Tat peptide-modified Ad”) is about 10 to 500 times stronger gene expression activity than conventional Ad regardless of CAR negative and positive cells As a result, the present invention has been completed.
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 introduction auxiliary agent of the present invention is more robust by binding an “NHS group” to an intracellular transit peptide (specifically, when the intracellular transit peptide is a Tat peptide, its lysine group). A high level of gene expression activity can be exhibited regardless of the presence or absence of a receptor (CAR, integrin, etc.).

  The above-described “gene transfer 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. It belongs to the target range. For example, as a chemical linker to be coupled to the intracellular translocation peptide, in addition to the NHS group, dicyclohexylcarbodiimide (DCC), maleimidobenzoyl-N-hydroxysuccinimide (MBS), N-ethyloxycarbonyl-2-ethyloxy-1,2, -Dihydroquinoline (EEDQ), N-isobutyloxy-carbonyl-2-isobutyloxy-1,2-dihydroquinoline (IIDQ) can be listed. 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.).

The gene transfer adjuvant of the present invention comprising an NHS group bound to an intracellular translocation peptide such as a Tat peptide as an active ingredient is the outer shell of a “virus (inactivated)” used as a gene transfer vector. By covalently binding to the surface of the protein, sufficient gene transfer effect is exerted even for some cancer cells and blood cells that have been difficult to transfer using conventional viral vectors (Ad, AdRGD, etc.). obtain.
As used herein, “virus” refers to an infectious microstructure that has either DNA or RNA as a genome and that grows only in infected cells. Viruses include retroviridae, togaviridae, coronaviridae, flaviviridae, paramyxoviridae, orthomyxoviridae, bunyaviridae, rhabdoviridae, poxviridae, herpesviridae, baculoviridae and hepa Although the virus etc. which belong to the family selected from the group which consists of Donnaviridae are mentioned, the virus used more preferably in this invention is an "adenovirus". Adenovirus can efficiently transfect a wide variety of cells and tissues regardless of mitotic or quiescent phase, and also exhibits transiently high gene transduction efficiency and expression efficiency as a gene transduction vector to mammals. Therefore, it is considered optimal for achieving the object of the present invention (expansion of application range / improvement of gene transfer efficiency, etc.) (also as a virus vector modified by the gene transfer aid of the present invention).
As used herein, “inactivation” refers to inactivation of a genome when referring to a virus (eg, adenovirus). Inactivated viruses are replication defective. Inactivation is accomplished by a variety of means normally available to those skilled in the art.

The “covalent bond” between the gene transfer adjuvant of the present invention and the virus used as the gene transfer vector is usually limited as long as it is an experimental method within the range that can be easily performed by those skilled in the art. Although it is not a thing, in order to produce efficiently the complex (for example, "Tat peptide modification Ad") of the said gene introduction adjuvant and a viral vector, 10-50 degreeC, 100-1000 rpm, 5 minutes-60 minutes It is desirable that (covalent bonding) be performed under the following conditions. Here, since the NHS group is bound to the intracellular translocation peptide, the gene introduction aid of the present invention is easily covalently bound to the viral vector (due to the reactivity of the NHS group) (see FIG. 3). ).
Whether or not the gene transfer adjuvant of the present invention is covalently bound to the surface of the outer shell protein of the virus used as a gene transfer vector is usually carried out 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, “conjugation confirmation by SDS-PAGE” (see FIG. 4) and intracellular transfer peptides such as Tat peptide are stagnant in “+”. The presence or absence of binding can be confirmed by (indirectly) the value (mV) ”or the like.

By applying the gene transfer adjuvant of the present invention covalently to the surface of the outer shell protein of the virus vector before gene transfer (proactively), the application range of the virus vector for gene transfer is expanded, and the gene transfer efficiency is increased. (See FIGS. 9 and 10).
That is, as a vector for gene transfer, compared with a vector obtained by simply mixing a cell translocation peptide and a virus vector (as described above), the gene transfer auxiliary 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 a peptide bound to the virus surface is involved in enhancement of gene expression.

  By using the gene transfer adjuvant of the present invention, the “gene transfer efficiency (activity)” is statistically significant (for example, the significance level p <0) compared to the case where transfection is performed without the gene transfer adjuvant. .05) (see FIGS. 5-8 and 10). That is, (at the stage before gene introduction), the gene introduction viral vector in which the gene introduction auxiliary agent of the present invention is covalently bonded to the surface of the outer protein of the virus has the effect of improving gene introduction efficiency (activity) in vivo. A typical drug delivery system (DDS). The measurement of gene transfer efficiency (activity) is not particularly limited as long as it is an experimental method within the range that can be easily carried out by those skilled in the art. Measurement by luciferase activity (luciferase assay) [RLU (relative light unit) / Well]. The increase in gene transfer efficiency by the gene transfer aid of the present invention can be confirmed (by luciferase activity [RLU / well]) by about 10 to 500 times compared to the case without the gene transfer aid. That is, in the stage before gene introduction, 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, Since the gene transfer efficiency is increased by about 10 to 500 times, for example, in the case of clinical application as a gene therapy vector, since it can be clinically applied at a low dose, side effects are reduced, which is very beneficial.

There are no particular limitations on the cell type to be introduced into the gene transfer adjuvant of the present invention and the viral vector for gene transfer to which the adjuvant is bound. That is, it may be any type of cell such as the small intestine, nasal mucosa, skin tissue, subcutaneous tissue, bone tissue, cartilage tissue, and the origin thereof is human cells, non-human animal cells, and other microorganisms. Cells of all species such as 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 introduction adjuvant of the present invention and the gene introduction viral 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).

Examples of the cell type to be a gene transfer target of the viral vector to which the gene transfer aid of the present invention is bound include commonly used cultured cells such as A549 cells (human alveolar epithelial cancer cells), B16BL6 cells, CHO. Cells, EL4 cells (mouse thymus-derived T cells), HEK293T cells, HT1080 cells (human fibrosarcoma cells), HeLa cells (human cervical cancer cells), KG-1a cells (human myeloid leukemia cells), NIH3T3 cells, etc. Although it can mention, it does not specifically limit.
However, since none of CAR / integrin as a receptor is expressed, some cancer cells and blood cells that have been difficult to introduce with existing viral vectors for gene transfer (Ad, AdRGD, etc.) Also in (see FIGS. 1 and 2), gene introduction is possible (can also exhibit sufficient gene expression activity) if it is a viral vector for gene introduction combined with the gene introduction auxiliary agent of the present invention. Therefore, “cell types that are poorly expressed or defective in both CAR and integrin (some cancer cells, blood cells, etc.)” have an advantageous effect not found in conventional viral vectors. From a viewpoint), it is a preferable cell type for gene transfer. Specific examples of cell types in which expression of both CAR and integrin cannot be confirmed include “KG-1a cells”.

  Furthermore, the viral vector to which the gene introduction auxiliary agent of the present invention is bound is a cell type that can be introduced by a conventional viral vector, that is, “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, etc.) Compared to vectors, it can exhibit much higher gene transfer activity (improves 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).

The gene introduction auxiliary agent of the present invention and the virus vector for gene introduction to which the auxiliary agent is bound can also be preferably used in the form of “lyophilized powder”.
The lyophilized powder can be obtained by lyophilizing the gene introduction auxiliary agent of the present invention and the gene introduction virus vector to which the auxiliary agent is bound, and lyophilization can use a known method, for example, After freezing in liquid nitrogen, it can be performed by 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 introduction adjuvant of the present invention and the gene introduction viral vector to which the adjuvant is bound can be regenerated with water at the time of use.

  As mentioned above, “the gene transfer adjuvant containing the intracellular translocation peptide bound with NHS group as an active ingredient” has been specially described, but it can be changed and modified, and these belong to the technical scope of the present invention. is there. In addition, the gene transfer aid of the present invention can be applied to all currently used viral vectors including adenovirus vectors.

  The gene introduction method using the gene introduction auxiliary agent of the present invention (hereinafter referred to as “the present method”) comprises the above gene on the surface of the outer protein of a virus used as a gene introduction vector in the stage before gene introduction. This method is characterized by the use of a viral vector covalently bound to an introduction aid. Compared with conventional Ad that carries out infection and gene expression in a CAR-dependent manner, this method is useful for the intracellular translocation of peptides. This is a gene transfer method based on a new idea of increasing gene transfer efficiency by transferring Ad into cells efficiently and rapidly using transfer activity. In other words, since it follows the CAR-independent infection / gene transfer pathway, it is a system that can efficiently transfer and express even many CAR-negative cells.

  Examples of genes to be treated in this method include genes encoding enzymes, hormones, lymphokines, receptors, growth factors, regulatory proteins, polypeptides that affect the immune system, immune regulatory factors, antibodies, and the like. But not limited to. 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: Examples include, but are not limited to, genes encoding C, calcitonin, thymidine kinase, interferon, granulocyte macrophage (GMCSF), erythropoietin (EPO), hepatocyte growth factor (HGF) and the like. These genes can 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.

  The gene transfer adjuvant of the present invention and the viral vector to which the adjuvant is bound include any sterile biocompatible pharmaceutical carrier (including but not limited to saline, buffered saline, dextrose and water). Not). Any of these molecules can be administered to a patient alone or in combination with other agents in a pharmaceutical composition mixed with suitable excipients, adjuvants, and / or pharmaceutically acceptable carriers. . In certain embodiments of the invention, the pharmaceutically acceptable carrier is pharmaceutically inert.

  Administration of the gene transfer adjuvant of the present invention and the viral vector for gene transfer to which the adjuvant is bound is 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.

  Examples are shown below, but the present invention is not limited to these examples.

(1) Preparation of Tat peptide-modified Ad As a gene transfer aid according to the present invention, a Tat peptide having 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. Then, mixing with a vortex mixer and incubating under conditions of 37 ° C., 300 rpm, 30 min to produce an adenoviral vector for gene transfer (hereinafter referred to as “Tat-Ad”) to which “Tat-NHS” is bound. (See [FIG. 3]).
Specific examples (Experimental Examples 1 to 3) for producing “Tat-Ad” are shown below.

[Experimental Example 1]
i) Tat-NHS
Amino acid sequence: Ac-GRKKRERRRRPPQG-GC-NHS
Molecular weight: about 2000
Concentration (amount): 5 mg / 50 μL / tube = 2.5 × 10 ⁻⁶ mol / tube
= 1.5 × 10 ¹⁸ molecules / tube
ii) Reaction conditions Lysine residues present in the outer shell protein of Ad [7500 / vp (virus particle)]: “Tat-NHS” = 1: 1000
The required number of Ad particles is 2 × 10 ¹¹ vp / tube
iii) “Ad”, “Tat-NHS” suspension (composition)
Ad (7 × 10 ¹² vp / ml): 28.6 μL
・ PBS: 121.4 μL
Tat-NHS: 50 μL
⇒ 2 × 10 ¹¹ vp / 200 μL
iv) Preparation of “Tat-Ad” Using “Tat-NHS” in i) in a frozen state, a suspension was prepared with the composition shown in iii) under “Reaction conditions” in ii), and a vortex mixer was prepared. Mixed with. Next, “Tat-Ad” was obtained by incubation under conditions of 37 ° C., 300 rpm, and 30 min.

[Experimental example 2]
i) Tat-NHS
Amino acid sequence: Ac-GRKKRERRRRPPQG-GC-NHS
Molecular weight: about 2000
Concentration (amount): 5 mg / 50 μL / tube = 2.5 × 10 ⁻⁶ mol / tube = 1.5 × 10 ¹⁸ molecules / tube
ii) Reaction conditions Lysine residue (7500 / vp) present in Ad outer shell protein: “Tat-NHS” = 1: 200
The required number of Ad particles is 1 × 10 ¹² vp / tube
iii) “Ad”, “Tat-NHS” suspension (composition)
Ad (2.3 × 10 ¹² vp / ml): 435 μL
・ PBS: 515 μL
Tat-NHS: 50 μL
⇒1 × 10 ¹² vp / 1mL
iv) Preparation of “Tat-Ad” Using “Tat-NHS” in i) in a frozen state, a suspension was prepared with the composition shown in iii) under “Reaction conditions” in ii), and a vortex mixer was prepared. Mixed with. Next, “Tat-Ad” was obtained by incubation under conditions of 37 ° C., 300 rpm, and 30 min.

[Experimental Example 3]
i) Tat-NHS
Amino acid sequence: Ac-GRKKRERRRRPPQG-GC-NHS
Molecular weight: about 2000
Concentration (amount): 2.5 mg / 100 μL / tube = 1.25 × 10 ⁻⁶ mol / tube = 7.5 × 10 ¹⁷ molecules / tube
ii) Reaction conditions Lysine residue (7500 / vp) present in the outer shell protein of Ad: “Tat-NHS” = 1: 1000
The required number of Ad particles is 1 × 10 ¹¹ vp / tube
iii) “Ad”, “Tat-NHS” suspension (composition)
Ad (2.5 × 10 ¹² vp / ml): 40 μL
・ PBS: 860 μL
Tat-NHS: 100 μL
⇒ 1 × 10 ¹¹ vp / 1mL
iv) Preparation of “Tat-Ad” Using “Tat-NHS” of i) in a frozen state, mixing with a vortex mixer under “Reaction conditions” of ii), under conditions of 37 ° C., 300 rpm, 45 min. And “Tat-Ad” was obtained by incubation.
v) Others “Tat-Ad” was prepared in the same manner as in Examples 1 and 2, except that the reaction time in incubation was changed to 45 min (from 30 min). Therefore, it is considered that there is almost no influence by the change.

(2) Confirmation of binding by SDS-PAGE Whether or not Tat-NHS was covalently bound to the surface of the outer shell protein of Ad was confirmed by SDS-PAGE from the viewpoint of molecular weight (kDa).
Specifically, 2 × 10 ¹⁰ vp (virus particles) of “Ad” and “Tat-Ad” were concentrated using an evaporator. Thereafter, loading buffer + 2ME was mixed at 95: 5, and 15 μL was added to the suspension (Ad, Tat peptide-modified Ad). And after pipetting, it incubated on 96 degreeC and the conditions for 5 minutes, and applied to the gel (PAG mini-first 4/20). The gel was electrophoresed at 20 mA, stained with a CBB staining solution for 2 hours, and decolorized with a CBB decoloring solution for 2 hours, and then a gel image was captured with a scanner. The sample of [Experimental Example 2] was used as “Tat-Ad”, and “FULL RANGE RAINBOW (Amersham Bioscience # RPN800)” was used as “marker”.
The results are shown in FIG. As is apparent from the gel image in FIG. 4, the molecular weight of hexon increased, suggesting Tat-NHS binding to the Ad surface. That is, in the Tat peptide-modified Ad, the molecular weight (kDa) corresponding to “Tat-NHS” was larger than that of normal Ad. Thereby, in “Tat-Ad” prepared in (1) above, it was considered that Tat-NHS was covalently bonded to the surface of the outer protein of Ad.

(3) Binding confirmation by surface charge (mV) [Zeta potential measurement]
Since “Ad” has a charge of (−), “Tat peptide” rich in basic amino acids has a positive charge (+). Therefore, in “Tat-Ad” prepared in (1) above, If Tat-NHS is bound (on the surface of Ad's outer shell protein), the surface charge should be shifted from (-) to (+). Therefore, it was confirmed by surface charge (mV) whether Tat-NHS was covalently bonded to the surface of the outer protein of Ad. Specifically, “Tat-Ad” as a sample was diluted with PBS, and the surface charge (mV) was measured by Zetasizer 3000HS (Malvern Instrument Ltd.). The sample of [Experimental Example 2] was used as “Tat-Ad”.
The results are shown in [Table 2]. Since the surface charge is shifted to (+), that is, “Tat−Ad” is charged to (+) compared to Ad, Tat. -NHS was considered to be covalently bound to the surface of the outer shell protein of Ad.

In order to evaluate / examine the usefulness of "Tat-Ad" from the viewpoints of "gene introduction target (/ application target area)" and "gene expression efficiency (/ gene expression activity)", the following experiments were conducted.
In the evaluation / examination, as a comparison target of Tat-Ad, a normal adenovirus vector (Ad) and an improved Ad (AdRGD) imparted with integrin directivity were used.
The cell types used for the evaluation / examination are adherent cells and floating cells. Specifically, cultured cells listed in [Table 3] below were used.

(4) Usefulness evaluation of “Tat-Ad” regarding gene expression efficiency (B16BL6 cells)
In B16BL6 cells (cell types in which CAR expression could not be confirmed and integrin expression could be confirmed), the gene expression efficiency (activity) of “Tat-Ad” was examined by a luciferase assay (luciferase assay).
In addition, it was also examined whether the gene expression efficiency (activity) of Tat-Ad is seen in an amount-dependent manner. Specifically, a luciferase assay was performed according to the following procedure.

[Procedure] Luciferase assay in B16BL6 cells
1) B16BL6 cells were seeded on a 48-well flat bottom plate at 10 ⁴ cells / well in 500 μL medium.
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 × 10 ⁷ vp / ml, 1 × 10 ⁸ vp / ml, 3 × 10 ⁸ vp / ml, 1 × 10 ⁹ vp / ml (“Tat-Ad” is the same as in Experimental Example 3 above) 100 μL / well was added (ie, 300 vp / cell, 1000 vp / cell, 3000 vp / cell, and 10,000 vp / cell were added).
5) Incubated for 24 hours.
6) The medium was aspirated and washed twice with PBS.
7) Cell Culture Lysis Reagent 5x (promega # E1531) diluted with MilliQ water was added at 100 μL / well.
8) 10 μL of the cell lysate was taken and placed in a lumino tube.
9) 10 μL of a reaction substrate (Promega # E1501 Luciferase Assay System) was added, vortexed lightly, and [RLU (relative light unit) / well] was measured with a luminometer.
For samples with RLU exceeding 10 ⁷ , the cell lysate was diluted 100-fold with cell lysis reagent, 100 μL of substrate was added to 10 μL 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 luciferase activity (RLU / well) in B16BL6 cells between “Ad”, “AdRGD” and “Tat-Ad” in order to evaluate the usefulness of Tat-Ad with respect to gene expression efficiency. It is the graph compared by.
The horizontal axis of FIG. 5 indicates the number of infected virus particles (virus particle / cell), and the vertical axis indicates the luciferase activity value [RLU / well] measured with a luminometer. In the (vp / cell) zone, “Tat-Ad” was found to show a gene transfer activity much higher than “Ad” and “AdRGD”. In particular, in B16BL6 cells in which CAR expression could not be confirmed, “Tat-Ad” exhibited 400 times the luciferase activity of “Ad”, that is, 400 times the gene transfer activity. Furthermore, even in cell types in which CAR expression could not be confirmed, the gene transfer activity was remarkably high as compared with “AdRGD”, which allows gene transfer.
Further, from the results of FIG. 5, it was also confirmed that the gene expression efficiency (activity) of Tat-Ad is seen in an amount-dependent manner (similar to Ad and AdRGD).
From the above, it was proved that Tat-Ad exhibits high gene transfer activity against B16BL6 cells with low CAR expression as compared with conventional virus vectors.

(5) Evaluation of usefulness of “Tat-Ad” for gene expression efficiency (adherent cells)
As various types of adherent cells, HeLa cells (human cervical cancer cells), A549 cells (human alveolar epithelial cancer cells), and HT1080 cells (human fibrosarcoma cells) (both cell types in which expression of CAR and integrin can be confirmed) ), The gene expression efficiency (activity) of “Tat-Ad” was examined by luciferase assay.
Specifically, a luciferase assay was performed according to the following procedure.

[Procedure] Luciferase assay in adherent cells
1) Various cells were seeded on a 48-well flat-bottom plate at 10 ⁴ cells / well in 500 μL medium.
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) 100 μL / well of various vectors diluted to 10 ⁸ or 10 ⁹ vp / ml (“Tat-Ad” was the sample of Experimental Example 1) was added.
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) Cell Culture Lysis Reagent 5x (promega # E1531) diluted with MilliQ water was added at 100 μL / well.
8) 10 μL of the cell lysate was taken and placed in a lumino tube.
9) 10 μL of a reaction substrate (Promega # E1501 Luciferase Assay System) was added and mixed gently by vortexing, and then RLU (relative light unit) / well was measured with a luminometer.
For samples with RLU exceeding 10 ⁷ , the cell lysate was diluted 100-fold with cell lysis reagent, 100 μL of substrate was added to 10 μL 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. 6 shows luciferase activity (RLU / well) in various adherent cells (HeLa cells, A549 cells, HT1080 cells) in order to evaluate the usefulness of Tat-Ad with respect to gene expression efficiency (activity). ”,“ AdRGD ”and“ Tat-Ad ”.
Since HeLa cells, A549 cells, and HT1080 cells are cell types in which CAR and integrin expression can be confirmed, there was no difference in gene transfer activity between “Ad” and “AdRGD”. However, there was a significant difference in the gene transfer activity between “Ad” and “AdRGD” and “Tat-Ad” (p <0.01).
It was 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.
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, it has been shown that Tat-Ad is a vector that can achieve an expanded range of application of Ad in cancer gene therapy and a reduction in dose in vivo (ie, reduction of side effects).

Next, FIG. 7 shows the results of examining 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 adherent cells (A549 cells, HT1080 cells, B16BL6 cells) in order to evaluate the usefulness of Tat-Ad with respect to gene expression efficiency. It is the graph compared between "AdRGD" and "Tat-Ad". A549 cells and HT1080 cells are cell types in which the expression of CAR and integrin can be confirmed, but there is a significant difference in gene transfer activity between “Ad” and “AdRGD” and “Tat-Ad”. Was observed (p <0.01). It was also shown that “Tat-Ad” has 30 times the luciferase activity (ie, gene transfer activity) of “Ad” in A549 cells and 40 times in HT1080 cells.
On the other hand, in B16BL6 cells, since CAR expression cannot be confirmed (integrin expression can be confirmed), a significant difference was observed between “Ad” and “AdRGD”, but “Tat-Ad”. Then, the gene introduction activity was further confirmed as compared with “AdRGD”, and the luciferase activity 500 times that of “Ad” was confirmed.

(6) Evaluation of usefulness of “Tat-Ad” for gene expression efficiency (floating cells)
In EL cells (mouse thymus-derived T cells) and KG-1a cells (human myeloid leukemia cells), the gene expression efficiency (activity) of “Tat-Ad” was examined by luciferase assay (RLU / well). The EL cell is a cell type in which expression of both CAR and integrin can be confirmed, and the KG-1a cell is a cell type in which expression of both CAR and integrin cannot be confirmed. Specifically, a luciferase assay was performed according to the following procedure.

[Procedure] Luciferase assay in suspension cells
1) Various cells were seeded on a 48-well flat bottom plate at 10 ⁴ cells / well in 400 μL 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” used the sample of Experimental Example 1) were added at 100 μL / well.
・ 10 ⁸ vp / well ・ 10 ⁴ vp / cell
4) Incubated for 24 hours.
5) 100 μL of the cell suspension was taken in a luminotube.
6) 100 μL of Bright-Glo (Promega # E2610) was added.
7) Left at room temperature for several minutes.
8) After lightly mixing by vortex, RLU (relative light unit) / well was measured with a luminometer.

The results are shown in FIG. FIG. 8 shows the luciferase activity (RLU / well) in various floating cells (EL cells, KG-1a cells) in order to evaluate the usefulness of Tat-Ad with respect to 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 the luciferase activity (gene transfer activity) between “Ad” and “AdRGD”. “Ad” showed much higher luciferase activity than “Ad” and “AdRGD”, and luciferase activity five times that of “Ad” was confirmed.
On the other hand, since KG-1a 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” is KG−. Even in the 1a cell, it was possible to show sufficient gene transfer activity, and it was confirmed that the cell had luciferase activity 10 times that of “Ad”.
Based on the above, Tat-Ad is sufficient for gene transfer in cell types (cell types in which expression of both CAR and integrin is poor or deficient) in which gene transfer was difficult with conventional viral vectors. It was shown that the effect can be demonstrated. At the same time, it has been shown that even cell types that can be transfected with conventional virus vectors can exhibit much higher gene transduction activity than conventional cells.

(7) Comparison of gene expression activity (efficiency) by covalently binding “Tat-NHS” to the surface of the outer protein of the virus 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 introduction, B16BL6 cells However, “Tat-Ad” was compared with “Tat peptide mixed Ad (non-specifically adsorbed by mixing Tat peptide not having NHS group and Ad)” (by luciferase assay) ( (See FIG. 9).
As “Tat-Ad”, the sample of [Experimental Example 3] is used, and the experimental procedure (luciferase assay, etc.) and “Tat-Ad” as a sample are as described above. In addition, “Ad” and “AdRGD” were also used as comparison targets of gene expression activity.

The examination result by the luciferase assay is shown in FIG.
FIGS. 9 and 10 show luciferase in B16BL6 cells in order to evaluate the usefulness of gene expression efficiency (activity) by covalently binding to the surface of adenovirus outer shell protein (positively) before gene introduction. FIG. 3 is a graph comparing “Ad”, “AdRGD”, “Tat peptide mixed Ad”, and “Tat-Ad” by assay.
As is clear from FIG. 10, “Tat-Ad” was significantly higher than the luciferase activity of “Tat 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 (in this case, Ad). That is, by covalently bonding “Tat-NHS” to the surface of the outer protein of the virus used as a vector before gene introduction, a conventional virus vector or simply a mixture of an intracellular translocation peptide and a virus vector can be obtained. It was shown that the activity of gene transfer was remarkably enhanced as compared with the above.
From the above, it was proved that “Tat-NHS” chemically bound to the surface of a virus used as a vector was involved in the enhancement of gene expression.

It is the figure which showed the intracellular penetration | invasion mode of Ad and AdRGD in relation to the receptor. It is the figure which showed the intracellular penetration | invasion mode of Ad, AdRGD, and Tat modification Ad in relation to the receptor. It is the figure which showed the specific example of Tat modification Ad preparation. It is the figure which confirmed confirmation of the coupling | bonding of Ad and Tat-NHS by SDS-PAGE. It is the graph which compared the gene expression efficiency in B16BL6 cell between "Ad, AdRGD, and Tat modification Ad". It is the graph which compared the gene expression activity in various adhesion cells (HeLa cell, A549 cell, HT1080 cell) with "Ad, AdRGD, and Tat modification Ad". It is the graph which compared the gene expression activity in various adhesion cells (A549 cell, HT1080 cell, B16BL6 cell) between "Ad, AdRGD, and Tat modification Ad". It is the graph which compared the gene expression efficiency in various floating cells (EL cell, KG-1a cell) with "Ad, AdRGD, and Tat modification Ad". It is the schematic diagram showing "Tat peptide-mixed Ad" and "Tat modification Ad". It is the graph which compared gene expression activity between "Ad, AdRGD, Tat peptide-mixed Ad, and Tat modification Ad".

Claims (13)

A gene transfer aid comprising a cell translocation peptide as an active ingredient, wherein an NHS (N-hydroxysuccinimidyl) group is bound to the cell transfer peptide. The gene transfer adjuvant according to claim 1, wherein the intracellular translocation peptide is a Tat peptide. A gene transfer auxiliary agent having 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 an NHS group is bound to the intracellular translocation peptide A gene transfer adjuvant characterized by the above. 4. The amino acid sequence of the intracellular translocation peptide is any one 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 aid described. The gene transfer auxiliary agent 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 gene transfer adjuvant according to claim 5, wherein the virus used as the gene transfer vector is an adenovirus. 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, and 5 to 60 minutes. The gene transfer aid described. 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 gene introduction auxiliary agent according to any one of claims 1 to 8, wherein the cell type to be introduced is a cell type in which expression of both CAR and integrin is poor or deficient. The gene transfer adjuvant according to any one of claims 1 to 9, which is in the form of a lyophilized powder. The gene introduction adjuvant according to any one of claims 1 to 10, which is used for gene therapy. A viral vector for gene transfer, wherein the gene transfer auxiliary agent according to any one of claims 1 to 11 is covalently bonded to the surface of a shell protein in a stage before gene transfer. 12. A gene introduction method, wherein 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 in a stage before gene introduction.

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