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WO2008068982A1 - Adjuvant pour transfert de gènes comprenant un peptide de migration cellulaire comme principe actif et procédé de transfert de gènes dans lequel est utilisé cet adjuvant pour un transfert de gènes - Google Patents

Adjuvant pour transfert de gènes comprenant un peptide de migration cellulaire comme principe actif et procédé de transfert de gènes dans lequel est utilisé cet adjuvant pour un transfert de gènes Download PDF

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Publication number
WO2008068982A1
WO2008068982A1 PCT/JP2007/071154 JP2007071154W WO2008068982A1 WO 2008068982 A1 WO2008068982 A1 WO 2008068982A1 JP 2007071154 W JP2007071154 W JP 2007071154W WO 2008068982 A1 WO2008068982 A1 WO 2008068982A1
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Prior art keywords
gene transfer
tat
gene
cells
peptide
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Japanese (ja)
Inventor
Shinsaku Nakagawa
Naoki Okada
Yasuo Yoshioka
Koichi Kawasaki
Mitsuko Maeda
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University of Osaka NUC
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Osaka University NUC
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10345—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/6045—RNA rev transcr viruses
    • C12N2810/6054—Retroviridae

Definitions

  • 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
  • Ad adenovirus vector
  • 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.
  • Penton consists of a Penton base and fiber, and the fiber is further divided into a tail, shaft and knob.
  • Ad 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 ⁇ -integrin on the cell surface.
  • CAR coxacki e-virus adenovirus receptor
  • adenoviral vectors can efficiently introduce genes into a wide variety of cells and tissues, regardless of whether they are dividing or stationary.
  • Ad Ad infection receptor
  • CAR Ad infection receptor
  • AdRGD Arg — Improved Ad
  • AdRGD is a cell that has been difficult to transduce with conventional Ad.
  • Non-patent Documents 1 and 2 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.
  • an oligonucleotide corresponding to the RGD sequence having affinity for ⁇ -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.
  • 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.
  • DDS Drug Delivery System
  • 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.
  • AdRGD is still sufficient for gene transfer.
  • 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
  • the present invention has poor expression of both CAR and integrin! / Or is defective! / In some cancer cells, blood cells, etc.
  • gene transfer is possible. It is an object to provide a new gene transfer vector having the following requirements (1) to (3).
  • 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.
  • 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.
  • 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.
  • 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 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 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.
  • 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
  • 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.
  • Ad, AdRGD, etc. conventional gene transfer vectors
  • sufficient gene transfer effects can be exerted even on cell types that are deficient (“expansion of application range”).
  • 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”). ).
  • 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).
  • gene transfer can be performed efficiently for any cell, it can be applied in basic research as a gene transfer tool.
  • 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).
  • a chemical linker bound to an intracellular translocation peptide such as a Tat peptide as a gene transfer aid
  • 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).
  • gene transfer activity is very strong (even in cell types that could be transferred with conventional virus vectors).
  • 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).
  • intracellular translocation peptide which is an essential component of the gene transfer adjuvant according to the present invention.
  • 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).
  • MPP membrane-permeable peptide
  • 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.
  • 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. .
  • one or more of these amino acid sequences 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”.
  • a chemical linker linked to the intracellular translocation peptide represented by the above-described Tat peptide as an auxiliary for “gene transfer”.
  • gene transfer means that a target polynucleotide sequence (nucleic acid) is introduced into a cell.
  • gene transfer may be to exert the action of the nucleic acid in the cell into which the nucleic acid has been introduced.
  • 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.
  • the gene 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.
  • the term “gene transfer activity” refers to the activity of “gene transfer” using a vector.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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.
  • virus eg, adenovirus
  • 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.
  • a complex for example, “Tat peptide-modified Ad”
  • 5 minutes to 60 minutes covalent bonding force is desirable.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • a conventional viral vector ie, a cell type that can sufficiently confirm the expression of both CAR and integrin.
  • 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).
  • 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.
  • 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
  • an NHS group such as MHS
  • the gene transfer aid of the present invention can be applied to any Winores betater that is currently widely used, including adenovirus vectors.
  • 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.
  • the present method 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.
  • 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.
  • 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.
  • 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.
  • the pharmaceutically acceptable carrier is pharmaceutically inert.
  • 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.
  • Examples are shown below, but the present invention is not limited thereto. Example 1
  • 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.
  • NHS 6-maleimidohexanoic acid N hydroxysuccinimide ester
  • Tat Ad adenoviral vector for gene transfer
  • the lysate was mixed with 4.6 mg of MHS (15.6 ⁇ mol) 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.
  • 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
  • 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
  • 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).
  • 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 ⁇ L 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).
  • 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.).
  • Tat—Ad 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.
  • EGF P End green fluorescent protein
  • EGFP gene introduction activity was observed according to the following procedure.
  • 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.
  • B16BL6 cells were seeded at 10 4 cells / well in 500 medium in a 48-well flat bottom plate.
  • reaction substrate Promega # E1501 Luciferase Assay System
  • 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.
  • 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.
  • “Tat-Ad” clearly showed a gene transfer activity much higher than “Ad” and “AdRGD”.
  • 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! /.
  • Tat-Ad exhibits high gene transfer activity against B16BL6 cells with low CAR expression as compared to conventional virus vectors.
  • Tit-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.
  • the amount of various vectors (“Tat-Ad” and “Ad”) was only lOOOvp / cell.
  • 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”.
  • FIG 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.
  • HeLa cells human cervical cancer cells
  • A549 cells human alveolar carcinoma cells
  • HT1080 cells human fibrosarcoma cells
  • luciferase assay was performed according to the following procedure.
  • 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 (113 ⁇ 4 ⁇ ) ''. There was a significant difference in the gene transfer activity between “Tat-Ad” and “Ad” and “AdRGD” and “Tat-Ad” (p ⁇ 0.01).
  • Tit-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.
  • Tat-Ad exhibits high gene transfer activity against various adherent cells (HeLa cells, A549 cells, HT1080 cells) as compared with conventional virus vectors.
  • 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).
  • 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 ( ⁇ ⁇ 0 ⁇ 01).
  • “Tat-Ad” is 30 times the luciferase activity (ie, gene transfer activity) of “Ad” in ⁇ 549 cells and 40 times in HT1080 cells. It has also been shown to have luciferase activity.
  • 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.
  • 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”.
  • 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.
  • 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.
  • EL cells mouse thymus-derived T cells
  • KG-la cells human myeloid leukemia cells
  • EL cells are cell types in which the expression of both CAR and integrin can be confirmed
  • KG-la cells are cell types in which the expression of both CAR and integrin cannot be confirmed.
  • luciferase assay was performed according to the following procedure.
  • 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 ”.
  • 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.
  • 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.
  • 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”.
  • FIG. 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.
  • 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.
  • Tit-Ad the sample of [Experimental Example 4] is used, and the experimental procedure (luciferase, etc.) and the sample “Tat-Ad” are as described above.
  • Ad and “AdRGD” were also used as comparison targets of gene expression activity.
  • 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.
  • Tat-Ad two types of modification ratios (1: 12 ⁇ 5, 1: 25) of the samples obtained in Experimental Example 5 above are used.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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”.
  • 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.
  • Tat Ad has the ability to avoid neutralizing antibodies.
  • R8-Ad an R8 peptide as an intracellular transit peptide (PTD) other than the Tat peptide
  • R8—NHS 6-maleimidohexanoic acid N-hydroxysuccinimide ester
  • R8-Ad a “R8_NH Sj-bound adenoviral vector” (hereinafter referred to as“ R8-Ad ”) was prepared in the same manner as“ Tat-Ad ”.
  • B16BL6 cells (a cell type in which CAR expression cannot be confirmed and integrin expression can be confirmed)! I reviewed it.
  • 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 Ad, Ta1-8 (1) and 3 ⁇ 48-8 ( It is a graph compared between "1".
  • 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.
  • 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.6 GFP fluorescence by EGFP gene transfer in B16BL6 cells, Ad and Tat—
  • 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”.
  • 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.

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Abstract

L'objectif de l'invention est d'obtenir un transfert de gènes y compris dans certaines cellules cancéreuses, des hémocytes etc., CAR et l'intégrine étant tous les deux très peu ou pas du tout exprimés. L'invention concerne un adjuvant pour transfert de gènes comprenant un peptide de migration cellulaire comme principe actif, se caractérisant par le fait qu'un lieur chimique a été fixé au peptide de migration cellulaire. L'invention concerne également un procédé de transfert de gènes se caractérisant par le fait qu'il consiste à fixer par une liaison covalente l'adjuvant de transfert de gènes décrit ci-dessus à la surface de la protéine de coque d'un virus utilisé comme vecteur du transfert de gènes. De préférence, le lieur chimique décrit ci-dessus est du MHS (ester N-hydroxysuccinimide d'acide 6-maléimidohexanoïque) comportant un groupe NHS (N-hydroxysuccinimidyle), etc.
PCT/JP2007/071154 2006-12-08 2007-10-30 Adjuvant pour transfert de gènes comprenant un peptide de migration cellulaire comme principe actif et procédé de transfert de gènes dans lequel est utilisé cet adjuvant pour un transfert de gènes Ceased WO2008068982A1 (fr)

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PCT/JP2007/071154 Ceased WO2008068982A1 (fr) 2006-12-08 2007-10-30 Adjuvant pour transfert de gènes comprenant un peptide de migration cellulaire comme principe actif et procédé de transfert de gènes dans lequel est utilisé cet adjuvant pour un transfert de gènes

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WO2005032593A1 (fr) * 2003-10-01 2005-04-14 Japan Science And Technology Agency Liposome a polyarginine modifiee pouvant etre transfere dans un noyau
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WO2004072289A1 (fr) * 2003-02-17 2004-08-26 Fuso Pharmaceutical Industries, Ltd. Nouveau vecteur de virus
WO2005032593A1 (fr) * 2003-10-01 2005-04-14 Japan Science And Technology Agency Liposome a polyarginine modifiee pouvant etre transfere dans un noyau
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