JP2007145761A - Cell membrane permeable peptide modified-polysaccharide-cholesterol or polysaccharide-lipid non-viral vector and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a non-viral vector having cell membrane permeability. <P>SOLUTION: The non-viral vector comprises a polysaccharide-cholesterol or a polysaccharide-lipid derivative bonded to a membrane permeable substance having a positive charge. The method for producing the same is provided. The complex comprises the vector and a useful substance. The composition for treating diseases comprises the complex. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a non-viral vector comprising a polysaccharide-cholesterol or a polysaccharide-lipid derivative chemically bound with a cell membrane permeable substance having a positive charge, its use, and a production method thereof.

  The vector of the present invention enables a nucleic acid that promotes or suppresses the expression of a specific gene to be efficiently introduced into a target cell.

  In recent years, nucleic acids, genes, synthetic peptides, proteins, etc. for the treatment and regenerative medicine of congenital diseases, cancer, AIDS, multiple sclerosis, myasthenia gravis, Parkinson's disease, rheumatoid arthritis, etc. The development of a technique for controlling the cell function by introducing a gene into a cell is expected, and in particular, there is a strong demand for the development of a gene delivery system for delivering a gene or nucleic acid to a target cell.

  In order to achieve a gene delivery system, an effective gene transfer vector is indispensable. This includes not only cytotoxicity but also safety and excellent gene transfer efficiency in the living body, as well as the quality of life of patients. In view of improvement (Quality of Life, QOL), a gene delivery system capable of controlling temporal and spatial patterns to control gene expression as needed is desired.

  Examples of gene transfer vectors include viral vectors that use retroviruses and adenoviruses, and non-viral vectors that use synthetic lipids such as cationic liposomes and cationic polymers. Viral vectors have the advantage of being able to introduce genes into non-dividing cells because of their ability to inactivate or attenuate viruses and use their ability to infect, but there are dangers such as recovery of pathogenicity and high immunogenicity. It also has sex. In contrast, it is known that non-viral vectors have the advantage of high safety but low gene transfer efficiency compared to viral vectors. However, this non-viral vector is characterized by gene transfer using electrostatic interaction between a positively charged substance such as a cationic liposome or cationic polymer and a negatively charged cell. Due to the large number, it is known that this positive charge sometimes causes cytotoxicity.

  Patent Document 1 describes a carrier for introducing a nucleic acid having specificity for a target cell and excellent gene expression. Patent Document 2 describes a cyclodextrin / dendrimer conjugate that functions as a gene transfer vector.

  It is necessary to use a material that is safe for the living body as the material for the gene transfer vector. Sunamoto et al. In Patent Document 3 show that a polysaccharide-cholesterol derivative, α-linolenic acid and water in which the side chain cholesterol of the polysaccharide is bound at a ratio of 0.1 to 10 per 100 monosaccharide units of the polysaccharide. The anticancer agent which uses the fat emulsion containing these as an active ingredient is described. In addition, Sunamoto et al. Described in Patent Document 4 an o / w type fat emulsion having anticancer activity composed of an oil layer containing a polysaccharide-cholesterol derivative, α-linolenic acid and medium chain fatty acid triglyceride. . Patent Document 5 describes a skin external preparation using a polysaccharide-cholesterol in combination with a preparation using an acrylic acid / alkyl methacrylate copolymer. However, these Patent Documents 3 to 5 do not describe the use of a polysaccharide-cholesterol derivative as a gene transfer vector.

  In order to efficiently express a gene in a target cell, introduction of the gene into the cell, escape from the endosome (or macropinosome) and endosome / lysosome after being taken into the cell, permeation of the nuclear membrane, It is necessary to perform transcription in the nucleus efficiently. The mechanism of uptake of genes and vectors into cells and their behavior in cells are being clarified, and they are called cell penetrating peptides (CPP) and protein transduction domains (PTDs). Intracellular translocation peptides with 10-20 amino acids necessary for permeation of the membrane, as well as nuclear localization signals (transcription factor) that have functions of translocation to the nucleus, localization in the nucleus, and transcription promotion. Has attracted attention (Non-Patent Document 1). Patent Document 6 describes a cell-permeable carrier peptide containing a polypeptide having a special sequence.

JP 2005-176830 A Japanese Patent Laid-Open No. 2001-103969 Japanese Unexamined Patent Publication No. 5-139967 Japanese Patent Laid-Open No. 5-26645 Japanese Patent Laid-Open No. 2003-73281 JP 2001-199997 Shiro Futaki, Chemistry and Biology 43 (10): 649-653 (2005)

  The construction of a non-viral vector that has a function of transferring genes into cells or nuclei using biocompatible materials and that promotes high gene transfer and gene expression is extremely important for establishing a gene delivery system. It is believed that.

  An object of the present invention is to efficiently deliver a useful substance such as a plasmid, a short ribonucleic acid or a deoxyribonucleic acid, a therapeutic protein, a peptide or a small molecule that can promote or suppress the expression of a specific protein to a target cell or tissue. It is to provide a polysaccharide-cholesterol or polysaccharide-lipid non-viral vector to which a positively charged cell-permeable substance is bound, which can be introduced, has high biocompatibility and low cytotoxicity.

Another object of the present invention is to provide a method for producing the above vector.
Still another object of the present invention is to provide a complex of the vector and a useful substance.

  As a result of intensive studies to solve the above problems, the present inventors have found that a conjugate obtained by chemically modifying a polysaccharide-cholesterol or a polysaccharide-lipid derivative with a membrane-permeable substance can target a useful nucleic acid or a target nucleic acid. The present invention was completed by finding efficient introduction into an organization.

  That is, the present invention can be summarized as follows.

In the first aspect, the present invention provides a non-viral vector comprising a polysaccharide-cholesterol or a polysaccharide-lipid derivative to which a positively charged membrane-permeable substance is bound.
In one embodiment thereof, the membrane permeable substance is a peptide, a basic amino acid, or a cationic polymer compound.
In another embodiment thereof, the membrane permeable substance is a substance having a cell migration property, a nuclear migration property, or a nuclear migration signal.
In another embodiment thereof, the membrane permeable substance is bound to at least one sugar residue of the polysaccharide.
In another embodiment thereof, the membrane permeable substance is a peptide containing a cysteine residue.
In another of its embodiments, wherein the peptide, in its C-terminus, is intended to include -Cys-Gly-NH _2.
In another embodiment thereof, the peptide is bound to the polysaccharide-cholesterol or polysaccharide-lipid derivative by a disulfide bond.
In another embodiment thereof, the vector of the present invention is characterized by forming nano-sized micelles in the presence of water.

In the second aspect, the present invention also provides a complex of the above-described vector of the present invention and a useful substance.
In one embodiment thereof, the useful substance is for treatment of a disease.
In another embodiment thereof, the useful substance is a nucleic acid, protein, peptide or small molecule.
In another embodiment thereof, the nucleic acid comprises a gene that can be expressed.
In another embodiment thereof, the nucleic acid containing a gene capable of expressing the nucleic acid is a plasmid.
In another embodiment thereof, the nucleic acid has a single-stranded or double-stranded structure, has a length in the range of about 15 to 30 bases, and has a function of suppressing the expression of a specific gene in a target cell. Or RNA.
In another embodiment thereof, the nucleic acid is a dsRNA, siRNA or ribozyme.
In another embodiment thereof, the nucleic acid is an aptamer.
In another embodiment thereof, the complex can be administered to a diseased or non-disease site in mammalian cells, tissues or organs, including humans.
In another embodiment thereof, the tissue is a mucosa.

In the third aspect, the present invention further provides a composition for treating a disease, comprising the above-described complex of the present invention and a pharmaceutically acceptable carrier.
In that embodiment, the composition further comprises a peptide having the action of a nuclear translocation signal.

The present invention further relates to a method for producing a non-viral vector comprising a polysaccharide-cholesterol or a polysaccharide-lipid derivative bound to a membrane-permeable peptide having a positive charge in the fourth embodiment, comprising: (i) polysaccharide-cholesterol Or a step of binding a linker having a mercapto (SH) group to at least one OH group of a sugar residue of a polysaccharide-lipid derivative, and (ii) an SH group of the linker of the product obtained in step (i) Providing a method comprising forming a disulfide bond with a cysteine (Cys) residue of a membrane-permeable peptide containing -Cys-Gly-NH ₂ at the C-terminus.
In that embodiment, the method may further comprise steps of protection and deprotection.

  The vector of the present invention can stably and quickly introduce useful substances such as nucleic acids and proteins into cells without being degraded by enzymes in the blood or cells. Useful substances such as nucleic acids can be introduced into cells to efficiently inhibit gene expression, and by using the vectors of the present invention, nucleic acids can be quickly transferred from the cytoplasm into the nucleus and efficiently In addition, the expression of the target gene can be promoted.

  The vector of the present invention can easily form a complex with a useful substance such as a nucleic acid and protein, and the vector of the present invention is not cytotoxic and has a risk of damaging a target cell or tissue. It can be said that it is safe. Furthermore, the vector of the present invention can be administered to mucous membranes such as the nose, vagina, and lung, and if necessary, by using an electroporation method or a sonoporation method in combination, a higher gene transfer and expression effect can be obtained. Can show.

1. Definitions The terms used herein include the following definitions.
The “vector” means a polysaccharide-cholesterol or a polysaccharide-lipid derivative having a function of transporting a useful substance such as a nucleic acid, a protein, and a small molecule into a cell, and can also be called a carrier. In particular, vectors for transporting nucleic acids can also be referred to as “gene transfer vectors”, “gene carriers”, and the like.

  “Membrane permeability” refers to the property that the non-viral vector of the present invention permeates through the cell membrane or nuclear membrane of animal cells, eg, mammalian cells including human cells, and into the cytoplasm or nucleus. Therefore, the “membrane permeable substance” refers to any small or high molecular substance having such characteristics and capable of transferring the vector into cells. A preferred membrane-permeable substance in the present invention is a peptide, which is sometimes referred to as a “carrier peptide”.

  “Has a positive charge” means that the molecule is electrically positive. Such a group imparting a positive charge is, for example, a group such as an amino group or a guanidino group, and these groups form a positively charged quaternary ammonium ion or guanidinium ion. The term “cationic” as used herein has a similar meaning.

  “Nanosize” refers to a nanometer size in which the diameter of the nanoparticles is less than about 1 μm.

  “Complex” refers to a substance in which a useful substance is noncovalently bound to or incorporated into the vector of the present invention. “Non-covalent” means relatively weak binding such as ionic, hydrophobic, physical interaction (ie van der Waals forces) or uptake such as embedding, inclusion, etc.

  “Disease site” refers to the site of a tissue or organ that has actually been affected by a disease or is subject to gene therapy.

2. Polysaccharide-cholesterol or polysaccharide-lipid derivative The polysaccharide-cholesterol or polysaccharide-lipid derivative, which is a component of the vector of the present invention, is obtained by covalently binding a polysaccharide to cholesterol or lipid.

  Polysaccharides include naturally occurring or chemically synthesized products such as pullulan, amylopectin, amylose, glycogen, cyclodextrin, dextran, hydroxyethyldextran, mannan, cellulose, starch, alginic acid, chitin, chitosan, hyaluronic acid and their derivatives. Although it can be used, it is not limited to these.

  Cholesterol and lipids include, in addition to cholesterol, lipids of natural origin or chemical synthesis, such as caprylic acid, pelargonic acid, capric acid, lauric acid, myristic acid, pentadecyl, which can form sugar fatty acid esters. Acids, palmitic acid, margaric acid, stearic acid, oleic acid, linoleic acid, linolenic acid and other fatty acids, capable of forming glycolipids, ceramide (N-acyl sphingosine), acyl glycerol, alkyl glycerol, steroids, etc. However, it is not limited to these.

  The polysaccharide and cholesterol or lipid may be bound directly or indirectly via a linker (or spacer), but are preferably bound via a linker.

The synthesis of the polysaccharide-cholesterol derivative is carried out, for example, by adding OHOC-NH (CH ₂ ) _n NH-COOH (where n = 1 to 18), HOOC- (CH ₂ ) as a linker to the OH group at the C3 position of cholesterol. the _n -COOH alkylene diisocyanates and alkylene dicarboxylic acids (where n = 1 to 18), such as esterified, and the other carboxyl group, can be obtained by esterification of the primary OH group of the polysaccharide.

  Specifically, the polysaccharide-cholesterol derivative can be synthesized as follows by a method described in JP-A-5-139967 (Patent Document 3).

  Cholesterol (10 mmol) is dissolved in toluene (100 ml), pyridine (4 ml) and alkylene diisocyanate (148 mmol) are added thereto and reacted at 80 ° C. for 24 hours, and then toluene and unreacted alkylene diisocyanate are removed under reduced pressure. To this is added petroleum ether (500 ml) and the product is extracted (45% yield). Next, the synthesized N- (isocyanatoalkyl) cholesterylcarbamate (0.19 mmol) was added to a solution in which polysaccharide (7.44 mmol per saccharide unit) was dissolved in anhydrous dimethyl sulfoxide (100 ml) and pyridine (8 ml) was added, React at 100 ° C. for 8 hours. After completion of the reaction, dimethyl sulfoxide is removed under reduced pressure, and ethanol (500 ml) is added thereto to precipitate the product (yield: about 75%).

  The synthesis of polysaccharide-lipid derivatives is performed by esterifying the carboxyl group to a primary OH group of a polysaccharide when the lipid is a fatty acid, and when the lipid is a ceramide, acylglycerol, alkylglycerol, or steroid. Then, after linking the above OH group to the OH group, the other carboxyl group can be obtained by esterification to a primary OH group of a polysaccharide. A polysaccharide-lipid derivative can be synthesized in the same manner as in the synthesis of the polysaccharide-cholesterol derivative.

  The polysaccharide-cholesterol or polysaccharide-lipid derivative of the present invention is a conjugate in which cholesterol or lipid is bound to the side chain of the polysaccharide at a ratio of 0.1 to 10 per 100 monosaccharide units of the polysaccharide. When the polysaccharide is, for example, pullulan, cholesterol is bound to the side chain of the polysaccharide at a ratio of 1 to 2 per 100 monosaccharide units (Patent Document 3).

  The polysaccharide-cholesterol or polysaccharide-lipid derivative of the present invention has a characteristic of forming monodisperse nanoparticles by self-association in water due to the structure having a hydrophobic group and a hydrophilic group. The charge is neutral, and a membrane-permeable substance having a positive charge is bound to it for use as the material of the vector of the present invention.

3. According to the present invention, a target vector can be obtained by covalently binding a positively charged membrane-permeable substance to a polysaccharide-cholesterol or a polysaccharide-lipid derivative.

  The membrane-permeable substance having a positive charge may be any substance as long as it has a positive charge and can permeate into the cells of animal cells. Preferred are low molecular or high molecular organic molecules. According to an embodiment of the present invention, such a low molecular or high molecular organic molecule is preferable. According to an embodiment of the present invention, such a membrane permeable substance is a peptide, a basic amino acid or a cationic polymer compound. Other possible membrane permeable materials include cationic lipids.

  Examples of basic amino acids include (DL-, D- or L-) arginine, lysine, histidine and the like, but are not limited thereto.

  Examples of the cationic polymer compound include, but are not limited to, polyethyleneimine, polyarginine, polylysine and the like.

  Cationic lipids include dimethyl dioctadecyl ammonium bromide (DDAB), trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA), N-1-2,3-dioleyloxypropyl-N, N, N- Examples include trimethylammonium chloride (DOTAP), cholesteryl 3β-N-dimethylaminoethylcarbamate hydrochloride (DC-Chol), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxylammonium bromide (DMRIE). However, it is not limited to these.

  Membrane-permeable peptides have the effect of rapidly introducing useful substances such as genes and proteins into cells, and are called cell penetrating peptide (CPP) or protein transduction domain (PTD). A basic peptide having ˜45 amino acids.

  Examples of the membrane-permeable peptide include a peptide containing an amino acid sequence rich in arginine at positions 48 to 57 of human immunodeficiency virus-1 (HIV-1) Tat protein showing cell membrane permeability, or a derivative thereof (JS Wadia and SF Dowdy, Adv. Drug Deliv. Rev. 57: 579 (2005)), R6-R8 peptide composed of 6-8 arginines and derivatives thereof (LR Wright et al., Curr. Protein Pept. Sci. 4: 105 ( 2003); S Futaki et al., J. Mol. Recobnit. 16: 260 (2003)), and HIV-1 Rev (34th-50th), FHV coat (35th-49th), BMG Gag (7-25) HTLV-II Rex (4th to 16th), P22 N (14th to 30th), IN (1st to 22nd), Yeast PRP6 (129th to 144th), Human U2AF (142th to 153th), Human cFos (139-164), Human cJun (252-279), Yeast GCN4 (231-252) and other peptides or derivatives that bind to DNA or RNA (edited by Yasuhiko Tabata, Drug Delivery System DDS Technology) New developments and their usage, p25, media Karudu, Osaka, 2003) include, but are not limited to these.

  The membrane-permeable substance that is a constituent component of the vector of the present invention has an action as a cell migration property, a nuclear migration property, or a nuclear migration signal.

  In particular, a special endocytosis called macropinocytosis has been proposed as a mechanism for permeation of membrane-permeable substances through cell membranes (Non-patent Document 1; JS Wadia et al., Nature Med., 10: 310 (2004). I Nakase et al., Mol. Ther., 10: 1011 (2004)). Macropinocytosis is a mechanism in which the actin filaments around the cell membrane are polymerized so that the membrane protrudes out of the cell and the extracellular fluid is taken into the cell (SD Conner and SL Schmid, Nature 422: 37 (2003)). In the case of macropinocytosis, it is said that endosomes (macropinosomes) often exceeding 1 μm are formed, so that substances with a diameter of 200 nm or more can be taken into cells. Cell membrane permeation mechanism of sex substances may be temperature-dependent or caveolae-mediated endocytosis (P. Jarver and U. Langel, Drug Discovery Today, 9 (9), 395 (2004), A. Fittipaldi, M. Giacca, Ad. Drug Del. Rev., 57, 597-608 (2005)), etc., and it has not yet been fully clarified.

  Furthermore, it is thought that the exchange of substances between the nucleus and the cytoplasm is performed through the nuclear pore complex (NPC) as a mechanism for the permeability of membrane-permeable substances through the nuclear membrane. It is said that molecules with a diameter of 9 nm or less (equivalent to a molecular weight of 40 to 90 kDa) can freely pass through the NPC. The nuclear translocation signal has the effect of selectively translocating large molecules with a diameter of about 9-40 nm (molecular weight of 90 kDa or more) that cannot freely pass through the NPC to the nucleus. Has functions of translocation, nuclear localization, and transcription promotion.

  Examples of the nuclear translocation signal include peptides such as transcription factors NF-κB (p50), NF-κB (p60), Mitosin, Myc, DNA helicase Q1, hNRNP, and derivatives thereof (edited by Yasuhiko Tabata, drug delivery). New development of system DDS technology and its utilization, p25, Medical Do, Osaka (2003)) are not limited to these.

  In addition, as the membrane-permeable peptide in the present invention, not only a peptide having cell transferability and nuclear transferability but also a pH-sensitive or pH-insensitive peptide having membrane fusion ability can be used.

Examples of pH-insensitive peptides include:
HIV-1 gp41 (N-terminus) (AVGVLGALFLGFLGAAGSTMGAASLTLT; SEQ ID NO: 4),
SIV gp32 (N-terminus) (GVFVLGFLGFLATAG; SEQ ID NO: 5),
Sendai virus F1 (N-terminus) (FFGAVIGTIALGVATSAQITAGIALAER; SEQ ID NO: 6)
Virus-derived peptides, such as
Examples of pH sensitive peptides include:
influenza (X31) HA2 (N-terminus) (GLFGAIAGFIENGWEGMIDGWYG; SEQ ID NO: 7),
influenza (X31) HA2 (N-terminus) (IFGIDDLIIGLLFVAIVEAGIG; SEQ ID NO: 8),
Polio1 vp1 (N-terminus) (GLGQMLESMIDNTVREVGGA; SEQ ID NO: 9),
Polio3 vp1 (N-terminus) (GIEDLISEVAQGALTLVP; SEQ ID NO: 10),
Vaccinia virus 14kD (N-terminus) (MDGTLFPGDDDLAIPATEFFSTKA; SEQ ID NO: 11),
Semliki Forest virus E1 (internal) (DYQCKVYTGVYPFMWGGAYCFCD; SEQ ID NO: 12)
Virus-derived peptides, such as
INF1 (GLFGAIAGFIENGWEGMIDGGGC; SEQ ID NO: 13),
INF2 (GLFEAIAGFIENGWEGMIDGGGC; SEQ ID NO: 14),
INF3 (GLFEAIEGFIENGWEGMIDGGGC; SEQ ID NO: 15),
INF4 (GLFEAIEGFIENGWEGnIDGCA; SEQ ID NO: 16),
INF5 (GLFEAIEGFIENGWEGnIDGC-ss-CGDINGEWGNEIFGEIAEFLG; SEQ ID NO: 17),
INF6 (GLFGAIAGFIENGWEGMIDGWYG; SEQ ID NO: 18),
INF7 (GLFEAIEGFIENGWEGMIDGWYG; SEQ ID NO: 19),
diINF7 (GLFEAIEGFIENGWEGMIDGWYGC-ss-CGYWGDIMGEWGNEIFGEIAEFLG; SEQ ID NO: 20),
NF8 (GLFEAIEGFIENGFEGMIDGGGC-s-sCGGGDIMGEFGNEIFGEIAEFLG; SEQ ID NO: 21),
INF9 (GLFELAEGLAELGAEGLAEGWYGC; SEQ ID NO: 22),
INF10 (GLFELAEGLAELGWEGLAEGWYGC; SEQ ID NO: 23),
D4 (GLFGAIADFIEGGWEGLIEG; SEQ ID NO: 24),
E5 (GLFEAIAEFIEGGWEGLIEG; SEQ ID NO: 25),
E5L (GLLEALAELLEGGWEGLLEG; SEQ ID NO: 26),
K5 (GLFKAIAKFIKGGWKGLIKG; SEQ ID NO: 27),
GALA (WEAALAEALAEALAEHLAEALAEALAEALAAGGSC; SEQ ID NO: 28),
GALA-GLF (GLFGALAEALAEALAEHLAEALAEALEALAAGGSC; SEQ ID NO: 29),
GALA-INF1 (GLFGAIAGFIENGWEGLAEALAEALEALAAGGSC; SEQ ID NO: 30),
GALA-INF3 (GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC; SEQ ID NO: 31),
KALA (WEAKLAKALAKALAKHLAKALAKALKACEA; SEQ ID NO: 32),
ppTG1 (GLFLALLKLLKSLWKLLLKA; SEQ ID NO: 33),
ppTG20 (GLFRALLRLLRSLWRLLLRA; SEQ ID NO: 34),
JTS-1 (GLFEALLELLESLWELLLEA; SEQ ID NO: 35),
JST-1-K13 (GLFEALLELLESLWELLLEAC; SEQ ID NO: 36, SEKKKKWKKKKKKKKAKYC; SEQ ID NO: 37)
Synthetic peptides such as, but not limited to.

4). Vector Production A method for conjugating a membrane permeable substance to a polysaccharide-cholesterol or polysaccharide-lipid derivative comprises chemically linking the membrane permeable substance to a hydroxyl group (OH) of at least one sugar residue of the polysaccharide. .

Binding can be done directly or indirectly. When the membrane-permeable substance is, for example, a basic amino acid, the amino group or guanidino group can be protected with an appropriate protecting group, and then the free carboxyl group can be directly bonded to the primary OH group of the sugar by an esterification reaction. If necessary, they may be linked via a linker (or spacer).

When the membrane-permeable substance is a peptide as exemplified above, it is desirable to modify it to include a cysteine (Cys) residue. According to an embodiment of the present invention, membrane permeable peptide, to the C-terminus is modified to include a -Cys-Gly-NH _2. When the peptide contains a Cys residue, a disulfide (-SS-) bond can be formed between the Cys group and the terminal mercapto (SH) group of the linker that is previously bonded to the OH group of the polysaccharide. Disulfide bonds are stable in the body circulatory system such as blood and taken into cells, then cleaved by enzymes, diffuse in the cytoplasm with basic peptides such as Tat, and move into the nucleus to efficiently promote gene expression It is expected that.

  Examples of the linker are, for example, ω-mercaptoalkanoic acid, such as 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 5-mercaptovaleric acid and the like, but are not limited thereto. When ω-mercaptoalkanoic acid is bonded to a polysaccharide, the mercapto group is protected with an appropriate protecting group such as a triphenylmethyl group, and then an ester bond is formed between the polysaccharide and the OH group (preferably a primary OH group). Form.

Therefore, the above method provides a method for producing the viral vector of the present invention comprising a polysaccharide-cholesterol or polysaccharide-lipid derivative to which a positively charged membrane-permeable peptide is bound, which comprises (i) a polysaccharide -Coupling a linker having a mercapto (SH) group to at least one OH group of a sugar residue of a cholesterol or polysaccharide-lipid derivative; and (ii) SH of the linker of the product obtained in step (i) comprising a base, a step of forming a disulfide bond between the cysteine (Cys) residues of the membrane-permeable peptide containing a -Cys-Gly-NH ₂ at the C-terminus.

When a linker is attached to polysaccharide-cholesterol or polysaccharide-lipid derivative polysaccharide, it is necessary to protect the mercapto group. Thereafter, when the membrane-permeable peptide is bound to the linker group of the polysaccharide, it is necessary to deprotect the protecting group of the mercapto group. Therefore, the above method of the present invention can further include protection and deprotection steps.
In the examples described later, production examples of Tat-modified cholesterol pullulan derivatives are specifically described.

  In any case, the method for binding the polysaccharide-cholesterol or polysaccharide-lipid derivative and the membrane-permeable substance is not limited to the above-described method for forming a disulfide bond, but an ester bond, an ether bond, an amide Includes methods commonly used in amino acid and sugar chemistry such as conjugation (C. Fernadez, O. Nieto, and A. Fernadez-Mayoralas and et al., Org. Biomol. Chem., 1, 767-771 (2003), Taku Mizuno and Kazutoshi Nishizawa, Illustrated Carbohydrate Chemistry Handbook, p187-286 (1971)).

5. Complex of vector and useful substance The vector of the present invention forms nano-sized micelles in the presence of water by making the cholesterol or lipid part a hydrophobic group, while the polysaccharide part is a hydrophilic group. The average particle size is about 50 nm to 3 μm, preferably about 50 nm to 1,000 nm, more preferably about 50 nm to 800 nm, more preferably less than 300 nm, such as about 50 nm to 200 nm. One of the important characteristics of the vector of the present invention is the ability to form nanoparticles. This improves the efficiency of introduction into cells and nuclei.

  The vector of the present invention also has extremely low cytotoxicity and high safety because 100% of cells survive in the cytotoxicity test. This property is very important when applied to the human body.

  The vector of the present invention can further incorporate or adsorb (or attach) useful substances inside or on the surface, and useful substances incorporated into cells or nuclei can be incorporated into cells or nuclei. Can fulfill the function.

Useful substances include, but are not limited to, nucleic acids, proteins, peptides, small molecules and the like. Preferred useful substances are for the treatment of diseases.
Diseases include, for example, cancer, inherited diseases caused by defective genes.

  Examples of nucleic acids include (i) a plasmid containing a foreign gene, for example, (a) a plasmid containing a foreign gene so that it can be expressed, and (b) a normal gene replacing a defective gene (defective enzyme gene, defective tumor suppressor gene, etc.). (Ii) plasmids containing DNA encoding nucleic acids such as dsRNA, siRNA, ribozyme, etc. (ii) having a single-stranded or double-stranded structure, about DNA or RNA (short-chain deoxyribonucleic acid and ribonucleic acid), such as dsRNA, siRNA, ribozyme, etc., having a length in the range of 15 to 30 bases and having a function of suppressing the expression of a specific gene in a target cell (iii) Aptamers and the like are included.

  In particular, dsRNA, siRNA or ribozyme is useful for inhibiting or blocking the function of genes specifically expressed in tumors such as angiogenic factor carcinogenesis-related genes and cancer metastasis-related genes. These RNAs and ribozymes can block protein translation by binding to or cleaving transcripts (mRNA) of tumor-associated genes. Such a disease-related gene sequence is registered in GenBank or the like and can be easily obtained. The sequence of dsRNA, siRNA or ribozyme can be designed based on the registered sequence information of the target gene.

  Both dsRNA and siRNA are nucleic acids involved in RNA interference, and double-stranded RNA (dsRNA) is processed by a dicer to form siRNA (small interfering RNA). siRNA specifically cleaves target mRNA. Target site selection criteria are also known (DM Dykxhoorn et al., Nature Rev. Mol. Cell Biol. 77: 7174-7181 (2003); A Khvorova et al., Cell 115: 209-216 (2003)) Therefore, based on this criterion, the target site of the target gene can be estimated and the siRNA sequence can be determined.

  Ribozymes are RNAs that have catalytic activity and cleave target mRNA, thereby inhibiting or preventing target gene expression. The target sequence is generally known to be a sequence containing NUX (where N = A, G, C, U; X = A, C, U), such as a GUC triplet. Ribozymes include hammerhead ribozymes and hairpin ribozymes.

  An aptamer is a nucleic acid having a relatively short chain length that binds to a protein, and consists of an RNA aptamer and a DNA aptamer. Aptamers are not only limited to proteins, but are artificially created by evolutionary engineering techniques called in vitro selection targeting a wide range of substances from low molecular substances such as metal ions, amino acids, and antibiotics to proteins, viruses, and cells. be able to. In particular, an aptamer that binds to an enzyme has an action of inhibiting the enzyme activity.

  The protein, peptide or small molecule is preferably for the treatment of a disease and is a substance that can act directly at the site of the disease, for example, an intracellular enzyme, antibody, signal transduction agent, inhibitor, agonist, antagonist, gene expression Examples include regulatory proteins or peptides (P. Jarver and U. Lougel, Drug Discovery Today, 9 (9), 395 (2004)), APC, tumor suppressor gene-producing proteins such as p53 and p73, and other small molecule drugs.

  The complex of the present invention is formed by mixing the vector of the present invention and a useful substance in a physiologically acceptable aqueous solution such as physiological saline or buffer. The ratio of useful substance to vector is 1: 2 to 1:15, preferably 1: 5 to 1:10 (mass ratio).

6). Therapeutic Composition The complex of the present invention can be combined with a carrier such as a pharmaceutically acceptable diluent and an additive used as appropriate to form a therapeutic composition.
Examples of the diluent include physiological saline, buffer solution and the like.

  Additives include, for example, polyethylene glycol, cationic lipids, sugars such as glucose, mannose and galactose, substances having surface activity such as cetyltrimethylammonium bromide (CTAB), poloxamer and Tween 80, tissues and cells involved in diseases And substances specific to its receptor or related factors, such as transferrin (TF), endothelial growth factor (EGF), low density lipoprotein (LDL), insulin, folic acid, galactose, mannose, etc. However, it is not limited to these.

  The composition of the present invention includes a peptide having the action of a nuclear translocation signal as described above, for example, transcription factors NF-κB (p50), NF-κB (p60), Mitosin, Myc, DNA helicase Q1 , Peptides such as hNRNP can be included. This increases the efficiency of transfer into the nucleus.

  Diseases that can be treated by the composition of the present invention are not particularly limited, but for example, cancer, genetic diseases, viral diseases caused by HIV, HCV, HPV, etc., myasthenia gravis, Parkinson's disease, rheumatoid arthritis, Alzheimer Disease, obstructive arteriosclerosis, angina pectoris, myocardial infarction, age-related macular disease, etc.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example and an experiment example, this invention is not limited to these Examples.

Example 1
The purpose of this example is to synthesize a Tat derivative having cell transferability and nuclear transferability, and an NF-κB derivative and MA (HIV-1) derivative which are nuclear transfer signals.

(Synthesis Example 1)
As represented by SEQ ID NO: 1 in Table 1, C of a polypeptide having an amino acid sequence of positions 48-57 of human immunodeficiency virus 1 (HIV-1) Tat, which is said to have cell membrane permeability and nuclear translocation A polypeptide with cysteine-glycine-amide (-Cys-Gly-NH ₂ ) added to the end is used to start NH ₂ -SAL-Trt (2-Cl) resin using ABI433A type peptide synthesizer manufactured by Applied Biosystems The raw material was synthesized by Fmoc solid phase synthesis.

  The resulting peptide was treated with 1 mL of a mixture of trifluoroacetic acid 1050 μL / thioanisole 62.5 μL / water 62.5 μL / triisopropylsilane 31.5 μL / crystalline phenol 100 mg and treated at room temperature for 3 hours. The product was cut out from the sample, purified using an ion exchange resin (TOYOPEARL CM-650, manufactured by TOSOH) and a gel filter (Sephadex G-25, manufactured by Amersham Biosciences), and then lyophilized. After performing mass spectrometry using a time-of-flight mass spectrometer (MALDI-TOFMS, Voyager DE, manufactured by PerSeptive Biosystems), adding an appropriate amount of 6N hydrochloric acid to the obtained peptide, and performing heat-sealing at 110 ° C for 24 hours Amino acid analysis was performed using a high-speed amino acid analyzer (L-8500, manufactured by HITACHI).

(result)
The mass of the obtained polypeptide was very close to the calculated value calculated from the molecular weight of each amino acid, and from the results of amino acid analysis, it was confirmed that the peptide had the amino acid sequence represented by SEQ ID NO: 1. In addition, since the amino acid sequence of HIV-1 Tat was partially modified by addition of cysteine and glycine, it is referred to as a Tat derivative in this example.

(Synthesis Examples 2 and 3)
As shown in SEQ ID NOs: 2 and 3 in Table 1, transcription factors (nuclear factor-κB, NF-κB) which are nuclear localization signals (NLS) and matrix peptides derived from matrix protein of HIV-1 Gly at the N-terminus of a polypeptide having an amino acid sequence, a polypeptide -Cys-Gly-NH ₂ are respectively added to the C-terminus, it was synthesized and purified in the same manner as in synthesis example 1, was recovered as a freeze-dried product. The obtained polypeptide was subjected to mass spectrometry and amino acid analysis in the same manner as in Synthesis Example 1.

(result)
As a result of mass spectrometry, the mass of the obtained polypeptide is very close to the calculated value calculated from the molecular weight of each amino acid, and from the results of amino acid analysis, it is a peptide having the amino acid sequence represented by SEQ ID NOs: 2 and 3. It was confirmed that there was. Since the amino acid sequences of the NF-eB and MA peptides were partially modified by the addition of cysteine and glycine, they are referred to as NF-eB derivatives and MA derivatives in this example.

(Example 2)
The purpose of this example is to synthesize a Tat-modified cholesterol pullulan derivative in which a cell-permeable and nuclear translocation peptide Tat derivative is chemically bound to cholesterol pullulan via a disulfide bond as a non-viral gene transfer vector. Yes.

(Synthesis Example 4)
A solution obtained by dissolving 56.6 mmol of 3-mercaptopropionic acid as a cross-linking agent in 50 mL of dichloromethane and dissolving 62.3 mmol of triphenylchloromethane corresponding to 1.1 equivalent of 3-mercaptopropionic acid in 30 mL of dichloromethane. After stirring at room temperature for 16 hours, the solvent was distilled off. Next, acetonitrile was added and dissolved by heating, followed by recrystallization. By suction filtration, 8.7189 g of white powder 3-trityl mercaptopropionic acid (yield 72.61%) was obtained.
This product was confirmed to be the target product by NMR spectrum analysis and thermal analysis.

Melting point 208.3 ° C, H ¹ -NMR (400 MHz, DMSO-d ₆ ) δ7.23-7.36 (m, 15H, C ₆ H _5- ), δ2.14-2.30 (d, 4H, -C H ₂ S- , -C H ₂ CO-); 13C NMR (100 MHz, DMSO-d6) δ 172.57 ( -C O-), δ 126.63-128.98, 144.25 ( C ₆ H _5- ), δ 66.07 (C ₆ H ₅ -C S-), δ32.79 ( -C H ₂ S-), δ 26.58 ( -C H ₂ CO-); m / z = 243.12 (C ₆ H ₅ ) 3C ⁺ ).

を有するコレステロールプルラン(MW 108,000; 0.9コレステロール単位/100グルコース単位) 150 mg（約1 mmol）と、上記生成物の3-トリチルメルカプトプロピオン酸365 mg（約1.05 mmol）とをジメチルホルムアミド4 mLに溶解し、さらに、水溶性カルボジイミド0.266 mL（1.5 mmol）と4-ジメチルアミノピリジン12.2 mg（0.1 mmol）を加えて室温で24時間撹拌し、3-トリチルメルカプトプロピオン酸をコレステロールプルランにエステル結合を介して導入した。反応終了後、生成されたコレステロールプルランと3-トリチルメルカプトプロピオン酸の結合体を、水を加えて沈殿させたのち吸引ろ過によって固体を回収した。これをアセトニトリルもしくは水/酢酸エチル混液で洗浄後、減圧乾燥を行い、精製された白色固体のコレステロールプルランと3-トリチルメルカプトプロピオン酸の結合体（保護基付）を得た。この生成物50 mgに対して、トリフルオロ酢酸／ジクロロメタン／トリイソプロピルシラン／結晶フェノール／水（50/47/1/1/1）の混合液2-5 mLを加え、15分間撹拌することによってトリチル基を脱保護し、さらに、この反応液を濃縮後、残渣にジエチルエーテルを加えて生成物を析出させ、洗浄後、減圧乾燥によりチオール基を有するコレステロールプルラン−リンカー（収率49%）を得た。 The following structure:

150 mg (approx. 1 mmol) of cholesterol pullulan (MW 108,000; 0.9 cholesterol units / 100 glucose units) and 365 mg (approx. 1.05 mmol) of 3-trityl mercaptopropionic acid of the above product dissolved in 4 mL of dimethylformamide Furthermore, 0.266 mL (1.5 mmol) of water-soluble carbodiimide and 12.2 mg (0.1 mmol) of 4-dimethylaminopyridine were added and stirred at room temperature for 24 hours, and 3-trityl mercaptopropionic acid was added to cholesterol pullulan via an ester bond. Introduced. After completion of the reaction, the produced cholesterol pullulan and 3-trityl mercaptopropionic acid conjugate was precipitated by adding water, and then the solid was collected by suction filtration. This was washed with acetonitrile or a water / ethyl acetate mixture, and then dried under reduced pressure to obtain a purified white solid cholesterol pullulan and 3-trityl mercaptopropionic acid conjugate (with protecting group). To 50 mg of this product, add 2-5 mL of a mixture of trifluoroacetic acid / dichloromethane / triisopropylsilane / crystalline phenol / water (50/47/1/1/1) and stir for 15 minutes. After deprotecting the trityl group, the reaction solution was concentrated, diethyl ether was added to the residue to precipitate the product, washed, and dried under reduced pressure to remove cholesterol pullulan-linker (yield 49%) having a thiol group. Obtained.

  1 mL of acetic acid / 2,2,2-trifluoroethanol / dichloromethane (20:20:60) is added to 50 mg of the Tat derivative-protected peptide resin in Synthesis Example 1 and reacted at room temperature for 30 minutes to release the protected peptide from the resin. Then, the solution was filtered to remove the resin. After completely distilling off the filtrate, the residue (Tat derivative-protected peptide) is dissolved in 1.5 ml of dichloromethane, and 4 to 5 times (molar ratio) chlorocarbonylsulfenyl chloride is added to the Tat derivative-protected peptide for 15 minutes at room temperature. Reacted. Next, while cooling, add 10 times (molar ratio) of methylaniline to chlorocarbonylsulfenyl chloride, react at room temperature for 15 minutes, distill off the solvent with nitrogen gas, and use ethyl acetate / distilled water. Extracted. After concentrating the ethyl acetate layer, a small amount of diethyl ether was added to precipitate a Tat derivative, washed 3 times by decantation, evaporated to dryness (recovery rate 53%). By the above operation, the protecting group of the cysteine group of the Tat derivative is substituted from the acetamidomethyl group (Acm) to the N-methyl-N-phenylcarbamoyl) sulfenyl group (Snm).

  10 mg of the cholesterol pullulan-linker was dissolved in dimethylformamide / N-methylmorpholine (99: 1). To this, 40 mg of a Tat derivative in which the cysteine protecting group was substituted from Acm to Snm was added and stirred overnight to disulfide bond the thiol group (—SH) of cholesterol pullulan-linker and the SH group of cysteine. After completion of the reaction, the reaction mixture is concentrated, and diethyl ether is added to the residue to precipitate a solid. After washing, the solvent is distilled off using nitrogen gas, dried under reduced pressure, and Tat-modified cholesterol pullulan (protective) Obtained). This Tat-modified cholesterol pullulan (with protecting group) is dissolved in 1 mL of a mixture of trifluoroacetic acid 1050 μL / thioanisole 62.5 μL / water 62.5 μL / triisopropylsilane 31.5 μL / crystalline phenol 100 mg, and deprotected for 3 hours. To cleave the protecting group. The reaction solution was concentrated, diethyl ether was added to the residue, and the precipitate was washed and dried under reduced pressure to obtain Tat-modified cholesterol pullulan as a white solid. The product was purified by dialysis treatment in distilled water using a dialysis membrane (Spectra / pore CE dialysis tube, manufactured by Spectrum Laboratories), and Tat-modified cholesterol pullulan (CP-Tat) purified by lyophilization was recovered. (Recovery rate 25%).

  The purified CP-Tat was dissolved in distilled water, and the solution was spotted on a silica gel thin-layer chromatography plate and subjected to a ninhydrin reaction (heated at 110 ° C. for 10 minutes after ninhydrin spraying). As a result, ninhydrin and amino The presence of Tat contained in CP-Tat was confirmed from the reaction of the group with a blue-violet color. In addition, after spraying with 5% phenol, concentrated sulfuric acid was sprayed and the phenol-sulfuric acid reaction was performed. As a result, the presence of polysaccharide sugar chains in CP-Tat was confirmed, and the target CP-Tat was synthesized. It was done.

(Experimental example 1)
CP-Tat synthesized in Synthesis Example 4 and cholesterol pullulan (CP) to which no Tat derivative is bound are each dispersed in distilled water, and a probe-type ultrasonic crusher (TAITEC ULTRASONIC PROCESSOR VP-5) is used. Sonicate for 5 minutes on ice, measure the zeta potential and particle size distribution of CP-Tat using a zeta potential and particle size distribution analyzer (NICOMP380ZLS, manufactured by Particle Sizing Systems), and calculate the average particle size in the number-based distribution Asked.

(result)
The average particle size measured by the DLS instrument is about 11 nm for CP and about 82 nm for CP-Tat, and the zeta potential is about 1.1 mV for CP and about 21.1 mV for CP-Tat. CP-Tat synthesized in Example 4 has a very small particle size and a high positive charge, and has been shown to be a useful gene transfer carrier.

(Experimental example 2)
In this experimental example, CP-Tat and luciferase protein were expressed for the purpose of measuring the gene expression effect using CP-Tat synthesized in Synthesis Example 4 and evaluating its usefulness as a gene transfer vector. An experiment was conducted using a complex with a circular plasmid (denoted as pCMV-Luc or pDNA).

COS7 cells, an African green monkey kidney-derived cell line, are suspended in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS), and 2 mL each in a 6-well culture flask at a density of 1 × 10 ⁵ cells / mL And pre-cultured for 24 hours under conditions of 37 ° C. and 5% CO ₂ . After pre-cultured COS7 cells were washed with phosphate buffered saline (PBS), 1.9 mL of serum-free medium Opti-MEM was added. Next, the naked pDNA solution obtained by dissolving 1 μg of pCMV-Luc in 100 μL of serum-free medium Opti-MEMI or CP-Tat (1 to 20 μg) synthesized in Synthesis Example 4 into 100 μL of serum-free medium Opti-MEMI After dissolution, add 1 μg of CMV-Luc, add the complex solution of CP-Tat and pCMV-Luc prepared by rotary stirring at 4 ° C for 30 minutes, and transfer for 4 hours at 37 ° C under 5% CO ₂ conditions. (Gene transfer). Thereafter, the cells were washed with PBS, the medium was replaced with 10% FBS-containing DMEM medium, and cultured at 37 ° C. under 5% CO ₂ for 20 hours. After washing the cells with PBS, add 100 μL of cultured cell lysate diluted 5-fold with sterilized water, leave it at room temperature for 15 minutes, collect this suspension, and centrifuge at 15,000 rpm for 3 minutes. The supernatant was collected as a cell extract. To 15 μL of the obtained cell extract, 30 μL of a substrate solution containing luciferin, coenzyme A, ATP and the like was added, and the amount of luminescence per minute was measured using a luminometer (MICROLUMAT PLUS LB96V, manufactured by Bertrand). In addition, 5 μL of the cell extract was diluted 160-fold with ultrapure water, 40 μL of protein staining reagent was added and stirred, and then reacted at room temperature for 1 hour. After completion of the reaction, the absorbance of the reaction solution at a wavelength of 595 nm was measured using a microplate reader (Safire Microplate Reader, manufactured by TECAN). A calibration curve was prepared using a 2 mg / mL bovine serum albumin solution as a standard solution, and the protein concentration in the cell extract was calculated. The relative luminescence amount (Relative Light Unit, RLU) obtained by converting the luminescence amount per minute of the sample solution per 1 mg of protein was defined as luciferase activity and used as an index of gene expression level.

(result)
When the CP-Tat and pCMV-Luc complex is transfected, luciferase activity is higher than that of pCMV-Luc alone, especially when the mass ratio of CP-Tat to pCMV-Luc is 1:10. It was shown to be active.

(Experimental example 3)
The purpose of this experimental example is to compare the gene expression effect when CP-Tat synthesized in Synthesis Example 4 is used as a gene transfer vector with other gene transfer vectors.

To the COS7 cells precultured in the same manner as in Experimental Example 2 and replaced with 1.9 mL of serum-free medium Opti-MEM, the naked pDNA solution used in Experimental Example 2 and the CP-Tat 10 synthesized in Synthetic Example 4 were used. μg or branched polyethyleneimine (PEI, Mw 25 kDa, manufactured by Sigma) was dissolved in 100 μL of serum-free medium Opti-MEMI, and then 1 μg of pCMV-Luc used in Experimental Example 2 was added, and 30 μg at 4 ° C. Dissolve CP-Tat / pDNA complex and PEI / pDNA complex prepared by rotating and agitating for 30 minutes and Lipofectamine (trademark) 16 μg on the market in 100 μL serum-free medium Opti-MEMI and leave at room temperature for 30 minutes Lipofectamine ™ / pCMV-Luc complex prepared by the above was added, and the cells were transfected at 37 ° C. under 5% CO ₂ for 4 hours. Thereafter, the cells were washed with PBS, the medium was replaced with 10% FBS-containing DMEM medium, cultured at 37 ° C. under 5% CO ₂ for 20 hours, and luciferase activity was measured in the same manner as in Experimental Example 2.

(result)
As shown in Fig. 1, when COS7 cells were transfected with a complex of CP-Tat and pCMV-Luc, the luciferase was extremely high compared to pCMV-Luc alone, although this was not possible with PEI or Lipofectamine (trademark). Showed activity. From this, it was shown that CP-Tat synthesized in Example 2 and Synthesis Example 4 serves as a gene transfer vector for nucleic acid such as a circular plasmid.

(Experimental example 4)
In this experimental example, an experiment was conducted for the purpose of evaluating the safety of the CP-Tat synthesized in Synthesis Example 4 as a gene transfer vector.

COS7 cells pre-cultured in the same manner as in Experimental Example 2 were suspended in DMEM medium containing 10% FBS, and seeded at 5 × 10 ³ cells / 100 μL in a 96-well culture plate. 100 μg of the naked pDNA solution, CP-Tat / pDNA complex solution, PEI / pDNA complex solution, Lipofectamine ™ / pDNA complex solution used in Experimental Example 3 and 10 μg of the Tat derivative synthesized in Synthetic Example 1 After dissolving in μL serum-free medium Opti-MEMI, 1 μg of pCMV-Luc was added, and the Tat / pDNA complex solution prepared by rotating and stirring at 4 ° C. for 30 minutes was added to each well of the culture plate. The transfection was performed at 5 ° C. under 5% CO ₂ for 4 hours. Thereafter, the cells were washed with PBS and the medium was changed, followed by culturing at 37 ° C. under 5% CO ₂ for 20 hours, and then the cell activation evaluation test (MTT test) described below was performed.

  MTT test: Add MTT reagent (3- [4,5-dimethylthyazol-2-yl] -2,5-diphenyl tetrazolium bromide) to the above COS7 cells and let stand for 4 hours, then add 100 μL of lysate at room temperature. Left for 24 hours. In viable cells, intracellular mitochondrial dehydrogenase cleaves the tetrazolium ring of MTT, producing purple formazan crystals that are insoluble in aqueous solution, so the sample in the 96-well culture plate treated with the above MTT reagent Absorbance was measured at a wavelength of 550 nm using a microplate reader. The absorbance of the cells alone without the addition of plasmid or complex is taken as the control (100%), the ratio of the absorbance of each sample to this is calculated as the cell viability, and the cytotoxicity is evaluated using this viability as an index. did.

(result)
When transfected using PEI or Lipofectamine (trademark), cell viability was 95% and 77%, respectively, and cytotoxicity was observed. In contrast, when the pCMV-Luc complex using the Tat derivative synthesized in Synthesis Example 1 and the CP-Tat synthesized in Synthesis Example 4 as a carrier was transfected, the cell viability was 100% and cytotoxicity was exhibited. Is a very low gene transfer vector.

(Experimental example 5)
In this experimental example, the pCMV-Luc complex was prepared by combining the CP-Tat synthesized in Synthesis Example 4 with the NF-κB derivative and MA derivative, which are the nuclear translocation signals (NLS) synthesized in Synthesis Examples 2 and 3. The experiment was conducted with the aim of examining the possibility of improving the gene expression effect.

COS7 cells precultured in the same manner as in Experimental Example 2 and replaced with 1.9 mL of serum-free medium Opti-MEM, the naked pDNA solution used in Experimental Example 2, the Tat / pDNA complex solution used in Experimental Example 4, 10 μg of CP-Tat synthesized in Synthesis Example 4 was dissolved in 100 μL of serum-free medium Opti-MEM, 1 μg of pCMV-Luc used in Experimental Example 2 was added, and the mixture was allowed to stand at room temperature for 15 minutes. CP-Tat / pDNA / NLS complex prepared by adding 10 μg of the NF-κB derivative synthesized in step 2 or 10 μg of the MA derivative and standing at room temperature for 15 minutes, and 10 μg of the NF-κB derivative or MA derivative 10 μg NLS / pDNA / CP-Tat prepared by adding 10 μg of CP-Tat and allowing to stand at room temperature for 15 minutes after adding pCMV-Luc 1 μg and dissolving in 100 μL of Opti-MEM medium Dissolve 10 μg of the complex, CP-Tat and 10 μg of NF-κB or MA derivative in 100 μL of Opti-MEM medium and let stand at room temperature for 15 minutes, then add 1 μg of pCMV-Luc and let stand at room temperature for 15 minutes CP-Tat / NLS / p prepared Each DNA complex was added, and the cells were transfected for 4 hours at 37 ° C. and 5% CO ₂ . Thereafter, the cells were washed with PBS, the medium was replaced with 10% FBS-containing DMEM medium, cultured at 37 ° C. under 5% CO ₂ for 20 hours, and luciferase activity was measured in the same manner as in Experimental Example 2.

(result)
The pDNA complex prepared by adding CP-Tat and NF-κB derivative or MA derivative, which is a nuclear translocation signal, shows significantly higher luciferase activity than Tat / pDNA complex. It has been shown that gene expression efficiency of pDNA is improved by complexing together nuclear translocation signals such as NF-κB derivatives and MA derivatives having the property.

(Experimental example 6)
In this experimental example, gene transfer of CP-Tat synthesized in Synthesis Example 4 to not only the nucleic acids (pDNA) described in Experimental Examples 2 to 5 but also short-chain deoxyribonucleic acid and ribonucleic acid having a gene expression suppressing action An experiment was conducted to evaluate the possibility of application as a vector.

Mouse-derived cancer cells Sarcoma180 (S180 cells) were suspended in serum-free medium RPMI1640, seeded at 1 × 10 ⁵ cells / mL in a 24-well culture plate, and pre-cultured for 24 hours. The medium was changed 1 hour before transfection. After that, CP-Tat synthesized with Synthesis Example 4 and 21 base pair short ribonucleic acid (small interference RNA, siRNA), which suppresses the production of mouse vascular endothelial growth factor (VEGF), were added to the siRNA. Add CP-Tat / siRNA complexes with different mass ratios or siRNA single solutions prepared by adding to serum-free medium RPMI1640 so that the ratio is 1, 2, 5, 10, 15 and let stand at 4 ° C for 30 minutes. Added to the cells and transfected for 12 hours. Next, an appropriate amount of the supernatant of the medium was collected, and the amount of VEGF in the medium was measured by enzyme immunoassay (ELISA method). The amount of VEGF per unit cell in a medium containing only cells was taken as 100%, and the ratio of the amount of VEGF per unit cell in each sample medium was calculated as the VEGF production inhibition rate (%). Using this VEGF production inhibition rate as an index, the effect of CP-Tat as a gene transfer vector on short-chain ribonucleic acid or deoxyribonucleic acid was evaluated.

(result)
As shown in Figure 2, the mass ratio of CP-Tat to siRNA in the complex of CP-Tat and siRNA is 31% when transfection with siRNA alone is extremely low. The VEGF production suppression rate markedly increased with the increase in the VEGF production. In particular, when the mass ratio of CP-Tat / siRNA was 10, the suppression rate of VEGF production was as high as 85%.

(Experimental example 7)
The anti-mouse VEGF siRNA (0.13 μg) used in Experimental Example 6 and the Tat derivative (1.3 μg) synthesized in Synthetic Example 1 were synthesized in S180 cells pre-cultured in the same manner as in Experimental Example 6. SiRNA / Tat complex, siRNA / CP-Tat complex, siRNA prepared by adding CP-Tat (1.3 μg) or Lipofectamine ™ (2 μg) to serum-free medium RPMI1640 and leaving at 4 ° C. for 30 minutes / Lipofectamine complex and siRNA alone solution were added and transfected for 12 hours. Thereafter, an appropriate amount of the supernatant of the medium was collected, and the inhibition rate (%) of VEGF production was measured in the same manner as in Experimental Example 6.

(result)
As shown in FIG. 3, in the complex of CP-Tat and siRNA, it was recognized that VEGF production was remarkably suppressed as compared with the case where a Tat derivative and Lipofectamine were used. This indicates that CP-Tat synthesized in Example 2 and Synthesis Example 4 is a gene carrier useful not only for nucleic acids but also for short-chain ribonucleic acids such as siRNA and deoxyribonucleic acid.

(Experimental example 8)
In this experimental example, an experiment was conducted for the purpose of evaluating the in vivo gene transfer effect of CP-Tat synthesized in Synthesis Example 4 using the tumor-bearing mouse and the siRNA used in Experimental Example 6.

Production of tumor-bearing mice: S180 cells (5 × 10 ⁶ cells / 300 μL) used in Experimental Example 6 suspended in serum-free medium RPMI1640 were injected subcutaneously into the back of 8-week-old ICR female mice and transplanted. Six days after transplantation, it was judged that a tumor-bearing mouse was produced when the volume of the formed tumor reached 50 mm ³ or more, and it was used in the following experiments.

  CP-Tat single solution, siRNA single solution, CP-Tat / siRNA complex prepared by mixing CP-Tat and siRNA, and PEI and siRNA in the tumor-bearing mouse tumor 6 days after S180 cell transplantation by the above method The PEI / siRNA complex prepared by mixing was administered, the major axis and minor axis of the tumor were measured every 2 days for 18 days, and the volume of the tumor was calculated using the following formula. The tumor volume of a tumor-bearing mouse to which nothing was administered was used as a control.

Tumor volume (mm ³ ) = (Tumor minor axis) ² × Tumor major axis / 2

(result)
As shown in FIG. 4, a rapid increase in tumor volume over time was confirmed in the control and CP-Tat single administration groups. In contrast, in the group administered with the complex of CP-Tat and siRNA, it was shown that the tumor volume was smaller and tumor growth was suppressed as compared with siRNA alone administration. The PEI and siRNA complex suppressed tumor growth compared to the CP-Tat / siRNA complex, which is thought to be due to the high cytotoxicity of PEI as shown in Experimental Example 4. From the above, it was shown that CP-Tat, which has low cytotoxicity, is a gene transfer vector that can exert its effect sufficiently in vivo.

COS7 cells were transfected with a complex of a circular plasmid (pDNA) expressing luciferase protein and the Tat-modified cholesterol pullulan (CP-Tat) synthesized in Synthesis Example 4 and a pDNA complex with other gene transfer reagents. It is the figure which showed the luciferase activity in the case (mean value ± SD, n = 3). When a complex of CP-Tat, a short ribonucleic acid (siRNA) that suppresses the production of anti-mouse vascular endothelial growth factor (VEGF), prepared at different mass ratios, is introduced into mouse-derived cancer cells (S180 cells) It is a figure which shows the VEGF production suppression rate from the cell of (average value +/- SD, n = 3). It is a figure which shows the VEGF production suppression efficiency from a cell at the time of introduce | transducing siRNA into S180 cell using CP-Tat, a Tat derivative | guide_body, and Lipofectamine (mean value +/- S.D., N = 3). It is a figure which shows the time-dependent tumor volume change after administering the composite_body | complex of CP-Tat and siRNA in the tumor of a tumor bearing mouse | mouth. The white triangle is the control group without siRNA administration, the white circle is the CP-Tat single administration group, the white square is the siRNA single administration group, the black circle is the CP-Tat / siRNA complex administration group, and the black square is the PEI / siRNA complex administration group (Mean ± SE, n = 5).

Claims (22)

  A non-viral vector comprising a polysaccharide-cholesterol or a polysaccharide-lipid derivative to which a positively charged membrane-permeable substance is bound.   The vector according to claim 1, wherein the membrane-permeable substance is a peptide, a basic amino acid, or a cationic polymer compound.   The vector according to claim 1 or 2, wherein the membrane-permeable substance is a substance having a cell migration property, a nuclear migration property, or a nuclear migration signal.   The vector according to any one of claims 1 to 3, wherein the membrane-permeable substance is bound to at least one sugar residue of the polysaccharide.   The vector according to any one of claims 2 to 4, wherein the membrane-permeable substance is a peptide containing a cysteine residue. Wherein the peptide, in its C-terminal, characterized in that it comprises a -Cys-Gly-NH _2, the vector of claim 5.   The vector according to claim 5 or 6, wherein the peptide is bound to the polysaccharide-cholesterol or polysaccharide-lipid derivative by a disulfide bond.   The vector according to any one of claims 1 to 7, wherein the vector forms nano-sized micelles in the presence of water.   A complex of the vector according to any one of claims 1 to 8 and a useful substance.   The complex according to claim 9, wherein the useful substance is used for treatment of a disease.   The complex according to claim 9 or 10, wherein the useful substance is a nucleic acid, a protein, a peptide, or a small molecule.   12. The complex according to claim 11, wherein the nucleic acid is a nucleic acid containing a gene that can be expressed.   13. The complex according to claim 12, wherein the nucleic acid is a plasmid.   The nucleic acid is a DNA or RNA having a single-stranded or double-stranded structure, a length of about 15 to 30 bases, and a function of suppressing the expression of a specific gene in a target cell. 12. The composite according to claim 11, wherein   14. Complex according to claim 13, characterized in that the nucleic acid is a dsRNA, siRNA or ribozyme.   12. The complex according to claim 11, wherein the nucleic acid is an aptamer.   17. The complex according to any one of claims 9 to 16, wherein the complex can be administered to a diseased or non-disease site of mammalian cells, including humans, tissues or organs.   18. Complex according to claim 17, characterized in that the tissue is a mucosa.   A composition for treating a disease, comprising the complex according to any one of claims 9 to 18 and a pharmaceutically acceptable carrier.   20. The composition according to claim 19, further comprising a peptide having an action of a nuclear translocation signal. The method for producing a non-viral vector according to any one of claims 1 to 8, comprising a polysaccharide-cholesterol or a polysaccharide-lipid derivative to which a positively charged membrane-permeable peptide is bound, comprising: (i) a polysaccharide -Coupling a linker having a mercapto (SH) group to at least one OH group of a sugar residue of a cholesterol or polysaccharide-lipid derivative; and (ii) SH of the linker of the product obtained in step (i) Forming a disulfide bond between the group and a cysteine (Cys) residue of a membrane-permeable peptide containing —Cys-Gly-NH ₂ at the C-terminus.   The method according to claim 21, further comprising the steps of protection and deprotection.

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