WO2013162054A1

WO2013162054A1 - Composition for controlling chromatin structure

Info

Publication number: WO2013162054A1
Application number: PCT/JP2013/062737
Authority: WO
Inventors: 浩良川上; 章一郎朝山; 健一硲; 祐太朗浅羽; 太甫野口
Original assignee: 公立大学法人首都大学東京
Priority date: 2012-04-24
Filing date: 2013-04-24
Publication date: 2013-10-31
Also published as: JPWO2013162054A1; JP6004499B2

Abstract

The purpose of the present invention is to provide a composition for controlling a chromatin structure. A composition of the present invention contains: (1) a polymer which is selected from the group consisting of amphoteric polymers, anionic polymers and nonionic polymers having a specific main chain skeleton; or (2) a carrier which encloses two or more kinds of substances selected from the group consisting of expression vectors containing a gene such as a gene encoding histone acetyltransferase and inhibitors such as histone acetylation inhibitors.

Description

Composition for controlling chromatin structure

The present invention relates to a composition for controlling a chromatin structure and a method for controlling a chromatin structure using the composition.

Epigenetics contributes to the expression of proteins that regulate the function of epigenetic cells (genetics), and epigenetics contributes to the regulation of gene transcription and translation. Furthermore, since epigenetics is controlled by proteins, it is thought that the genetic-epigenetics-protein integrated regulation mechanism related to life functions plays a central role in life phenomena.

A fertilized egg proliferates and differentiates, and the process of forming tissues / organs and an individual occurs.The individual grows and then ages with time, sometimes causing diseases such as cancer and lifestyle-related diseases. You may suffer. However, even if cell or tissue abnormality occurs, it may be regenerated by receiving natural healing or medical treatment. Individuals also have a genetic phenomenon that passes the genome to the next generation through germ cells. These can basically be understood as epigenetic life phenomena in which cells having the same genome change into cells having different properties.

It is considered that the individuality of a wide variety of cells is determined by the gene expression pattern of the cells. When the total number of genes on the genome is 30,000, about 10,000 genes are expressed in one differentiated cell, and the remaining genes are inactivated. In other words, by selectively utilizing genes on the genome, it means that cell personality is established, maintained, and erased epigenetically.

In the epigenetic control system, DNA methylation, chromatin, protein modification / demodification, and transcriptional regulators play important roles in gene regulation. To date, new molecular groups and functional complexes such as DNA methylases, methylated DNA binding proteins, histone modifying enzymes, chromatin structure factors, chromatin remodeling factors, chromatin insulators (chromatin boundaries), etc. Has been discovered. These epigenetic mechanisms lead to gene regulation and formation of chromatin structures. Furthermore, expression of individual genes on the genome in a tissue-specific, differentiation-specific, and situation-specific manner indicates that there is a mechanism in which the genes are independently controlled.

Chromatin Structure Eukaryotic chromosomal DNA has a higher-order structure called chromatin. Chromatin is a structure in which nucleosome repeat structures are connected in a spiral. Nucleosomes are connected to each other via a linker DNA not wound around histones, and become packed 30 nm chromatin fibers. The nucleosome has a structure in which DNA of 146 base pairs is wound around a histone octamer composed of two molecules of H2A, H2B, H3, and H4 histone proteins. For example, when DNA in a single human cell is stretched, it becomes about 1.8 m, and this DNA is wound around a histone protein to form a chromatin structure. Chromatin is condensed and stored in the nucleus of the cell. Highly condensed chromatin is called heterochromatin.

In order for the gene to be activated, that is, turned on, such a condensed chromatin structure must be loosened, the histones may be removed or displaced, and the DNA double helix must be unwound. By unwinding, the base sequence of the gene is transcribed and is read as RNA.

It is known that histone proteins have been modified with various chemical substances such as acetyl and methyl groups, but their roles are not known and have not received much attention. In recent years, it has been shown that when an acetyl group is attached to a specific place of histone, the chromatin structure is loosened and the gene can be turned on. It has been discovered that a complex mechanism works in the process of histone methylation and the formation of heterochromatin by recognizing methyl groups. Two types of fission yeast HP1 proteins that are slightly different in shape are known. One has a function of promoting heterochromatinization and the other has a function of suppressing heterochromatinization. It has been found that heterochromatin cannot be formed and maintained unless two types of HP1 having opposite functions are gathered in a certain balance. It is not clear why such a complicated mechanism is necessary.

In higher organisms such as humans, there is a mechanism that strongly suppresses genes by directly methylating DNA instead of histones. This DNA methylation has also been found to be closely related to histone modifications.

Histone-modified histones are proteins having many basic amino acids such as lysine and arginine, and are tightly bound to anionic DNA. The N-terminus of histone is called histone tail and exists a little away from the nucleosome core. Histones undergo various modifications mainly at this N-terminal part.

In recent years, it has been known that chromatin structural changes due to histone modifications play an important role in the induction of transcription. First, when a DNA-binding transcription activator binds to a target gene, transcription coactivators such as PCAF and CBP / p300 are recruited. The transcription coactivator has histone acetylase (Histone Acetyl Transferase: HAT) activity and acetylates surrounding histones. This triggers the recruitment of chromatin remodeling factors, induces chromatin remodeling, and initiates transcription by basic transcription factors and RNA polymerase. In particular, acetylation of histones H3K9 and K14 is known as acetylation that correlates well with transcription induction. It is believed that when the amino group of a lysine residue of histone is acetylated, the positive charge of the amino group is neutralized and the interaction between nucleosomes is relaxed. In addition to his acetylation, histones are known to undergo modifications such as methylation and phosphorylation to cause transcriptional control, silencing, and chromatin condensation.

As an enzyme that modifies histone, there is histone deacetylase (HDAC) that catalyzes deacetylation in addition to HAT described above. The activity of HDAC is dominant in the cell, and chromatin is normally kept in a deacetylated state, and is thought to be acetylated and opened only when necessary by the action of a transcription factor.

Histone methylation is induced by histone methyltransferase (HMT). Currently, the methylation of histone H3K9 is well analyzed. HP1 (Heterochromatin Protein 1) recognizes and binds methylated K9. HP1 recruits DNA methylase and HDAC, and HP1 aggregates to convert the surrounding chromatin region into a closed state. It is known that gene silencing occurs through such a mechanism. On the other hand, the methylation of histone H3K4 is known to correlate with the activation of transcription.

In general, it was thought that a demethylation reaction opposite to the methylation reaction hardly occurred, but in recent years, a histone demethylase was also reported.

DNA methylation Methylation modification of eukaryotic genomic DNA has evolved as a mechanism to control the expression of genetic information. Methylation of genomic DNA is one of the epigenetic factors, but its state is the process of newly methylating the base sequence to draw a pattern, maintaining it in the process of cell growth, and deleting it as necessary Determined as the sum. In particular, five genes have been reported so far as DNA methyltransferases and homologous molecules involved in DNA methylation modification (Dnmt1, Dnmt2, Dnmt3a, Dnmt3b, Dnmt3L). However, there are many problems to be solved in the future regarding how and what regions of the genome are recognized and methylated.

DNA methylation modification controls its expression without changing the base sequence that is the substance of the gene, that is, without changing the amino acid sequence information encoded by the gene. In addition, the methylation pattern on the genome once attached is stably inherited to the next generation cells. On the other hand, methylation modification also has reversibility that can be removed if necessary. In understanding the mechanism of expression control of genetic information, it is important to understand how methylation of genomic DNA is regulated.

In eukaryotes, with the exception of some organisms, the 5th position of cytosine base (C) of genomic DNA is subjected to methylation modification. In animals, the degree of cytosine methylation modification in the genome increased at the same time that the base amount of the genome increased when it evolved into a vertebrate. Vertebrate genomic DNA methylation is added to cytosine bases in the CpG sequence followed by cytosine bases followed by guanine bases. In mouse genomic DNA, about 80% of the CpG sequence is methylated. This methyl group is transferred from S-adenosyl-L-methionine by the action of DNA methyltransferase.

When looking at the mouse genome, regions with relatively high G + C content are scattered in islands, which are called CpG islands. In many cases, they become promoters of housekeeping genes and become hypomethylated. is there. In general, the promoter region of a transcribed gene is in a hypomethylated state, and the inactive gene is highly methylated. When a transcription factor binding motif present in the promoter region is methylated, most transcription factors except for some transcription factors such as Sp1 cannot bind to DNA. There is also a protein that specifically recognizes and binds to methylated DNA, and transcription is inhibited through this methylated DNA binding protein and histone modification. DNA methylation functions as a kind of memory for silencing genes over the long term.

Many proteins that specifically recognize and bind to methylated DNA are either members of the transcriptional repression complex that contains histone deacetylases, or bind to these complexes and eventually deacetylate histones. Transcription is suppressed by methylation and methylation. Although it is certain that there is a close relationship between DNA methylation and histone modification, there are still many unknowns regarding the molecular mechanisms involved in the interaction between them, and this is an important issue to be solved.

As described above, it is considered that the control of chromatin structural change involves histone or DNA modification, but the details are not clear. No means for artificially controlling the chromatin structure has been established.

The object of the present invention is to provide a method for artificially controlling the chromatin structure and a composition for use in the method.

As a result of diligent research to solve the above problems, the present inventors have found that the chromatin structure is formed by administering a carrier containing a specific polymer or two or more substances that control acetylation or methylation of histones. As a result, the present invention has been conceived.

The present invention includes the following embodiments as preferred embodiments.
[Aspect 1] The following

1) A polymer selected from the group consisting of amphoteric polymers, anionic polymers, and nonionic polymers, and having one of the following main chain skeletons:

Or
2)
i) an expression vector comprising a gene encoding a histone acetylase;
ii) an expression vector comprising a gene encoding a histone methylase;
iii) an expression vector comprising a gene encoding a histone deacetylase;
iv) an expression vector comprising a gene encoding a histone demethylase;
v) histone acetylation inhibitors;
vi) histone deacetylation inhibitors;
vii) a histone methylation inhibitor; and viii) a composition for controlling a chromatin structure, comprising a carrier containing two or more substances selected from the group consisting of histone demethylation inhibitors.
[Aspect 2]

The composition according to aspect 1, for relaxing chromatin structure.
[Aspect 3]

Polymer
a) carboxymethylated polyvinylimidazole,
b) from polyacrylic acid, polymethacrylic acid, polyglutamic acid, carboxylmethylated polyhistidine, or polyaspartic acid, or c) PEG or sugar chain, or a block copolymer, graft copolymer, or dendrimer thereof. The polymer according to

aspect

1 or 2, which is selected.
[Aspect 4]

The composition according to any one of aspects 1-3, wherein the molecular weight of the polymer is 10,000-2,000,000.
[Aspect 5]

The composition according to any one of aspects 1-4, wherein the carrier comprises polyester, calcium phosphate, or polyamino acid.
[Aspect 6]

Any of aspects 1-5, wherein the carrier further comprises an expression vector comprising a gene encoding a DNA demethylase, an expression vector comprising a gene encoding a DNA methylase, or a DNA methylase inhibitor The composition according to claim 1.
[Aspect 7]

A method for controlling a chromatin structure by administering the composition according to any one of aspects 1-6 in vitro, ex vivo, or in vivo.

The present invention makes it possible to artificially control the chromatin structure. Thereby, the expression can be controlled more easily without manipulating the base sequence of the gene.

FIG. 1 shows the results of examining the formation of an artificial chromatin model (DNA / core histone complex) by agarose electrophoresis.
[FIG. 2] FIG. 2 shows the results of examining the relaxation effect of the chromatin structure by the polymer by the fluorescence intensity after agarose electrophoresis.
[FIG. 3] FIG. 3 shows the results of examining the translation activity for the relaxation effect of chromatin structure by polymer PAA.
[FIG. 4] FIG. 4 shows the results of examining the translational activity for the relaxation effect of the chromatin structure by the polymers CM-PVIm and PMAA.
FIG. 5 shows the content of coenzyme A (CoA) in the calcium phosphate complex of the present invention.
[FIG. 6] FIG. 6 shows the result of examining the change in the amount of acetyllysine in each histone by Western blotting when the calcium phosphate complex of the present invention was used.
CaP: calcium phosphate complex;
CoA: Coenzyme A
SIRT2: Plasmid DNA containing the SIRT2 gene
DNA: Plasmid DNA
AA: Anacardic acid [FIG. 7A] FIG. 7 shows the results of confocal microscope observation of Example 12. 7A-D respectively show (A) fluorescence image by DAPI staining cell nuclei, (B) fluorescence image by FITC-labeled PLD-EDA-β-CD, (C) differential interference image, and (D) A- C images are combined.
[FIG. 7B] FIG. 7 shows the results of confocal microscope observation of Example 12. 7A-D respectively show (A) fluorescence image by DAPI staining cell nuclei, (B) fluorescence image by FITC-labeled PLD-EDA-β-CD, (C) differential interference image, and (D) A- C images are combined.
[FIG. 7C] FIG. 7 shows the result of confocal microscope observation of Example 12. 7A-D respectively show (A) fluorescence image by DAPI staining cell nuclei, (B) fluorescence image by FITC-labeled PLD-EDA-β-CD, (C) differential interference image, and (D) A- C images are combined.
[FIG. 7D] FIG. 7 shows the result of confocal microscope observation of Example 12. 7A-D respectively show (A) fluorescence image by DAPI staining cell nuclei, (B) fluorescence image by FITC-labeled PLD-EDA-β-CD, (C) differential interference image, and (D) A- C images are combined.

The present invention relates to a composition for controlling chromatin structure and a method for controlling chromatin using the composition.

The composition of the present invention is
1) A polymer selected from the group consisting of amphoteric polymers, anionic polymers, and nonionic polymers, and having any of the following main chain skeletons;

Or
2)
i) an expression vector comprising a gene encoding a histone acetylase;
ii) an expression vector comprising a gene encoding a histone methylase;
iii) an expression vector comprising a gene encoding a histone deacetylase;
iv) an expression vector comprising a gene encoding a histone demethylase;
v) histone acetylation inhibitors;
vi) histone deacetylation inhibitors;
vii) a histone methylation inhibitor; and viii) a carrier that includes two or more substances selected from the group consisting of histone demethylation inhibitors.

1. Polymer selected from the group consisting of amphoteric polymers, anionic polymers, and nonionic polymers The polymer of the present invention is a group consisting of amphoteric polymers, anionic polymers, and nonionic polymers. Selected from.

The polymer of the present invention has a repeating structure of any of the following main chain skeletons.

A particularly preferred skeleton structure is — (CH ₂ —CH) —. From the applicability to living bodies, the following skeletal structure

Is also useful.

The number of repeats of the main chain skeleton is not particularly limited. It is determined appropriately according to the skeleton structure, side chain structure, and the like.

"Amphoteric polymer" is a polymer that has both anionic and cationic substituents in the side chain and is neutral as a whole, or both an anionic part and a cationic part in the side chain. It is a polymer having a substituent having a neutrality as a whole. Since an amphoteric polymer has both an anionic part and a cationic part in one molecule, it has both characteristics. In addition, by balancing the charge of the anion and cation, it may have a nonionic characteristic (giving biocompatibility). The type of the anionic part and the cationic part contained in the amphoteric polymer may be one each or plural kinds.

The “anionic group” as a side chain in the amphoteric polymer may have the following structure, for example.

A is a linker part that binds the anionic part and the main chain part. The linker moiety may be branched, an alkyl group having 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms, more preferably 1 carbon atom; a carbocyclic ring, preferably a carbocyclic ring having 5 to 7 carbon atoms. Preferred is a 6-membered ring; or a heterocycle containing nitrogen, oxygen or sulfur, preferably a 5-7-membered heterocycle, more preferably a 5-membered heterocycle. The alkyl group, carbocycle or heterocyclic ring may be optionally substituted with one or more alkyl groups, aryl groups, halogen groups, various anionic groups and the like. For example, an embodiment in which a ring such as a carbocycle or a heterocycle is substituted with an anionic group such as —COOH is also included.

In one embodiment, the linker is selected from an ethyl group, an imidazolyl group, or other functional group having a quaternary nitrogen.

The “cationic group” as a side chain in the amphoteric polymer includes the following embodiments.

[Wherein A is a linker as defined for an anionic group;
R ₁ , R ₂ and R ₃ are an optionally branched alkyl group having 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms]

The “substituent having both an anionic part and a cationic part of the side chain” includes, for example, the following embodiments.

As an example, a nitrogen-containing heterocyclic ring, for example, an embodiment in which one nitrogen atom of an imidazole group is bonded to the main chain and the other nitrogen atom is substituted with an anionic group. The anionic group can be appropriately selected from those described above. One embodiment of the anionic substituent of the nitrogen atom in the amphoteric polymer is a carboxymethyl group.

The amphoteric polymer includes carboxymethylated polyvinyl imidazole as one embodiment.

“Anionic polymer” is a polymer having an anionic group in the side chain. The “anionic group” in the anionic polymer can be appropriately selected from the anionic groups described above for the amphoteric polymer.

As an embodiment, the anionic group includes a carboxyl group, a carboxy group, a phosphate group, a sulfonate group, a nitrate group, and a boronic acid group.

As an embodiment, the anionic polymer contains polyacrylic acid, polymethacrylic acid, polyglutamic acid, carboxylmethylated polyhistidine, or polyaspartic acid.

When an anionic polymer is used, a spacer may be included in order to prevent repulsion of the main chain skeleton and side chain substituents in the anionic polymer. The spacer is not particularly limited as long as it has a property of not having an anionic group or having a large hydrophobic group. For example, polyethylene diamine, polyethylene glycol (PEG) and the like can be used.

“Nonionic polymer” is a polymer having neither an anionic part nor a cationic part. A nonionic polymer can also act on chromatin and control the structure of chromatin as long as it satisfies the condition that it has hydrogen bonding properties or hydrophobic bonding properties. Nonionic polymers include polymers having hydrogen bonding properties such as polyethylene glycol (PEG) and sugar chains.

The polymer contained in the composition of the present invention may be a block copolymer, graft copolymer, or dendrimer of the above-identified amphoteric polymer, anionic polymer, or nonionic polymer. . The “block copolymer” means a copolymer having a molecular structure in which two or more kinds of polymers are connected by covalent bonds to form a long chain. The “graft copolymer” means a copolymer having a molecular structure in which another polymer is bonded in a branched manner to a certain polymer. The “dendrimer body” means a dendritic polymer having a structure regularly branched from the center. The dendrimer body is composed of a central molecule called a core and a side chain part called a dendron.

The high molecular weight of the present invention is not particularly limited, but is preferably 10,000-2,000,000, more preferably 25,000-1,000,000.

Although not necessarily limited, it is more preferable that cyclodextrin derivatives such as cyclodextrin and Meβ cyclodextrin are bound to the polymer used in the present invention.

As an additional component, a cationic polymer may be used together with an amphoteric polymer, an anionic polymer, a nonionic polymer, or a carrier. A “cationic polymer” is a polymer having a cationic group in the side chain. The “cationic group” in the cationic polymer can be appropriately selected from the cationic groups described above for the amphoteric polymer. In one embodiment, the cationic polymer is a single-chain stearylamine, a double-chain cationic lipid, N- [1- (2,3-dioleoyloxy) propyl] -N, N, N- Contains trimethylammonium methylsulfate (DOTAP).

2. Carrier The composition of the present invention comprises:
i) an expression vector comprising a gene encoding a histone acetylase;
ii) an expression vector comprising a gene encoding a histone methylase;
iii) an expression vector comprising a gene encoding a histone deacetylase;
iv) an expression vector comprising a gene encoding a histone demethylase;
v) histone acetylation inhibitors;
vi) histone deacetylation inhibitors;
viii) a histone methylation inhibitor; and viii) a carrier that includes two or more substances selected from the group consisting of histone demethylation inhibitors.

The composition of the present invention includes an embodiment containing both the polymer and the carrier.

I) -viii) are all substances involved in acetylation, deacetylation, methylation, or demethylation of histones of chromatin. Histones are thought to be neutralized when the amino group of lysine residues is acetylated, and the interaction between nucleosomes is relaxed. Therefore, the chromatin structure is relaxed by histone acetylation, and transcription is activated. In contrast, deacetylation condenses the chromatin structure. i) and vi) have the effect of relaxing the chromatin structure. On the other hand, iii) and v) have structures that condense chromatin structures. On the other hand, histone methylation is known to condense chromatin and form heterochromatin when H3K9 is methylated, and is thought to depend on position. On the other hand, transcription is activated when histone H3K4 is methylated.

In the present invention, two or more of the above substances are administered using a carrier. “A carrier that includes two or more substances” includes an embodiment that includes two or more substances in one carrier and an embodiment that includes two or more substances in two or more carriers. . By administering two or more substances that control chromatin, more powerful or complicated control becomes possible. For example, the composition may contain two or more genes, or a combination of a gene and a compound (inhibitor).

The carrier of the present invention has the condition that it is non-toxic or low-toxic and can be applied to a living body, can contain an expression vector and / or an inhibitor, and can carry the substance i) -viii) to the nucleus of the cell. Any known carrier can be used as long as it satisfies the requirements. In embodiments, the carrier includes polyester, calcium phosphate, or polyamino acid.

“Polyester” is a polycondensate of polyhydric carboxylic acid and polyalcohol, and is generally produced by reacting (dehydrating and condensing) polyalcohol and polycarboxylic acid. In one embodiment, the polyester includes a biodegradable polymer such as polylactic acid, polyglycolic acid, and polylactic acid-polyglycolic acid copolymer.

“Calcium phosphate” has low toxicity and biodegradability. In particular, it is preferable to include an expression vector containing a gene in a carrier. Sodium phosphate and the like can be used in the present invention as having the same function.

“Polyamino acid” is a polymer in which a plurality of amino acids are amide-bonded, and is also called a polypeptide. Amino acids are components of biological proteins, and polyamino acids are non-toxic or hypotoxic. Acetylated polylysine (AcPLL), hyperbranched polylysine (highly branched polylysine), dendritic polylysine (DPK) as described in the examples herein may also be used as the carrier of the present invention. Furthermore, polylysine can also act as a histone acetylation inhibitor by the action of histone acetylase on polylysine in the nucleus of the cell. In contrast, acetylated polylysine can act as a histone deacetylation inhibitor.

"Histone acetylase" (HAT) is an enzyme that promotes acetylation of histone lysine side chains. HAT is known, and for example, the amino acid sequence of a protein and the base sequence of a gene encoding the protein are known in humans and the like. For example, the nucleotide sequence of the human histone acetylase (KAT2B) gene is disclosed in NCBI Data Bank accession number NM_003884. KAT2B may also be called “CAF”.

“Histone methylase” (HMT) is an enzyme that promotes methyl groups in histones. Histone methylases are known, and for example, the amino acid sequence of a protein and the base sequence of a gene encoding the protein are known in humans and the like. For example, the nucleotide sequence of the mutant (G9a) gene of human histone methylase (EHMT2) is disclosed in accession number NM_006709 of NCBI Data Bank.

“Histone deacetylase” (HDAC) is an enzyme that hydrolyzes the acetyl group of the lysine side chain of histone and converts it into lysine. HDAC is known. For example, in humans and the like, the amino acid sequence of a protein and the base sequence of a gene encoding the protein are known. For example, the nucleotide sequence of the human histone deacetylase (HDAC3) gene is disclosed in NCBI Data Bank Accession No. NM — 029678. The nucleotide sequence of the human histone deacetylase (SIRT2) gene is disclosed in NCBI Data Bank, Accession No. NM — 034146.

“Histone demethylase” is an enzyme that hydrolyzes a methyl group bound to histone.

The amino acid sequence of the enzyme encoded by each gene is preferably a natural protein sequence. Alternatively, as long as the function of each enzyme is maintained, mutants thereof can also be used in the present invention. Specifically, it preferably has at least 80%, 85%, 90%, 95%, 97%, 99% sequence homology to the natural amino acid sequence or the base sequence encoding the amino acid.

The percent identity between two sequences may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences can be determined by Needleman, S .; B. And Wunsch, C.I. D. (J. Mol. Biol., 48: 443-453, 1970) and determined by comparing sequence information using the GAP computer program available from the University of Wisconsin Genetics Computer Group (UWGCG). May be. Preferred default parameters for the GAP program include: (1) Henikoff, S .; And Henikoff, J. et al. G. (Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992), scoring matrix, blossum 62; (2) 12 gap weights; (3) 4 gap length weights; And (4) no penalty for end gaps.

Alternatively, a natural amino acid sequence may have an amino acid sequence in which one or more amino acid residues are deleted, added, or substituted. A gene encoding a protein having an amino acid sequence homologous to such a natural protein and having the same function as a natural enzyme can also be used in the present invention. The number of amino acids that can be changed is, but is not limited to, 1 to 100 amino acid residues, 1 to 80 amino acid residues, 1 to 50 amino acid residues, 1 to 30 amino acid residues, 1 to 20 amino acid residues, 1 to 15 amino acid residues, 1 to 10 amino acid residues, and 1 to 5 amino acid residues. The number of amino acid residues that can be modified by a known site-directed mutagenesis method, for example, 1 to 10 amino acid residues, 1 to 8, 1 to 5, 1 to 3 amino acid residues is more preferable.

As the “expression vector”, a known vector such as a plasmid vector, a virus vector, or a cosmid vector for expressing a gene can be used. For the purpose of administration to a living body, a vector that is non-toxic or less toxic to the living body is preferable. When the composition of the present invention is intended to express two or more types of genes, two or more types of expression vectors containing each gene may be used, or two or more types of genes may be used in one expression vector. You may use what was included.

As the “histone acetylation inhibitor”, a known one can be used. Examples include coenzyme A (CoA), anacardic acid (AA), curcumin, MB-3.

As the “histone deacetylation inhibitor”, a known one can be used. For example, valproic acid (VPA), trichostatin A (TSA), vorinostat, MS-275. Histone deacetylation inhibitors are known to exert activity on cancer cells such as cell cycle arrest, differentiation induction, and apoptosis, and affect the proliferation of cancer cells.

As the “histone methylation inhibitor”, a known one can be used. For example, ketocin, BIX-01294, DZNep, AMI-1.

As the “histone demethylation inhibitor”, a known one can be used. For example, tranylcypromine.

The inhibitor may be included in a carrier together with the expression vector to constitute a composition, or may be administered directly (for example, in a medium or in a living body) separately from the carrier.

3. Method for Controlling Chromatin Structure The present invention includes a method for controlling chromatin structure by administering the above-described composition of the present invention in vitro, ex vivo, or in vivo.

A composition containing both the polymer and the carrier may be administered, or an embodiment in which two types of compositions, a composition containing the polymer and a composition containing the carrier, are administered is also included.

Control of chromatin structure refers to relaxation or condensing of chromatin structure. The phrase “relaxing” the chromatin structure means that the higher-order structure in a state where the chromatin is condensed is loosened, and the transcription of the gene is facilitated, that is, the transcription is activated and turned on. “Condensation” of the chromatin structure means that, contrary to relaxation, it takes a higher-order structure in which chromatin is condensed and is in an off state in which transcription is difficult to occur.

Chromatin “relaxation” and “condensation” can be examined, for example, by measuring gene transcription and / or translation activity. The transcriptional and / or translational activity of the gene can be confirmed using a known method. For example, fluorescence can be measured by expression of a fluorescent protein. Or you may confirm by observing a chromatin structure more directly.

The dosage of the polymer and carrier of the present invention is not particularly limited. Those skilled in the art can appropriately determine the amount depending on the type of polymer, carrier, substance contained in the carrier, the condition of the administration target, and the like. When the amphoteric polymer and the anionic polymer of the present invention are used, the number of anions and the ratio of DNA in the chromatin structure may affect the magnitude of the effect of relaxation of the chromatin structure. Although not limited, when the amphoteric polymer and the anionic polymer of the present invention are used in an in vitro cell-free system, the number of anions of the polymer is twice that of the DNA in the chromatin structure. It was observed that the chromatin structure was most relaxed.

4). Control of DNA methylation Apart from the modification of histones in the chromatin structure, DNA methylation is also known to be involved in the control of the expression of genetic information. Specifically, transcription is inactivated by methylation of the chromatin structure, and transcription is activated by demethylation. Thus, in addition to histone modification, more powerful, complex or flexible control over gene expression is possible by controlling DNA methyl modification.

In the present invention, the carrier may further contain an expression vector containing a gene encoding a DNA demethylase, an expression vector containing a gene encoding a DNA methylase, or a DNA methylase inhibitor. Good.

Regarding DNA methylation modification, five genes have been reported so far as DNA methyltransferase and its homologous molecules (Dnmt1, Dnmt2, Dnmt3a, Dnmt3b, Dnmt3L). The base sequences and encoded amino acid sequences of these genes are known and disclosed as NCBI data bank accession numbers NM_001130823, NM_176085, NM_022552, NM_001207055, and NM_013369, respectively.

As the expression vector, the known expression vectors described above regarding the gene encoding histone acetylase and the like can be used.

A known “DNA methylase inhibitor” can be used. For example, 5-azacytidine, 5-azadeoxycytidine, zebraline, hydralazine.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and change the present invention based on the description of the present specification, and these are included in the technical scope of the present invention.

Example 1 Production of Artificial Chromatin In this example, an artificial chromatin model imitating the chromatin structure was produced.

The artificial chromatin used plasmid DNA called pGL3 as an alternative to DNA, and the purchased histone used HeLa cell Core Histone. Plasmid DNA differs from chromosomal DNA in that it is circular and has a low molecular weight. However, since it is easy to handle and the gene to be expressed can be incorporated arbitrarily, it has advantages such as easy evaluation of gene expression, so it was used in this example.

Also, the ratio of DNA and core histone in the in vitro chromatin formation reaction is important, and in general, it is necessary to perform titration every time new DNA and histone are prepared. In this example, using agarose gel electrophoresis, the histone / DNA weight ratio at which no free DNA was observed and only the complex band could be confirmed was determined as the optimum weight ratio.

In Example 3 and later, the artificial chromatin produced in this example was used. At that time, the optimum concentration for relaxing the chromatin structure of the polymer was determined by agarose gel electrophoresis. Then, the relaxation activity of the chromatin structure of each polymer was indirectly evaluated by evaluating the translation activity at the optimum concentration.

1-1) Purification of plasmid DNA

The pGL3 plasmid DNA was cultured in large quantities using E. coli. Purification was performed using a plasmid DNA replication kit (Qiagen), and the amount of the obtained plasmid was calculated from the absorbance. Absorbance was measured using a UV / Vis spectrophotometer (manufactured by JASCO Corporation). The UV / Vis spectrophotometer is a device that performs spectral absorption using light having a wavelength from the ultraviolet to the visible region (200 nm to 780 nm) causing electronic energy transition of molecules.

The obtained plasmid DNA concentration was as follows when three samples were measured. In the experimental system using the translation system of Example 3 and later, the experiment was performed using the higher concentration (0.6 mg / mL), and in the experimental system using agarose gel electrophoresis, the lower concentration (0.24 mg / mL). Went.

1-2) Production of artificial chromatin The plasmid DNA (T7 Luciferase control DNA (0.6 mg / mL)) purified in 1-1) was used. Artificial chromatin was prepared by adjusting each weight ratio (histone / DNA = 0.213, 0.43, 0.85, 1.7, 2, 4) and incubating for 1 hour. The optimal weight ratio was determined by agarose gel electrophoresis. The results are shown in FIG.

When DNA / histone was forming a complex, the charge was neutralized, and the experiment was conducted on the assumption that a band was seen near the injection site without much flow. As shown in FIG. 1, a complex band was observed at a weight ratio of 1.7 and 2, but a free DNA band was also observed. On the other hand, only the composite band was observed at a weight ratio of 4.

Using these three samples, transcription / translation was attempted by an in vitro translation system. When DNA / histone is properly formed like in vivo chromatin, the expression level was considered to be lower than that of naked DNA. However, when DNA / histone weight ratio is 1.7 or 2, DNA is a positive control. An expression level equivalent to that of the only sample was obtained. However, when the weight ratio was 4, the expression level was 1/10 ⁴ times, so that this sample was most suitable as a transcription-inactive chromatin structure used in the relaxation experiment of this study. Experiments after Example 3 were performed.

Example 2 Synthesis of Polymer In this example, a polymer for use in chromatin control was synthesized. Considering the fact that it has a carboxyl group that allows efficient endosome escape by the proton sponge effect and cell membrane fusion activity, the following five anionic and amphoteric polymers were selected in this example.

2-1) Synthesis of carboxymethylated polyvinyl imidazole (CM-PVIm) Synthesis of polyvinyl imidazole (PVIm) V-65 (2,2'-azobis (2,4-dimethylvaleronitrile)) was used as a radical polymerization initiator. Radical polymerization of polyvinyl imidazole (PVIm) by radical polymerization. Since V65 has an unpaired electron in a temperature response at 45 ° C., radical polymerization was performed by adding 1-vinylimidazole having a C═C bond.

300 mg (3.19 mmol, density 1.04 g / mL, 288.5 μL) of 1-vinylimidazole was dissolved in 2400 μL of N, N-dimethylformamide (DMF). V65 15.8 mg (0.064 mmol) was dissolved in 300 μL of DMF. After mixing the two kinds of solutions, it was necessary to remove oxygen in the solution in order to prevent the disappearance of radicals during the radical reaction, so bubbling with nitrogen was performed for 40 min. It incubated without stirring for 24 hours with the water bath preset to 45 degreeC which is radical generation temperature. Reprecipitation was performed by dropping into 80 mL of acetone. Centrifugation was performed at 2600 rpm for 5 min to remove the supernatant acetone. After dissolving in a small amount of DMF, reprecipitation with 80 mL of acetone, centrifugation, and removal of the supernatant were performed again. Acetone was volatilized by allowing to stand for 24 hours in a fume hood. The obtained product was dissolved in H ₂ O (20 mL), and then dialyzed for 2 days using a MWCO: 1000 dialysis membrane. The material obtained by lyophilization was recovered. The yield was 57.97 mg and the yield was 19.32%.

Synthesis of Carboxymethylated Polyvinylimidazole (CM-PVIm) Polyvinylimidazole (PVIm) (30 mg) synthesized as described above was dissolved in H ₂ O (8 mL). 40 mg of iodoacetic acid was dissolved in H ₂ O (2 mL). Triethylamine (TEA, 44 μL, 0.32 mmol) was added to the PVIm aqueous solution, and then an iodoacetic acid aqueous solution was added, followed by stirring at 40 ° C. for 24 hours (FIG. 2-2). After dialysis using MWCO: 1000 for 2 days, the substance obtained by lyophilization was recovered.

PVIm and CM-PVIm each confirmed the structure by ¹ H-NMR. Specifically, 3 mg of PVIm was dissolved in 700 μL of heavy water and measured.

CM-PVIm was measured as follows. 3 mg of CM-PVIm was added to heavy DMSO, and a few drops of trifluoroacetic acid were added to break hydrogen bonds. Furthermore, in order to exchange counter ions, ammonium hexafluorophosphate (NH ₄ PF ₆ ) was added and measured. In either case, the synthesis of the target compound was confirmed.

2-2) Synthesis of polymethacrylic acid (PMAA) 16.73 mL of methacrylic acid (Kanto Chemical Co., Inc.) distilled under reduced pressure was mixed with 133.7 mL of dimethylformamide (DMF). 88-1.6 mg of V-65, a radical polymerization initiator dissolved in 17 mL of DMF, was added to the mixture, and nitrogen bubbling was performed overnight with stirring. Then, it was made to react at 50 degreeC under nitrogen atmosphere for 24 hours.

The synthesized polymer was purified by reprecipitation by dropping it into acetone. Then, it vacuum-dried at 50 degreeC for 12 hours. However, the polymer is very easy to gel and encloses acetone as a reprecipitation solvent. Therefore, the dried polymer was dissolved in deionized water, dialyzed in water for 2 days using a membrane having a molecular weight cut-off (MWCO) of 1,000, and recovered by lyophilization.

2-3) Synthesis of carboxymethylated polyhistidine (CM-PLH) Poly-L-histidine (PLH) (50 mg) and iodoacetic acid (I-CH ₂ COOH) (PLH × 0.8 times amount: 40 mg), respectively The solution was dissolved in 8 mL of H ₂ O and 2 mL, and the aqueous solution was adjusted to pH 4.5 to 5 with 1N NaOH and stirred at room temperature for 24 hours. Thereafter, iodoacetic acid (40 mg) dissolved in H ₂ O (0.2 mL) was further added, and the aqueous solution was kept at pH 4.5 to 5 and again stirred at room temperature for 24 hours. Then, the same operation was performed and reaction was performed for a total of 3 days. The resulting aqueous solution was dialyzed against a MWCO: 1000 dialysis membrane for 3 days (adding 5 mL PBS (−) (× 20) in 1 L of H ₂ O), and then with H ₂ O alone before collecting the sample. Time dialysis was performed. Thereafter, a white powder was recovered by lyophilization.

GFC was measured with carrier: 0.5 M CH ₃ COOH, 0.3 M Na ₂ SO ₄ , flow rate: 1.0 mL / min, column: Shodex OHpak SB-804 HQ. ¹ H NMR was measured with D ₂ O (700 μL) added with hydrochloric acid (5 μL).

The number average molecular weight was estimated to be about 6000 by GFC, and the carboxymethyl group modification rate was estimated to be about 50 mol% by ¹ H NMR.

Example 3 Artificial Chromatin Relaxation Experiment with Polymer In this example, an artificial chromatin relaxation experiment with a polymer was performed.

3-1) Materials As the polymer, carboxymethylated polyvinylimidazole (CM-PVIm), polymethacrylic acid (PMMA), and carboxymethylated polyhistidine (CM-PLH) synthesized in Example 2 were used. Polyglutamic acid (PLE) was purchased from Sigma Aldrich. Polyacrylic acid (PAA: molecular weight MW = 25,000 or 1,000,000) was purchased from Wako Pure Chemical Industries, Ltd.

3-2) Evaluation by Agarose Gel Electrophoresis In 2.2 μL of the artificial chromatin prepared in Example 1, each polymer was compared with the number of anions of DNA so that the number of anions was 2, 4 or 8 times. Added and incubated for 15 minutes. The reaction product was subjected to electrophoresis for 20 minutes under a voltage of 50 V using Mupid (registered trademark) -2x using 1% agarose gel, pH 7.4 (solvent 50 mM phosphate buffer (PB)). Thereafter, the optimum concentration for relaxing each polymer was determined by Quantity One. Each polymer was added so that the number of anions was 2, 4, and 8 times that of DNA.

The results are shown in FIG. As shown in FIG. 2, it was found that when the anionic polymer (PAA, PMAA, CM-PLH and PLE) was added, the fluorescence intensity of the complex band was stronger than when it was not added. This fact suggests that artificial chromatin is relaxed by all anionic polymers.

In the amphoteric polymer CM-PVIm, a tendency different from that of the anionic polymer was observed. The artificial chromatin added with CM-PVIm had lower fluorescence intensity than that without addition. It is considered that the amphoteric polymer is relaxed by a mechanism different from that of the anionic polymer. Anionic polymers interact only with histones and weaken DNA-histone interactions, whereas amphoteric polymers interact with histones at anion sites and between DNA and histones by interacting with cation sites on DNA. Relax the interaction. For this reason, it is presumed that the amphoteric polymer is unlikely to intercalate ethidium bromide and the fluorescence intensity has been lowered.

In addition, it was suggested that when the number of anions was added twice as much as that of DNA in all polymers, the fluorescence intensity became maximum and was most relaxed.

3-3) Translation activity evaluation The translation activity evaluation was performed using the TNT (registered trademark) Quick Coupled Translation / Translation System to evaluate the relaxation ability as follows.

Reaction component (translation system)
TNT (registered trademark) Quick Master Mix 40 μL, methionine 1 mM 1 μL, artificial chromatin 2.2 μL,
Each polymer (CMPVIm (1 mg / mL), PAA (0.1 mg / mL), PMAA (0.15 mg / mL)),
Add nuclease-free water for a total volume of 50 μL

The amount of each polymer added and the amount of Nuclease-free-water

The reaction components were put into an Eppendorf tube, and all components were pipetted. Thereafter, the cells were incubated at 30 ° C. for 90 minutes, and the relaxation ability was evaluated by measuring the luminescence intensity with a luminometer.

Translational activity was evaluated at a concentration suggested to be most relaxed by agarose gel electrophoresis. First, it was confirmed that the translation activity of artificial chromatin was lower than that of the positive control naked DNA, and it was found that the transcription-inactive artificial chromatin was successfully produced. If the transcriptionally inactive form, that is, the aggregated heterochromatin can be relaxed, the exposed DNA region increases, and as a result, transcription and translation are promoted, and the relaxation ability was evaluated by this experimental system. The results are shown in FIGS.

FIG. 3 shows the results of PAA having two molecular weights of molecular weights MW 25,000 and 1,000,000. Both showed high translation activity, but MW 1,000,000 was slightly higher. FIG. 4 shows the translational activity of CM-PVIm and PMAA. All showed translational activity, but a significantly higher effect was confirmed particularly with CM-PVIm. Regarding these anionic polymers, it is considered that the higher the anionic charge density, the more relaxed.

On the other hand, the improvement in translational activity was seen in CM-PVIm with a low charge density, which is an interesting result. Since this polymer is an amphoteric polymer, it is more efficient by a mechanism different from that of an anionic polymer. It is thought that it is relaxed.

As described above, we have succeeded in constructing an artificial chromatin model using histones, and have established a useful system for evaluating relaxation of polymers. It was revealed that all macromolecules (PLE, CM-PLH, PAA, PMAA, CM-PVIm) tested in the established system relax the chromatin structure. Among the chromatin relaxed polymers, it was revealed that PAA and CM-PVIm improve the translation efficiency in the cell-free translation system.

Example 4 Calcium Phosphate Complex This example describes the preparation of a calcium phosphate complex comprising a plasmid containing the gene encoding histone deacetylase (HDAC) and coenzyme A, a histone acetylase inhibitor.

A TE solution (pH 7.6) was prepared by dissolving 1 mM Tris-HCl and 0.1 mM EDTA in millipore. A plasmid solution containing the plasmid at a concentration of 0.1 mg / ml in TE solution was prepared. As the plasmid, a plasmid in which a gene encoding histone deacetylase HDAC3 is inserted into the pCMV-SPORT6 vector and a plasmid in which a gene encoding DNA methylase (DNMT3a) is inserted into the pCMV-SPORT6 vector are used. It was. The base sequence of the pCMV-SPORT6 vector is shown in SEQ ID NO: 2.

Then, 2.5M CaCl ₂ solution and 0.1 mg / ml plasmid solution were mixed at a ratio of 1: 9 (solution A). Polyglutamic acid and coenzyme A were added to 100 μl of solution A so as to be 10 μg and 0, 1, 2, 5, 10 μg, respectively (solution B).

140 mM NaCl, 50 mM HEPES, and 6 mM Na ₂ HPO ₄ were dissolved in millipore to prepare a Hepes / phosphate solution (pH 7.1). Next, an equal amount of Hepes / phosphate solution was added to Solution B, stirred for several minutes, and then allowed to stand at 37 ° C. for 24 hours to receive a calcium phosphate complex. The prepared complex was recovered by centrifugation (10000 rpm, 15 minutes).

All calcium phosphate complexes recovered by centrifugation were suspended in excess PBS, and the particle size and zeta potential were measured using ELSZ-2 (Otsuka Electronics). The results are shown in the table below.

Furthermore, the coenzyme A content in the calcium phosphate complex was evaluated. Specifically, first, all prepared complexes were suspended in sterile PBS, and the particle surface was washed by centrifuging (10000 rpm, 15 minutes). Next, all the washed complexes were dispersed in dilute hydrochloric acid (pH = 3) and thoroughly pipetted to break the complexes. CPM into solution (7-diethylamino-3- (4'-maleimidyl-phenyl) -4-methyl coumarin) was added DTX 800 (BECKMAN COULTER) by measuring the fluorescence _{_{(E X = 360, E M}} = 469) By doing so, the coenzyme A contained in the particles was quantified. The results are shown in FIG.

Example 5 Gene Expression Evaluation Using Calcium Phosphate Complex In this example, gene expression evaluation when the calcium phosphate complex prepared in Example 4 was used was examined.

For gene expression evaluation, pGL3 encoding a luciferase gene as a plasmid species was included in the calcium phosphate complex. First, 1 × 10 ⁴ Cells of HepG2 cells were seeded on a 96-well plate and cultured for 24 hours. After exchanging the medium, the prepared complex was added to the medium and cultured for 24 hours so that the amount of plasmid was about 200 ng / well. Lipofectin, a commercially available gene transfer carrier, was used as a control. After the medium change, the cells were further cultured for 48 hours.

After removing the medium and washing 3 times with sterile PBS, cell lysate was obtained by adding 100 μl of cell lysis to all wells and pipetting. 100 μl of lysate luciferase substrate was added, and luminescence intensity was measured using AccuFLEX Lumi400 (ALOKA).

Example 6 Western Blot Measurement to HepG2 Cells In this example, histone acetylation when the calcium phosphate complex of the present invention and histone deacetylation inhibitor were administered to cells was measured by Western blot. The complex used this time contains a gene encoding histone deacetylase (SIRT2) instead of the plasmid containing the gene encoding histone deacetylase (HDAC) in "Example 4 calcium phosphate complex". A plasmid is used.

First, HepG2 cells were seeded on 96-well plates at 5 × 10 ⁵ cells, cultured for 24 hours, then changed to a medium containing 3 μM of histone deacetylation inhibitor valproic acid (VPA), and further cultured for 24 hours. Next, the prepared complex was added to Well and cultured for 24 hours. Anacardic acid (AA) was used as a control.

After removing the medium from all the wells and washing with sterile PBS three times, NP40 buffer was added to all the wells by 100 μ and pipetting was performed. Subsequently, cell lysate was obtained by centrifuging (15000 rpm, 5 minutes) to remove insoluble substances and collecting the supernatant. The collected supernatant solution was mixed with 2 × SB buffer at a ratio of 1: 1 and boiled at 95 ° C. for 5 minutes to prepare a sample for Western blotting.

Using a mini-PROTEAN Tetra cell (Bio-Rad), a mini-trans blot cell (Bio-Rad), and various acetylated histone antibodies (H3-K9, -K18, -K27, H4-K5, -K8, -K12: CST) Western blot measurement was performed.

The result of Western blot measurement is shown in FIG. The leftmost lane is a plasmid DNA containing a gene encoding calcium phosphate (CaP), coenzyme A (CoA), SIRT2, a plasmid DNA that has no effect on this experiment, and a sample to which no anacardic acid is added. is there. The second lane from the left is a sample to which plasmid DNA was added using CaP. Compared with the sample to which nothing was added, it was confirmed that CaP had no effect on histone acetylation since no difference was observed. The middle lane is a sample to which only AA is added as a control. Since the bands of acetylated histones H3 and H4 are thinner than the two samples on the left, it was confirmed that the evaluation of the amount of acetylation in this experiment was established. The second sample from the right is a sample into which plasmid DNA containing a gene encoding SIRT2 is introduced using CaP. It was confirmed that the amount of acetylated histone decreased due to the expression of SIRT2. Finally, the rightmost lane is a sample into which a plasmid containing a gene encoding CoA and SIRT2 was introduced using CaP. Compared with all samples, the band was the thinnest, and it was confirmed that CoA and SIRT2 acted to strongly deacetylate. In this experiment, since H3-K9, -K18, -K27, H4-K5, -K8, and -K12 were used as acetylated histone antibodies, it is considered that the amount of acetylation decreased.

Example 7 Synthesis of Acetylated Polylysine (AcPLL) This example describes the synthesis of acetylated polylysine (AcPLL).

7-1) Synthesis of AcPLL (5% target) 9.58 mg of PLL was dissolved in 0.95 mL of DMSO. 20 μL of TEA was put into this solution, and 50 μL of a sample for amino group determination before reaction was collected (solution (1)). Acetic anhydride (4.5 μL) was dissolved in DMSO (1.0 mL), and 50 μL was taken from the solution and added to the solution (1). This solution was reacted in a water bath (40 ° C.) for 2 hours. After the reaction, 50 μL of a sample for amino group determination was collected.

Using a fluorescence spectrophotometer, the amino group was quantified to confirm that the reaction was progressing. As a result of measuring ¹ H NMR and calculating the acetylation rate, it was 8.5%. That is, the reaction progress rate was suggested to be almost 100%.

7-2) Synthesis of AcPLL (75% target) + maltose (25% target) PLL 11.2 mg was dissolved in DMSO 1 mL. 10 μL of TEA was put into this solution, and 50 μL of a sample for amino group determination before reaction was collected (solution (1)). 4 μL of acetic anhydride was added to the solution (1). This solution was reacted in a water bath (40 ° C.) for 2 hours. After the reaction, 50 μL of a sample for amino group determination was collected (solution (2)). Maltose 6.28mg was added to the said solution (2), and it was made to react in a water bath (40 degreeC) for 24 hours. 50 μL of the solution after the reaction was collected for amino group determination.

The amino group was quantified using a fluorescence spectrophotometer to confirm that the reaction was progressing.

7-3) Synthesis of AcPLL (30% target) + maltose (70% target) PLL (10 mg) was dissolved in DMSO (0.99 mL). 10 μL of TEA was added to this solution. (Solution (1)). 150 μL of acetic anhydride was dissolved in 1.0 mL of DMSO, 10 μL was collected from the solution, and added to the solution (1). This solution was reacted in a water bath (40 ° C.) for 2 hours (solution (2)). To the solution (2), 226 mg of maltose was added and reacted in a water bath (40 ° C.) for 2 days.

^The acetylation rate and maltose binding rate were calculated by ¹ H NMR. As a result, the acetylation rate was calculated to be 29.3%, and the binding rate of amino group and maltose was calculated to be 39%.

Example 8 Synthesis of Hyperbranched Polylysine This example describes the synthesis of hyperbranched polylysine (DMF solvent).

HBTU (379 mg, 1.0 mmol) and HOBt · H ₂ O (153 mg, 1.0 mmol) were added to a mixed solution of L-lysine (208 mg, 1 mmol), TEA (154 μL, 1.1 mmol) and DMF in 15 mL, and water was added for 15 hours. The bath was stirred under the condition of 40 ° C. Then, it stirred at room temperature for 14 days.

The pre-reaction solution was also white and cloudy, but even after stirring for 15 hours using a water bath, the reaction mixture was suspended in white. DMF and DMSO were added to the reaction mixture obtained by stirring for 15 hours, and it was confirmed whether or not it dissolved. Dilution with DMF did not change the solubility. On the other hand, when DMSO was added and diluted, the solution was successfully dissolved when the solution was diluted 4 times.

The DMF solvent was dialyzed and lyophilized to confirm that it was ready, and a white powder was obtained. Since hyperbranched PLL is water-soluble, in order to remove impurities insoluble in water, the obtained white powder was dissolved in water and freeze-dried again to recover the product. When the molecular weight of the synthesized product was measured by the aqueous GFC, it was suggested that the hyperbranched PLL was synthesized although it was a small amount. Further, when ¹ H NMR analysis was performed, a peak different from that of L-Lycine was observed.

Example 9 Synthesis of Dendritic Polylysine (DPK) This example describes a synthesis example of dendritic polylysine (DPK).

It was confirmed that the synthesis of KG2 was successful when it was confirmed that the synthesis was completed by comparing the measured values of FAB-MS and literature values.

Example 10 Gene Introduction Using a Carrier Containing Four Elements of Histone Acetylase DNA, Histone Deacetylase Inhibitor, Polylactic Acid, and Cationic Lipid In this example, histone acetylase DNA, histone deacetylation Gene transfer was performed using a carrier containing four elements of an enzyme inhibitor, polylactic acid, and cationic lipid, and the function of the carrier, histone acetylation amount and cell differentiation rate were evaluated.

10-1 Preparation of polylactic acid carrier Histone acetylase DNA / histone deacetylase inhibitor / polylactic acid complex / cationic lipid was prepared by the following procedure.

First, there are compounds having various alkyl chain lengths as cationic lipids. In this study, however, a single chain having an alkyl chain length of 16 to 18 has low cytotoxicity and excellent intracellular introduction efficiency. Alternatively, a double-chain cationic lipid was selected. In this example, single-chain stearylamine or double-chain cationic lipid N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium methylsulfate An example using (DOTAP) is shown. A DNA encoding histone acetylase (CAF) and a cationic lipid were dissolved in ethanol so that the charge ratio was 1: 1, and incubated at 37 ° C. for 1 h.

Next, 50 nM histone deacetylase inhibitor (tricostatin A (TSA)) is added to the DNA / cationic lipid complex, and polylactic acid (PLA) is added in an amount 5 times that of the DNA / cationic lipid. In addition, it was incubated at 37 ° C. for 1 h. After the incubation, the sample was dialyzed for 4 days using a MWCO1000 dialysis membrane. Finally, the dialyzed sample was freeze-dried for 3 days to obtain a white powder. The presence of DNA in the carrier was confirmed by gene expression evaluation by luciferase assay.

The luciferase assay was performed according to the following procedure. HepG2 cells were seeded at 1 × 10 ⁵ c cells / ml in 96-well plates and incubated at 37 ° C. for 24 hours. A carrier containing plasmid DNA encoding luciferase was added to HepG2 cells and incubated at 37 ° C. for 48 hours. After completion of the incubation, the HepG2 cells were collected and lysed with a cell lysis solution (manufactured by Promega, attached to the luciferase assay system, “Cell Culture lysis reagent”). The gene expression level was measured by adding a substrate to the lysate and using a luminometer. Inhibitors in the carrier were evaluated for activity using an HDAC activity analysis kit.

10-2 Cell Line (1) Carrier Introduction Human myeloid leukemia cells (HL60) were seeded in 12-well plates at a cell concentration of 1 × 10 ⁵ cells / ml and incubated at 37 ° C. for 24 hours. The prepared carrier was added to HL60 cells and incubated at 37 ° C. for 48, 96, 192 hours. One of the cells after completion of the incubation was evaluated for cell differentiation rate by NBT staining, which is a technique for specifically staining granulocytes. On the other hand, the cells were lysed with a cell lysis solution to obtain a protein solution.
(2) Evaluation of histone acetylation (Western blot analysis)
Changes in the amount of histone acetylation were evaluated by Western blotting using the protein solution obtained above. The recovered protein solution was incubated at 95 ° C. for 5 minutes with a reducing agent in order to cleave the disulfide bond of the protein. The prepared sample was subjected to SDS-PAGE using a 15% polyacrylamide gel. Next, the PVDF membrane hydrophilized with methanol and the migrated gel were superposed, and the protein was transferred from the gel to the PVDG membrane. The transferred PVDF membrane was immersed in 5% skim milk and allowed to stand for 1 hour. Thereafter, the PVDF membrane was cleaned using a cleaning solution. The washed PVDF membrane was subjected to a primary antibody reaction. As primary antibodies, Ac-H3K9, K18, K27 (manufactured by Cell Signaling technology Japan) and β-actin as a control were used. The primary antibody was used after being diluted 1000 times with a washing solution. The PVDF membrane was immersed in the diluted antibody solution and left for 6 hours. Thereafter, the PVDF membrane was cleaned using a cleaning solution. Next, a secondary antibody reaction was performed. As the secondary antibody, horseradish peroxidase (HRP) was used. The HRP antibody was used after being diluted 5000 times with a washing solution. The PVDF membrane was immersed in the diluted antibody solution and left for 6 hours. Thereafter, the PVDF membrane was cleaned using a cleaning solution. Finally, ECL Prime Western Blotting Detection System (manufactured by GE Healthcare Japan) was used as a detection reagent to detect luminescence. The detection reagent was dropped on the PVDF membrane and allowed to stand for 5 minutes. Thereafter, the PVDF film was subjected to chemiluminescence detection using an imaging device AE-9300 Ez-Capture MG manufactured by ATTO.
(3) Cell differentiation rate evaluation (NBT staining)
A nitro blue tetrazolium (NBT) solution was prepared to be 0.2% NBT, 20% FBS. HL60 cells after completion of the incubation were suspended in RPMI medium so as to be 1 × 10 ⁶ cells / ml. Next, an NBT solution equivalent to RPMI medium was added, and phorbol myristyl acetate (PMA) was further added to 2 × 10 ⁻⁶ M, followed by incubation at 37 ° C. for 30 minutes. After completion of the incubation, the mixture was centrifuged at 1200 rpm for 5 minutes, and the supernatant was removed. Finally, it was resuspended in PBS and the cells were counted using a hemocytometer. The differentiation rate (%) was calculated as 100 × number of stained cells / total number of cells.

10-3 Results (1) Functional evaluation of polylactic acid carrier using DOTAP as cationic lipid In gene expression evaluation by luciferase assay, gene expression was confirmed in cells introduced with polylactic acid carrier. When the activity was evaluated using the HDAC activity analysis kit, the inhibitory activity was 65% as compared with the control to which no drug was added. From the above results, it was confirmed that the DNA and the inhibitor were encapsulated in the carrier.
(2) Evaluation of histone acetylation amount The amount of histone acetylation of cells after 2 days is 1.4 times higher when histone deacetylase inhibitor alone is added than when nothing is added. When only acetylase DNA was added, it increased by 1.25 times. On the other hand, when a carrier using DOTAP as a cationic lipid was introduced, it increased 1.65 times. The same tendency was observed after 4 and 8 days, and the acetylation amount of the cells to which the carrier using DOTAP as the cationic lipid was added was increased by 2 times compared to the control after 8 days.
(3) Cell differentiation rate evaluation The differentiation rate into granulocytes after 2, 4, and 8 days was 29, 33, and 35%, respectively, when only a histone deacetylase inhibitor was added. On the other hand, the differentiation rates of cells introduced with carriers using DOTAP as the cationic lipid were 45, 51 and 59%, respectively.

Summary In this example, epigenetics modification was controlled by simultaneously promoting epigenetics modification by gene introduction and suppressing reverse reaction by an inhibitor. It was found that anti-cancer therapy is possible by promoting histone acetylation among epigenetic modifications.

Example 11 Gene Introduction Using a Carrier Containing Four Elements of Histone Acetylase DNA, Histone Deacetylase Inhibitor, Calcium Phosphate, and Polyglutamic Acid In this example, histone acetylase DNA, histone deacetylase inhibition Genes were introduced using a carrier containing 4 elements of a drug, polylactic acid, and cationic lipid, and the function of the carrier, histone acetylation amount and cell differentiation rate were evaluated.

11-1 HepG2 cell line CAF plasmid and TSA introduction experiment (1) Preparation of complex 2.5M CaCl ₂ solution, 10 mg / ml Polyglutamic acid (PLE) solution is a plasmid DNA solution encoding CAF which is histone acetylase Mixed with. The final concentration of Ca ²⁺ was 250 mM, and the final amount of PLE was 5 μg. Next, the above-mentioned mixed solution was mixed with an equal amount of Hepes / Phosphate solution, stirred for several minutes, and then incubated at 37 ° C. for 24 hours. After completion of the incubation, the complex was collected by centrifugation at 10,000 rpm for 5 minutes.
(2) Complex introduction HepG2 cells were seeded in a 96-well plate at 1 × 10 ⁵ cells / ml and incubated at 37 ° C. for 24 hours. The prepared complex and histone deacetylase inhibitor TSA (final amount 1 μg) were added to HepG2 cells and incubated at 37 ° C. for 24 hours. After the incubation, the medium was changed to remove the complex that was not taken up by the cells. Thereafter, the medium was changed twice every 24 hours. Cells after incubation were lysed with a cell lysis solution to obtain a protein solution.
(3) Evaluation of histone acetylation (Western blot analysis)
Changes in the amount of histone acetylation were evaluated by Western blotting. The evaluation of acetylated histone was performed using Ac-H3K9, K18, K27 antibodies (manufactured by Cell Signaling technology Japan).

Specifically, the recovered protein solution was incubated at 95 ° C. for 5 minutes using a reducing agent in order to cleave the disulfide bond of the protein. The prepared sample was subjected to SDS-PAGE using a 15% polyacrylamide gel. Next, the PVDF membrane hydrophilized with methanol and the migrated gel were superposed, and the protein was transferred from the gel to the PVDG membrane. The transferred PVDF membrane was soaked in 5% skim milk and allowed to stand for 1 hour. Thereafter, the PVDF membrane was cleaned using a cleaning solution. The washed PVDF membrane was subjected to a primary antibody reaction. As primary antibodies, Ac-H3K9, K18, and K27 antibodies (Cell Signaling Technology Japan) and a control anti-β-actin antibody were used.

The primary antibody was used after being diluted 1000 times with a washing solution. The PVDF membrane was immersed in the diluted antibody solution and left for 6 hours. Thereafter, the PVDF membrane was cleaned using a cleaning solution. Next, a secondary antibody reaction was performed. As the secondary antibody, an anti-horseradish peroxidase (HRP) antibody was used. The HRP antibody was used after being diluted 5000 times with a washing solution. The PVDF membrane was immersed in the diluted antibody solution and left for 6 hours. Thereafter, the PVDF membrane was cleaned using a cleaning solution. Finally, luminescence detection was performed using ECL Prime Western Blotting Detection System (manufactured by GE Healthcare Japan) as a detection reagent. The detection reagent was dropped on the PVDF membrane and allowed to stand for 5 minutes. Thereafter, the PVDF film was subjected to chemiluminescence detection using an ATTO imaging device AE-9300 Ez-Capture MG.

11-2 HL60 cell line (1) Carrier introduction Human myeloid leukemia cells (HL60) were seeded in 12-well plates at a cell concentration of 1 × 10 ⁵ cells / ml and incubated at 37 ° C. for 24 hours. CaP / CAF plasmid DNA / TSA / PLE complex was added to HL60 cells and incubated at 37 ° C. for 48 hours. One of the cells after completion of the incubation was evaluated for cell differentiation rate by NBT staining, which is a technique for specifically staining granulocytes. On the other hand, the cells were lysed with a cell lysis solution to obtain a protein solution. Changes in the amount of histone acetylation were evaluated by Western blotting using this protein solution.
(2) Evaluation of histone acetylation amount The change in histone acetylation amount was evaluated by Western blotting. The evaluation of acetylated histone was performed using Ac-H3K9, Ac-H3K18, and Ac-H4K12 antibodies (manufactured by Cell Signaling technology Japan).
(3) Cell differentiation rate evaluation (NBT staining)
A nitro blue tetrazolium (NBT) solution was prepared to be 0.2% NBT, 20% FBS. HL60 cells after completion of the incubation were suspended in RPMI medium so as to be 1 × 10 ⁶ cells / ml. Next, an NBT solution equivalent to RPMI medium was added, and phorbol myristyl acetate (PMA) was further added to 2 × 10 ⁻⁶ M, followed by incubation at 37 ° C. for 30 minutes. After completion of the incubation, the mixture was centrifuged at 1200 rpm for 5 minutes, and the supernatant was removed. Finally, it was resuspended in PBS and the cells were counted using a hemocytometer. The differentiation rate (%) was calculated as 100 × number of stained cells / total number of cells.

11-3 Results (1) HepG2 cell line-Experiment using CAF plasmid DNA and trichostatin A Compared with cells to which nothing was added, cells to which only CAF plasmid DNA was added had a histone acetylation level of 1 10-fold, and 1.21 times higher in cells to which only trichostatin A was added. Furthermore, the amount of acetylation increased 2.51 times in the cells into which the calcium phosphate carrier was introduced.
(2) HL60 cell line (a) Evaluation of histone acetylation amount Compared with cells to which nothing was added, cells to which only CAF plasmid DNA was added had a histone acetylation amount of 1.74 times, and trichostatin The number of cells added with A alone was 1.75 times. Furthermore, in the cells into which the calcium phosphate carrier was introduced, the acetylation amount was changed 1.89 times.
(B) Cell differentiation rate evaluation In cells to which only trichostatin A was added, 27% of the cells differentiated into granulocytes. On the other hand, 36% of cells added with calcium phosphate carrier were differentiated into granulocytes.

Example 12 Control of Chromatin Structure Using Anionic Polymer In this example, engineering control of chromatin structure by anionic polymer in cells is described. Polyaspartic acid-ethylenediamine-β-cyclodextrin (PLD-EDA-β-CD), an anionic polymer, was introduced into the nucleus, and the relaxation of the chromatin structure and the increase in the amount of histone acetylation accompanying the relaxation of the chromatin structure were observed. It was issued.

12-1
(1) Synthesis of 6-O-Ts-β-CD (Org. Biomol. Chem., 2011, 9, 7799)
5 g (12.3 mmol) of β-cyclodextrin (CD) was added to 41 mL of water, and 1.7 mL of an aqueous sodium hydroxide solution in which 0.6 g (41 mmol) of sodium hydroxide was dissolved was added over 15 minutes. Thereafter, the reaction solution was transferred to ice, and 1.02 g (14.8 mmol) of TsCl (p-toluenesulfonyl chloride) dissolved in 2.5 mL of acetonitrile was added over 25 minutes. Thereafter, the mixture was stirred in ice for 4 hours, the precipitate was filtered through Kiriyama filter paper (No. 5), and the filtrate was allowed to stand at 4 ° C. overnight. The resulting crystals were collected with Kiriyama filter paper (No. 4) and washed with water and ethanol. Ethanol was dried in a desiccator to obtain 1.05 g of 6-O-Ts-β-CD.
(2) Synthesis of PLD-EDA and PLD-EDA-β-CD PLD (polyaspartic acid) 20 mg (0.2 mmol / COOH), HOBt (1-hydroxybenzotriazole) 27.7 mg (0.2 mmol), PyBOP ( 105.1 mg (0.2 mmol) of (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate) was dissolved in 10 mL of dehydrated DMF and incubated for 10 minutes while irradiating with ultrasonic waves. Next, 320 mg (2.0 mmol) of N-Boc-EDA (N-butoxycarbonylethylenediamine) dissolved in 2 mL of dehydrated DMF was added, and the mixture was stirred in a 60 ° C. oil bath for 48 hours in a closed system. The reaction solution was allowed to cool to room temperature, precipitated in 20 times the amount of ethanol, and centrifuged at 3500 rpm for 30 minutes. The supernatant was discarded, 40 mL of ethanol was newly added and centrifuged at 3500 rpm for 30 minutes, and this operation was repeated twice.

The remaining precipitate was poured into a vial using ethanol, and ethanol was dried overnight at 70 ° C. and normal pressure. 1 mL of TFA (trifluoroacetic acid) was added to the resulting powder and stirred for 1 hour, and the reaction solution was precipitated in 20 mL of diethyl ether. After discarding 40% or more of the supernatant, diethyl ether was added again to 20 mL, and this operation was repeated 5 times. After diethyl ether was dried overnight at room temperature, the remaining solid was dissolved in 20 mL of ultrapure water and lyophilized for 5 days to obtain 3.9 mg of PLD-EDA.

3.9 mg (0.04 mmol / EDA) of the obtained PLD-EDA and 50 mg (0.4 mmol) of 6-O-Ts-β-CD were dissolved in 4 mL of dehydrated DMSO and stirred in a 70 ° C. oil bath for 48 hours. The reaction solution was allowed to cool to room temperature and then dialyzed in water using a regenerated cellulose membrane having a molecular weight cut off of 3500. Thereafter, lyophilization was performed for 48 hours to obtain 5.0 mg of PLD-EDA-β-CD.
(3) Identification of PLD-EDA-β-CD PLD-EDA-β-CD confirmed that there was no free CD removal and no molecular weight loss by GFC. From ¹ H-NMR, CD-derived peak and PLD-derived A peak was confirmed.
(4) FITC labeling of PLD-EDA-β-CD 2.5 mg (3 μmol / NH ₂ ) of PLD-EDA-β-CD synthesized in the previous section was replaced with 1.17 mg (3 μmol) of FITC (fluorescein isothiocyanate). In addition, the mixture was stirred at room temperature for 2 hours while being shielded from light in 10 mM K ₂ CO ₃ to perform FITC labeling. Then, using a membrane with a molecular weight cut off of 3500, dialyzed for 6 days while shielding from water, removed the remaining insoluble material by centrifuging at 3000 rpm for 10 minutes, and freeze-dried the supernatant for 2 days to be labeled with FITC PLD-EDA-β-CD was obtained.

12-2 Observation of intracellular introduction of PLD-EDA-β-CD by confocal microscope HeLa cells were seeded on a slide glass at a concentration of 3 × 10 ⁴ cells / well and incubated at 37 ° C. under 5% CO _2. did. After 8 hours, the medium was changed, and 30 μg / well of PLD-EDA-β-CD was added. After further incubation for 16 hours at 37 ° C. under 5% CO ₂ , all the medium was aspirated, washed with PBS (−), fixed with 4% paraformaldehyde, and then cell nuclei with DAPI solution (1 / 10,000 dilution). Was stained. After washing twice with PBS (−), the sample was mounted with 50% glycerol and stored protected from light.

Confocal microscope observation was performed using FV1000-D from Olympus. Each wavelength was excited and observed with a wavelength optimized for FITC / DAPI.

12-3 Chromatin structure relaxation evaluation by gene transfer and Western blotting When chromatin structure is relaxed by introduction of anionic polymer into nucleus and histone is exposed, the accessibility of histone acetylase (HAT) to histone tail is improved. However, the amount of acetylated histone is thought to increase. Therefore, the amount of acetyl histone is quantified by Western blotting.

HeLa cells were seeded in 12 well plates at 5 × 10 ⁴ cells / well and incubated at 37 ° C., 5% CO ₂ . After 8 hours, the medium was changed, and 100 μg / well of PLD-EDA-β-CD was added. After further incubation for 16 hours under the same conditions and aspiration of all the medium, plasmid DNA encoding histone acetylase (HAT) was mixed with lipofectin at a charge ratio (+/−) of 1 and in phosphate buffer for 1 hour. The complex prepared by incubating with was added (DNA amount 1 μg / well). Thereafter, the cells were incubated for 48 hours under the same conditions, the medium was aspirated, washed with PBS (−), and cell lysate was recovered by adding 75 μL / well of 5 × cell lysis solution. 200 μL of PBS (−) was added to the collected lysate, and centrifugation was performed at 5000 × g for 10 minutes to remove insoluble protein. 200 μL of the supernatant was incubated for 5 minutes at 95 ° C. using a reducing agent to cleave the disulfide bonds of the protein.

30 μL of the sample was loaded and SDS-PAGE was performed under a constant condition of 30 mA. After the electrophoresis for 30 minutes, the PVDF membrane was blotted for 90 minutes under a constant condition of 100 V, and the PVDF membrane was blocked for 1 hour. The PVDF membrane after blocking was washed by shaking with 1 × TBS-T for 5 minutes at 400 rpm, and this operation was repeated three times. Thereafter, the PVDF membrane was incubated at 4 ° C. for 12 hours in 3 mL of 1 × TBS-T containing 3 μL each of acetyl-H3 antibody, acetyl-H4 antibody and β-actin antibody derived from rabbit. Further, the PVDF membrane was washed with 1 × TBS-T as described above, and incubated in 3 mL of 1 × TBS-T containing 3 μL of HRP-conjugated secondary antibody derived from rabbit for 6 hours. After washing with 1 × TBS-T, color development with alkaline phosphatase was performed, and a band was detected with a CS analyzer at an exposure time of 4 minutes.

12-4 Result (1) Result of confocal microscope observation The result of confocal microscope observation is shown to FIG. 7A-FIG. 7D. 7A-D respectively show (A) fluorescence image by DAPI staining cell nuclei, (B) fluorescence image by FITC-labeled PLD-EDA-β-CD, (C) differential interference image, and (D) A- C images are combined. FIG. 7 shows that PLD-EDA-β-CD is localized in the cell nucleus region. In addition, PLD-EDA not subjected to CD modification was not observed to be localized in the nucleus. Without limitation, the “EDA” moiety is a spacer to prevent repulsion between the anionic polymer (PLD) and the intercellular membrane, and the “CD” moiety is rich in lipids through interaction with plasma membrane cholesterol. It is thought that it is functioning because the polymer stays in the region.
(2) Change in the amount of acetyl histone by Western blot (KSB102)
The amount of acetylation of histones H3 and H4 in HeLa cells is 3.9 times higher in the case of adding HAT / lipofectin than in the case of adding no anionic polymer / HAT / lipofectin complex. The amount increased, and the addition of HAT / lipofectin after addition of the anionic polymer showed a 4.7-fold increase in acetylation relative to the control.

Claims

1) A polymer selected from the group consisting of amphoteric polymers, anionic polymers, and nonionic polymers, and having any of the following main chain skeletons:

Or
2)
i) an expression vector comprising a gene encoding a histone acetylase;
ii) an expression vector comprising a gene encoding a histone methylase;
iii) an expression vector comprising a gene encoding a histone deacetylase;
iv) an expression vector comprising a gene encoding a histone demethylase;
v) histone acetylation inhibitors;
vi) histone deacetylation inhibitors;
vii) a histone methylation inhibitor; and viii) a composition for controlling a chromatin structure, comprising a carrier containing two or more substances selected from the group consisting of histone demethylation inhibitors.
The composition according to claim 1 for relaxing chromatin structure.
Polymer
a) carboxymethylated polyvinylimidazole,
b) from polyacrylic acid, polymethacrylic acid, polyglutamic acid, carboxylmethylated polyhistidine, or polyaspartic acid, or c) PEG or sugar chain, or a block copolymer, graft copolymer, or dendrimer thereof. The polymer according to claim 1 or 2, which is selected.
The composition according to any one of claims 1 to 3, wherein the polymer has a molecular weight of 10,000 to 2,000,000.
The composition according to any one of claims 1 to 4, wherein the carrier comprises polyester, calcium phosphate, or polyamino acid.
The carrier further comprises an expression vector containing a gene encoding a DNA demethylase, an expression vector containing a gene encoding a DNA methylase, or a DNA methylase inhibitor. The composition according to any one of the above.
A method for controlling a chromatin structure by administering the composition according to any one of claims 1 to 6 in vitro, ex vivo, or in vivo.