JP2003500052A - Inhibition of histone deacetylase - Google Patents

Abstract

  (57) [Summary] The present invention relates to the inhibition of histone deacetylase expression and enzymatic activity, and particularly to the inhibition of specific histone deacetylases. The present invention also relates to a composition comprising the antisense oligonucleotide and a method for inhibiting histone deacetylase using the composition. Also disclosed are a method for identifying a histone deacetylase involved in the induction of cell proliferation and a method for identifying a compound that interacts with such a histone deacetylase and reduces its enzymatic activity.

Description

Detailed Description of the Invention

RELATED APPLICATION This application claims priority from U.S. Provisional Patent Application No. 60 / 132,287, filed March 3, 1999, which is incorporated by reference in its entirety. There is.

[0001] FIELD The present invention BACKGROUND OF THE INVENTION relates to the inhibition of the expression and enzymatic activity of a histone deacetylase (histone deacethylase). Summary of Related Art Deacetylation of core histones H1-H4 is mediated by two related families of enzymes called histone deacetylases. 1 of histone deacetylase
Two families include HDAC-1, HDAC-2, HDAC-3. The second family of histone deacetylases is HDAC-4 (formerly HDAC-A), HDAC-5 (formerly HDAC-B).
, HDAC-C, HDAC-D and HDAC-E. Histone deacetylase activity is believed to regulate accessibility to transcription factor enhancers and promoters.
Indeed, an enrichment of acetylated histone H4 has been found in the transcriptional silence region of the genome (Taunton et al., Science 272: 408-411, 1996).

Functional histone deacetylases have been mentioned as a requirement in the cell cycle of both normal and tumor cells. Trichostatin A (TCA), an antibiotic isolated from Tostreptomyces, has been shown to suppress histone deacetylase activity and block cell cycle progression in the G1 and G2 phases (Yoshida Et al., J. Biol. Chem. 265: 17174-17179, 1990; Yoshida et al., Exp. C.
ell Res. 177: 122-131, 1988). Trichostatin C, trapoxin, depudecin
Other inhibitors of histone deacetylase activity, such as, suberoylanilide, hydroxamic acid (SAHA), FR901228 (Fujisawa) and butyrate, have been shown to similarly inhibit intracellular cell cycle progression. (Taunton et al., Science 272: 40.
8-411, 1996; Kijima et al., J. Biol. Chem. 268 (30): 22429-22435, 1993; Kwon et al., Proc. Natl. Acad. Sci. USA 95 (7): 3356-61, 1998).

All known inhibitors of histone deacetylase are natural products, all small molecules that suppress the activity of histone deacetylase at the protein level. Furthermore, known histone deacetylase inhibitors are also non-specific for specific histone deacetylases and inhibit all histone deacetylases of either family to a greater or lesser extent.

Therefore, there remains a need to develop a reagent that suppresses histone deacetylase at the gene level and suppresses the expression of a specific histone deacetylase. There is also a need to develop methods used to identify and suppress specific histone deacetylases involved in tumorigenesis using these reagents.

BRIEF SUMMARY OF THE INVENTION The present invention provides methods and reagents for suppressing histone deacetylase at the nucleic acid level and suppressing the expression of specific histones by suppressing the expression at the nucleic acid level. . According to the present invention, it is possible to identify and specifically suppress a specific histone deacetylase involved in tumor formation.

Therefore, in a first aspect, the present invention provides an antisense oligonucleotide that suppresses the expression of histone deacetylase. In one embodiment of this aspect, the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HD.
AC-5, HDAC-C, HDAC-D or HDAC-E. In other embodiments, the oligonucleotide inhibits more than one histone deacetylase or the oligonucleotide inhibits all histone deacetylases. The oligonucleotides are preferably chimeric or hybrid oligonucleotides.

In one preferred embodiment of the first aspect of this invention, the oligonucleotide represses transcription of a nucleic acid molecule encoding a histone deacetylase. The nucleic acid molecule may be genomic DNA (eg a gene), cDNA or RNA. In another embodiment, the oligonucleotide inhibits translation of histone deacetylase.

In various embodiments of the first aspect of the invention, the antisense oligonucleotide comprises a phosphorothionate, a phosphorodithionate (p
hosphorodithionnate), alkylphosphonate, alkylphosphonothionate, phosphotriester
photriester, phosphoramidate, siloxane
), Carbonate, carboxylmethylester
), Acetamidate, carbamate, thioether, bridgd phosphramidate, bridged methylene phosphonate, bridged phosphorothionate, and sulfone interfacial. At least 1 selected from the group consisting of nucleotide internucleotide linkages
With an internucleotide linkage of In some embodiments, the oligonucleotide has ribonucleotides or 2'-O-substituted ribonucleotide regions and deoxyribonucleotide regions.

In a second aspect, the invention provides a method of inhibiting histone deacetylase in a cell, the method comprising contacting the cell with the antisense oligonucleotide of the first aspect of the invention. provide. In one preferred embodiment of this second aspect, cell proliferation is suppressed in contact cells. In a preferred embodiment, the cells are tumor cells that are present in animals, including humans, and are in tumor growth. In certain preferred embodiments, the method of the second aspect of the invention comprises contacting the cell with a histone deacetylase protein repressor that interacts with histone deacetylase and reduces the enzymatic activity of histone deacetylase. Including further. The histone deacetylase protein repressor is preferably operably linked to the antisense oligonucleotide.

In a third aspect, the present invention is a method of inhibiting tumor cell growth in an animal, wherein the antisense oligo of the first aspect of the invention is applied to an animal having at least one tumor cell in its body. Methods are provided that include administering a therapeutically effective amount of a nucleotide with a pharmaceutically acceptable carrier for an effective therapeutic period.

In one preferred embodiment of the third aspect of this invention, the method comprises administering a therapeutically effective amount of a histone deacetylase protein inhibitor that interacts with histone deacetylase and reduces its enzymatic activity, With a pharmaceutically acceptable carrier,
Further comprising administering to the animal for a therapeutically effective period. The histone deacetylase inhibitor is preferably operably linked to the antisense oligonucleotide.

In a fourth aspect, the present invention is a method for identifying a histone deacetylase involved in the induction of cell proliferation, which comprises contacting cells with an antisense oligonucleotide that suppresses the expression of the histone deacetylase. And a method of identifying the histone deacetylase as a histone deacetylase involved in the induction of cell proliferation, which suppresses cell proliferation of contact cells. In certain preferred embodiments, the cells are tumor cells and the induction of cell proliferation is tumorigenesis. In a preferred embodiment, the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4,
HDAC-5, HDAC-C, HDAC-D or HDAC-E.

In a fifth aspect, the present invention is a method for identifying a histone deacetylase protein inhibitor that suppresses histone deacetylase involved in the induction of cell proliferation, which is the method of the fourth aspect of the present invention. Contact of a histone deacetylase identified by the method with a candidate compound, and a decrease in the enzymatic activity of the contacted histone deacetylase suppresses the histone deacetylase involved in the induction of proliferating cells Histone deacetylase protein inhibition A method of identifying the candidate compound as a factor is provided. In certain preferred embodiments, histone deacetylase protein inhibitors interact with less than all histone deacetylases and reduce their enzymatic activity.

In a sixth aspect, the present invention is a method for identifying a histone deacetylase involved in inducing cell differentiation, which comprises contacting a cell with an antisense oligonucleotide that suppresses the expression of histone deacetylase. And a method for identifying the histone deacetylase as a histone deacetylase involved in cell differentiation, wherein the induction of differentiation of contact cells is provided. In certain preferred embodiments, the cells are tumor cells. In a preferred embodiment, histone deacetylase is HDAC-1,
HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D or HDAC-E.

In a seventh aspect, the present invention is a method for identifying a histone deacetylase protein inhibitor that inhibits histone deacetylase involved in inducing cell differentiation, which is identified by the method of the sixth aspect. The contacted histone deacetylase is contacted with a candidate compound, and the enzymatic activity of the contacted histone deacetylase is measured. A decrease in the enzymatic activity of the contacted histone deacetylase is involved in the induction of cell differentiation. The present invention provides a method for identifying a candidate compound as a histone deacetylase protein inhibitor that suppresses histone deacetylase. In certain preferred embodiments, histone deacetylase protein inhibitors interact with less than all histone deacetylases and reduce their enzymatic activity.

In an eighth aspect, the invention provides a histone deacetylase protein inhibitor identified by the method of the fifth or seventh aspect of the invention. The histone deacetylase protein inhibitor is preferably substantially pure.

In a ninth aspect, the present invention relates to a method for suppressing cell growth in cells, which comprises suppressing an antisense oligonucleotide that suppresses histone deacetylase, a histone deacetylase protein suppressor, and a DNA methyltransferase. And an at least two reagents selected from the group consisting of anti-oligonucleotides, and DNA methyltransferase protein repressors. In one embodiment, the inhibition of cell growth of contacted cells is greater than the cell growth of cells contacted with a single reagent. In certain embodiments, each reagent selected from the group is substantially pure. In a preferred embodiment, the cells are tumor cells. In a yet further preferred embodiment, the reagent selected from the group is operably linked.

According to the present invention, the reagent found to specifically inhibit histone deacetylase involved in tumor formation can be used as a therapeutic agent for suppressing tumor cell growth in a patient suffering from tumor formation. For example, antisense oligonucleotides that inhibit the expression of histone deacetylase can be used in a pharmaceutically acceptable carrier (eg, physiologically sterile saline) to reduce any illness symptoms that occur (eg, death). ), By any route of administration, neoplasia or hyperplasia (h
yperplasia) is administered to patients suffering from. Similarly, antisense oligonucleotides that suppress the expression of histone deacetylase are used in gene therapy expression vectors (
Phage particles carrying such vectors, for example incorporated into replication-defective adenoviruses, are delivered directly to tumorigenic or hyperplastic cells by pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers and formulations thereof are well known and are generally described, for example, in Remington's Pharmaceutical Science (18th Edition, A. Gen.
Naro, Mack Publishing Co., Easton, PA, 1990).

Detailed Description of the Preferred Embodiments The present invention provides methods and reagents for inhibiting histone deacetylase at the nucleic acid level as well as inhibiting the expression of specific histone deacetylases at the nucleic acid level. The reagents described here inhibit histone deacetylase at the nucleic acid level (that is, inhibit transcription and translation), and this reagent can identify a specific histone deacetylase involved in tumorigenesis. Further, therapeutic compositions for treating and / or alleviating tumorigenic conditions are developed using the reagents of this invention that specifically inhibit the specific histone deacetylases involved in tumorigenesis.

The reagents according to the invention are useful as therapeutic tools, including analytical tools and genetic tools. The invention also provides methods and compositions that can be manipulated and fine-tuned to the condition being treated while producing fewer side effects. The patent and scientific literature referred to herein is knowledge that is certainly available to those with skill in the art. The references cited in this specification, including issued patents, applications, and GenBank database sequences, are cited as part of this specification as much as each is specifically and individually referenced. ing.

In a first aspect, the present invention provides an antisense oligonucleotide that suppresses the expression of histone deacetylase. In some embodiments of this aspect, the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC.
-C, HDAC-D or HDAC-E. In certain embodiments, the oligonucleotide is
Inhibits more than one histone deacetylase or the oligonucleotide inhibits all histone deacetylases.

The antisense oligonucleotide according to the present invention is complementary to the region encoding the histone deacetylase of RNA or double-stranded DNA. For the purpose of this invention,
The term "oligonucleotide" includes polymers of two or more deoxyribonucleosides, ribonucleosides, or 2'-O-substituted ribonucleoside residues, or any combination thereof. Such oligonucleotides are preferably about 8 to about 50.
Of about 12 to about 30 nucleotide residues, most preferably about 12 to about 30 nucleotide residues. The nucleoside residues are linked to each other by any of the many well-known internucleoside linkages. Such internucleoside linkages include, but are not limited to, phosphorothioates, phosphorodithioates, alkylphosphonates, alkylphosphonothioates, phosphotriesters, phosphoramidates, siloxanes, carbonates, Carboxymethyl ester, acetamidate,
Carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, sulfone internucleotide linkages. In certain preferred embodiments, these internucleoside linkages may be phosphodiester, phosphotriester, phosphorothioate or phosphoramidate linkages, or combinations thereof. The term oligonucleotide refers to a polymer having a chemically modified base or sugar and / or a polymer having additional substituents including, but not limited to, lipophilic groups, intercalating agents, diamines and adamantane. Including. For purposes of this invention, the term "2-O-substitution" refers to a pentose moiety.
The 2'position is a -O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or 2-
Means substituting by -O-aryl or allyl group having 6 carbon atoms,
Where such alkyl, aryl or allyl groups are unsubstituted or substituted with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxy, carbalkoxy or amino groups; or such The 2'substitution has a hydroxyl group (which produces a ribonucleoside), an amino or a halo group, but no 2'-H group.

For the purposes of this invention, the term “complementary” means having the ability to hybridize to a genomic region, gene or RNA transcript thereof under physiological conditions. Such hybridizations are usually the result of base-specific hydrogen bonding between complementary strands, and similar to base stacking, Watson-Crick or Hoogsteen bases, despite other forms of hydrogen bonding leading to hybridization. It is preferable to form a pair. As a particular matter, such hybridization can be inferred from the observation of specific gene expression repression at the transcriptional or translational (or both) level.

Particularly preferred antisense oligonucleotides utilized in this aspect of the invention include chimeric and hybrid oligonucleotides.

For the purposes of this invention, “chimeric oligonucleotide” refers to an oligonucleotide having one or more internucleoside linkages. One of the preferred embodiments of such chimeric oligonucleotides comprises a phosphorothioate, phosphodiester or phosphorodithioate region and an alkylphosphonate or alkylphosphonothioate region, preferably comprising from about 2 to about 12 nucleotides. (See Pederson et al., US Pat. Nos. 5,635,377 and 5,366,878). Such chimeric oligonucleotides include at least 3 selected from phosphodiesters, phosphorothioates or combinations thereof.
It is preferable to include continuous internucleoside linkages of

For the purposes of this invention, the term “hybrid oligonucleotide” refers to an oligonucleotide having one or more nucleosides. One of the preferred embodiments of such hybrid oligonucleotides is a ribonucleotide or a 2'-O-substituted ribonucleotide region and a deoxyribonucleotide region, preferably consisting of about 2 to about 12 2'-O-substituted ribonucleotide regions. Contains nucleotide regions. Such hybrid oligonucleotides comprise at least 3 contiguous deoxyribonucleosides and preferably also ribonucleosides, 2'-O-substituted ribonucleosides, or combinations thereof (Metelev and Agrawal, U.S. Patent No. 5,652,355.
No.).

The exact nucleotide sequence and chemical structure of the antisense oligonucleotides utilized in this invention may vary, so long as the oligonucleotide retains the ability to suppress histone deacetylase expression. This is quantification of the amount of mRNA encoding histone deacetylase, quantification of the amount of histone deacetylase protein, quantification of histone deacetylase activity, all described in detail in this specification. , Or in vitro or in vivo cell growth assays, by quantifying the ability of histone deacetylase to inhibit cell growth, and immediately determining by testing whether a particular antisense oligonucleotide is active. It

Antisense oligonucleotides utilized in the present invention have traditionally been H-phosphonate chemistry, phosphoramidate chemistry, or a combination of H-phosphonate chemistry and phosphoramidate chemistry (ie, for some cycles It is synthesized on a suitable solid support using well known chemistries including H-phosphonate chemistry, phosphoramidate chemistry for other cycles). Suitable solid supports may be any standard solid support used for solid phase oligonucleotide synthesis, such as controlled pore glass (CGP) (eg Pon, RT, Methods in Molec. Biol. 20).
: 465-496, 1993).

The antisense oligonucleotide according to the present invention is useful for various purposes.
For example, by suppressing the activity of histone deacetylase in an experimental cell culture or animal system and using it to evaluate the effect of suppressing such histone deacetylase activity, the physiological function of histone deacetylase can be probe"
Can be used as This can be done by administering to cells or animals an antisense oligonucleotide that suppresses histone deacetylase expression according to the invention and observing the phenotypic effect. In this application, the antisense oligonucleotides according to the invention are preferred over conventional "gene knockout" approaches due to their ease of use and can be used to suppress histone deacetylase activity at selected stages of development or differentiation. .. Therefore, the method according to the invention serves as a probe to test the role of histone deacetylation at various stages of development.

Preferred antisense oligonucleotides of this invention inhibit either transcription of a nucleic acid molecule encoding a histone deacetylase or translation of a nucleic acid molecule encoding a histone deacetylase. Nucleic acid that encodes histone deacetylase is RNA or double-stranded DNA, and not only the coding sequences for histone deacetylase family member genes, but also intron sequences, 5'and 3'untranslated regions, and intron-exon boundaries Including without exception. For human sequences, Yang et al., Pro
c. Natl. Acad. Sci. USA 93 (23): 12845-12850, 1996; Furukawa et al., Cytogenet.
Cell. Genet. 73 (1-2): 130-133, 1996; Yang et al., J. Biol. Chem. 272 (44): 2
8001-28007, 1997; Betz et al., Genomics 52 (5): 245-246, 1998; Taunton et al., Scie.
nce 272 (5260): 408-411, 1996; and Dangond et al., Biochem. Biophys. Res. Co.
mmun. 242 (3): 648-652, 1998).

Particularly preferred non-limiting examples of antisense oligonucleotides of this invention are:
Regions of RNA or double-stranded DNA encoding histone deacetylase (eg HDAC
-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D or HDAC-E). Antisense oligonucleotides according to the present invention include HDAC-1, HD
It is complementary to a region of RNA or double-stranded DNA encoding AC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D and / or HDAC-E. The sequence of human HDAC-1 is Ge
It is found in nBank Accession No. U50079 (amino acid sequence of SEQ ID NO: 24; nucleic acid sequence of SEQ ID NO: 25). The sequence of human HDAC-2 is found in GenBank Accession No. U31814 (amino acid sequence of SEQ ID NO: 26; nucleic acid sequence of SEQ ID NO: 27). The sequence of human HDAC-3 is found in GenBank Accession No. U75697 (amino acid sequence of SEQ ID NO: 28; nucleic acid sequence of SEQ ID NO: 29). The sequence of human HDAC-4 (formerly human HDAC-A) is GenBan
k Accession No. AB006626 (amino acid sequence of SEQ ID NO: 30; SEQ ID NO: 30
31 nucleic acid sequences). The sequence of human HDAC-5 (formerly human HDAC-B) is GenBank Accessio
n No. AB011172 (amino acid sequence of SEQ ID NO: 32; nucleic acid sequence of SEQ ID NO: 33). The sequence of human HDAC-C is found in GenBank Accession No. AC004994 (amino acid sequence of SEQ ID NO: 34; nucleic acid sequence of SEQ ID NO: 35). The sequence of human HDAC-D is GenB
found in ank Accession No. AC004466 (nucleic acid sequence of SEQ ID NO: 36).

Histone deacetylase coding sequences in many animal species other than human are also known (for example, as a mouse histone deacetylase, GenBank Accessio
n No. AF006603, AF006602, and AF074882). Thus, the antisense oligonucleotides of this invention are complementary to regions of RNA or double-stranded DNA that encode histone deacetylases from non-human animals. Particularly preferred oligonucleotides have a nucleotide sequence of about 13 to about 35 nucleotides, including the nucleotide sequences set forth below as SEQ ID NOs: 1-18. Yet another particularly preferred oligonucleotide has a nucleotide sequence of about 15 to about 26 nucleotides of the nucleotide sequence shown below. Most preferably, the oligonucleotide shown below has a phosphorothioate backbone, a length of 20-26 nucleotides, at the terminal 4 nucleotides at the 5'end of the oligonucleotide and at the 3'end of the oligonucleotide. A 2'-O-methyl group is attached to each of the sugar residues of a terminal 4 nucleotides.

[0033]   Antisense oligonucleotide specific for human HDAC-1 (MG2608): 5'-GAA ACG TGA GGG ACT CAG CA-3 '(SEQ ID NO: 1).

An antisense oligonucleotide (MG2610) specific for both human HDAC-1 and human HDAC-2 is a 25/25/25/25 mixture of four oligonucleotides: 5'-CAG CAA ATT ATG GGT. CAT GCG GAT TC-3 '(SEQ ID NO: 2); 5'-CAG CAA GTT ATG AGT CAT GCG GAT TC-3' (SEQ ID NO: 3); 5'-CAG CAA ATT ATG AGT CAT GCG GAT TC-3 '( SEQ ID NO: 4); and 5'-CAG CAA GTT ATG GGT CAT GCG GAT TC-3 '(SEQ ID NO: 5).

[0035]   Antisense oligonucleotides specific for human HDAC-2: 5'-TGC TGC TGC TGC TGC TGC CG-3 '(MG2628; SEQ ID NO: 6); 5'-CCT CCT GCT GCT GCT GCT GC-3 '(MG2633; SEQ ID NO: 7); 5'-GCT TCC TTT GGT ATC TGT TT-3 '(MG2635; SEQ ID NO: 8); and 5'-CTC CTT GAC TGT ACG CCA TG-3 '(MG2636; SEQ ID NO: 9).

Antisense oligonucleotides according to the present invention are optionally formulated with any of the well known pharmaceutically acceptable carriers or diluents (eg, Remington's).
Pharmaceutical Sciences, 18th Edition, edited by A. Gennaro, Mack Publishing Co. Easton, PA, 1990, see Preparation of Pharmaceutically Acceptable Formulations).

In a second aspect, the invention is a method of inhibiting histone deacetylase in a cell comprising contacting the cell with an antisense oligonucleotide that inhibits expression of histone deacetylase. I will provide a. Preferably, cell proliferation is suppressed in contact cells. Therefore, the antisense oligonucleotide according to the present invention is useful in a treatment method for human diseases including benign and malignant tumors by suppressing cell growth in cells contacted with the antisense oligonucleotide. The expression "suppressing cell proliferation" refers to the growth of cells in contact with a histone deacetylase antisense oligonucleotide or histone deacetylase protein inhibitor (or combination thereof), which is contacted with the oligonucleotide or protein inhibitor. It means the ability to delay compared to cells that are not. Such an assessment of cell proliferation is performed by the Coulter cell counter (Coulter,
(Miami, FL) or a hemacytometer to count contact and non-contact cells. When cells are solid growing (eg solid tumors or organs), such an assessment of cell proliferation measures growth with calipers,
This can be done by comparing the growth size of contact and non-contact cells.
Preferably, the term includes retardation of cell proliferation which is at least 50% of non-contact cells. More preferably, the term includes retardation of cell proliferation which is 100% of non-contacted cells (ie contacted cells do not increase in number or size). This term
More preferably, it comprises a reduction in the number or size of contact cells as compared to non-contact cells. Therefore, histone deacetylase antisense oligonucleotides, or histone deacetylase protein inhibitors that suppress cell proliferation in contact cells, may cause growth retardation, growth arrest, programmed cell death (ie apoptosis), or tumor growth in contact cells. Induces cell death.

Conversely, the expression “induces cell proliferation” is used to describe the presence of histone deacetylase or the requirement of enzymatic activity for cell proliferation in normal (ie non-tumor) cells. Therefore, overexpression of histone deacetylase, which induces cell proliferation, may or may not lead to increased cell proliferation; however, inhibition of histone deacetylase that induces cell proliferation results in inhibition of cell proliferation. Connect

The expression “inducing cell differentiation” means that histone deacetylase antisense oligonucleotides or histone deacetylase protein inhibitors (or combinations thereof) induce differentiation in contact cells as compared to non-contact cells. Used to express the ability to induce. Thus, tumor cells are induced to differentiate when contacted with histone deacetylase antisense oligonucleotides of the invention or histone deacetylase protein repressors (or both) and, as a result, are more systematically more systematic than contact cells. Generate evolved daughter cells.

The ability of the antisense oligonucleotides according to the invention to suppress cell proliferation enables the synchronization of a group of a-synchronous growing cells. For example, the antisense oligonucleotides of the invention are used to arrest a group of non-tumor cells grown in vitro at the G1 or G2 phase of the cell cycle. Such synchronization can, for example, identify genes and / or gene products expressed during the G1 or G2 phases of the cell cycle. Such synchronization of cultured cells tests its efficacy in a new transfection protocol in which the efficacy of transfection varies depending on the particular cell cycle phase of the transfected cells. It is also useful. The use of the antisense oligonucleotides of this invention allows for the synchronization of a panel of cells and thus serves to detect an increase in transfection efficacy.

The anti-tumor utility of antisense oligonucleotides according to the present invention is elaborated elsewhere in this specification.

In another preferred embodiment, the cells contacted with a histone deacetylase antisense oligonucleotide are also contacted with a histone deacetylase protein repressor.

As used herein, a “histone deacetylase protein inhibitor” is an activity capable of interacting with histone deacetylase at the protein level and reducing the activity of that histone deacetylase. Shows the part. Histone deacetylase protein inhibitors include, without limitation, trichostatin A, trichostatin B, trichostatin C, depudecin, trapoxin, butyrate, suberoylanilide, hydroxamic acid (SAHA), FR901228 (Fujisawa Pharmaceutical), acetyldina Phosphorus (el
-Beltagl et al., Cancer Res. 53 (13): 3008-3014, 1993). Histone deacetylase protein repressors are molecules that reduce the activity of histone deacetylase to a greater extent than the activity of unrelated proteins. In a preferred embodiment, such a decrease in activity of histone deacetylase is at least 5-fold, more preferably at least 10-fold, most preferably at least 50-fold. In another embodiment, the activity of histone deacetylase is reduced 100-fold. Histone deacetylase protein inhibitors interact with fewer enzymes than all histone deacetylases and reduce their enzymatic activity. By "all histone deacetylases" is meant all members of both histone deacetylase families of proteins by animals of a particular species, including, without limitation, HDAC-1, HDAC-2, HDAC-3, HD.
It includes AC-4, HDAC-5, HDAC-C, HDAC-D or HDAC-E, all of which are considered "related proteins" as used herein. For example, a preferred histone deacetylase protein suppressor interacts with HDAC-1 and HDAC-2,
They suppress them, but they do not interact with HDAC-3 and do not suppress them. More preferably, the histone deacetylase protein inhibitor is a histone deacetylase (
For example, it interacts with HDAC-2) and reduces its activity, but does not interact with the other histone deacetylases (eg HDAC-1 and HDAC-3) and does not reduce its activity. As described below, a preferred histone deacetylase protein inhibitor is a histone deacetylase protein inhibitor that interacts with histone deacetylase involved in tumorigenesis and reduces its enzymatic activity.

The histone deacetylase protein repressor is preferably operably linked to the antisense oligonucleotide. As mentioned above, the antisense oligonucleotides according to the present invention are optionally formulated with well known pharmaceutically acceptable carriers or diluents. The formulation further comprises one or more one or more additional histone deacetylase antisense oligonucleotides, and / or one or more histone deacetylase protein regulators, or other pharmacological An activator may be included.

In a particularly preferred embodiment of this invention, the antisense oligonucleotide is operably linked to a histone deacetylase protein repressor. An "operable linkage" is an antisense oligonucleotide that enables an antisense oligonucleotide to suppress the expression of a nucleic acid that encodes histone deacetylase, allowing a histone deacetylase protein inhibitor to suppress histone deacetylase activity. Includes all binding between the oligonucleotide and the histone deacetylase protein repressor. One or more antisense oligonucleotides of this invention are operably linked to one or more histone deacetylase protein repressors. Antisense oligonucleotides that target a particular histone deacetylase (eg, HDAC-2) are preferably operably linked to a histone deacetylase protein repressor that targets the same histone deacetylase. The preferred operable bond is hydrolysable. The hydrolysable bond is preferably a covalent bond between the antisense oligonucleotide and the histone deacetylase protein repressor. Such covalent bonds are preferably hydrolysable by esterases and / or amidases. Such hydrolyzable bonds are known in the art. Phosphate esters are particularly preferred.

In certain preferred embodiments, the covalent bond may directly link the antisense oligonucleotide and the histone deacetylase protein inhibitor so that the histone deacetylase protein inhibitor can be integrated together. Alternatively, the covalent linkage may be by an extended structure, in which the antisense oligonucleotide is histone by attaching both the antisense oligonucleotide and the histone deacetylase protein inhibitor to a carrier molecule such as a carbohydrate, peptide or lipid or glycolipid. It can also be produced by covalently binding to a deacetylase protein inhibitor. Other preferred operable linkages include lipophilic linkages, such as the formation of liposomes containing antisense oligonucleotides and histone deacetylase protein inhibitors that are covalently attached to lipophilic molecules and thus to liposomes. . Such lipophilic molecules include, without limitation, phosphotidylcholine, cholesterol, phosphatidylethanolamine, synthetic neoglycolipids such as sialylac NAc-HDPE. In certain preferred embodiments, the operable linkage is not a physical linkage, but is simply co-existing in the body, for example when the antisense oligonucleotide is attached to one liposome and the protein agent is attached to another liposome. You can just do it.

In a third aspect, the invention is a method of inhibiting tumor cell growth in an animal, wherein the animal has at least one tumor cell in its body and the antisense oligonucleotide of the first aspect of the invention is used. And a therapeutically effective amount of the is administered with a pharmaceutically acceptable carrier for an effective treatment period. The animal is preferably a mammal, especially a domestic animal. More preferably, the animal is a human.

The term “tumor cell” refers to a cell that exhibits abnormal cell growth. Preferably the abnormal cell growth of tumor cells is increased cell growth. Tumor cells are hyperplastic cells,
It is a cell showing a deficiency of contact inhibition of growth in vitro, a benign tumor cell that cannot metastasize in vivo, and a cancer cell that can metastasize in vivo and may recur even after the removal trial. The term "tumor formation" is used to refer to the induction of cell proliferation leading to the development of tumor growth.

The terms “therapeutically effective amount” and “effective treatment duration” are used to describe a known treatment at a dose, duration effective to reduce tumor cell growth. Preferably, such administration is parenteral, oral, sublingual, transdermal, topical, intranasal, or rectal. When administered systemically, the therapeutic composition is preferably administered in an amount sufficient to reach a blood concentration of antisense oligonucleotide of from about 0.1 μM to about 10 μM. For topical administration, much lower concentrations are sufficient and much higher concentrations are tolerated. Those skilled in the art are aware that such therapeutic effects, which occur at lower effective concentrations of histone deacetylase inhibitors, will vary greatly depending on the tissue, organ, or particular animal or patient treated according to this invention. Should be.

In a preferred embodiment, the therapeutic compositions of this invention are administered to the tissue at a dose sufficient to achieve a blood concentration of antisense oligonucleotide of about 0.01 μM to about 20 μM. In a particularly preferred embodiment, the therapeutic composition is from about 0.05 μM to about 1.
It is administered at a dose sufficient to achieve a blood concentration of 5 μM antisense oligonucleotide. In a more preferred embodiment, the blood concentration of antisense oligonucleotide is from about 0.1 μM to about 10 μM.

For topical administration, much lower concentrations are therapeutically effective. Preferably, the total dose of antisense oligonucleotide will range from about 0.1 g to about 200 mg of oligonucleotide per kg body weight per day. In a more preferred embodiment,
The total dose of antisense oligonucleotide will range from about 1 mg to about 20 mg of oligonucleotide per kg of body weight daily. In a more preferred embodiment, the total dose of antisense oligonucleotide will range from about 2 mg to about 10 mg oligonucleotide per kg body weight per day. In a particularly preferred embodiment, the therapeutically effective amount of histone deacetylase antisense oligonucleotide is about 0.5 mg of oligonucleotide per kg of body weight per day.

In one preferred embodiment of the third aspect of the invention, the method comprises providing a therapeutically effective amount of a histone deacetylase protein inhibitor with a pharmaceutically acceptable carrier to the animal for a therapeutically effective period. Further comprising administering. The histone deacetylase inhibitor is preferably operably linked to the antisense oligonucleotide. Methods for operably linking histone deacetylase inhibitors with histone deacetylase antisense oligonucleotides have been described above.

The therapeutic composition containing a histone deacetylase protein inhibitor of the present invention is systemically administered at a dose sufficient to achieve a blood level of the histone deacetylase protein inhibitor of about 0.01 μM to about 10 μM. Be administered to. In a particularly preferred embodiment, the therapeutic composition is administered in a sufficient dose to achieve a blood level of histone deacetylase protein inhibitor of about 0.05 μM to about 10 μM. In a more preferred embodiment, the blood concentration of histone deacetylase protein inhibitor is about 0.1 μM to about 7 μM. For topical administration, much lower concentrations are effective. Preferably, the total dose of histone deacetylase protein inhibitor is 1
It ranges from about 0.01 mg to about 5 mg protein agent per kg body weight daily. In a more preferred embodiment, the total dose of histone deacetylase protein inhibitor will range from about 0.1 mg to about 4 mg protein agent per kg body weight per day. In the most preferred embodiment, the total dose of histone deacetylase protein inhibitor will range from about 0.1 mg to about 1 mg protein agent per kg body weight per day.
In a particularly preferred embodiment, the therapeutically effective synergistic amount of histone deacetylase protein inhibitor (when administered with an antisense oligonucleotide) is
The daily dose is 0.1 mg / kg body weight.

This aspect of the invention enhances the inhibitory effect so that a nucleic acid level inhibitor (ie, an antisense oligonucleotide) required to obtain a given inhibitory effect.
And the therapeutically effective concentration of either or both of the protein level inhibitors (ie histone deacetylase protein inhibitors) is lower than that required when either is used individually.

Furthermore, one of skill in the art will recognize that the therapeutically effective synergistic amount of either the antisense oligonucleotide or the histone deacetylase protein inhibitor will be increased or decreased by fine tuning or altering the amounts of the other ingredients. Will. The present invention thus provides a method of tailoring the administration / treatment to a particular emergency specific to a given animal species or a particular patient. The therapeutically effective range is relatively easily determined empirically, for example, by starting with a relatively small dose or increasing the steps by simultaneous assessment of inhibition.

In a fourth aspect, the invention provides a method of investigating the role of specific histone deacetylases in cell proliferation, including tumor cell proliferation. In this method, as described in the fifth aspect of the present invention, when the target cell type is contacted with an antisense oligonucleotide in an amount that suppresses the expression of a histone deacetylase, the histone decellularization in the cell is stopped. The expression of acetyl enzyme is suppressed. Contact cells in which the expression of histone deacetylase is suppressed also show suppression of cell growth, and thus the histone deacetylase is involved in the induction of cell growth. In this scenario, the down-regulated histone deacetylase is the histone deacetylase involved in tumorigenesis when the contacted cells are tumor cells and the contacted tumor cells show inhibition of cell growth. The histone deacetylase is preferably HDAC-1, HDAC
-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D or HDAC-E.

Therefore, in order to suppress cell proliferation and induce differentiation by identifying the histone deacetylase involved in the induction of cell proliferation, only that particular histone deacetylase is used for the antisense oligonucleotide. Need to be targeted. As a result, cell proliferation can be effectively suppressed by the lower therapeutically effective amount of antisense oligonucleotides. Moreover, the unwanted side effect of inhibiting all histone deacetylases is involved in the induction of cell proliferation 1
It is prevented by specifically inhibiting the (and above) histone deacetylase.

Once such histone deacetylases involved in cell proliferation have been identified using the antisense oligonucleotides of the first aspect of the present invention, then other histone deacetylases not involved in the induction of cell proliferation are present. A histone deacetylase protein inhibitor that specifically suppresses histone deacetylase involved in the induction of cell proliferation is derived without inhibiting acetyl fermentation. Therefore, in a fifth aspect, the present invention provides a method for identifying a histone deacetylase protein inhibitor that suppresses histone deacetylase involved in the induction of cell proliferation. This method involves contacting a candidate compound with a histone deacetylase identified as being involved in inducing cell proliferation and measuring the enzymatic activity of the contacted histone deacetylase. The reduction in the enzymatic activity of the contacted histone deacetylase identifies the candidate compound as a histone deacetylase protein inhibitor that suppresses histone deacetylase involved in the induction of cell proliferation.

The enzyme activity of histone deacetylase can be measured using a known method. For example, Yoshida et al. (J. Biol. Chem. 265: 17174-17179, 1990) describe the evaluation of the enzymatic activity of histone deacetylase by detecting acetylated histones in trichostatin A-treated cells. Taunton et al. (Science 272: 408-411, 1
996) similarly describes a method for measuring the enzymatic activity of histone deacetylase using endogenous and recombinant HDAC-1. Yoshida et al. (J. Biol. Chem. 26
5: 17174-17179, 1990) and Taunton et al. (Science 272: 408-411, 1996) are both cited as part of this specification.

Histone deacetylase protein repressors that suppress histone deacetylases involved in the induction of cell proliferation interact with less than all histone deacetylases and reduce their enzymatic activity. It is preferably an enzyme protein suppressor.

In a sixth aspect, the present invention is a method for identifying a histone deacetylase involved in inducing cell differentiation, which comprises contacting a cell with an antisense oligonucleotide that suppresses the expression of histone deacetylase. And a method for identifying the histone deacetylase as a histone deacetylase involved in cell differentiation, wherein the induction of contact cell differentiation is provided. The cells are preferably tumor cells. In a preferred embodiment, the histone deacetylase is HDAC-1, HDAC-2, HDAC-3,
HDAC-4, HDAC-5, HDAC-C, HDAC-D or HDAC-E.

In a seventh aspect, the present invention is a method for identifying a histone deacetylase protein inhibitor that inhibits histone deacetylase involved in the induction of cell differentiation, which is identified by the method of the sixth aspect. The contacted histone deacetylase is contacted with a candidate compound, and the enzymatic activity of the contacted histone deacetylase is measured. The present invention provides a method for identifying a candidate compound as a histone deacetylase protein inhibitor that inhibits histone deacetylase. In certain preferred embodiments, histone deacetylase protein inhibitors interact with less than all histone deacetylases and reduce their enzymatic activity.

[0063] In an eighth aspect, the invention provides a histone deacetylase protein inhibitor identified by the method of the fifth or seventh aspects. The histone deacetylase protein inhibitor is preferably substantially pure.

Substantially purified proteins include, without limitation, recombinant protein expression, affinity chromatography, antibody-based affinity generation, high performance liquid chromatography (
HPLC; eg Fisher (1980) Laboratory Techniques in Biochemistry and M.
olecular Biology. Work and Burdon (eds. Elsevier)). Preferably, the substantially purified protein is at least 80% by weight pure and is pure in that it is free of other proteins or naturally occurring organic molecules. More preferably, the substantially purified protein is at least 90% by weight pure. Most preferably, the substantially purified protein is at least 95% pure by weight.

In a ninth aspect, the present invention relates to a method for suppressing cell growth in cells, which comprises suppressing an antisense oligonucleotide that suppresses histone deacetylase, a histone deacetylase protein suppressor, and a DNA methyltransferase. And an at least two reagents selected from the group consisting of anti-oligonucleotides, and DNA methyltransferase protein repressors. In one embodiment, the inhibition of cell growth of contacted cells is greater than the cell growth of cells contacted with a single reagent. In certain embodiments, each reagent selected from the group is substantially pure. In a preferred embodiment, the cells are tumor cells. In a yet further preferred embodiment, the reagent selected from the group is operably linked.

Antisense oligonucleotides that suppress DNA methyltransferase are described in Szyl and von Hofe, US Pat. No. 5,578,716, the entire contents of which are incorporated by reference. As a DNA methyltransferase protein suppressor, without exception, 5-aza-2'-deoxycytidine (5-aza-dC)
, 5-Fluoro-2'-deoxycytidine, 5-aza-cytidine (5-aza-C) or 5,6-
Dihydro-5-aza-cytidine may be mentioned.

The following examples are intended to further illustrate the preferred embodiments of this invention and are not intended to limit the invention. One of ordinary skill in the art would be able to recognize or ascertain numerous materials and procedures equivalent to the specific materials and procedures described herein using only routine experimentation. Such equivalent materials and procedures are considered within the scope of this invention and are covered by the claims.

Example 1 Screening of antisense oligonucleotides In order to identify the antisense oligonucleotides that suppress the specific histone deacetylase most efficiently, GenBank Accession No. U50079 was used for HDAC-1.
For HDAC-2, a number of oligonucleotides were created based on the sequence provided in GenBank Accession No. U31814. Some of the screened oligonucleotides are described in Besterman et al., U.S. Patent Application No. 60 /
Nos. 104,804, Tables 2 and 3, the entire disclosures of which are incorporated herein by reference.

In addition, oligonucleotides complementary to both HDAC-1 and HDAC-2 were made.

To screen for the ability of these oligonucleotides to suppress target histone deacetylases, Northern blotting analysis was first performed.
For this purpose, T24 human bladder cancer cells (purchasable from American Type Culture Collection (ATCC), Manassas, Vanidia) were cultured under the indicated conditions. The cells were washed with PBS (phosphate buffered saline) before adding the oligonucleotide. Next, 6.25 μg / ml Lipofectin transfection reagent (Gibco-BRL, Mississauga, Ontario) was added to serum-free OPTIMEN medium (GIBCO / BRL), which was then added to the cells. The oligonucleotides to be screened were added to various wells of cells (ie 1 oligonucleotide per cell well). The same concentration of oligonucleotide (eg 50 nM) was used per cell well. Cells were incubated with lipofectin and oligonucleotide in a cell culture incubator for 4 hours at 37 ° C. The cells were then washed with PBS and returned to medium containing total serum. After 24 hours, cells were harvested and HDAC mRNA was analyzed by Northern blotting analysis.
Determined the level.

To determine mRNA levels by Northern blot, guanidinium isothiocyanate standard procedures (eg Ausubel et al., Current Protocol in Molecular.
Biology, John Wiley & Sons, New York, NY. 1994) to prepare total RNA from cells. However, to purify RNA from cellular DNA contamination, 4 ° C, 2 mL
An additional step was added to precipitate overnight in iCl. Northern blotting analysis
Performed according to standard procedures. The HDAC-1 and HDAC-2 probes are PCRs from known sequences (eg, GenBank Accession No. U50079 and U31814, respectively).
It was a full-length cDNA clone generated by amplification. These probes are ³² P-AT
Radiolabeled with P. Northern Blot is based on Alpha Imager (Alpha Innovotec
h) was used to scan and quantify.

Oligonucleotides that have demonstrated the ability to reduce target histone deacetylase mRNA expression (ie, are capable of repressing histone deacetylase mRNA transcription) then express the target histone deacetylase protein. Were screened for their ability to suppress To do this, T24 cells were transfected with oligonucleotides using lipofectin as described above. After 24 hours, cells were lysed according to standard procedures. 7-15% gradient S of whole cell extract (50 μg)
Degraded by DS / PAGE, transferred to PVDF membrane (Amersham, Arlington Heights, IL) and rabbit polyclonal HDAC-1 and HDAC-2 specific antibodies (1: 500, Santa Cruz Biot
ech., Santa Cruz, CA). The detection is the second anti-rabbit IgG-H
R peroxidase antibody and advanced chemiluminescence detection kit (Amersham) were used according to the manufacturer's instructions.

Based on our results, the following antisense oligonucleotides were confirmed to be most effective in suppressing target histone deacetylase expression, as determined by both mRNA and protein expression blotting analysis. These oligonucleotides are: In case of inhibition of HDAC-1, oligonucleotide No. MG2608 has the following sequence: 5'- GAA A CG TGA GGG ACT C AG CA -3 '(SEQ ID NO: 10 ).

In the case of inhibition of both HDAC-1 and human HDAC-2, oligonucleotide No. MG2610
Is a 25/25/25/25 mixture of four oligonucleotides with the following sequences: 5'- CAG C AA ATT ATG GGT CAT GCG G AU UC -3 '(SEQ ID NO: 11); 5'- CAG C AA GTT ATG AGT CAT GCG G AU UC -3 '(SEQ ID NO: 12); 5'- CAG C AA ATT ATG AGT CAT GCG G AU UC -3' (SEQ ID NO: 13); 5'- CAG C AA GTT ATG GGT CAT GCG G AU UC -3 '(SEQ ID NO: 14).

In the case of inhibition of HDAC-2, the antisense oligonucleotides of Table 1 are shown to be most effective.

[0076]

[Table 1]

To evaluate the specificity of the second generation histone deacetylase antisense oligonucleotides, HDAC-1 (MG2608) and HDAC-1 / 2 (MG2610) mismatch control oligonucleotides were generated. These mismatch control oligonucleotides are made primarily by substituting four 5'and 3'nucleotide bases. Here, the highest affinity occurs with the nucleic acid encoding the target histone deacetylase. The HDAC-1 mismatch control (MG2609) has the following sequence: 5'- CAA U CG TCA GAG ACT C CG AA -3 '(SEQ ID NO: 19). The HDAC-1 / 2 mismatch control (MG2637) is a 225/25/25/25 mixture of four oligonucleotides with the following sequences: 5'- AAG G AA GTC ATG ATT GAT GCC C AU UG -3 '. (SEQ ID NO: 20); 5'- AAG G AA ATC ATG GAT GAT GCC C AU UG -3 '(SEQ ID NO: 21); 5'- AAG G AA GTC ATG GAT GAT GCC C AU UG -3' (SEQ ID NO: 22 ); 5'- AAG G AA ATC ATG AAT GAT GCC C AU UG -3 '(SEQ ID NO: 23).

These oligonucleotides (ie having SEQ ID NOs: 10-23) are second generation oligonucleotides (ie 4x4 hybrids). That is, SEQ ID NO: 10-
The oligonucleotide with 23 is chemically modified as follows; A corresponds to 2'-deoxyriboadenosine; C corresponds to deoxyriboribocytidine; G
Corresponds to deoxyriboriboguanosine; T corresponds to 2'-deoxyribothymidine; A corresponds to riboadenosine; U corresponds to uridine; C corresponds to ribocytidine; G corresponds to riboguanosine. Underlined bases are 2'-methoxyribose substituted nucleotides. Bases without underlining indicate deoxyribose nucleosides. The backbone of each oligonucleotide consisted of phosphorothioate linkages between adjacent nucleotides.

A number of oligonucleotides complementary to HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D, and HDAC-E are then created. These oligonucleotides are based on the known histone deacetylase nucleic acid sequence (eg GenB for HDAC-3).
ank Accession No. U75697). Antisense oligonucleotides, specific for one of these histone deacetylases, are effective in suppressing mRNA and protein expression, as described above for HDAC-1, HDAC-1 / 2 and HDAC-2. Screen for sex. Furthermore, antisense oligonucleotides that suppress two or more histone deacetylases (for example, HDAC-1 / 3 / C-specific) are also prepared by mixing antisense oligonucleotides specific to each histone deacetylase, Screen for efficacy.

Example 2 Inhibition of histone deacetylase mRNA expression by antisense oligonucleotides To determine the specificity and dose requirements of antisense oligonucleotides specific for nucleic acids encoding histone deacetylase. We investigated the dose-dependent suppression of these oligonucleotides on histone deacetylase mRNA expression.

To do this, T24 cells were treated with lipofectin for 10, 25, 50 or 100.
Transfected with nM oligonucleotide (as described in Example 1)
. Cells were harvested 24 hours after transfection, RNA was prepared and Northern blotting analysis was performed with radiolabeled HDAC-1 and HDAC-2 cDNAs as probes as described in Example 1.

FIG. 1 shows a dose-dependent suppression of HDAC-1 mRNA expression by both HDAC-1 and HDAC-1 / 2 antisense oligonucleotides at 50-100 nM. On the other hand, HDAC-2
mRNA expression is 50-100 nM HDAC-1 / 2 antisense oligonucleotide (MG2610)
HDAC-1 antisense oligonucleotide (MG2608), which was suppressed only by
Has no effect. The oligonucleotides used in this experiment, the results of which are shown in FIG. 1, were first generation oligonucleotides (ie, not chemically modified).
The oligonucleotides used to obtain the results in Figure 1 had SEQ ID NOs: 1-5.

FIG. 2 shows dose-dependent suppression of HDAC-2 mRNA by HDAC-2 antisense oligonucleotides. Four HDAC-2 antisense oligonucleotides (MG2628, MG2
633, MG2635, MG2636) were all able to reduce the level of HDAC-2 mRNA expression at 50-100 nM. In this experiment, MG2628 appeared to be particularly effective in reducing HDAC-2 mRNA expression.

These data show that exposure to oligonucleotides by targeting histone deacetylase at the nucleic acid level with antisense oligonucleotides.
After 24 hours it was demonstrated that a reduction in mRNA expression occurred.

Example 3 Histone Deacetylase Tampa with Second Generation Antisense Oligonucleotides
In order to determine the ability of histone deacetylase oligonucleotides to suppress protein expression, 2nd generation antisense oligonucleotides of HDAC-1, HDAC-1 / 2 and HDAC-2 antisense oligonucleotides were used. Created. Each of these second generation antisense oligonucleotides had a backbone consisting of phosphorothioate linkages between each adjacent nucleotide. Furthermore, the four terminal nucleotides at both the 5'and 3'ends of the oligonucleotide had a sugar residue consisting of a 2'-O-methyl group. Such modifications to the terminal nucleotides were useful to improve the binding affinity of the oligonucleotide to the target nucleic acid and suppress the nuclease sensitivity to improve the stability of the oligonucleotide.

FIG. 3 shows the ability of second generation HDAC-2 antisense oligonucleotides to suppress HDAC-2 protein expression. T24 cells were transfected with 0, 25 or 50 nM MG2628 or MG2636 using lipofectin as described in Example 1. Twenty-four hours later, cells were transfected a second time with the same amount of the same oligonucleotide. 24 hours after this (ie 48 hours after the first transfection), cellular proteins were prepared and resolved by 7-15% gradient SDS-PAGE,
As described in Example 1, rabbit polyclonal HDAC2 specific antibody (1: 500, Santa Cr
uz Biotech) and subjected to Western blot analysis. Second anti-rabbit IgG-HR
Blotting with peroxidase antibody and advanced chemiluminescence detection kit (Amer
blots were stripped and reprobed with an antibody specific for actin to confirm equal loading of all wells (data not shown).

As shown in FIG. 3, 50 μM of second generation MG2628 or MG2836 was able to suppress HDAC-2 protein expression.

FIG. 4 shows that HDAC-1 / 2 and HDAC-1 antisense oligonucleotides suppress both HDAC-1 and HDAC-2 or HDAC-1 protein expression, respectively, when compared to the mismatch control. It shows a specific ability. T24 cells were twice transfected with 50 nM oligonucleotide as described above. Cell lysates were prepared 24 h after the second transfection, lysed on a 7-15% gradient SDS-PAGE and PDV.
Transferred to F membrane. PDVF membrane blots were first blotted with anti-HDAC-1 antibody. After detection with horseradish peroxidase labeled antibody and high chemiluminescence, blots were stripped and reprobed with anti-HDAC-2 antibody. After detection, blots were stripped a second time and reprobed with an actin-specific antibody to ensure equal loading of lanes.

As shown in FIG. 4, neither HDAC-1 and HDAC-1 / 2 mismatch control oligonucleotides were able to suppress HDAC-1 or HDAC-1 and HDAC-2 protein expression, respectively. It was In contrast, HDAC-1 antisense oligonucleotides efficiently reduced HDAC-1 protein expression, and HDAC-1 / 2 antisense oligonucleotides reduced both HDAC-1 and HDAC-2 protein expression. did.

Example 4 Identification of Histone Deacetylase Involved in Induction of Cell Proliferation In order to identify a histone deacetylase which induces cell proliferation in cultured cells, antisense which suppresses the expression of various histone deacetylases. Screen oligonucleotides.

To identify histone deacetylases that induce division of normal (ie, non-tumor) cells, antisense oligonucleotides that suppress the expression of histone deacetylases are transferred to cultured normal human fibroblasts. Action. CaPO ₄
Any standard transfection protocol can be used without limitation, including precipitation, electroporation, DEAD-dextran, but the use of lipofectin transfection reagent (Gibco-BRL) is preferred. After transfection with lipofectin and histone deacetylase antisense oligonucleotides, cells were harvested by trypsinization at each time point and counted using a hemacytometer or Coulter cell counter. Mock-transfected control cells (ie treated with lipofectin plus control non-specific oligonucleotides) were also collected and counted. Both antisense oligonucleotides and mock-transfected cells were visually inspected under the microscope for phenotypic changes (eg induction of apoptosis). An antisense oligonucleotide that suppresses the expression of histone deacetylase, which is known to suppress cell growth when transfected into normal cells, contains a histone deacetylase involved in the induction of cell growth in normal cells. Identify.

To identify histone deacetylases that induce tumor cell proliferation, the histone deacetylase antisense oligonucleotides according to the invention were transfected into T24 bladder cancer cells, their growth patterns were observed and non-transfected. Comparison with the growth pattern of the control cells. For this, one day before transfection, T24 cells (ATCC No. HTB-4) were seeded in 10 cm plates at a concentration of 4x10 ⁵ cells / dish. At the time of transfection, cells were washed with phosphate buffered saline (PBS) and 5 ml Opti-MEM containing 6.25 μg / ml lipofectin transfection reagent.
Medium (Gibco-BRL, Mississauga, Ontario) was added. The antisense oligonucleotides to be tested were diluted to the desired concentration from a 0.1 mM stock solution of transfection medium. After incubation for 4 hours at 37 ° C. in a 5% CO ₂ incubator, plates were washed with PBS and 10 ml of fresh cell culture medium was added. T24 cells were transfected for a total of 3 days and split every other day to ensure optimal transfection conditions. At each time point, cells were harvested by trypsinization and pelleted by centrifugation at 1100 rpm and 4 ° C for 5 minutes. Cells were resuspended in PBS and counted on a Coulter particle counter to determine total cell number. Mock-transfected T24 cells (transfected with lipofectin and control oligonucleotides) were similarly cultured, harvested and counted. Antisense oligonucleotides that suppress the expression of histone deacetylase, which is known to suppress cell growth when transfected into tumor cells, identify histone deacetylases involved in the induction of cell growth in tumor cells.

By screening a number of various histone deacetylase antisense oligonucleotides in normal cells and tumor cells, the histone deacetylases involved in the induction of cell proliferation are immediately identified. More preferably, the histone deacetylase antisense oligonucleotide of the present invention is a histone deacetylase antisense oligonucleotide that suppresses cell growth of tumor cells, but does not suppress cell growth of normal cells.

Example 5 Histone Deacetylase Protein Suppressor Factor That Interacts With Histone Deacetylase Involved In Inducing Cell Proliferation And Reduces Its Enzyme Activity Identified as Involved In Inducing Cell Proliferation (eg, Example) The histone deacetylase (identified by method 4) can be used as a target for candidate compounds designed to interact with the histone deacetylase and reduce its enzymatic activity. FR901228 (available from Fujisawa Pharmaceutical) is used as a positive control.

Candidate compounds are derived from any source and may be of natural or synthetic type and may have natural or synthetic components. Candidate compounds are, without exception, chemically bound to any of the known histone deacetylase protein inhibitors, including trichostatin A, trichostatin C, trapoxin, depudecin, suberoylanilide, hydroxamic acid (SAHA), FR901228, butyrate. May be designed to be similar to each other.

Once candidate compounds have been identified, pools of such compounds can be added to histone deacetylases. Such a histone deacetylase is preferably a histone deacetylase specified as a histone deacetylase involved in the induction of cell proliferation using the antisense oligonucleotide of the present invention. Histone deacetylases are administered, for example, using an antibody specific for that particular histone deacetylase (eg, an anti-HDAC-1 antibody available from Santa Cruz Biotech) or in prokaryotic or eukaryotic cells. It can be produced by recombinant production of an acetyl enzyme. Histone deacetylase can also be present in cells that normally express histone deacetylase.

A pool of candidate compounds is added to histone deacetylase and the histone deacetylase enzymatic activity is measured. A pool of candidate compounds showing such histone deacetylase inhibitory activity is subdivided to have a histone deacetylase inhibitory activity 1.
The splits are tested until one candidate compound is isolated. Once a pool of candidate compounds is identified as having the ability to suppress histone deacetylase activity, various methods of confirming their presence within the pool or one or more histone deacetylase protein suppressor compounds, The pool can be screened. For example, in cells that have histone deacetylase, if the pool is first screened, the pool is then screened for purified histone deacetylase.

The candidate compound found to be a histone deacetylase protein repressor signature preferably suppresses less enzyme activity than all histone deacetylases. More preferably, such a candidate compound suppresses only histone deacetylase involved in the induction of cell proliferation. Still more preferably, the candidate compound identified as a histone deacetylase protein inhibitor is a candidate compound that inhibits only one histone deacetylase involved in the induction of cell proliferation.
More preferably, the candidate compound identified as a histone deacetylase protein inhibitor is involved in the induction of cell proliferation in tumor cells, but only one histone deacetylase that is not involved in the induction of cell proliferation in normal cells. It is a candidate compound to suppress.

In another method of identifying candidate compounds that are histone deacetylase protein inhibitors, purified histone deacetylase is attached to the well bottom of a 96-well microtiter plate. A candidate compound (or pool thereof) modified by covalently attaching a detectable marker (eg biotin label) is then added to the plate. The binding of the candidate compound to the plate-bound histone deacetylase is a detectable marker (eg streptavidin-labeled fluorophore).
Is detected by adding a second reagent bound to the enzyme and subsequently analyzing the plate on a microtiter plate reader. The candidate compounds thus identified that interact with histone deacetylase are then screened for their ability to suppress the enzymatic activity of histone deacetylase.

Example 6 Histone Deacetylase Antisense Oligonucleotide Tumors In Vivo
Antitumor effect on ulcer cells The purpose of this example is to demonstrate the ability of the histone deacetylase antisense oligonucleotides of the invention to treat diseases corresponding to histone deacetylase inhibition in animals, especially mammals. This example further provides evidence of the ability of the methods and compositions of this invention to inhibit tumor growth in livestock. 8-10 week old female BALB / c nude mice (Taconic Labs, Great Barrington, NY)
The flank region was subcutaneously injected with 2 × 10 ⁶ A549 human lung cancer cells preconditioned. The preconditioning of these cells is
It is performed by continuously transplanting tumors to nude mice of the same lineage three or more times. Then, a tumor section of about 30 mg was cut out and subcutaneously transplanted into the left flank of a mouse subjected to Forene anesthesia (Abbott Labs., Geneva, Switzerland). When the average tumor volume reached 100mm ^3, 0.1~6mg / kg oligonucleotide saline preparations containing antisense oligonucleotides (Sigma, St. Louis, MO) using a daily, by bolous infusion into the tail vessels , The mice are treated intravenously. The optimal final concentration of oligonucleotide is defined by dose response experiments according to standard protocols. Tumor volume was calculated every 2 days after infusion according to standard methods (eg M
eyer et al., Int. J. Cancer 43: 851-856 (1989)). Treatment with an oligonucleotide according to the present invention results in a tumor weight and volume that is higher than that of a control treated with saline alone (ie no oligonucleotide) or a control treated with saline and a control non-specific oligonucleotide. Diminished. In addition, the measurement of histone deacetylase activity is expected to be significantly reduced compared to saline treated controls.

Example 7 In vivo histone deacetylase antisense oligonucleotides and histone deacetylases
Antitumor Synergistic Effect of Stone Deacetylase Protein Suppressor on Tumor Cells The purpose of this example was to demonstrate the ability of histone deacetylase antisense oligonucleotides and histone deacetylase protein suppressors to suppress tumor growth in mammals. It is to show. Transplanted A549 tumors (mean volume) as described in Example 6
Mice with 100 mm ³ ) are treated daily with a saline preparation containing about 0.1 mg to about 30 mg / kg body weight of histone deacetylase antisense oligonucleotides. A second group of mice is treated daily with a pharmaceutically acceptable preparation comprising from about 0.01 mg to about 5 mg / kg body weight histone deacetylase protein inhibitor. Some mice receive both histone deacetylase antisense oligonucleotides and histone deacetylase protein repressors. Of these mice, one group is co-administered with antisense oligonucleotides and histone deacetylase protein inhibitors via tail vein intravenous injection. Another group receives antisense oligonucleotides via the tail vein and histone deacetylase protein inhibitors by subcutaneous injection. Another group receives both antisense oligonucleotides and histone deacetylase protein inhibitors simultaneously by subcutaneous injection. A control group of mice is created as well. There are groups that are not treated (eg, saline only), groups that are mismatch antisense oligonucleotides only, groups that are control compounds that do not suppress histone deacetylase activity, and groups that are mismatch antisense oligonucleotides and control compounds.

Tumor volume is measured with calipers. Treatment with the antisense oligonucleotides of this invention and histone deacetylase protein inhibitors significantly reduced tumor weight and volume compared to controls. It is preferable that the antisense oligonucleotide and the histone deacetylase protein inhibitor suppress the expression and activity of the same histone deacetylase.

[Sequence list]

[Brief description of drawings]

FIG. 1 is a schematic representation of Northern blotting analysis, showing specific binding to both HDAC-1 encoding nucleic acid or both HDAC-1 and HDAC-2 encoding nucleic acids. Figure 4 shows the dose-dependent activity of representative and non-limiting synthetic oligonucleotides according to the invention that suppress the expression of both mRNA or HDAC-1 mRNA as well as HDAC-2 mRNA.

FIG. 2 is a schematic representation of Northern blotting analysis, which is a representative and non-limiting representative of the present invention that specifically binds to a nucleic acid encoding HDAC-2 and suppresses the expression of HDAC-2 mRNA. Shows the dose-dependent activity of various synthetic oligonucleotides.

FIG. 3 is a schematic representation of Western blotting analysis, which is a representative and non-limiting representation of the present invention, which specifically binds to a nucleic acid encoding HDAC-2 and represses HDAC-2 protein expression. Shows the activity of various synthetic oligonucleotides.

FIG. 4 is a schematic representation of a Western blotting analysis showing specific binding to nucleic acid encoding HDAC-1, or to both HDAC-1 and HDAC-2.
Figure 5 shows representative and non-restrictive synthetic oligonucleotide activities according to the invention that suppress the expression of HDAC-1 protein or both HDAC-1 protein as well as HDAC-2 protein, respectively. A mismatched synthetic oligonucleotide was used as a negative control. Equal loading of all lanes is evidenced by similarly expressed actin.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/80 C12Q 1/02 4C086 C12Q 1/02 1/34 1/34 G01N 33/15 Z G01N 33 / 15 33/50 Z 33/50 C12N 15/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC , NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID , IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Invention The Bestman Jeffrey M. H9X 3V3 Canada Quebec by e Durufe gray Crescent 51 F-term (reference) 2G045 AA40 FB01 FB02 FB03 FB07 FB08 4B024 AA01 BA11 BA80 CA04 EA04 HA17 4B050 CC03 DD11 LL01 4B063 QA01 QA18 QR10 QR31 QR72 4C084 AA13 AA17 MA02 NA14 ZB26 4C086 AA01 AA02 AA03 EA16 MA01 MA02 MA04 NA14 ZB26

Claims (39)

[Claims] 1. An antisense oligonucleotide that suppresses the expression of histone deacetylase. 2. The histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC.
-4, HDAC-5, HDAC-C, HDAC-D, and HDAC-E.
Antisense oligonucleotide. 3. The antisense oligonucleotide according to claim 1, wherein the oligonucleotide suppresses two or more histone deacetylases. 4. The antisense oligonucleotide according to claim 3, wherein the oligonucleotide suppresses all histone deacetylases. 5. The antisense oligonucleotide according to claim 1, wherein the oligonucleotide represses transcription of a nucleic acid molecule encoding a histone deacetylase. 6. The antisense oligonucleotide of claim 5, wherein the nucleic acid molecule is selected from the group consisting of genomic DNA, cDNA, and RNA. 7. The antisense oligonucleotide according to claim 1, wherein the oligonucleotide suppresses translation of histone deacetylase. 8. The oligonucleotide is phosphorothionate, phosphorodithionate, alkylphosphonate, alkylphosophonothionate, phosphotriester, phosphoramidate, siloxane, carbonate, carboxylmethyl ester, acetamidate, carbamate. The antisense oligonucleotide of claim 1 having at least one internucleotide linkage selected from the group consisting of a thioether, a bridged phosphoramidate, a bridged methylene phosphonate, a bridged phosphorothionate, and a sulfone internucleotide linkage. 9. The antisense oligonucleotide according to claim 1, wherein the oligonucleotide is a chimeric oligonucleotide or a hybrid oligonucleotide. 10. The oligonucleotide has a ribonucleotide or a 2′-O-substituted ribonucleotide region and a deoxyribonucleotide region.
Antisense oligonucleotide. 11. A method for inhibiting histone deacetylase in a cell, comprising contacting the cell with the antisense oligonucleotide of claim 1. 12. The method of claim 11, wherein cell proliferation is suppressed in the contact cells. 13. The method of claim 11, wherein the cells are tumor cells. 14. The method of claim 13, wherein the tumor cells are in an animal. 15. The method of claim 14, wherein the tumor cells are in tumor growth. 16. The method of claim 11, further comprising contacting the cell with a histone deacetylase protein inhibitor that interacts with histone deacetylase and reduces its enzymatic activity. 17. The method of claim 16, wherein the histone deacetylase protein repressor is operably linked to the antisense oligonucleotide. 18. A method of inhibiting tumor growth in an animal, wherein the body of the animal has at least one tumor cell, wherein a therapeutically effective amount of the antisense oligonucleotide of claim 1 is pharmaceutically acceptable. A method comprising administering with a carrier for a therapeutically effective period. 19. The method of claim 18, wherein the animal is a mammal. 20. The method of claim 19, wherein the mammal is a human. 21. A therapeutically effective amount of a histone deacetylase protein inhibitor that interacts with histone deacetylase and reduces the enzyme activity in an animal,
19. The method of claim 18, further comprising administering for a therapeutically effective period with a pharmaceutically acceptable carrier. 22. The method of claim 21, wherein the histone deacetylase protein repressor is operably linked to the antisense oligonucleotide. 23. A method for identifying a histone deacetylase involved in the induction of cell proliferation, which comprises contacting the cell with an antisense oligonucleotide that suppresses the expression of the histone deacetylase. A method for identifying a histone deacetylase as a histone deacetylase involved in the induction of cell proliferation in which cell growth suppression is involved. 24. The method of claim 23, wherein the cells are tumor cells and the induction of cell proliferation is tumorigenesis. 25. The histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HD.
24. The method of claim 23, selected from the group consisting of AC-4, HDAC-5, HDAC-C, HDAC-D, and HDAC-E. 26. A method for identifying a histone deacetylase protein inhibitor that inhibits histone deacetylase involved in the induction of cell proliferation, wherein the histone deacetylase identified by the method of claim 23 is a candidate compound. And measuring the enzymatic activity of the contacted histone deacetylase, wherein the decrease in the enzymatic activity of the contacted histone deacetylase inhibits the histone deacetylase involved in the induction of cell proliferation. A method for identifying a candidate compound as a deacetylase protein inhibitor. 27. The method of claim 26, wherein the histone deacetylase protein inhibitor interacts with less than all histone deacetylase enzymes and reduces their enzymatic activity. 28. A method for identifying a histone deacetylase involved in the induction of cell differentiation, which comprises contacting the cell with an antisense oligonucleotide that suppresses the expression of histone deacetylase, the differentiation of the contacted cell. A method wherein induction identifies the histone deacetylase as a histone deacetylase involved in cell differentiation. 29. The method of claim 28, wherein the cells are tumor cells. 30. Histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HD
29. The method of claim 28, selected from the group consisting of AC-4, HDAC-5, HDAC-C, HDAC-D, and HDAC-E. 31. A method for identifying a histone deacetylase protein inhibitor that inhibits histone deacetylase involved in the induction of cell differentiation, wherein the histone deacetylase identified by the method of claim 28 is a candidate compound. The histone deacetylase, which is involved in the induction of cell differentiation, including the measurement of the enzymatic activity of the contacted histone deacetylase. A method for identifying a candidate compound as a deacetylase protein inhibitor. 32. The method of claim 31, wherein the histone deacetylase protein inhibitor interacts with less than all histone deacetylases and reduces their enzymatic activity. 33. A histone deacetylase protein inhibitor identified by the method of claim 26 or 31. 34. A histone deacetylase that is substantially pure. 35. A method for suppressing cell growth in a cell, which comprises an antisense oligonucleotide that suppresses histone deacetylase, a histone deacetylase protein suppressor, an antisense oligonucleotide that suppresses DNA methyltransferase, and DNA. A method comprising contacting a cell with at least two reagents selected from the group consisting of methyltransferase protein inhibitors. 36. The method of claim 35, wherein the inhibition of cell growth of the contacted cells is greater than that of cells contacted with a single reagent. 37. The method of claim 3, wherein each reagent selected from the group is substantially pure.
Method 5 38. The method of claim 35, wherein the cells are tumor cells. 39. The method of claim 35, wherein the reagent selected from the group is operably linked.