RU2575608C2 - Treatment of diseases, associated with protein of prostate apoptotic response par4, by inhibition of natural antisense transcript of par4 gene - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. Disclosed are methods for increasing expression of PAR4 polynucleotide, including realisation of contact with at least one oligonucleotide from 15 to 30 nucleotides long, which is aimed at natural antisense sequence for PAR4 polynucleotide, and said natural antisense sequence has sequence, represented in SEQ ID NO: 2. Claimed is synthetic modified oligonucleotide, from 15 to 30 nucleotides long, and pharmaceutical composition, containing said oligonucleotide. disclosed is method for apoptosis induction in biological system, expressing or with acquired ability to express products of PAR4 genes, which includes introduction of oliginucleotide from 15 to 30 nucleotides long into said system, which is aimed as natural antisense sequence SEQ ID NO: 2 of PAR4 polynucleotide and increases expression of said PAR4 polynucleotide.

EFFECT: invention makes it possible to increase PAR4 polynucleotide expression.

28 cl, 6 dwg, 1 tbl, 3 ex

Description

FIELD OF TECHNOLOGY

[0001] This application claims priority based on provisional application for US patent No. 61/334933, filed May 14, 2010, the full contents of which are incorporated into this description by reference.

[0002] Embodiments of the present invention include oligonucleotides that modulate the expression and / or function of the PAR4 gene and its associated molecules.

BACKGROUND

[0003] Hybridization of DNA-RNA and RNA-RNA is important for many aspects of the functioning of nucleic acids, including DNA replication, transcription and translation. The hybridization process is also fundamental in many technologies that allow either the detection of a specific nucleic acid or altering its expression. Antisense nucleotides, for example, disrupt gene expression due to hybridization with the target RNA, thereby interfering with RNA splicing, transcription, translation and replication. Antisense DNA has the additional feature that DNA-RNA hybrids act as a substrate for enzymatic cleavage by ribonuclease H, the activity of which is present in most cell types. Antisense molecules can be delivered to cells, as is the case for oligodeoxynucleotides (ONE), or they can be the products of expression of endogenous genes in the form of RNA molecules. The US Federal Drug Control Agency (FDA) recently approved an antisense drug, Vitravene ™ (for the treatment of cytomegalovirus retinitis), which reflects the usefulness of this antisense drug for therapeutic purposes.

SUMMARY OF THE INVENTION

[0004] This brief description provides a brief description of the present invention in order to briefly describe the subject and essence of the present invention. A brief description is presented with the understanding that it should not be used to interpret or limit the scope or meaning of the claims.

[0005] In one embodiment, the invention provides methods for inhibiting the action of a natural antisense transcript by using an antisense oligonucleotide (oligonucleotides) targeting any region of a natural antisense transcript, leading to increased expression of the corresponding sense gene. The authors of this application also suggest that inhibition of the natural antisense transcript can be achieved using small interfering RNAs (siRNAs), ribozymes, and small molecules contemplated by the present invention.

[0006] According to one embodiment, a method for modulating the function and / or expression of a PAR4 polynucleotide in a patient’s cells or tissues in vivo or in vitro, comprising contacting said cells or tissues with an antisense oligonucleotide of 5 to 30 nucleotides in length, wherein said oligonucleotide has at least 50% sequence identity with a sequence that is the reverse complement of a polynucleotide containing 5 to 30 consecutive nucleotides within nucleotides 1-354 corresponding to SEQ ID NO: 2, which leads to modulation of the function and / or expression of the PAR4 polynucleotide in the cells or tissues of the patient in vivo or in vitro.

[0007] In one embodiment, the oligonucleotide targets a natural antisense sequence for the PAR4 polynucleotide, for example, against nucleotides indicated in the sequence corresponding to SEQ ID NOS: 2, and any variants, alleles, homologs, mutants, derivatives, fragments and sequences that represent their complement. Examples of antisense oligonucleotides are indicated as sequences corresponding to SEQ ID NOS: 3-9.

[0008] According to another embodiment, a method for modulating the function and / or expression of a PAR4 polynucleotide in a patient’s cells or tissues in vivo or in vitro, comprising contacting said cells or tissues with an antisense oligonucleotide of 5 to 30 nucleotides in length, said oligonucleotide having at least 50% sequence identity with a sequence that is the reverse complement of the antisense sequence for the PAR4 polynucleotide; resulting in modulation of the function and / or expression of the PAR4 polynucleotide in the cells or tissues of the patient in vivo or in vitro.

[0009] According to another embodiment, a method for modulating the function and / or expression of a PAR4 polynucleotide in a patient’s cells or tissues in vivo or in vitro, comprising contacting said cells or tissues with an antisense oligonucleotide of 5 to 30 nucleotides in length, said oligonucleotide having at least 50% sequence identity with the antisense oligonucleotide for the antisense polynucleotide of the PAR4 gene; resulting in modulation of the function and / or expression of the PAR4 polynucleotide in the cells or tissues of the patient in vivo or in vitro.

[0010] In one embodiment, the composition comprises one or more antisense oligonucleotides that bind to sense and / or antisense polynucleotides of the PAR4 gene.

[0011] In one embodiment, the oligonucleotides comprise one or more modified or substituted nucleotides.

[0012] In one embodiment, the oligonucleotides comprise one or more modified bonds.

[0013] In another embodiment, the modified nucleotides comprise modified bases, including molecules of phosphothioate, methylphosphonate derivatives of nucleic acids, peptidic nucleic acids, 2'-O-methyl, fluoro or carbon, methylene or other closed nucleic acids ( ZNA; locked nucleic acids, LNA). Preferably, the modified nucleotides are closed nucleic acid molecules, including α-L-ribo configurations (α-L-LNAs).

[0014] In one embodiment, the oligonucleotides are administered to the patient subcutaneously, intramuscularly, intravenously or intraperitoneally.

[0015] In one embodiment, the oligonucleotides are administered in a pharmaceutical composition. The treatment regimen includes at least a single administration of antisense compounds to a patient; however, this treatment may be modified prior to the administration of multiple doses over a period of time. Treatment may be combined with one or more other types of therapy.

[0016] In one embodiment, the oligonucleotides are encapsulated in a liposome or attached to a carrier molecule (eg, cholesterol, TAT peptide).

[0017] Other aspects are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 illustrates the fold and standard deviation of the number of copies of the PAR4 gene mRNA in HepG2 cells 48 hours after treatment with oligonucleotides containing phosphothioate groups introduced using the Lipofectamine 2000 transfection agent (Lipofectamine 2000), compared to the control. Real-time polymerase chain reaction (PCR) analysis results indicate that PAR4 mRNA levels in HepG2 cells significantly increase 48 hours after treatment with two of all tested oligonucleotides that target the jortybo.aApr07 antisense sequence for the PAR4 gene. The columns of the diagram designated as CUR-1564 to CUR-1570 correspond to samples treated with oligonucleotides corresponding to SEQ ID NOS: 3-9, respectively.

[0019] Figure 2 illustrates the fold and standard deviation of the number of copies of the PAR4 gene mRNA in A549 cells 48 hours after treatment with oligonucleotides containing phosphothioate groups administered using the Lipofectamine 2000 transfection agent compared to the control. Real-time PCR analyzes indicate that the PAR4 gene mRNA levels in A549 cells significantly increase 48 hours after treatment with the CUR-1566 oligonucleotide, which targets the jortybo.aApr07 antisense sequence for the PAR4 gene. The column of the diagram designated as CUR-1566 corresponds to a sample treated with an oligonucleotide corresponding to SEQ ID NO: 5.

[0020] Figure 3 illustrates the increase in apoptosis in HEK293 cells 48 hours after treatment with CUR-1566 oligonucleotide administered using the Lipofectamine 2000 transfection agent compared to the control. The column of the diagram designated as CUR-1566 corresponds to a sample treated with an oligonucleotide corresponding to SEQ ID NO: 5.

[0021] Figure 4 illustrates the increase in the level of apoptosis in keratinocytes 48 hours after treatment with the CUR-1566 oligonucleotide administered using the Lipofectamine 2000 transfection agent compared to the control. The column of the diagram designated as CUR-1566 corresponds to a sample treated with an oligonucleotide corresponding to SEQ ID NO: 5.

[0022] Figure 5 illustrates the increase in apoptosis in HepG2 cells 48 hours after treatment with oligonucleotides containing phosphotioate groups administered using the Lipofectamine 2000 transfection agent compared to the control. The columns of the diagram designated as CUR-1565, CUR-1566 and CUR-1568 correspond to samples treated with oligonucleotides corresponding to SEQ ID NO: 4, 5 and 7.

[0023] Figure 6 illustrates the increase in the level of apoptosis in MCF-7 cells 48 hours after treatment with the CUR-1566 oligonucleotide administered using the Lipofectamine 2000 transfection agent compared to the control. The columns of the diagram designated as CUR-1565, CUR-1566 and CUR-1568 correspond to samples treated with oligonucleotides corresponding to SEQ ID NO: 4, 5 and 7.

[0024] Description of the Sequence Listing - SEQ ID NO: 1: Regulator of protein kinase C (PRKC), apoptosis, human Wilms tumor suppressor protein (WT1) (PAWR), mRNA (sequence number in the NCBI database (National Center for Biotechnological Information, USA) : NM_002583); SEQ ID NO: 2: Natural antisense sequence (jortybo.aApr07) for the PAR4 gene; SEQ ID NOs: 3-9: Antisense Oligonucleotides. The symbol "*" indicates a phosphotioate bond.

DETAILED DESCRIPTION

[0025] Some aspects of the invention are described below with reference to examples of use for illustration. It should be understood that numerous specific details, communications, and methods are provided in order to provide a thorough understanding of the invention. However, one skilled in the art will readily find that the invention can be practiced without one or more specific details or with other methods. The present invention is not limited to the ordering of actions or events, as some actions can be carried out in different orders and / or simultaneously with other actions or events. In addition, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

[0026] All genes, gene names, and gene products described herein correspond to homologs from organisms of any species for which the compositions and methods provided herein are applicable. Thus, these terms include, but are not limited to, genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is proposed, this element of the invention is provided solely as an example, and should not be construed as limiting unless the context in which it appears clearly requires otherwise. Accordingly, for example, the genes described in this application, which in some embodiments relate to the nucleic acids and amino acid sequences of mammals, encompass homologous and / or orthologous genes and gene products from organisms of other animals, including, but not limited to, other mammals, fish amphibians, reptiles and birds. In one embodiment, the genes or nucleotide sequences are human genes or sequences.

Definitions

[0027] The terminology used in this application serves solely to describe specific embodiments and does not limit the invention. In the present application, the indefinite and definite singular forms also include the plural forms unless the context clearly dictates otherwise. In addition, to the extent that the terms “including,” “includes,” “having,” “has,” “c,” or variants thereof are used in the detailed description and / or claims, such terms are intended to include, like the term "containing".

[0028] The term “about” or “approximately” means that an acceptable error is within a range for a specific quantity determined by one of ordinary skill in the art, which will depend in part on how the quantity is measured or determined, i.e. from the limitations of the measuring system. For example, the term “about” may mean that the value is within 1 or more than 1 standard deviation, as practice in the art shows. Alternatively, the term “about” may mean an error range of up to 20%, preferably up to 10%, more preferably up to 5% and even more preferably up to 1% of a predetermined value. Alternatively, with respect to biological systems or processes, the term may mean being within an order of magnitude, preferably within a fivefold value and more preferably a twofold value. In those cases where specific values are given in the application and claims, then unless otherwise indicated, the term "about" should be assumed to mean that the acceptable error is within a range for a specific value.

[0029] In the present application, the term "mRNA" refers to the currently known mRNA transcript (s) of the target gene and any additional transcripts whose origin can be explained.

[0030] Using the terms "antisense oligonucleotides" or "antisense compound" refers to an RNA or DNA molecule that binds to another RNA or DNA (target RNA, DNA). For example, if the antisense compound is an oligoribonucleotide, it binds to another target RNA through RNA-RNA interactions and changes the activity of the target RNA. An antisense oligonucleotide may enhance or inhibit the expression and / or function of a particular polynucleotide. The definition includes any foreign DNA or RNA molecule that is suitable from a therapeutic, diagnostic or other point of view. Such molecules include, for example, antisense DNA or RNA molecules, interfering RNA (RNAi) molecules, microRNAs, mock RNAs, siRNAs, enzymatic RNAs, RNAs used in corrective therapy, and RNAs having the properties of agonists and antagonists, antisense oligomeric compounds, antisense oligonucleotides, oligonucleotides of an external guide sequence (EGS), splice variants, primers, probes, and other oligomeric compounds that hybridize with at least a portion of the target nucleic acid. In this regard, these compounds can be introduced in the form of single-chain, double-stranded, partially single-stranded or ring oligomeric compounds.

[0031] In the context of the present invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or their mimetics. The term “oligonucleotide” also includes linear or ring oligomers of natural and / or modified monomers or bonds, including deoxyribonucleosides, ribonucleosides, their substituted and alpha-anomeric forms, peptide nucleic acids (PNAs), closed nucleic acids (LNAs), locked nucleic acids, LNA), phosphothioate, methylphosphonate derivatives and the like. Oligonucleotides are capable of specific binding to the target polynucleotide due to regular interactions between monomeric units, such as the Watson-Cricova type of base pairing, the Hoogsteen or reverse Hoogsteen type of base pairing, etc.

[0032] The oligonucleotide may be "chimeric", that is, composed of different regions. In the context of the present invention, “chimeric” compounds are oligonucleotides that contain two or more chemically distinct regions, for example, a region (s) of DNA, a region (s) of RNA, a region (s) of PNA, etc. Each chemically distinct region consists of at least one monomer unit, i.e. nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region in which the oligonucleotide is modified to exhibit one or more desired properties. Desired oligonucleotide properties include, but are not limited to, for example, increased resistance to degradation by nucleases, increased cellular uptake, and / or increased ability to bind to a target nucleic acid. Different regions of an oligonucleotide may thus have different properties. Chimeric oligonucleotides according to the present invention can be obtained in the form of mixed structures from two or more oligonucleotides, modified oligonucleotides, oligonucleosides and / or analogs of the oligonucleotides described above.

[0033] The oligonucleotide can be composed of regions that can be connected in a “row”, that is, when the monomers are connected in series, as in native DNA, or connected by spacers. Spacers form a covalent “bridge” between regions and, in preferred cases, have a length not exceeding about 100 carbon atoms. Spacers can have different functional characteristics, for example, carry a positive or negative charge, possess the properties of specific binding of nucleic acids (intercalators, substances that bind to grooves, toxins, fluorophores, etc.), be lipophilic, and induce the formation of specific secondary structures, such as for example, alanine-containing peptides that induce the formation of alpha helices.

[0034] In the present application, the term "PAR4 gene" includes all family members, mutants, alleles, fragments, species, coding and non-coding sequences, sense and antisense polynucleotide chains, etc.

[0035] In the present description, the words PAR4, par-4, Par-4, protein kinase C regulator protein (PRKC), apoptosis, Wilms tumor suppressor protein (WT1), prostate apoptotic response protein 4 are considered to have the same meaning in the literature and are used interchangeably in this application.

[0036] As used herein, the term “oligonucleotide specific for” or “oligonucleotide aimed at” refers to an oligonucleotide containing a sequence (I) capable of forming a stable complex with a portion of a target gene or (ii) capable of forming a stable duplex with a portion of an mRNA transcript of the target gene. The stability of complexes and duplexes can be determined by theoretical calculations and / or in vitro analyzes. Typical assays for determining the stability of hybridization complexes and duplexes are described in the Examples below.

[0037] In the present application, the term "target nucleic acid" encompasses DNA, RNA (containing pre-mRNA and mRNA) transcribed from such DNA, and also cDNA obtained on the matrix of such RNA, coding, non-coding sequences, sense or antisense polynucleotides . The specific hybridization of the oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of the function of the target nucleic acid using compounds that specifically hybridize with the latter is generally referred to as “antisense technology”. Affected DNA functions include, for example, replication and transcription. Affected RNA functions include all vital functions, such as, for example, translating RNA to a protein translation site, translating proteins on an RNA matrix, splicing RNA to form one or more types of mRNA, and catalytic activity that may be present on RNA or which RNA may contribute to . The overall effect of such an obstruction of the function of the target nucleic acid is to modulate the expression of the encoded product or oligonucleotides.

[0038] RNAi RNA interference is mediated by double-stranded RNA molecules (dsRNAs) that have sequences homologous to their target nucleotide sequences. In some embodiments, the mediators are duplexes of “small interfering” RNAs (siRNAs) of 5-25 nucleotides in length. siRNAs are formed by cutting dsRNA with an RNAse enzyme known as Dicer. Duplex siRNA products are incorporated into a multi-protein complex called RISC (RNA-induced silencing complex). Without reference to any specific theory, the RISC complex is believed to be targeted at the target nucleic acid (the role of which is suitable for mRNA), while the miRNA duplex interacts with mRNA due to the complementarity of their sequences, causing the mRNA to cleave catalytically. Small interfering RNAs that can be used according to the present invention can be synthesized and used in accordance with methods that are well known in the art and which will be familiar to one skilled in the art. Small interfering RNAs for use in accordance with the methods of the present invention suitably contain from about 1 to about 50 nucleotides (nt). In examples of non-limiting embodiments, siRNAs may contain from about 5 to about 40 nt, from about 5 to about 30 nt, from about 10 to about 30 nt, from about 15 to about 25 nt, or about 20-25 nucleotides.

[0039] The selection of suitable oligonucleotides is facilitated by the use of computer programs that automatically align nucleotide sequences and mark identical or homologous regions. Such programs are used to compare nucleotide sequences obtained, for example, by searching databases such as GenBank, or by sequencing PCR products. Comparison of nucleotide sequences from organisms of a number of species allows the selection of nucleotide sequences that exhibit a suitable degree of identity between species. In the case of genes that have not been sequenced, Southern blots are performed to determine the degree of identity between the genes of the target species and other species. By conducting Southern blotting at varying degrees of stringency of hybridization conditions, as is well known in the art, an approximate measure of identity can be obtained. These procedures allow the selection of oligonucleotides that show a high degree of complementarity to the target nucleotide sequences of the subject to be controlled, and a lower degree of complementarity to the corresponding nucleotide sequences of other species. One skilled in the art will find that there is considerable freedom in choosing suitable gene regions for use in accordance with the present invention.

[0040] By the term "enzymatic RNA" is meant an RNA molecule having enzymatic activity (Cech, (1988) J. American. Med. Assoc. 260, 3030-3035). Enzymatic nucleic acids (ribozymes) act due, firstly, to binding to the target RNA. Such binding occurs through a portion of the enzymatic nucleic acid that binds the target RNA, which is in close proximity to the enzymatic portion of the molecule that catalyzes the cleavage of the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds the target RNA by base pairing, and as soon as binding to the desired site occurs, it exhibits enzymatic activity by cutting the target RNA.

[0041] By the term "false RNA" is meant an RNA molecule that mimics the natural binding domain for a ligand. False RNA thus competes with a natural binding target for binding to a particular ligand. For example, it has been shown that overexpression of the HIV transactivation response (TAR) RNA element can act as a “false” and effectively binds HIV tat (transcriptional transactivator) protein, thereby inhibiting the binding of this protein to TAR sequences encoded by HIV RNA. The information provided is intended to be a specific example. Those skilled in the art will find that this is just one example, and other implementations can be easily developed using techniques well known in the art.

[0042] In the present application, the term "monomers" generally refers to monomers connected by phosphodiester bonds or their analogues to form oligonucleotides, the sizes of which vary from several monomer units, for example, from about 3-4 to about several hundred monomer units. Analogues of phosphodiester bonds include: phosphothioate, phosphodithioate, methylphosphonate, phosphoselenate, phosphoramidate bonds and the like, as described in more detail below.

[0043] The term "nucleotide" refers to naturally occurring nucleotides as well as non-naturally occurring nucleotides. One skilled in the art will appreciate that various nucleotides that were previously considered "not found in nature" were subsequently found in nature. Thus, the term "nucleotides" includes not only known molecules containing purine and pyrimidine heterocycles, but also their heterocyclic analogues and tautomers. Illustrative examples of other types of nucleotides are molecules containing adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurin, 8-oxo-N6-methyladenine, 7-deazaxanthin, 7-deazaguanin, N4, N4-ethanocytosine, N6, N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5- (C3-C6) alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine and "non-naturally occurring" nucleotides described in Benner et al., US Patent No. 5,432,272. The term "nucleotide" applies to each and all of these examples, as well as their analogues and tautomers. Particularly interesting nucleotides are those that contain adenine, guanine, thymine, cytosine and uracil, which are considered naturally occurring nucleotides for therapeutic and diagnostic use in humans. Nucleotides contain naturally occurring 2'-deoxy and 2'-hydroxysugars, for example as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992), as well as their analogues.

[0044] The term "analogues" as applied to nucleotides includes synthetic nucleotides containing modified bases and / or modified sugars (see, for example, analogues described mainly in Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, (1997) Nucl. Acid. Res., 25 (22), 4429-4443, Toulmé, JJ, (2001) Nature Biotechnology 19: 17-18; Manoharan M., (1999) Biochemica et Biophysica Acta 1489 : 117-139; Freier SM, (1997) Nucleic Acid Research, 25: 4429-4443, Uhlman, E., (2000) Drug Discovery & Development, 3: 203-213, Herdewin P., (2000) Antisense & Nucleic Acid Drug Dev., 10: 297-310); 2'-O, 3'-C-linked [3.2.0] bicycloarabinonucleosides. Such analogs include synthetic nucleotides aimed at enhancing binding properties, for example, duplex or triplex stability, specificity, and the like.

[0045] In the present application, the term "hybridization" refers to the pairing of almost complementary chains of oligomeric compounds. One pairing mechanism involves the formation of hydrogen bonds, which can be Watson-Cricova, Hoogsteen or inverse Hoogsteen hydrogen bonds between complementary bases of nucleosides or nucleotides (nucleotides) of the chains of oligomeric compounds. For example, adenine and thymine are complementary nucleotides that mate due to the formation of hydrogen bonds. Hybridization can occur under various conditions.

[0046] An antisense compound is "specifically hybridizable" if the binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid, modulating the function and / or activity of the latter, and a sufficient degree of complementarity is provided to avoid non-specific binding of the antisense compound to the nucleotide sequences, non-targets under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment and under the conditions under which the analyzes are carried out in the case of in vitro assays.

[0047] In the present application, the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which a compound of the invention will hybridize with its target sequence, but with a minimum number of other sequences. The stringent conditions are sequence dependent and will vary in different circumstances, and, in the context of the present invention, the “stringent conditions” under which the oligomeric compounds hybridize to the target sequence are determined by the nature and composition of the oligomeric compounds and the types of assays by which they are examined. In general, stringent hybridization conditions include low concentrations (<0.15 M) of salts containing inorganic cations, such as Na ⁺⁺ or K ⁺⁺ (i.e. low ionic strength), temperatures above a 20 ° point C-25 ° C below the _Tm complex oligomeric compound: target sequence, and the presence of denaturants such as formamide, dimethylformamide, dimethyl sulfoxide or detergent sodium dodecyl sulfate (SDS). For example, the hybridization rate is reduced by 1.1% for each added 1% formamide. An example of hybridization conditions with a high degree of stringency is hybridization using 0.1X sodium chloride-sodium citrate (SSC, standard citrate-salt) / 0.1% (w / v) SDS buffer at 60 ° C. for 30 minutes.

[0048] As used herein, the term "complementary" refers to the ability to precisely mate between two nucleotides or one or two oligomeric chains. For example, if a nucleic base at a specific position in an antisense compound chain is capable of forming hydrogen bonds with a nucleic base at a specific position of a target nucleic acid chain, wherein said target nucleic acid is a DNA, RNA, or oligonucleotide molecule, then the position at which the formation of hydrogen bonds between the oligonucleotide and the target nucleic acid is considered a complementary position. An oligomeric compound and an additional DNA, RNA or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleotides capable of forming hydrogen bonds with each other. Thus, the terms “specifically hybridizable” and “complementary” are used to mean a sufficient degree of exact pairing, or complementarity, within a sufficient number of nucleotides, so that a stable and specific binding occurs between the oligomeric compound and the target nucleic acid.

[0049] As those skilled in the art understand, the sequence of an oligomeric compound does not need to be 100% complementary to its target nucleic acid sequence in order for the compound to be capable of specific hybridization. In addition, the oligonucleotide can hybridize on top of one or more segments, so that the intermediate or adjacent segments are not involved in the hybridization process (e.g., loop structure, lack of complementarity or hairpin structure). The oligomeric compounds of the present invention comprise a sequence complementary to at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the target region within the target nucleotide sequence on which these compounds are targeted. For example, an antisense compound in which 18 of the 20 nucleotides of the antisense compound are complementary to the target region and, therefore, would specifically hybridize with it, would be characterized by 90% complementarity. In this example, the remaining non-complementary nucleotides can be located in clusters of complementary nucleotides or alternate with complementary nucleotides and do not have to be adjacent to each other or to complementary nucleotides. For this reason, an 18-nucleotide antisense compound containing 4 (four) non-complementary nucleotides that are flanked by two regions that are completely complementary to the target nucleic acid would generally have been 77.8% complementary to the target nucleic acid and would thus fall within the scope of the present invention. The percentage of complementarity of the antisense compound with the region of the target nucleic acid can be determined in the usual way using BLAST programs (search tools for basic local alignment) and PowerBLAST programs known in the art. The percentage of homology, sequence identity, or complementarity can be determined using, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) Using the default parameters, which uses Smith-Waterman algorithm (Adv. Appl. Math., (1981) 2, 482-489).

[0050] In the present application, the term "melting point (T _PL )" refers to the temperature, under conditions of a specific ionic strength, pH and nucleic acid concentration, at which 50% of the oligonucleotides complementary to the target sequence hybridize to the target sequence in equilibrium . Typically, stringent hybridization conditions will differ in that the salt concentration is at least about 0.01 to 1.0 M concentration of Na ions (or other salts) at a pH of 7.0-8.3, and the temperature is at least about 30 ° C for short oligonucleotides (e.g., 10 to 50 nucleotides long). Strict conditions can also be achieved by adding destabilizing agents such as formamide.

[0051] In the present application, the term "modulation" means either an increase (stimulation) or a decrease (inhibition) of gene expression.

[0052] The term "variant", as applied to a polynucleotide sequence, may encompass a sequence of a polynucleotide related to a wild-type gene. This definition may also include, for example, “allelic”, “splice”, “species” or “polymorphic” variants. The splice variant may be substantially identical to the original molecule, but will generally contain more or less polynucleotides due to alternative exon splicing during mRNA processing. The corresponding polypeptide may have additional functional domains or differ in the absence of domains. Species variants are sequences of polynucleotides that vary from one species to another. Especially useful according to the present invention are variants of wild type gene products. Variants may result from at least one mutation in the nucleotide sequence and may lead to the appearance of altered mRNAs or polypeptides, the structure or function of which may or may not be altered. Any single natural or recombinant gene may not have or have one or many allelic forms. The usual mutational changes that give rise to variants are generally attributed to natural divisions, insertions or nucleotide substitutions. Each of these types of changes can occur individually or in combination with other types, one or more times in a given sequence.

[0053] The resulting polypeptides in the General case will be characterized by a significant identity of amino acid sequences relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individual individuals of a given species. Polymorphic variants may also encompass "single nucleotide polymorphisms" (SNPs), or mutations affecting one base, in which the polynucleotide sequence changes to one base. The presence of SNPs may indicate, for example, a certain population with a tendency to a disease state, which reflects susceptibility versus resistance.

[0054] Derivatives of polynucleotides include nucleic acids subjected to chemical modification, for example, substitution of hydrogen with an alkyl, acyl or amino group. Derivatives, for example, oligonucleotide derivatives, may contain non-naturally occurring fragments, such as altered sugar residues or bonds between sugar residues. Typical among them are phosphothioate and other sulfur containing types of bonds that are known in the art. Derivatives of nucleic acids may also contain labels, including radionucleotides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles, and the like.

[0055] A “derivative” polypeptide or peptide is one that is modified, for example, by glycosylation, pegylation, phosphorylation, sulfation, reduction / alkylation, acylation, chemical coupling or mild formalin treatment. The derivative can also be modified by the addition of a detectable label, either directly or indirectly, including, but not limited to, a radioisotope, fluorescent and enzymatic label.

[0056] As used herein, the term “animal” or “patient” includes, for example, humans, sheep, moose, deer, black tailed deer, mink, mammals, monkeys, horses, cattle, pigs, goats, dogs, cats, rats , mice, birds, chicken, reptiles, fish, insects and arachnids.

[0057] The term "mammal" extends to warm-blooded mammals, which are typically provided with medical care (eg, humans and domesticated animals). Examples include feline, canine, equine, bovine, and human, as well as human only.

[0058] The term "treatment" refers to the treatment of a disease state in a mammal and includes: (a) preventing the occurrence of a disease state in a mammal, in particular when such a mammal is predisposed to a disease state, but has not yet been diagnosed in a subject; (b) inhibiting a disease state, for example, arresting its development; and / or (c) alleviating the disease state, for example, inducing remission of the disease state, until the desired result is achieved. Treatment also includes a decrease in the intensity of the symptom of the disease (e.g., a decrease in pain or discomfort), while such a decrease in the intensity of the symptoms may or may not directly affect the disease (e.g., cause, transmission, manifestation, etc.).

[0059] As used herein, the term "cancer" refers to all types of cancer or neoplasms or malignant tumors found in mammals, including but not limited to leukemia, lymphoma, melanoma, carcinoma and sarcoma. Cancer manifests itself as a “tumor” or tissue containing malignant cancer cells. Examples of tumors include sarcomas and carcinomas, such as, but not limited to: fibro sarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chondroma, angiosarcoma, endothelio sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, leukangiotheliosarcoma, sarcoma, leukangiotheliosarcoma, colon, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat carcinoma, carcinoma sal gland, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, carcinoma of the cell, carcinoma, carcinoma, tumor lung, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, auditory nerve neuroma, oligodendrogli ma, meningioma, melanoma, neuroblastoma and retinoblastoma. Additional types of cancer that can be treated using the proposed composition according to the invention include, but are not limited to, for example, Hodgkin’s disease, non-Hodgkin’s lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia , small cell lung tumors, primary brain tumors, gastric cancer, colon cancer, pancreatic cancer insulin malignant, carcinoid cancer, bladder cancer, ventricular cancer of tract precancerous skin disease, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and prostate cancer. The term "apoptosis" refers to programmed cell death. It also means "cell death" or "directed cell death" that occurs when an antisense oligonucleotide described herein is administered to treat or alleviate diseases or disorders associated with uncontrolled cell growth and / or impaired normal apoptosis and / or a function of the signaling pathways of apoptosis.

[0060] As used herein, the term “neurological disease or disorder" refers to any disease or disorder of the nervous system and / or visual system. A “neurological disease or disorder” includes a disease or disorders that affect the central nervous system (brain, brain stem and cerebellum), the peripheral nervous system (including cranial nerves) and the autonomic nervous system (parts of which are located in the central and peripheral nervous system ) A neurological disease or disorder includes, but is not limited to, acquired epileptiform aphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy; age-related macular degeneration; agenesis of the corpus callosum; agnosia; Aycardi syndrome; Alexander's disease Alper's disease; alternating hemiplegia; Alzheimer's disease; vascular dementia; amyotrophic lateral sclerosis; anencephaly; Angelman syndrome; angiomatosis; anoxia aphasia; apraxia; arachnoid cysts; arachnoiditis; Arnold-Chiari malformation; arteriovenous malformation; Asperger Syndrome; ataxia-telangiectasia; attention deficit hyperactivity disorder; autism; autonomic dysfunction; backache; Batten's disease; Behcet's disease; Bell palsy; benign essential blepharospasm; benign focal epilepsy; amyotrophy; benign intracranial hypertension; Binswanger's disease; blepharospasm; Bloch-Sulzberger syndrome; damage to the brachial plexus; brain abscess brain injury; brain tumors (including glioblastoma multiforme); spinal cord tumor; Brown-Secar syndrome; Canavan disease carpal tunnel syndrome; causalgia; central pain syndrome; central pontine myelinolysis; cephalic syndrome; cerebral vascular aneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism; cerebral paralysis; Charcot-Marie-Tooth disease; neuropathy and neuropathic pain caused by chemotherapy; Chiari malformation; chorea; chronic inflammatory demyelinating polyneuropathy; chronic pain chronic regional pain syndrome; Coffin-Lowry syndrome; coma, including a steady vegetative state; congenital facial diplegia; corticobasal degeneration; temporal arteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulative traumatic disorders; Cushing's syndrome; inclusion cytomegaly; cytomegalovirus infection; syndrome of dancing eye-dancing legs; Dandy Walker syndrome; Dawson's disease; De Morsier syndrome; Dejerine-Klumpke paralysis; dementia dermatomyositis; diabetic neuropathy; diffuse sclerosis; vegetative dystonia; dysgraphia; dyslexia dystonia; early infantile epileptic encephalopathy; empty Turkish saddle syndrome; encephalitis; encephalocele; encephalotrigeminal angiomatosis; epilepsy Erb's palsy; essential tremor; Fabry disease; Farah's syndrome; loss of consciousness; familial spastic paralysis; febrile seizures; Fisher's syndrome; ataxia of Friedreich; frontotemporal dementia and other "taupathy"; Gaucher disease Gerstmann syndrome; giant cell arteritis; giant cell inclusive disease; globoid cell leukodystrophy; Guillain-Barré syndrome; HTLV-1-associated myelopathy (myelopathy associated with human T-cell leukemia virus); Gallerwarden-Spatz disease; head injury; headache; hemifacial spasm; hereditary spastic paraplegia; hereditary polyneuropathic ataxia; crankshaft syndrome; shingles; Hirayama syndrome; HIV-associated dementia and neuropathy (also neurological manifestations of AIDS); holoproencephaly; Huntington's disease and other diseases caused by the presence of polyglutamine repeats; hydroencephaly; hydrocephalus; hypercorticism; hypoxia; immune mediated encephalomyelitis; myositis with inclusions of the Taurus; pigment incontinence; infantile form of phytanic acid accumulation disease; infantile Refsum disease; infantile cramps; inflammatory myopathy; intracranial cyst; intracranial hypertension; joubert's syndrome; Kearns-Sayre syndrome; Kennedy's disease; Kinsboume syndrome; Klippel-Feil syndrome; crabbe disease; Kugelberg-Velander disease; Kuru; Lafora disease; Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome; lateral medullary syndrome (Wallenberg syndrome); learning disabilities; lei's disease; Lennox-Gastaut syndrome; Lesch-Nyhen syndrome leukodystrophy; dementia with Levy bodies; lissencephaly; environmental syndrome; Lou Gehrig's disease (for example, motor neuron disease, or amyotrophic lateral sclerosis); lumbar disc disease; Lyme disease - neurological complications; Machado-Joseph disease; macroencephaly; megacephaly; Melkerssson-Rosenthal syndrome; Meniere's disease; meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly; migraine; Miller-Fisher syndrome; mini-strokes; mitochondrial myopathies; Moebius syndrome; monomelic amyotrophy; motor neuron disease; moya-moya disease; mucopolysaccharidoses; multi-infarct dementia; motor multifocal neuropathy; multiple sclerosis and other demyelinating disorders; multiple systemic atrophy with orthostatic hypotension; muscle dystrophy; severe pseudoparalytic myasthenia gravis; diffuse myelinclastic sclerosis; early myoclonic encephalopathy; myoclonus; myopathy congenital myotonia; narcolepsy; neurofibromatosis; malignant antipsychotic syndrome; neurological manifestations of AIDS; neurological complications after lupus; neuromyotonia; neuronal ceroid lipofuscinosis; impaired neuronal migration; Nimann-Peak disease; O'Sullivan-MacLeod syndrome; occipital neuralgia; hidden dysraphy of the spinal cord; Okhtakhar syndrome; olivopontocerebellar atrophy; opsoclonus-myoclonus syndrome; optic neuritis; orthostatic hypotension; overload syndrome; paresthesia; neurodegenerative disease or disorder (Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), dementia, multiple sclerosis and other diseases and disorders associated with neuronal death); congenital paramiotonia; paraneoplastic diseases; paroxysmal seizures; Parry-Romberg syndrome; Peliceus-Merzbacher disease; periodic paralysis; peripheral neuropathy; painful neuropathy and neuropathic pain; steady vegetative state; pervasive developmental disorders; light reflex of sneezing; phytic acid accumulation disease; Peak's disease; pinched nerve; pituitary tumors; polymyositis; porencephaly; post poliomyelitis syndrome; postherpetic neuralgia; postinfectious encephalomyelitis; orthostatic hypotension; Prader-Willi syndrome; primary lateral sclerosis; prion diseases; progressive hemifacial atrophy; progressive multifocal leukoencephalopathy; progressive sclerosing polyodystrophy; progressive supranuclear palsy; benign intracranial hypertension; Ramsay-Hunt syndrome (types I and II); Rasmussen encephalitis; reflex sympathetic dystrophy syndrome; Refsum disease; recurring movement disorders; tunnel syndrome; restless legs syndrome; myelopathy associated with retroviral infection; rett syndrome; Reye's syndrome; St. Witt's dance; Sandhoff disease; Schilder's disease; schizencephaly; septo-optical dysplasia; baby concussion syndrome; shingles; Shay-Drager syndrome; Sjogren's syndrome; sleep apnea; Sotos syndrome; spasticity; split spine; spinal cord injury; spinal cord tumors; spinal muscular atrophy; muscle stiffness syndrome; stroke; Sturge-Weber syndrome; subacute sclerosing panencephalitis; subcortical atherosclerotic encephalopathy; Sydenham's chorea; fainting syringomyelia; tardive dyskinesia; Tay-Sachs disease; temporal arteritis; fixed spinal cord syndrome; Thomsen's disease; upper chest aperture syndrome; trigeminal neuralgia; Todd's palsy; Tourette's syndrome; transient ischemic attack; transmissible spongioform encephalopathies; transverse myelitis; traumatic brain injury; tremor; trigeminal neuralgia; tropical spastic paraparesis; tuberous sclerosis; vascular dementia (multi-infarction dementia); vasculitis, including temporal arteritis; Hippel-Lindau disease; Wallenberg syndrome; Werdnig-Hoffmann disease; West syndrome; whiplash injury; Williams syndrome; Wilson’s disease and Zellweger syndrome.

[0061] A “proliferative disease or disorder" includes, but is not limited to, hematopoietic neoplastic disorders involving hyperplastic / neoplastic cells of hematopoietic origin originating from myeloid, lymphoid or erythroid cells or their progenitor cells. These diseases include, but are not limited to, erythroblastic leukemia; acute promyelocytic leukemia (OPL); chronic myeloid leukemia (CML); malignant lymphomas, including but not limited to acute lymphoblastic leukemia (ALL), which includes B-cell ALL and T-cell ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL); hairy cell leukemia (ON) and Waldenstrom macroglobulinemia (MB). Additional forms of malignant lymphomas include, but are not limited to, non-Hodgkin’s lymphoma and its variants, peripheral T-cell lymphomas, adult T-cell leukemia lymphoma (ATL), cutaneous T-cell lymphoma (KTKL), large-granular lymphocytic leukemia (LGF), disease Hodgkin and Reed-Sternberg disease.

Polynucleotide and Oligonucleotide Compositions and Molecules

[0062] Targets: In one embodiment, the targets include nucleotide sequences of the PAR4 gene, including, without limitation, sense and / or antisense non-coding and / or coding sequences associated with the PAR4 gene.

[0063] The Prostate Apoptotic Response 4 (PAR4) protein is a 38 kDa protein, originally identified as a gene product, the expression of which specifically increases in prostate tumor cells undergoing apoptosis. In accordance with the important role of the PAR4 protein in apoptosis, PAR4 gene induction in cultured cells was detected exclusively during apoptosis, and ectopic expression of the PAR4 gene in N1H-3T3 cells, neurons, prostate cancer cells, and melanoma cells was shown to increase the sensitivity of these cells to apoptotic stimuli. In addition, a decrease in PAR4 gene expression is crucial for gas-protein-induced tumor survival and progression, and suppression of PAR4 protein synthesis by antisense technology prevents apoptosis in several systems, including different models of neurodegenerative disorders, further emphasizing the important role of PAR4 protein in apoptosis . At the C-terminus, PAR4 protein contains both a domain containing leucine zipper and a partially overlapping death domain (Par4DD, amino acid residues 258-332). Removal of this C-terminal portion cancels the proapoptotic function of the PAR4 protein. On the other hand, overexpression of a PAR4 protein fragment containing a leucine zipper / death domain in a dominantly negative manner prevents apoptosis induced by the full-length PAR4 protein. The PAR4 protein fragment containing the leucine zipper / death domain mediates the interaction of the PAR4 protein with other proteins by recognizing two different types of motifs: the zinc finger domain of the Wilms tumor suppressor protein WT1 and typical isoforms of protein kinase C and the arginine-rich domain from Dik kinase similar to death-associated protein (DAP). Among these interactions, the binding of PAR4 protein to atypical protein kinases C (aPKCs) and the resulting inhibition of their enzymatic activity is of particular functional significance, since aPKCs are known to play a key role in cell survival and their overexpression has been shown to nullify the ability of the protein PAR4 induce apoptosis.

[0064] In one embodiment, antisense oligonucleotides are used to prevent or treat diseases or disorders associated with members of the PAR4 family. Typical diseases and disorders mediated by the PAR4 protein that can be treated with cells / tissues regenerated from stem cells obtained using antisense compounds include: a disease or disorder associated with abnormal function and / or expression of the PAR4 gene, cancer, aberrant apoptosis , a proliferative disease or disorder, a cardiovascular disease or disorder, an inflammatory disease or disorder, a neurological disease or disorder, and aging.

[0065] In one embodiment, the modulation of the PAR4 gene with one or more antisense oligonucleotides is the administration of the latter to a patient in need thereof to prevent or treat any disease or disorder associated with abnormal expression, function, or activity of the PAR4 protein compared to normal control.

[0066] In one embodiment, the oligonucleotides are specific for the PAR4 gene polynucleotides that contain, without limitation, non-coding regions. PAR4 gene targets include PAR4 gene variants; mutants of the PAR4 gene, including SNPs; non-coding sequences of the PAR4 gene; alleles, fragments, etc. Preferably, the oligonucleotide is an antisense RNA molecule.

[0067] In accordance with embodiments of the invention, the target nucleic acid molecule is not limited to PAR4 polynucleotides alone, but extends to any of the isoforms, receptors, homologs, non-coding regions of the PAR4 gene, and the like.

[0068] In one embodiment, the oligonucleotide targets a natural antisense sequence (a natural antisense sequence for coding and non-coding regions) for PAR4 targets, including, without limitation, variants, alleles, homologs, mutants, derivatives, fragments and sequences that represent them complement. Preferably, the oligonucleotide is an antisense RNA or DNA molecule.

[0069] In one embodiment, the oligomeric compounds of the present invention also include variants in which different bases are at one or more nucleotide positions in the compound. For example, if the first nucleotide is adenine, variants containing thymidine, guanosine, cytidine, or other natural or unnatural nucleotides at this position can be obtained. This can be done in any of the positions of the antisense compound. These compounds are further investigated using the methods described in this application, in order to determine their ability to inhibit the expression of the target nucleic acid.

[0070] In some embodiments, the homology, sequence identity, or complementarity between the antisense compound and the target is from about 50% to about 60%. In some embodiments, homology, sequence identity, or complementarity is from about 60% to about 70%. In some embodiments, homology, sequence identity, or complementarity is from about 70% to about 80%. In some embodiments, homology, sequence identity, or complementarity is from about 80% to about 90%. In some embodiments, homology, sequence identity, or complementarity is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0071] The antisense compound is capable of specific hybridization when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid, causing loss of activity, and a sufficient degree of complementarity is provided to avoid non-specific binding of the antisense compound to non-target nucleotide sequences when conditions in which specific binding is desired. Such conditions include, for example, physiological conditions in the case of in vivo assays or therapeutic treatment, and the conditions under which the analyzes are performed in the case of in vitro assays.

[0072] The antisense compound, whether DNA, RNA, a chimeric compound, a substituted compound, etc., is specifically hybridizable when the binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, causing loss functions, and a sufficient degree of complementarity is ensured to avoid nonspecific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment; and in the case of in vitro assays, under the conditions in which the assays are performed.

[0073] In one embodiment, the targeted effect on the PAR4 gene, including, without limitation, antisense sequences identified and disclosed using, for example, PCR, hybridization, etc., one or more sequences corresponding to SEQ ID NOS: 2, and the like, modulates the expression or function of the PAR4 gene. In one embodiment, expression or function is increased compared to a control. In one embodiment, expression or function is suppressed compared to control.

[0074] In one embodiment, the oligonucleotides comprise nucleotide sequences corresponding to SEQ ID NOS: 3-9, including antisense sequences identified and disclosed using, for example, PCR, hybridization, etc. These oligonucleotides may contain one or more modified nucleotides, shorter or longer fragments, modified bonds, and the like. Examples of modified bonds or internucleotide bonds include phosphothioate, phosphodithioate bonds and the like. In one embodiment, the nucleotides comprise a phosphate derivative. The phosphate derivative (or modified phosphate group) that can be attached to the sugar residue or an analog of the sugar residue in the modified oligonucleotides of the present invention can be monophosphate, diphosphate, triphosphate, alkyl phosphate, alkanephosphate, phosphotioate and the like. Obtaining the above phosphate analogues and their inclusion in the composition of nucleotides, modified nucleotides and oligonucleotides, in fact, is also known and does not have to be described in this application.

[0075] The specificity and sensitivity of the antisense sequence are also used by those skilled in the art for therapeutic use. Antisense oligonucleotides have been used as therapeutic agents for the treatment of disease states in animals and humans. Antisense oligonucleotides have been safely and effectively administered to humans, and numerous clinical trials are currently underway. Thus, it has been found that oligonucleotides can be suitable therapeutic agents that can be configured to suit treatment regimens for treating cells, tissues and animals, especially humans.

[0076] In embodiments of the present invention, oligomeric antisense compounds, in particular oligonucleotides, bind to target nucleic acid molecules and modulate the expression and / or function of the molecules encoded by the target gene. Affected DNA functions include, for example, replication and transcription. Affected RNA functions include all vital functions, such as, for example, translating RNA to a protein translation site, translating proteins on an RNA matrix, splicing RNA to form one or more types of mRNA, and catalytic activity that may be present on RNA or which RNA may contribute to . Functions may be enhanced or inhibited depending on the desired functions.

[0077] Antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, oligonucleotides of an external guide sequence (EGS), splice variants, primers, probes, and other oligomeric compounds that hybridize with at least a portion of the target nucleic acid. By themselves, these compounds can be introduced in the form of single chain, double chain, partially single chain or ring oligomeric compounds.

[0078] Targeting an antisense compound to a particular nucleic acid molecule, in the context of the present invention, may be a multi-step process. The process usually begins with the identification of the target nucleic acid, the function of which is modulated. This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed on a gene matrix), the expression of which is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. According to the present invention, the target nucleic acid encodes the PAR4 gene.

[0079] The targeting process usually also includes determining at least one target region, segment or site within the target nucleic acid so that antisense interaction can occur, so that the desired effect, for example, modulation of expression, is achieved. of the present invention, the term "region" is defined as part of a target nucleic acid having at least one identifiable structure, function or characteristic. Segments are located within the regions of the target nucleic acids. "Segments" are defined as smaller regions or portions of regions within a target nucleic acid. “Sites” in the present invention are defined as positions within a target nucleic acid.

[0080] In one embodiment, the antisense oligonucleotides bind to the natural antisense sequences of the PAR4 gene and modulate the expression and / or function of the PAR4 gene (corresponding to SEQ ID NO: 1). Examples of antisense sequences include sequences corresponding to SEQ ID NOS: 2-9.

[0081] In one embodiment, antisense oligonucleotides bind to one or more PAR4 polynucleotide segments and modulate the expression and / or function of the PAR4 gene. Segments contain at least five consecutive nucleotides of sense or antisense PAR4 polynucleotides.

[0082] In one embodiment, the antisense oligonucleotides are specific for the natural antisense sequences for the PAR4 gene, wherein the binding of oligonucleotides to the natural antisense sequences for the PAR4 gene modulates the expression and / or function of the PAR4 gene.

[0083] In one embodiment, the oligonucleotide compounds comprise sequences corresponding to SEQ ID NOS: 3-9, antisense sequences identified and disclosed using, for example, PCR, hybridization, etc. These oligonucleotides may contain one or more modified nucleotides, shorter or longer fragments, modified bonds, and the like. Examples of modified bonds or internucleotide bonds include phosphothioate, phosphodithioate bonds and the like. In one embodiment, the nucleotides comprise a phosphate derivative. The phosphate derivative (or modified phosphate group) that can be attached to the sugar residue or an analogue of the sugar residue in the modified oligonucleotides of the present invention can be monophosphate, diphosphate, triphosphate, alkyl phosphate, alkane phosphate, phosphotioate and the like. Obtaining the above phosphate analogues and their inclusion in the composition of nucleotides, modified nucleotides and oligonucleotides, in fact, is also known and does not have to be described in this application.

[0084] Since, as is known in the art, the initiator codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the initiator codon is also often referred to as the “AUG codon”, “start codon” or “AUG start codon”. A smaller portion of the genes has an initiator codon containing a 5'-GUG, 5'-UUG or 5'-CUG RNA sequence; and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms “initiator codon” and “start codon” can encompass multiple codon sequences, although the initiator amino acid in each case is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). Eukaryotic and prokaryotic genes can contain two or more alternative start codons, any of which can preferably be used to initiate translation in a particular type of cell or tissue, or under a specific set of conditions. In the context of the present invention, “start codon” and “initiator codon” refer to a codon or codons that are used in vivo to initiate translation of mRNA transcribed on a matrix of a gene encoding a PAR4 protein, regardless of the sequence (s) of such codons. A translation termination codon (or “stop codon”) of a gene can have one of three sequences, i.e. 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively).

[0085] The terms “start codon region” and “initiator codon region” refer to a portion of such an mRNA or gene that spans from about 25 to about 50 consecutive nucleotides in any direction (ie, 5 ′ or 3 ′) from the initiator codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene, spanning from about 25 to about 50 consecutive nucleotides in any direction (i.e., 5 ′ or 3 ′) from the termination codon broadcasts. Therefore, the "start codon region" (or the "initiator codon region") and the "stop codon region" (or the "translation termination codon region") are both regions that the antisense compounds of the present invention can effectively target.

[0086] An open reading frame (OPC) or "coding region", which, as is known in the art, refers to the region between the initiator codon and the translation termination codon, is also an area that can be effectively targeted. In the context of the present invention, the target region is an intragenic region, including an initiator codon or translation termination codon of an open reading frame (OPC) of a gene.

[0087] Another target region includes a 5'-untranslated region (5'-UTR) denoting in the art the portion of mRNA in the 5'-direction from the initiator codon and, thus, containing nucleotides between the 5'-capping site and the initiator codon of mRNA (or the corresponding nucleotides of the gene). Another target region includes a 3'-untranslated region (3'-UTR), denoting in the art a portion of mRNA in the 3'-direction from the translation termination codon, and thus containing nucleotides between the translation termination codon and the 3'-end mRNA (or the corresponding nucleotides of the gene). The 5'-site of mRNA capping contains an N7-methylated guanosine residue attached to the outermost 5'-residue of mRNA via a 5'-5'-triphosphate bond. The region of the 5'-cap of mRNA is believed to include the actual structure of the 5'-cap, as well as the first 50 nucleotides adjacent to the capping site. Another target region of the present invention is a 5′-cap region.

[0088] Although some eukaryotic mRNA transcripts are directly translated, many of them contain one or more regions known as "introns" that are excised from the transcript before it is translated. The remaining (and thus translated) regions are known as “exons,” and they bind to each other to form a continuous mRNA sequence. In one embodiment, targeting to splicing sites, i.e. to the intron-exon junction or the exon-intron junction, is particularly suitable in cases in which aberrant splicing is involved in the development of the disease or in which the supersynthesis of a particular splicing product is involved in the development of the disease. An aberrant merger due to rearrangement or deletion is another embodiment of the target site. mRNA transcripts obtained from the splicing process of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. Introns can be effectively used as a target using antisense compounds aimed at, for example, DNA or pre-mRNA.

[0089] In one embodiment, antisense oligonucleotides bind to the coding and / or non-coding regions of the target polynucleotide and modulate the expression and / or function of the target molecule.

[0090] In one embodiment, antisense oligonucleotides bind to naturally occurring antisense polynucleotides and modulate the expression and / or function of the target molecule.

[0091] In one embodiment, antisense oligonucleotides bind to sense polynucleotides and modulate the expression and / or function of the target molecule.

[0092] Alternative RNA transcripts can be obtained from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts derived from the same genomic DNA that differ from other transcripts derived from the same genomic DNA by the position of their start or stop codon and contain both intron and and exon sequences.

[0093] After removal of one or more exon or intron regions or parts thereof during the splicing process, pre-mRNA variants become sources of smaller “mRNA variants”. Therefore, mRNA variants are processed pre-mRNA variants, and each unique pre-mRNA variant should always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If splicing of the pre-mRNA variant does not occur, then the pre-mRNA variant is identical to the mRNA variant.

[0094] Variants can be obtained by using alternative signals to start or stop transcription. Pre-mRNA and mRNA can have more than one start codon or stop codon. Variants that originate from pre-mRNAs or mRNAs using alternative start codons are known as “alternative start-ups” of these pre-mRNAs or mRNAs. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of these pre-mRNAs or mRNAs. One particular type of alternative stop-variant is the “polyA-variant”, in which multiple transcripts obtained are the result of the alternative selection of one of the “polyA-stop-signals” by a transcription apparatus, which results in transcripts that end up at unique polyA-sites. In the context of the present invention, the types of variants described in this application are also embodiments of target nucleic acids.

[0095] The regions in the target nucleic acid with which antisense compounds hybridize are defined as part of the target region of at least 5 nucleotides in length that the active antisense compound is targeted at.

[0096] While the specific sequences of some exemplary target segments are provided herein, as one skilled in the art will recognize, these sequences serve to illustrate and describe specific embodiments within the scope of the present invention. Additional target segments are easily identifiable by a person skilled in the art in light of the present description.

[0097] 5-100 nucleotide target segments containing a chain of at least five (5) consecutive nucleotides selected from nucleotides within the illustrative preferred target segments are also believed to be suitable for targeting.

[0098] Target segments can include DNA or RNA sequences that contain at least 5 consecutive nucleotides from the 5'-end of one of the illustrative preferred target segments (the remaining nucleotides are a sequence of the same DNA or RNA, starting immediately above from the 5'-end of the target segment and extending from about 5 to about 100 nucleotides). Similarly, preferred target segments are represented by DNA or RNA sequences that contain at least 5 consecutive nucleotides from the 3 ′ end of one of the illustrative preferred target segments (the remaining nucleotides are a sequence of the same DNA or RNA starting immediately below 3 ′ the end of the target segment and lasting from about 5 to about 100 nucleotides). A person skilled in the art having target segments illustrated in this application will be able to identify further preferred target segments without further experimentation.

[0099] Once one or more target regions, target segments, or target sites are identified, antisense compounds that are sufficiently complementary to the target are selected, i.e. hybridize quite well and with sufficient specificity to obtain the desired effect.

[00100] In embodiments of the present invention, oligonucleotides bind to the antisense strand of a particular target. Oligonucleotides have a length of at least 5 nucleotides and can be synthesized in such a way that each oligonucleotide targets overlapping sequences so that the oligonucleotides are synthesized to cover the entire length of the target polynucleotide. These targets also include coding and non-coding regions.

[00101] In one embodiment, it is preferred to target specific nucleic acids with antisense oligonucleotides. Targeting an antisense compound to a specific nucleic acid is a multi-step process. The process usually begins by identifying a nucleic acid sequence whose function is to be modulated. This sequence may be, for example, a cell gene (or mRNA transcribed on a matrix of that gene), the expression of which is associated with a particular disorder or disease state, or a non-coding polynucleotide, such as, for example, non-coding RNA (ncRNA).

[00102] RNA molecules can be classified into (1) messenger RNAs (mRNAs), which are translated into proteins, and (2) RNAs that do not encode proteins (NK RNAs). ncRNAs include microRNAs, antisense transcripts, and other transcription units (ETs) that contain high density stop codons and do not have any extended “open reading frames”. Many ncRNAs apparently start at the initiation sites in the 3'-untranslated regions (3'-UTR) of protein loci encoding. ncRNAs are generally rare and at least half of the ncRNAs that have been sequenced by the FANTOM International RNA Analysis Consortium are not likely to be polyadenylated. Most researchers, for obvious reasons, have focused on polyadenylated mRNAs that are processed and exported to the cytoplasm. It has recently been shown that the collection of unpolyadenylated nuclear RNAs can be very large and that many such transcripts appear from the so-called intergenic regions. The mechanism by which ncRNAs can regulate gene expression is through base pairing with target transcripts. RNAs that function by base pairing can be grouped into (1) cis-encoded RNAs that are encoded in the same portion of a gene, but on the opposite chain to the RNAs that they act, thus showing perfect complementarity of their target, and (2) trans-encoded RNAs, which are encoded in chromosome regions other than the RNA regions on which they act, and do not show a perfect degree of potential base pairing with their targets.

[00103] Without reference to any particular theory, modification of an antisense polynucleotide with antisense oligonucleotides described herein may alter the expression of corresponding sense information RNAs. However, such regulation can be either inconsistent (antisense “knockdown” leads to an increase in the level of information RNA) or consistent (antisense “knockdown” leads to a concomitant decrease in the level of information RNA). In these cases, the antisense oligonucleotides can be targeted at the overlapping or non-overlapping parts of the antisense transcript, resulting in its "knockdown" or sequestration. Coding as well as non-coding antisense {molecules} can be targeted in the same way, and both categories are able to regulate the corresponding semantic transcripts - either in a coordinated or inconsistent manner. Strategies that are used to identify new oligonucleotides for interacting with targets can be based on the “knockdown” of antisense RNA transcripts with antisense oligonucleotides or any other modulation of the desired target.

[00104] Strategy 1: In the case of inconsistent knockdown regulation of the antisense transcript, the expression of the normal (sense) gene is increased. If this gene encodes a known or suspected drug target, then the “knockdown” of its antisense equivalent can presumably mimic the action of a receptor agonist or enzyme stimulator.

[00105] Strategy 2: In the case of coordinated regulation, it is possible to simultaneously “knock down” both antisense and sense transcripts and thereby achieve a synergistic decrease in the expression of the usual (sense) gene. If, for example, an antisense oligonucleotide is used to achieve a “knockdown”, then this strategy can be used to target a single antisense oligonucleotide to a sense transcript and another antisense oligonucleotide to a corresponding antisense transcript or a single energetically symmetric antisense oligonucleotide transcripts.

[00106] According to the present invention, antisense compounds include antisense oligonucleotides, ribozymes, external guide sequence oligonucleotides (EGS), siRNA compounds, single or double stranded RNA interfering (RNAi) compounds, such as siRNA compounds, and other oligomeric compounds that hybridize with at least a portion of the target nucleic acid and modulate its function. Accordingly, they may be DNA, RNA, DNA-like compounds, RNA-like compounds or mixtures thereof, or may be mimetics of one or more of these compounds. Such compounds may be single-stranded, double-stranded, ring or hairpin oligomeric compounds and may contain structural elements, such as internal or terminal loops, non-complementary positions or loops. Antisense compounds are usually prepared as linear compounds, but they can be combined or otherwise prepared to produce ring and / or branched compounds. Antisense compounds may include constructs such as, for example, two hybridized chains forming a fully or partially double-stranded compound or a single chain with self-complementarity sufficient to hybridize and form a fully or partially double-stranded compound. These two chains can be connected by internal bonds, having free 3'- or 5'-ends, or can be connected with the formation of a continuous hairpin-like structure or loop. The specified hairpin-like structure may contain a sticky end at either the 5'- or 3'-end, forming an extension of a single-chain nature. These double-stranded compounds may optionally contain sticky ends. Other modifications may include conjugated groups attached to one of the ends, nucleotides at specific positions, sugars at certain positions, or to one of the internucleoside bonds. Alternatively, these two chains may be linked via a non-nucleic fragment or linker group. Formed by a single strand of dsRNA, it can take the form of a self-complementary hairpin molecule, folding with itself to form a duplex. Thus, these dsRNAs can be fully or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines, however, in some embodiments, these gene expression or function is increased. When formed from two chains or a single chain, which takes the form of a self-complementary hairpin molecule, folding with itself to form a duplex, these two chains (or duplex-forming regions of a single chain) are complementary RNA chains that pair with Watson-Crick bases.

[00107] When introduced into the system, the compounds of the present invention can cause the action of one or more enzymes or structural proteins by cleavage or other modifications of the target nucleic acid, or they can work by the occupancy-based mechanism. In general, nucleic acids (including oligonucleotides) can be described as “DNA-like” (that is, containing, as a rule, one or more 2'-deoxysugars and, as a rule, predominantly T rather than U bases ) or "RNA-like" (i.e., containing, as a rule, one or more 2'-hydroxy or 2'-modified Sugars and, as a rule, mainly U rather than T bases). Nucleic acid helices can be represented by structures of more than one type, most often A- and B-forms. It is believed that, in the general case, oligonucleotides that have a structure similar to the B form are "DNA-like" and oligonucleotides that have a structure similar to the A-form are "RNA-like." In some (chimeric) embodiments, the antisense compound may contain both A- and B-form regions.

[00108] In one embodiment, the desired oligonucleotides or antisense compounds comprise at least one of the following: antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides containing modified bonds, interfering RNAs (RNAs), small interfering RNAs (miRs); micro interfering RNA (micro RNA); small transient RNA (mvRNA); or short hairpin RNAs (kshRNA); small RNA-induced gene activation (RNA); small activating RNAs (mRNAs) or combinations thereof.

[00109] dsRNA can also activate gene expression, a mechanism called "small RNA-induced gene activation" or RNA. dsRNAs targeted at gene promoters induce powerful transcriptional activation of associated genes. RNA has been demonstrated in human cells using synthetic dsRNAs called “small activating RNAs” (mRNAs). It is currently unknown whether RNA is conserved in other organisms.

[00110] It has been found that small double-stranded RNAs (dsRNAs), such as small interfering RNAs (miRNAs) and miRNAs (microRNAs), trigger an evolutionarily conserved mechanism known as RNA interference (RNAi). RNAi inevitably leads to silencing of genes due to chromatin remodeling, which leads to inhibition of transcription, degradation of complementary mRNA, or block protein translation. Nevertheless, in the examples described in detail in the subsequent experimental part, it was shown that oligonucleotides cause an increase in the expression and / or function of PAR4 polynucleotides and the products encoded by them. dsRNA can also act as small activating RNA (mRNA). Without reference to any particular theory, by targeting sequences in the promoters of target genes, mRNAs induce expression of the target gene as a result of a phenomenon called dsRNA-induced transcriptional activation (RNA).

[00111] According to another embodiment, the “preferred target segments” defined herein can be used to select additional compounds that modulate the expression of PAR4 polynucleotides. "Modulators" are compounds that lower or increase the expression of a nucleic acid molecule encoding PAR4 and contain at least a 5-nucleotide region complementary to a preferred target segment. The selection method includes the steps of contacting a preferred target segment of a nucleic acid molecule encoding sense or natural antisense PAR4 polynucleotides with one or more potential modulators, and selecting one or more potential modulator lowering or increasing the expression of a nucleic acid molecule encoding PAR4 polynucleotides, for example, SEQ ID NO: 3-9. After the ability of the potential modulator (s) to modulate (e.g., decrease or increase) the expression of a nucleic acid molecule encoding PAR4 polynucleotides has been confirmed, this modulator can be used for further studies of the functions of PAR4 polynucleotides, or for use as an agent for research, diagnosis or treatment according to the present invention.

[00112] Targeted exposure via a natural antisense sequence preferably modulates the function of the target gene. For example, the PAR4 gene (in particular, under access number NM_019113). In one embodiment, the target is an antisense polynucleotide of the PAR4 gene. According to one embodiment, the antisense oligonucleotide is aimed at sense and / or natural antisense sequences of PAR4 polynucleotides (in particular, under accession number NM_019113), their variants, alleles, isoforms, homologs, mutants, derivatives, fragments and complementary sequences. Preferably, said oligonucleotide is an antisense molecule, and said targets include coding and non-coding regions of antisense and / or sense PAR4 polynucleotides.

[00113] Preferred target segments of the present invention can also be combined with the corresponding complementary antisense compounds of the present invention to form stabilized double-stranded (duplex) oligonucleotides.

[00114] It has been shown in the art that such double-stranded oligonucleotide fragments modulate target expression and regulate translation, as well as RNA processing, through an antisense mechanism. In addition, these double-stranded fragments can undergo chemical modifications. For example, it has been shown that such double-stranded fragments inhibit the target by classical hybridization of the antisense strand of the indicated duplex with the target, thereby triggering enzymatic cleavage of the target.

[00115] According to one embodiment, the antisense oligonucleotide targets PAR4 polynucleotides (in particular, access number NM_019113), their variants, alleles, isoforms, homologs, mutants, derivatives, fragments, and complementary sequences. Preferably, said oligonucleotide is an antisense molecule.

[00116] According to embodiments of the present invention, the target nucleic acid molecule is not limited to PAR4 per se and can be represented by any isoforms, receptors, homologs, and the like. PAR4 molecules.

[00117] According to one embodiment, the oligonucleotide targets a natural antisense sequence of PAR4 polynucleotides, for example, polynucleotides shown in SEQ ID NO: 2, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereof. Examples of antisense oligonucleotides are represented by the sequences of SEQ ID NO: 3-9.

[00118] According to one embodiment, said oligonucleotides are complementary or bind to nucleic acids of PAR4 antisense sequences, including, but not limited to, non-coding sense and / or antisense sequences associated with PAR4 polynucleotides, and modulate the expression and / or function of PAR4 molecules.

[00119] According to one embodiment, said oligonucleotides are complementary to or bind to the natural antisense PAR4 nucleic acid sequences of SEQ ID NO: 2 and modulate the expression and / or function of PAR4 molecules.

[00120] According to one embodiment, the oligonucleotides comprise sequences of at least 5 consecutive nucleotides of the sequences SEQ ID NO: 3-9, and modulate the expression and / or function of PAR4 molecules.

[00121] These polynucleotide targets include PAR4, including members of this family, variants of PAR4; mutants PAR4, including SNP; non-coding sequences of PAR4; PAR4 alleles; species options, fragments, etc. Preferably, said oligonucleotide is an antisense molecule.

[00122] In one embodiment, an oligonucleotide that targets PAR4 polynucleotides includes: antisense RNA, interfering RNA (mRNA), small interfering RNA (siRNA); micro-interfering RNA (miRNA), small, transient RNA (mvRNA), or short hairpin RNA (kshRNA); small RNA-induced gene activation (RNA), or small activating RNA (mRNA).

[00123] In one embodiment, the targeted action on PAR4 polynucleotides, for example, SEQ ID NO: 2, modulates the expression or function of such targets. In one embodiment, expression or function is up-regulated as compared to control. In one embodiment, expression or function is down-regulated as compared to control.

[00124] In one embodiment, antisense compounds include the sequences shown in SEQ ID NO: 3-6. Such oligonucleotides may contain one or more modified nucleotides, shorter or longer fragments, modified bonds, and the like.

[00125] According to one embodiment of the sequence, SEQ ID NO: 3-9 comprise one or more ZNA nucleotides. Table 1 shows typical antisense oligonucleotides suitable for use in the methods of the present invention.

Table 1 Sequence ID The name of the antisense sequence Sequence SEQ ID NO: 3 CUR-1564 G * T * G * C * T * T * C * T * C * T * T * T * C * T * T * C * C * T * G * A SEQ ID NO: 4 CUR-1565 G * C * A * G * C * A * C * C * A * C * A * G * A * C * T * T * C * C * T * G SEQ ID NO: 5 CUR-1566 C * T * G * G * A * G * G * A * G * T * G * G * A * A * G * G * C * G * A * A SEQ ID NO: 6 CUR-1567 A * G * G * T * C * G * T * C * T * C * C * T * G * G * C * A * T * C * C * T SEQ ID NO: 7 CUR-1568 C * A * G * G * A * A * G * G * A * A * G * A * G * G * A * G * G * C * A * G SEQ ID NO: 8 CUR-1569 A * A * T * C * T * T * A * A * C * A * C * C * T * C * A * G * C * C * C SEQ ID NO: Cur- C * C * T * T * T * G * G * G * A * A * T * A * T * G * G * C * G * A * C * C * G SEQ ID NO: 9 CUR-15 70 C * C * T * T * T * G * G * G * A * A * T * A * T * G * G * C * G * A * C * C * G

[00126] Modulation of the desired target nucleic acid can be accomplished by several methods known in the art. For example, antisense oligonucleotides, siRNAs, etc. Enzymatic nucleic acid molecules (e.g., ribozymes) are nucleic acid molecules capable of catalyzing one or more of a variety of reactions, including the ability to repeatedly cleave other individual nucleic acid molecules in a sequence-specific manner in a sequence-sensitive manner. Such enzymatic nucleic acid molecules can be used, for example, for targeted action on almost any RNA transcript.

[00127] Due to sequence specificity, trans-cleaving enzymatic nucleic acid molecules are promising potential therapeutic agents for treating human diseases. Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets in a common pool of cellular RNA. This cleavage makes these mRNAs non-functional and cancels the expression of the protein from this RNA. Thus, protein synthesis associated with a disease state can be selectively blocked.

[00128] In general, enzymatic nucleic acids with RNA cleavage activity act, firstly, by binding to the target RNA. This binding occurs through a target-binding fragment of the enzymatic nucleic acid, which is located in close proximity to the enzymatic fragment of the specified molecule that cleaves the target RNA. Thus, this enzymatic nucleic acid first recognizes the target RNA, and then binds to it by complementary base pairing, and after binding to the corresponding site enzymatically cleaves the target RNA. The strategic cleavage of such a target RNA will destroy its ability to directly synthesize the encoded protein. After the enzymatic nucleic acid has bound and cleaved the target RNA, it is disconnected from this RNA, freed for another target and can re-bind and cleave new targets.

[00129] A number of approaches, such as in vitro selection (extraction) strategies (Orgel, (1979) Proc. R. Soc. London, B 205, 435), have been used to produce novel nucleic catalysts capable of catalyzing various reactions, such as cleavage and ligation of phosphodiester bonds and amide bonds.

[00130] Obtaining ribozymes with optimal catalytic activity will make a significant contribution to any strategy involving the use of RNA-cleaving ribozymes for regulating gene expression. The hammerhead type ribozyme, for example, functions at a catalytic reaction rate (kcat) of about 1 min-1 in the presence of saturating (10 mM) Mg2 + cofactor concentrations. It was shown that the synthetic “RNA-ligase” ribozyme catalyzes the corresponding self-change reaction at a rate of approximately 100 min-1. In addition, it is known that certain modified hammerhead-type ribozymes with substrate-binding DNA consisting of “handles” catalyze RNA cleavage at speeds of up to 100 min-1. Finally, substitution of a specific residue of the catalytic core of the “hammer head” with specific nucleotide analogues gives modified ribozymes that demonstrate an increase in the catalytic reaction rate up to 10-fold. These results show that ribozymes can provide chemical transformations with catalytic rates far exceeding those demonstrated in vitro by most natural self-cleaving ribozymes. In this case, it is possible that the structures of some self-cleaving ribozymes can be optimized in order to obtain maximum catalytic activity, or that completely new RNA motifs can be obtained that demonstrate significantly increased rates of phosphodiester cleavage of RNA.

[00131] Intramolecular cleavage of an RNA substrate by an RNA catalyst corresponding to the hammerhead model was first shown in 1987 (Uhlenbeck, O.S. (1987) Nature, 328: 596-600). The specified RNA catalyst was isolated and reacted with various RNA molecules, confirming that it really is a catalyst.

[00132] Catalytic RNAs constructed on the basis of the hammerhead motif were used to cleave specific target sequences by carrying out suitable base pairing in the catalytic RNA to provide the necessary base pairing with the target sequences. This made it possible to use the indicated catalytic RNA to cleave specific target sequences and show that catalytic RNAs constructed according to the hammerhead model are thought to be capable of cleaving specific substrate RNAs in vivo.

[00133] RNA interference (RNAi) has become a powerful tool for modulating gene expression in mammals and mammalian cells. This approach involves the production of small interfering RNAs (siRNAs) as RNA itself or as DNA, using an expression plasmid or virus and the coding sequence for small hairpin RNAs that are processed in siRNAs. This system provides efficient transport of pre-miRNAs to the cytoplasm, where they are active, and allows the use of regulated and tissue-specific gene expression promoters

[00134] In one embodiment, the oligonucleotide or antisense compound comprises an oligomer or polymer of ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA), or a mimetic, chimera, analog or homolog thereof. This term includes oligonucleotides consisting of naturally occurring nucleotides, sugars and covalent internucleoside (skeletal) bonds, as well as oligonucleotides containing non-naturally occurring fragments that function in a similar way. Such modified or substituted oligonucleotides are often preferable to the native forms, as they possess the necessary properties, such as, for example, increased uptake by cells, increased binding capacity for the target nucleic acid, and increased stability in the presence of nucleases.

[00135] According to the present invention, said oligonucleotides or “antisense compounds” include antisense oligonucleotides (eg, RNA, DNA, their mimetics, chimeras, analogues or homologs), ribozymes, external auxiliary sequence oligonucleotides (EGS), siRNA compounds, single or double stranded RNA interfering (RNAi) compounds, such as siRNA compounds, kRNAs, arRNAs, and other oligomeric compounds that hybridize with at least a portion of the target nucleic acid and modulate its function. Accordingly, they may be DNA, RNA, DNA-like compounds, RNA-like compounds or mixtures thereof, or may be mimetics of one or more of these compounds. Such compounds may be single-stranded, double-stranded, ring or hairpin oligomeric compounds and may contain structural elements, such as internal or terminal loops, non-complementary positions or loops. Antisense compounds are usually prepared as linear compounds, but they can be combined or otherwise prepared to produce ring or / and branched compounds. Antisense compounds may include constructs such as, for example, two hybridized chains forming a fully or partially double-stranded compound, or a single chain with sufficient self-complementarity to hybridize and form a fully or partially double-stranded compound. These two chains can be connected by internal bonds having free 3 'or 5' ends, or can bind to form a continuous hairpin structure or loop. The specified hairpin-like structure may contain a sticky end at the 5 'or 3' end, forming an extension of a single-chain nature. These double-stranded compounds in some cases may contain sticky ends. Other modifications may include conjugated groups attached to one of the ends, nucleotides at specific positions, sugars at certain positions, or to one of the internucleoside bonds. Alternatively, these two chains can be linked via a non-nucleic fragment or linker group. The dsRNA formed by a single chain can take the form of a self-complementary hairpin molecule, folding with itself with the formation of a duplex. Thus, these dsRNAs can be fully or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines. When forming from two chains or a single chain, which takes the form of a self-complementary hairpin molecule, folding with itself with the formation of a duplex, these two chains (or duplex-forming sections of a single chain) are complementary RNA chains that pair according to Watson-Crick.

[00136] When introduced into the system, the compounds of the present invention can lead to the activation of one or more enzymes or structural proteins, affecting cleavage or other modifications of the target nucleic acid, or they can act through “filling” mechanisms. Typically, nucleic acids (including oligonucleotides) can be called "DNA-like" (ie, typically containing one or more 2'-deoxy sugar and, as a rule, predominantly T rather than U, bases) or "RNA-like" (ie, typically containing one or more 2'-hydroxyl or 2'-modified sugars and, as a rule, predominantly T rather than U, bases). Nucleic acid helices can be represented by structures of more than one type, most often A- and B-forms. Assume that, as a rule, oligonucleotides having a structure of type B-form, "DNA-like", and having a structure of type A-form, "RNA-like." In some embodiments (with chimeras), the antisense compound may contain both A- and B-shaped regions.

[00137] The antisense compounds of the present invention may comprise an antisense fragment of about 5 to about 80 nucleotides (ie, from about 5 to about 80 linked nucleosides) in length. This refers to the length of said antisense chain or part of said antisense compound. In other words, the single-stranded antisense compound proposed in the present invention contains from 5 to about 80 nucleotides, and the double-stranded antisense compound proposed in the present invention (such as, for example, dsRNA) contains a sense and antisense strand or fragments from 5 to about 80 nucleotides long. It will be clear to a person skilled in the art that by this are meant antisense fragments of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleotides, or belonging to any range within the specified.

[00138] In one embodiment, the antisense compounds of the present invention comprise antisense fragments of 10-50 nucleotides long. It will be clear to a person skilled in the art that oligonucleotides are included that contain antisense fragments of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides, or belonging to any range within the specified. In some embodiments, the length of said oligonucleotides is 15 nucleotides.

[00139] According to one embodiment, the antisense or oligonucleotide compounds of the present invention comprise antisense fragments of 12 or 13 to 30 nucleotides in length. It will be clear to a person skilled in the art that antisense compounds containing antisense fragments 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 in length are included here. , 27, 28, 29 or 30 nucleotides, or belonging to any range within the specified.

[00140] In one embodiment, the oligomeric compounds of the present invention also include those in which different bases are present at one or more nucleotide positions of the compound. For example, if the first nucleotide is adenosine, variants that contain thymidine, guanosine or cytidine at this position can be obtained. This can be done at any position of said antisense or dsRNA compound. Such compounds are then tested using the methods described in this application to determine their ability to inhibit the expression of the target nucleic acid.

[00141] In some embodiments, the degree of homology, sequence identity, or complementarity between said antisense compound and target is from about 40% to about 60%. In some embodiments, the degree of homology, sequence identity, or complementarity is from about 60% to about 70%. In some embodiments, the degree of homology, sequence identity, or complementarity is from about 70% to about 80%. In some embodiments, the degree of homology, sequence identity, or complementarity is from about 80% to about 90%. In some embodiments, homology, sequence identity, or complementarity is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[00142] In one embodiment, antisense oligonucleotides, such as, for example, the nucleic acid molecules shown in SEQ ID NOS: 2-9, include one or more substitutions or modifications. In one embodiment, said nucleotides are replaced by closed nucleic acids (NNAs).

[00143] According to one embodiment, said oligonucleotides specifically target one or more regions of nucleic acid molecules of sense and / or antisense coding and / or non-coding sequences associated with PAR4 and the sequences shown in SEQ ID NO: 1 and 2. Oligonucleotides also target overlapping regions corresponding to SEQ ID NO: 1 and 2.

[00144] Some preferred oligonucleotides of the present invention are chimeric oligonucleotides. "Chimeric oligonucleotides" or "chimeras" in the context of the present invention are oligonucleotides that contain two or more chemically distinct regions, each of which consists of at least one nucleotide. Such oligonucleotides typically contain at least one region of modified nucleotides that provides one or more useful properties (such as, for example, increased resistance to nucleases, increased absorption by cells, increased binding ability with respect to the target) and a site that represents substrate for enzymes capable of cleaving RNA: DNA or RNA: RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA chain of RNA: DNA duplex. Activation of RNase H, thus, leads to cleavage of the target RNA, thereby significantly increasing the efficiency of antisense modulation of gene expression. Accordingly, when using chimeric oligonucleotides, comparable results can often be obtained with shorter oligonucleotides compared to using phosphorothioate deoxy oligonucleotides hybridizing with the same target region. The cleavage of the target RNA can be determined in a standard way by gel electrophoresis and, if necessary, the concomitant nucleic acid hybridization techniques known in the art. In one embodiment, the chimeric oligonucleotide comprises at least one region modified to increase its ability to bind to the target, and typically a region that functions as an RNase H substrate. The binding ability of the oligonucleotide with respect to the target (in this case, nucleic acid, encoding ras) is determined in a standard way by measuring the melting point of an oligonucleotide / target pair, which is the temperature at which the oligonucleotide and target dissociate; dissociation is determined spectrophotometrically. The higher the melting point, the greater the binding ability of the indicated oligonucleotide with respect to the target.

[00145] The chimeric antisense compounds of the present invention can be prepared as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and / or oligonucleotide mimetics according to the above descriptions. Such compounds are also known in the art as hybrids or hapmers. Examples of US patents describing the preparation of such hybrid structures include, but are not limited to, US Pat. No. 5,013,830; 5,149,797; 522,0007; 5,256,775; 5,366,878; 5,407,711; 5,491,133; 5,565,350; 5,623,065; 5652355; 5652356; and 5700922, each of which is incorporated into this application by reference.

[00146] According to one embodiment, the modifiable portion of said oligonucleotide comprises at least one nucleotide modified at the T position of sugar, most preferably 2'-O-alkyl, 2'-O-alkyl-O-alkyl or 2'-fluoro- modified nucleotide. In another embodiment, RNA modifications include 2'-fluoro, 2'-amino and 2'-O-methyl modifications of pyrimidine ribose, nitrogen-free residues or a reverse base at the 3'-end of said RNA. Such modifications of the oligonucleotides are carried out by standard methods; it was shown that such oligonucleotides have a higher melting point (i.e., greater ability to bind to the target) than 2'-deoxyoligonucleotides relative to a specific target. As a result of this increased binding capacity, the inhibition of gene expression of the RNAi oligonucleotide is significantly enhanced. RNase H is a cellular endonuclease that cleaves the RNA RNA chain: duplex DNA; activation of this enzyme, thus, leads to cleavage of the target RNA, and, therefore, can significantly increase the efficiency of RNAi inhibition. The cleavage of the target RNA can be confirmed in the usual way by gel electrophoresis. In one embodiment, said chimeric oligonucleotide is also modified to increase nuclease resistance. Cells contain many exo- and endonucleases capable of degrading nucleic acids. It was shown that some nucleotide and nucleoside modifications give the oligonucleotide, which they are part of, greater resistance to nuclease cleavage compared to the native oligodeoxynucleotide. Resistance to nucleases is measured in the usual way, incubating oligonucleotides with cell extracts or solutions with isolated nucleases and measuring the content of intact oligonucleotides after a certain time, usually by gel electrophoresis. Oligonucleotides modified to increase resistance to nucleases remain intact for a longer time compared to unmodified oligonucleotides. It has been shown that a number of modifications of oligonucleotides enhance or confer resistance to nucleases. Oligonucleotides that contain at least one phosphorothioate modification are currently more preferred. In some cases, oligonucleotide modifications that enhance the ability to bind to the target are also, independently, able to increase resistance to nucleases.

[00147] Specific examples of certain preferred oligonucleotides of the present invention include modified backbones, for example phosphorothioates, phosphotriethers, methylphosphonates, short chain alkyl or cycloalkyl bonds between sugars or short chain heteroatomic or heterocyclic bonds between sugars. Most preferred are oligonucleotides with phosphorothioate backbones and heteroatom backbones, in particular CH2-NH-O-CH2, CH, -N (CH3) -O-CH2 [known as methylene (methylimino) or MMI backbone], CH2-ON ( CH3) -CH2, CH2-N (CH3) -N (CH3) -CH2 and ON (CH3) -CH2-CH2 backbones, where the native phosphodiester backbone is presented as O-P-O-CH,). Amide cores described in De Mesmaeker et al. (1995) Ace. Chem. Res. 28: 366-374 are also preferred. Also preferred are oligonucleotides containing morpholine backbones (Summerton and Weller, US Pat. No. 5,034,506). According to another embodiment, such as a peptide nucleic acid (PNA) backbone, the oligonucleotide phosphodiester backbone is replaced by a polyamide backbone, the nucleotides are directly or indirectly bound to the nitrogen atoms of the azagroups of the polyamide backbone. Oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides contain one of the following substituents at position 2 ': OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 O (CH2) n CH3, O (CH2) n NH2 or O (CH2) n CH3, where n accepts values from 1 to about 10; C1-C10 lower alkyls, alkoxyalkoxy, substituted lower alkyls, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleavage group; reporter group; intercalator; a group that improves the pharmacokinetic properties of the oligonucleotide; or a group that improves the pharmacodynamic properties of the oligonucleotide, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy [2'-O-CH2 CH2 OCH3, also known as 2'-O- (2-methoxyethyl)]. Other preferred modifications include 2'-methoxy (2'-O-CH3), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions of the oligonucleotide, in particular at the 3 'position of the sugar 3' of the terminal nucleotide and 5 'position of the 5' terminal nucleotide. Oligonucleotides may also contain sugar mimetics, such as cyclobutyls, instead of the pentofuranosyl group.

[00148] Oligonucleotides may also contain, optionally or alternatively, modifications or substitutions of the nucleic base (often referred to simply as “base” in the art). As used herein, the terms “unmodified” or “natural” nucleotides include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleotides include nucleotides found rarely or temporarily in natural nucleic acids, for example, hypoxanthine, 6-methyladenine, 5-Me pyrimidines, in particular 5-methylcytosine (also called 5-methyl-2 'deoxycytosine and often referred to in the art such as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosil HMC, as well as synthetic nucleotides, for example, 2-aminoadenine, 2- (methylamino) adenine, 2- (imidazolylalkyl) adenine, 2- (aminoalkylamino adenine or other heterosubstituted alkyladenins, 2-thiouracil, 2 -thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine and 2,6-diaminopurin. A “universal” base known in the art, for example, inosine, may also be included. It has been shown that 5-Me-C substitutions increase the stability of nucleic acid duplexes by 0.6-1.2 ° C and are currently preferred base substitutions.

[00149] Another modification of the oligonucleotides of the present invention includes chemically binding one or more fragments or conjugates to said oligonucleotide to increase the activity or uptake of said oligonucleotide by cells. Such fragments include, but are not limited to, lipid moieties such as cholesterol moieties, cholesteryl moieties, aliphatic chains, for example, dodecanediol or undecyl moieties, a polyamine or polyethylene glycol chain, or adamantane-acetic acid. Oligonucleotides containing lipophilic fragments and methods for preparing such oligonucleotides are known in the art, see, for example, US Pat. Nos. 5,138,045, 5,218,105 and 5,459,255.

[00150] It is not necessary that all positions of a particular nucleotide be modified uniformly, and in fact, more than one of the above modifications may be contained in one oligonucleotide, or even in one nucleoside in the oligonucleotide. The present invention also includes oligonucleotides that are chimeric oligonucleotides as defined above.

[00151] According to another embodiment, the nucleic acid molecule of the present invention is conjugated to another fragment, including, but not limited to, baseless nucleotides, polyesters, polyamines, polyamides, peptides, hydrocarbons, lipids or polycarbon compounds. It will be clear to those skilled in the art that such molecules can be linked to any one or more than one nucleotide containing a nucleic acid molecule in several positions on a sugar, base or phosphate group.

[00152] Oligonucleotides used according to the present invention can be conveniently and simply obtained using the well-known solid-phase synthesis technique. Equipment for such synthesis is commercially available from a number of suppliers, including Applied Biosystems. You can also apply any other methods of such synthesis; the actual synthesis of the indicated oligonucleotide is not difficult for the average person skilled in the art. It is also well known that similar techniques can be used to obtain other oligonucleotides, such as phosphorothioate and alkylate derivatives. It is also well known that similar techniques and commercially available modified amidites and glass-based products with controlled pore size (CPG), such as biotin-, fluorescein-, acridine- or psoralen-modified amidites and / or CPG (available from Glen Research, Sterling, are available) VA) for the synthesis of fluorescently labeled, biotinylated or other modified oligonucleotides, such as cholesterol-modified oligonucleotides.

[00153] In accordance with the present invention, the use of modifications is contemplated, for example, the use of ZNA monomers to enhance the strength, specificity and duration of action and extend the range of routes of administration of oligonucleotides consisting of specific components such as MOE, ANC, FANK, FT, etc. . This can be achieved by replacing certain monomers in specific oligonucleotides with LNA monomers. The ZNA-modified oligonucleotide may have a size corresponding to the parent compound, or may be larger, or, preferably, smaller. Preferably, such ZNA-modified oligonucleotides contain less than about 70%, more preferably less than about 60%, most preferably less than about 50% of ZNA monomers, and their sizes are from about 5 to 25 nucleotides, more preferably from about 12 up to 20 nucleotides.

[00154] Preferred modified oligonucleotide backbones include, but are not limited to: phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriethers, aminoalkylphosphotriethers, methyl and other alkylphosphonates, including 3'alkylene phosphonates, and chiral phosphonates, amido phosphonates, aminosulfonates, , thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriethers, and boranophosphates, including normal 3'-5 'bonds, 2'-5' linked analogues, and having polar reversed Th, where adjacent pairs of nucleoside units are linked 3'-5 'to 5'-3' or 2'-5 'to 5'-T. Various salts, mixed salts and free acids are also included.

[00155] Examples of US patents disclosing the preparation of the above phosphorus-containing bonds include, but are not limited to, US patents 3,687,808; 4,469,863; 4,476,301; 50,23243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5453496; 5455233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5550111; 5,563,253; 5,571,799; 5,587,361; and 5625050, each of which is incorporated into this application by reference.

[00156] Preferred modified oligonucleotide backbones that do not include phosphorus atoms are formed by short chain alkyl or cycloalkyl internucleoside bonds, mixed heteroatom and alkyl or cycloalkyl internucleoside bonds, or one or more short chain heteroatomic or heterocyclic internucleols. These include skeletons with morpholine bonds (partially formed by nucleoside sugar); siloxane cores; sulfide, sulfoxide and sulfone cores; formacetyl and thioformacetyl backbones; methyleneformyl and thioform acetyl backbones; alkene-containing cores; sulfamate skeletons; methyleneimine and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide cores; and others containing mixed N, O, S, and CH2 components.

[00157] Examples of US patents containing guidance on obtaining the above oligonucleotides include, but are not limited to, US patents 5034506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5405938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5602240; 5,610,289; 5602240; 5,608,046; 5,610,289; 5,618,704; 5,630,070; 5,663,312; 5,633,360; 5,677,437; and 5677439, each of which is incorporated into this application by reference.

[00158] In other preferred oligonucleotide and sugar mimetics, and an internucleoside bond, i.e. the backbone, nucleotide units replaced by new groups. The bases are kept for hybridization with a suitable target nucleic acid compound. One of these oligomeric compounds, an oligonucleotide mimetic that has demonstrated excellent hybridization ability, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced by an amide-containing backbone, for example, an aminoethyl glycine backbone. The nucleic acid bases are stored and bound directly or indirectly to the nitrogen atoms of the azagroups of the amide part of the core. Examples of US patents that describe the preparation of PNA compounds include, but are not limited to, US patents 5539082; 5,714,331; and 5719262, each of which is incorporated into this application by reference. Further description of PNA compounds can be found in Nielsen, et al. (1991) Science 254, 1497-1500.

[00159] According to an embodiment of the invention, oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular CH2-NH-O-CH2-, -CH2-N (CH3) -O-CH2-, known as methylene (methylimino) or MMI backbone, - CH2-ON (CH3) -CH2-, -CH2N (CH3) -N (CH3) CH2- and -ON (CH3) -CH2-CH2-, where the native phosphodiester backbone is represented as -O-P-O -CH2- according to the aforementioned US patent 5489677, and amide cores according to the aforementioned US patent 5602240. Oligonucleotides containing morpholine cores according to the aforementioned US patent are also preferred. 5034506.

[00160] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides contain one of the following substituents at position 2 ′: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O alkyl-O-alkyl, wherein said alkyl, alkenyl and alkynyl may be substituted or unsubstituted C → CO alkyl or C2 → CO alkenyl and alkynyl. In particular, O (CH2) n OmCH3, O (CH2) n, OCH3, O (CH2) nNH2, O (CH2) nCH3, O (CH2) nONH2 and O (CH2nON (CH2) nCH3) 2, where n and m can range from 1 to about 10. Other preferred oligonucleotides contain one of the following substituents at position 2 ′: C → CO, (lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, improving group pharmacokinetic properties of the oligonucleotide, or a group that improves the pharmacodynamic properties of the oligonucleotide, and other substituents with similar properties.The preferred modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O- (2-methoxyethyl) or 2 '-MOE), i.e. an alkoxyalkoxy group, Another preferred modification includes a 2'-dimethylaminooxyethoxy, i.e., O (CH2) 2ON (CH3) 2 group, also known as 2'-DMAOE, as described in the above examples below, and 2'-dimethylaminoethoxyethoxy (also from estno in the art as 2'-O-dimetilaminoetoksietil or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N (CH2) 2.

[00161] Other preferred modifications include 2'-methoxy (2'-O CH3), 2'-aminopropoxy (2'-O CH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions of the oligonucleotide, in particular at the 3 'position of the sugar 3' terminal nucleotide or in the 2'-5 'linked oligonucleotides and 5' position 5 'of the terminal nucleotide. Oligonucleotides may also contain sugar mimetics, for example, cyclobutyl fragments instead of pentofuranosyl sugar. Examples of US patents describing the preparation of such modified sugar structures include, but are not limited to, US patents 4,981,957; 5,118,800; 5319080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5658873; 5,670,633; and 5700920, each of which is incorporated into this application by reference.

[00162] Oligonucleotides may also contain modified or substituted nucleic bases (often referred to simply as “bases” in the art). As used herein, “unmodified” or “natural” nucleotides include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleotides include other synthetic and natural nucleotides, such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halogenouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil 4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halogen, in astnosti, 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenin, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[00163] Further, nucleotides include those described in US Pat. No. 3,687,808, described in 'The Concise Encyclopedia of Polymer Science And Engineering', pp. 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, described by Englisch et al., 'Angewandle Chemie, International Edition', 1991, 30, p. 613, and described by Sanghvi, YS, Chapter 15, 'Antisense Research and Applications', p. 289 -302, Crooke, ST and Lebleu, B. ea., CRC Press, 1993. Some of these nucleotides are suitable, in particular, to increase the binding capacity of the oligomeric compounds of the present invention. Such include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase the stability of nucleic acid duplexes by 0.6-1.2 ° C (Sanghvi, YS, Crooke, ST, Lebleu, B., eds, 'Antisense Research and Applications', CRC Press, Boca Raton, 1993, pp. 276-278) and are currently preferred base substitutions, in particular in combination with 2'-O-methoxyethyl sugar modifications.

[00164] Examples of US patents that describe the preparation of the aforementioned modified nucleotides as well as other modified nucleotides include, but are not limited to, US patents 3,687,808, as well as 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5502177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5750692 and 5681941, each of which is incorporated into this application by reference.

[00165] Another modification of the oligonucleotides of the present invention is to chemically bind to the oligonucleotide one or more moieties or conjugates that stimulate activity, distribution in cells, or uptake of said oligonucleotide by cells.

[00166] Such fragments contain, but are not limited to lipid fragments, such as cholesterol fragments, cholic acid, a thioether, for example hexyl-S-tritylthiol, thiocholesterol, aliphatic chains, for example, dodecandiol or undecyl residues, for example, di- hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or polyethylene glycol chain, or adamantane-acetic acid, a palmityl moiety, or octadecylamino or hexylamino-oxolesteryl t-carbonyl fragment.

[00167] Examples of US patents that describe the preparation of such oligonucleotide conjugates include, but are not limited to, US patents 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5578717, 5580731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,441,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,588,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5082830; 5,112,963; 5,214,136; 5082830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,371,098; 5371241, 5391723; 5,416,203; 5451463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5599928 and 5688941, each of which is incorporated into this application by reference.

[00168] Search for new drugs: The compounds proposed in the present invention can also be used to search for new drugs and validation of targets. The present invention includes the use of the compounds described herein and preferred target segments in the search for new drugs to elucidate the relationships between PAR4 polynucleotides and a disease, phenotype or condition. Such methods include determining or modulating PAR4 polynucleotides, including contacting a sample, tissue, cell or organism with the compounds of the present invention, measuring the level of a nucleic acid or protein of the PAR4 polynucleotides and / or evaluating the corresponding phenotypic or chemical end state some time after treatment, and, in some cases, comparison of the measured value with untreated samples or samples treated with other proposed in the present invention is connected iai. These methods can be used in parallel or in combination with other experiments to determine the functions of unknown genes in the process of target validation or to determine the validity of using a particular gene product as a target for treating or preventing a specific disease, condition or phenotype.

Evaluation of up-regulation or inhibition of gene expression:

[00169] Transfer of exogenous nucleic acid to a host cell or to a host organism can be evaluated by directly determining the presence of said nucleic acid in said cell or organism. Such a determination can be made by several methods known to those skilled in the art. For example, the presence of exogenous nucleic acid can be determined by southern blotting or polymerase chain reaction (PCR) using primers that specifically amplify the nucleotide sequences associated with the specified nucleic acid. The expression of these exogenous nucleic acids can also be measured using conventional methods, including analysis of gene expression. For example, mRNA derived from exogenous nucleic acid can be detected and quantified using Northern blotting and reverse transcription PCR (RT-PCR).

[00170] The expression of RNA from the indicated exogenous nucleic acid can also be determined by measuring the enzymatic activity or activity of the reporter protein. For example, antisense modulating activity can be indirectly measured by decreasing or increasing the expression of a target nucleic acid as an indicator that exogenous nucleic acid produces effector RNA. Using sequence conservatism, primers can be designed and used to amplify the coding regions of these target genes. First, you can use the most actively expressed coding region of each gene to build a model of the control gene, although any coding or non-coding region can be used. Each control gene is obtained by inserting each coding region between the coding region of the reporter region and its poly (A) signal. These plasmids produce mRNA with a reporter gene in the overlying part of the gene and a potential target for RNAi in the 3 'non-coding region. The effectiveness of individual antisense oligonucleotides can be analyzed by modulating the reporter gene. Reporter genes suitable for use in the methods of the present invention include genes for the synthesis of acetohydroxy acids (AGCS), alkaline phosphatase (ALP), betagalactosidase (LacZ), beta glucuronidase (GUS), chloramphenicol acetyltransferase (HAT), green protein fluorescent (GFP) ), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (CFP), horseradish peroxidase (POX), luciferase (Luc), nopaline synthase (NOS), octopin synthase (OCS) and their derivatives. Various selective markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinotricin, puromycin and tetracycline. Methods for determining the modulation of a reporter gene are well known in the art and include, but are not limited to: fluorometric methods (e.g., fluorescence spectroscopy, sorting of fluorescence-activated cells (FACS), fluorescence microscopy), determination of antibiotic resistance.

[00171] The expression of PAR4 protein and mRNA can be analyzed using methods known to those skilled in the art as described in any of the sections of this application. For example, immunoassay methods such as ELISA can be used to measure protein levels. PAR4 assay ELISA kits are commercially available, for example, from R&D Systems (Minneapolis, MN).

[00172] According to embodiments, the expression of PAR4 (eg, mRNA or protein) in a sample (eg, in cells or tissues in vivo or in vitro) processed using the antisense oligonucleotide of the present invention is evaluated by comparison with PAR4 expression in the control sample. For example, expression of a protein or nucleic acid can be compared using methods known to those skilled in the art with that of a placebo sample or an untreated sample. Alternatively, a comparison can be made with a sample treated with a control antisense oligonucleotide (for example, having an altered or different sequence), depending on what information needs to be obtained. According to another embodiment, the difference in the expression of PAR4 protein or nucleic acid between the untreated and treated samples can be compared with the difference in the expression of various nucleic acids (including any standard, recognized by the researcher as suitable, for example, a constitutive gene) in the processed and untreated samples.

[00173] The observed differences can be expressed in a convenient form, for example, as a proportion or proportion, for comparison with a control. In embodiments, the level of mRNA or PAR4 protein in the sample treated with the antisense oligonucleotide of the present invention increases or decreases by about 1.25-fold-10-fold or more relative to the untreated sample or the sample treated with the control nucleic acid. In embodiments, the level of mRNA or PAR4 protein rises or falls at least about 1.25 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 2 times, at least about 2.5 times, at at least about 3 times, at least about 3.5 times, at least at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 5.5 times, at least about 6 times, at least about 6.5 at least about 7 times, at least about 7.5 times, at least about 8 times, at least about 8.5 times, at least about 9 times, at least about 9.5 times, or at least about 10 times or more.

Kits, research reagents, diagnostics and therapy

[00174] The compounds of the present invention can be used for diagnosis, treatment and prevention, as well as research reagents and kit components. In addition, antisense oligonucleotides capable of inhibiting gene expression with high specificity are often used by those skilled in the art to ascertain the functions of individual genes or to recognize the functions of various components of a biological pathway.

[00175] For use in kits for both diagnostics and various biological systems, the compounds of the present invention, individually or in combination with other compounds or therapeutic agents, are suitable as a differential and / or combinatorial analysis tool for determining patterns of expression of a portion or the entire set of genes expressed in cells and tissues.

[00176] As used herein, the term “biological system” or “system” is defined as any organism, cell, cell culture or tissue expressing, or with acquired ability to express, PAR4 gene products. These include, but are not limited to: human, transgenic animals, cells, cell cultures, tissues, xenografts, grafts, and combinations thereof.

[00177] According to one non-limiting example, expression patterns in cells or tissues treated with one or more antisense compounds are compared with control cells or tissues not treated with antisense compounds, and differential levels of gene expression are analyzed based on the patterns obtained, since they are related, for example, with disease, signaling pathways, localization in cells, expression levels, size, structure or function of the genes under study. These studies can be performed on stimulated or non-stimulated cells in the presence or absence of other compounds that affect expression patterns.

[00178] Examples of gene expression analysis methods known in the art include DNA or microarray analysis, SAGE (serial analysis of gene expression), READS (restriction enzyme amplification of cleavable cDNAs), TOGA (complete analysis of gene expression), protein chips and proteomics, express sequence label sequencing (EST), RNA subtracting fingerprinting (SuRF), cloning subtracting, differential display (DD), comparative genomic hybridization, FISH (in situ fluorescence hybridization) and mass spectrometric meshes Toda.

[00179] The compounds of the present invention are suitable for research and diagnosis, since such compounds hybridize to nucleic acids encoding PAR4. For example, oligonucleotides that hybridize with efficiency and under the conditions described in this application, being effective PAR4 modulators, are effective primers or probes under conditions favorable for amplification or gene determination, respectively. Such primers and probes are suitable for methods that require specific determination of nucleic acid molecules encoding PAR4, and for amplification of these nucleic acid molecules for determination or for use in further studies of PAR4. Hybridization of these antisense oligonucleotides, in particular the primers and probes of the present invention, with a nucleic acid encoding PAR4 can be determined using methods known in the art. Such methods may include conjugating an enzyme to said oligonucleotide, radioactive labeling of said oligonucleotide, and any other suitable detection methods. Kits can also be prepared for determining PAR4 levels in a sample using such determination methods.

[00180] The specificity and sensitivity of antisense compounds are also used by those skilled in the art for therapeutic purposes. Antisense compounds have been used as therapeutic components in the treatment of disease states in animals, including humans. Antisense oligonucleotide-based drugs have been safely and effectively administered to humans, and numerous clinical trials are currently under way. Thus, it was found that antisense compounds can be used as therapeutic agents, which can be modified under therapeutic regimens for the treatment of cells, tissues and animals, especially humans.

[00181] In the case of therapeutic agents, an animal, preferably a person who is suspected to have a disease or disorder that can be treated by modulating the expression of PAR4 polynucleotides, is treated with the introduction of antisense compounds in accordance with the present invention. For example, in one non-limiting embodiment, said methods include the step of administering to the animal in need of treatment a therapeutically effective amount of a PAR4 modulator. The PAR4 modulators of the present invention effectively modulate PAR4 activity or modulate the expression of PAR4 protein. In one embodiment, the activity or expression of PAR4 in an animal is down-regulated by about 10% compared to a control. Preferably, said animal activity or expression of PAR4 is inhibited by approximately 30%. More preferably, said activity or expression of PAR4 in an animal is suppressed by about 50% or more. Thus, these oligomeric compounds modulate the expression of PAR4 mRNA by at least 10%, at least 50%, at least 25%, at least 30%, at least 40%, at least 50 %, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 98%, at least 99%, or 100% relative to the control.

[00182] According to one embodiment, said animal activity and / or PAR4 expression is increased by about 10% compared to the control. Preferably, said animal activity or expression of PAR4 is increased by about 30%. More preferably, said animal activity or expression of PAR4 is increased by 50% or more. Thus, these oligomeric compounds alter the expression of PAR4 mRNA by at least 10%, at least 50%, at least 25%, at least 30%, at least 40%, at least 50 %, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 98%, at least 99%, or 100% relative to the control.

[00183] For example, a decrease in PAR4 expression can be detected in serum, blood, adipose tissue, liver, or any other body fluid, tissue, or organ of a specified animal. Preferably, the cells contained in said test liquids, tissues or organs contain a nucleic acid molecule encoding the PAR4 peptides and / or the PAR4 protein itself.

[00184] The compounds of the present invention are used in pharmaceutical compositions by adding an effective amount of the compound to a suitable pharmaceutically acceptable diluent or carrier. The use of the compounds and methods of the present invention may also be suitable for prophylaxis.

Conjugates

[00185] Another modification of the oligonucleotides of the present invention includes chemically binding one or more fragments or conjugates to said oligonucleotide that increase activity, enhance cell distribution or uptake of said oligonucleotide by cells. Such fragments or conjugates may include conjugated groups covalently linked to functional groups, such as primary or secondary hydroxyl groups. The conjugated groups of the present invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, pharmacodynamic properties of oligomers, and pharmacokinetic properties of oligomers. Exemplary conjugated groups include cholesterol, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins and dyes. Pharmacodynamic enhancing groups in the context of the present invention include absorption enhancing groups, resistance to degradation, and / or enhancing sequence-specific hybridization with the target nucleic acid. Pharmacokinetic enhancing groups in the context of the present invention include groups that improve the uptake, distribution, metabolism or excretion of the compounds of the present invention. Exemplary conjugated groups are described in international patent application PCT / US92 / 09196, filed October 23, 1992, and US patent 6287860, incorporated into this application by reference. Conjugated fragments include, but are not limited to: lipid fragments, such as cholesterol fragments, cholic acid, a thioether, for example, hexyl-5-tritylthiol, thiocholesterol, aliphatic chains, for example, dodecandiol or undecyl residues, phospholipid, for example, di-hexade -glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-N-phosphonate, a polyamine or polyethylene glycol chain, or adamantane-acetic acid, a palmityl moiety, octadecylamine or a hexylamino-carbonyl-hydroxycholesterol moiety. The oligonucleotides proposed in the present invention can also be conjugated with active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S) - (+) - pranoprofen, carprofen, 2,3, sansarcosin 5-triiodobenzoic acid, flufenamic acid, folinic acid, benzothiadiazide, chlortiazide, diazepine, indomethicin, barbiturate, cephalosporin, sulfa drug, antidiabetic agent, antibacterial agent or antibiotic .

[00186] Examples of US patents that describe the preparation of such oligonucleotide conjugates include, but are not limited to: US patents 4828979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5578717, 5580731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,441,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,588,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5082830; 5,112,963; 5,214,136; 5082830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,371,098; 5371241, 5391723; 5,416,203; 5451463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5599928 and 5688941.

Compositions

[00187] the Compounds proposed in the present invention can also be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecular structures or mixtures of compounds, for example, liposomes, target molecules of receptors, compositions for oral, rectal, local or other routes of administration, to facilitate absorption, distribution and / or absorption. Examples of US patents that describe the preparation of such absorption, distribution, and / or absorption promoting compositions include, but are not limited to: US Pat. No. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,165; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5013556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5595756, each of which is incorporated into this application by reference.

[00188] Although these antisense oligonucleotides do not have to be introduced using a vector to modulate the expression and / or function of a target, embodiments of the present invention include vector constructs for expression of antisense oligonucleotides, including promoters, gene sequences of compound promoters and having pronounced constitutive promoter activity, or promoter activity, which can be induced if necessary.

[00189] In one embodiment, the invention includes the administration of at least one of the above antisense oligonucleotides using a suitable nucleic acid delivery system. In one embodiment, such a system comprises a non-viral vector operably linked to said polynucleotide. Examples of such non-viral vectors include the oligonucleotide itself (for example, any of the sequences SEQ ID NO: 3-9) or a combination thereof with a suitable protein, polysaccharide or lipid composition.

[00190] Additionally, suitable nucleic acid delivery systems include viral vectors, typically based on the sequence of one or more adenoviruses, adeno-associated virus (AAB), helper-dependent adenovirus, retrovirus, or liposomal complex with Japanese hemagglutinating virus (HVJ). Preferably, said viral vector contains a strong eukaryotic promoter operably linked to said polynucleotide, for example, a cytomegalovirus (CMV) promoter.

[00191] Additional preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney murine leukemia viruses and HIV viruses. One of the preferred HIV virus vectors contains at least two vectors, where the gag and pol genes originate from the HIV genome and the env gene from another virus. DNA viral vectors are preferred. Such vectors include pox vectors, such as orthopox or avipox vectors, herpesvirus vectors, such as herpes simplex virus (HSV) I vectors, adenovirus vectors and adeno-associated virus vectors.

[00192] The antisense compounds provided by the present invention include any pharmaceutically acceptable salts, esters or salts of such esters, or any other compound which, when administered to an animal, including humans, is capable of producing (directly or indirectly) a biologically active metabolite or its component.

[00193] The term "pharmaceutically acceptable salts" refers to the physiologically and pharmaceutically acceptable salts of the compounds of the present invention: i.e. salts retaining the necessary biological activity of the starting compound and, in addition, not giving undesirable toxic effects. Preferred examples of pharmaceutically acceptable salts of oligonucleotides and their use are described in more detail in US patent 6287860, incorporated into this application by reference.

[00194] The present invention also includes pharmaceutical compositions and compositions containing antisense compounds proposed in the present invention. The pharmaceutical compositions of the present invention may be administered in various ways, depending on whether topical or systemic treatment is required and on which area to be affected. The introduction can be local (including through the eyes and through the mucous membranes, including vaginal and rectal administration), through the lungs, for example, by inhaling or injecting powders or aerosols, including using a nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, for example, intrathecal or intraventricular, administration.

[00195] In the treatment of central nervous system tissues, administration can be carried out, for example, by injection or infusion into the cerebrospinal fluid. The introduction of antisense RNA into the cerebrospinal fluid is described, for example, in US patent application published under number 2007/0117772, "Methods for slowing familial ALS disease progression", which is incorporated into this application by reference in fullness.

[00196] If it is contemplated that the antisense oligonucleotide of the present invention is introduced into the cells of the central nervous system, it can be administered in conjunction with one or more agents capable of penetrating the antisense oligonucleotide through the blood-brain barrier. Injection can be done, for example, in the entorhinal cortex or hippocampus. The delivery of neurotrophic factors by introducing an adenoviral vector into the motor neurons of muscle tissue is described, for example, in US Pat. No. 6,632,427, "Adenoviral-vector-mediated gene transfer into medullary motor neurons" included in the adenoviral vector mediated) The present application is by reference Delivery of vectors directly to the brain, for example, to the striatum, thalamus, hippocampus or black substance, is known in the art and is described, for example, in US Pat. No. 6,756,523, “Adenovirus vectors for the transfer of foreign genes into cells of the cent ral nervous system particularly in brain "(" Adenoviral vectors for transferring foreign genes into cells of the central nervous system, in particular brain cells "), incorporated herein by reference. The introduction can be quick in case of injection or over a period of time in the case of a slow infusion or administration of sustained release formulations.

[00197] Considered antisense oligonucleotides can be associated or conjugated with agents that provide the necessary pharmaceutical or pharmacokinetic properties. For example, said antisense oligonucleotide can be combined with any substance that is known in the art to penetrate or transport through the blood-brain barrier, such as an antibody to transferrin receptor, and administered by intravenous injection. The indicated antisense compound may be associated with a viral vector, for example, giving the specified antisense compound greater efficiency and / or enhancing the transport of the indicated antisense compound through the blood-brain barrier. Osmotic overcoming of the blood-brain barrier can also be achieved, for example, by infusion of sugars, including, but not limited to, mesoerythritis, xylitol, D (+) galactose, D (+) lactose, D (+) xylose, dulcite, myoinositol, L (- ) fructose, D (-) mannitol, D (+) glucose, D (+) arabinose, D (-) arabinose, cellobiose, D (+) maltose, D (+) raffinose, L (+) rhamnose, D (+ ) melibiosis, D (-) ribose, adonite, D (+) arabite, L (-) arabite, D (+) fucose, L (-) fucose, D (-) lyxose, L (+) lyxose and L (- ) lyxose, or amino acids, including, but not limited to, glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine and taurine. Methods and materials for increasing penetration through the blood-brain barrier are described, for example, in US Pat. No. 4,866,042, Methods for the delivery of genetic material across the blood brain barrier, US 6294520, Material for passage through the blood-brain barrier ”and“ US Pat. No. 6,936589, “Parenteral delivery systems”, incorporated herein by reference in their entirety.

[00198] Considered antisense compounds can be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecular structures or mixtures of compounds, for example, liposomes, target molecules of receptors, compositions for oral, rectal, local or other route of administration, to facilitate absorption, distribution and / or absorption. For example, cationic lipids may also be included in the composition to facilitate the uptake of oligonucleotides. One such composition as shown to promote absorption is LIPOFECTIN (available from GIBCO-BRL, Bethesda, MD).

[00199] Oligonucleotides containing at least one 2'-O-methoxyethyl modification are believed to be suitable, in particular, for oral administration. Topical pharmaceutical compositions and formulations may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmacological carriers, aqueous, powder or oil bases, thickeners, and the like. may be necessary or desirable. Suitable applications also include coatings for condoms, gloves, and the like.

[00200] The pharmaceutical compositions of the present invention, which can be presented in a convenient unit dosage form, can be prepared according to generally accepted methods well known in the pharmaceutical industry. Such techniques include the step of combining the active ingredients with pharmaceutical carriers and excipients. Typically, these formulations are prepared by uniformly deeply combining the active ingredients with liquid carriers or with finely ground solid carriers, or both of them, and then, if necessary, shape the product.

[00201] The compositions of the present invention can be prepared in any of many possible dosage forms, including, but not limited to tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories and enemas. The compositions proposed in the present invention can also be obtained in the form of suspensions based on aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.

[00202] The pharmaceutical compositions of the present invention include, but are not limited to: solutions, emulsions, foams, and liposome-containing formulations. The pharmaceutical compositions and compositions proposed in the present invention may contain one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.

[00203] Emulsions are typically heterogeneous systems where one liquid is distributed in another in the form of droplets, typically more than 0.1 microns in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug substance may be present in solution in the aqueous phase, the oil phase, or be a separate phase. Microemulsions also relate to embodiments of the present invention. Emulsions and their use are well known in the art and are described in more detail in US patent 6287860.

[00204] the Formulations proposed in the present invention include liposomal formulations. In the context of the present invention, the term "liposome" means vesicles consisting of amphiphilic lipids forming one or more spherical bilayers. Liposomes are monolayer or multilayer vesicles with a membrane formed by lipophilic substances and an aqueous internal phase, which contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are thought to interact with negatively charged DNA molecules to form a stable complex. pH-sensitive or negatively charged liposomes are thought to capture DNA rather than form complexes with it. Both cationic and non-cationic liposomes have been used to deliver DNA to cells.

[00205] Liposomes also include "sterically stabilized" liposomes, which when used in this application refers to liposomes containing one or more specialized lipids. After incorporation into liposomes, these specialized lipids form liposomes with an extended lifespan compared to liposomes that do not contain such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the bubble forming lipid component of the liposome contains one or more glycolipids or is derivatized with one or more hydrophilic polymers such as a polyethylene glycol (PEG) fragment. Liposomes and their use are described in more detail in US patent 6287860.

[00206] The pharmaceutical compositions and compositions provided by the present invention may also include surfactants. The use of surfactants in medicinal products, compositions and emulsions is well known in the art. Surfactants and their use are described in more detail in US patent 6287860, incorporated into this application by reference.

[00207] In one embodiment, the present invention includes various penetration enhancers that facilitate the efficient delivery of nucleic acids, in particular oligonucleotides. In addition to promoting the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers increase the penetration of lipophilic drugs. Penetration enhancers can be divided into five broad categories, namely surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their use are described in more detail in US Pat. No. 6,287,860, incorporated herein by reference.

[00208] One skilled in the art will understand that the formulations are prepared according to standard procedures in accordance with their intended use, i.e. route of administration.

[00209] Preferred topical formulations include those in which the oligonucleotides of the present invention are mixed with topical agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants . Preferred lipids and liposomes include neutral (e.g., dioleylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolphosphatidylcholine) negatively charged (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleyltetolamine-di-tetramidetol-tetramidetol-tetramidetamide di-methyltropylamide di-tetramidetolamine tetramide).

[00210] For topical or other uses, the oligonucleotides of the present invention can be encapsulated in liposomes or form part of liposome complexes, in particular with cationic liposomes. Alternatively, oligonucleotides can be part of lipid complexes, in particular with cationic lipids. Preferred fatty acids and esters, their pharmaceutically acceptable salts, and their use are described in more detail in US patent 6287860.

[00211] Compositions and compositions for oral administration include powders or granules containing microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, portioned packets, tablets or mini-tablets. The presence of thickening agents, flavoring agents, diluents, emulsifiers, dispersing agents or binders may be desirable. Preferred oral formulations are those in which the oligonucleotides of the present invention are administered together with one or more penetration enhancers, surfactants and chelating agents. Preferred surfactants include fatty acids and / or esters or their salts, bile acids and / or their salts. Preferred bile acids / salts and fatty acids and their use are described in more detail in US patent 6287860, incorporated into this application by reference. Also preferred are combinations of penetration enhancers, for example, fatty acids / salts in combination with bile acids / salts. In particular, a combination of the sodium salt of lauric acid, capric acid and UDCA is preferred. Other penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The oligonucleotides of the present invention can be administered orally, in granular form, including spray dried particles, or as part of complexes of micro- or nanoparticles. Complexing agents for oligonucleotides and their use are described in more detail in US patent 6287860, incorporated into this application by reference.

[00212] Compositions and compositions for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions, which may also contain buffers, diluents and other suitable additives, including, but not limited to, penetration enhancers, carriers and other pharmaceutically acceptable carriers or excipients .

[00213] Certain embodiments of the present invention include pharmaceutical compositions comprising one or more oligomeric compounds and one or more other chemotherapeutic agents that act by a non-antisense mechanism. Examples of such chemotherapeutic agents include, but are not limited to, chemotherapeutic drugs for treating cancer, such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, ezorubicin, bleomycin, mafosfamide, ifosfamino-azithozitozitozitozitozitozitozitozitozitozitozitozitozitozitozitozitozitozitozitozitozinitozitozinitozitozinitozitozinitozitozinito mitomycin C, actinomycin D, mitramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambuci , methylcyclohexyl nitrosourea, mustargen, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxy formoficin, 4-hydroxyperoxycyclo-phosphoramide, 5-fluorouracil-5-F-5-f-5-f-5-f-5-f-5-f-5-d-5-f-5-d-5-f-5-d-5-f-5-d-5-f-5-d-5-f-5-d-5-5-fluoride-5-d-5-5-d-l-5-5-d-d-dl (5-fluorourazide) , methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimerexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the present invention, such chemotherapeutic agents can be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for some time, then MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiation therapy and oligonucleotide). Anti-inflammatory drugs, including, but not limited to, non-steroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including, but not limited to, ribavirin, vidarabine, acyclovir, and ganciclovir, can also be combined in the compositions of the present invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of the present invention. Two or more combined compounds may be used together or sequentially.

[00214] According to another similar embodiment, the compositions of the present invention may contain one or more antisense compounds, in particular oligonucleotides targeting the first nucleic acid, and one or more additional antisense compounds targeting the second nucleic acid. For example, the first target may be a specific antisense sequence of PAR4, and the second target may be a portion of another nucleotide sequence. Alternatively, the compositions of the present invention may contain two or more antisense compounds targeting different regions of the same target PAR4 nucleic acid. Numerous examples of antisense compounds are provided herein, others may be selected from suitable compounds known in the art. Two or more combined compounds may be used together or sequentially.

Dosage:

[00215] The preparation of pharmaceutical compositions and their subsequent administration (dosing) should be familiar to those skilled in the art. Doses depend on the severity and sensitivity to treatment of the disease state, the target of treatment, the course of treatment can last from several days to several months, or until cure, or to alleviate the disease state. The optimal dosage regimen can be calculated based on measurements of drug accumulation in the patient’s body. The specialist will easily determine the optimal dosage, dosage methodology and frequency of repetition. Optimal doses may vary depending on the relative efficacy of specific oligonucleotides, and, as a rule, can be calculated based on EC50 effective for animal models in vitro and in vivo. As a rule, the dose is from 0.01 μg to 100 g per kg of body weight, and can be administered once or more daily, weekly, monthly or yearly, or even every 2-20 years. One skilled in the art can easily calculate the frequency of dosing based on retention time and drug concentrations in body fluids or tissues. After effective treatment, it may be desirable to use maintenance therapy in a patient to prevent recurrence of the disease, wherein said oligonucleotide is administered in maintenance doses ranging from 0.01 μg to 100 g per kg body weight, from a single or more frequent daily intake to a single dose once every 20 years.

[00216] In embodiments, the patient receives a dosage of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least e about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 mg / kg body weight. Some administration dosages of antisense oligonucleotides are described, for example, in US Pat. No. 7,563,884, “Antisense modulation of PTP1B expression”, which is incorporated herein by reference in its entirety.

[00217] Although various embodiments of the present invention have been described above, it should be understood that they are presented only as examples, and not for limitation. Numerous modifications of the disclosed embodiments may be carried out as described herein, without departing from the spirit and scope of the present invention. Thus, the scope and spirit of the present invention is not limited to any of the above embodiments.

[00218] All documents referred to herein are incorporated into this application by reference. All publications and patent documents referred to in this application are incorporated by reference to the same extent as if each publication or each patent document were individually mentioned. Citing the various sources in this document, the applicants do not recognize that any of the sources represents the "prior art" regarding the present invention. Embodiments of the compositions and methods of the invention are illustrated by the following examples.

EXAMPLES

[00219] The following non-limiting Examples are intended to illustrate certain embodiments of the present invention. It should be borne in mind that variations in the content and variations of the elements of the described components are obvious to experts in this field and are included in the scope of the present invention.

Example 1: Construction of antisense oligonucleotides specific for a nucleic acid molecule antisense to PAR4 and / or sense strand of a PAR4 polynucleotide

[00220] As indicated above, the term “specific oligonucleotide” or “targeted oligonucleotide” refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a fragment of a target gene or (ii) capable of forming a stable duplex with a fragment mRNA transcript of the target gene.

[00221] The selection of suitable oligonucleotides is simplified by the use of computer programs (for example, IDT AntiSense Design, IDT OligoAnalyzer) that automatically identify, in each particular sequence, sub-sequences of IP-25 nucleotides capable of forming hybrids with the target polynucleotide sequence having the desired melting point (typically , 50-60 ° C) and not forming homodimers or other complex secondary structures.

[00222] The selection of suitable oligonucleotides is performed using computer programs that automatically align nucleic acid sequences and determine regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, from databases such as GenBank, or by sequencing PCR products. Comparison of the nucleic acid sequences of different genes and intergenic regions of a particular genome allows you to select nucleic acid sequences that are characterized by the necessary degree of specificity for the desired gene. Such procedures allow selection of oligonucleotides exhibiting a significant degree of complementarity to target nucleic acid sequences and a lower degree of complementarity to other nucleic acid sequences from a particular genome. One skilled in the art will appreciate that there is considerable freedom of choice of suitable gene regions for use in accordance with the present invention.

[00223] An antisense compound is “specifically hybridizable” if the binding of the compound to the target nucleic acid interferes with the normal functioning of the target nucleic acid, modulating the function and / or activity, and the degree of complementarity is sufficient to avoid non-specific binding of the antisense compound to non-target sequences nucleic acid under conditions that require specific binding, i.e., under physiological conditions in the case of in vivo analysis methods or therapeutic effects, and under the conditions under which the analysis is carried out, in the case of in vitro studies.

[00224] The hybridization characteristics of the oligonucleotides described herein can be determined using one or more in vitro assay methods known to those skilled in the art. For example, the indicated characteristics of the oligonucleotides described in this application can be obtained by determining the binding strength of the target natural antisense sequences and potential drug molecules using analysis of melting curves.

[00225] The binding strength of the target natural antisense sequences and the potential drug molecule (hereinafter referred to as the Molecule) can be evaluated using any standard method for measuring the strength of intermolecular interactions, for example, analysis of melting curves.

[00226] An analysis of the melting curves determines the temperature at which a rapid transition from double-stranded to single-stranded conformation of the natural antisense sequence / Molecule complex occurs. This temperature is widely used as a reliable measure of the strength of the interaction between two molecules.

[00227] Analysis of the melting curves can be performed using a cDNA copy of the actual natural antisense RNA molecule or a synthetic DNA or RNA nucleotide corresponding to the binding site of the Molecule. Numerous kits are available that include all the necessary reagents for analysis (for example, the MeltDoctor kit from Applied Biosystems Inc.). Such kits contain a suitable buffer solution containing one of the double-stranded DNA (dsDNA) binding dyes (such as HRI dyes from ABI, SYBR Green, SYTO, etc.). The properties of dsDNA dyes are such that they practically do not fluoresce in free form, but fluoresce very much upon binding to dsDNA.

[00228] For analysis, said cDNA or corresponding oligonucleotide is mixed with the Molecule at concentrations determined according to the protocols of particular manufacturers. The mixture is heated to 95 ° C to dissociate the preformed dsDNA complexes, then slowly cooled to room temperature or another lower temperature specified by the kit manufacturer to renature the DNA molecules. The newly formed complexes are then slowly heated to 95 ° C, while continuously measuring the fluorescence intensity as a result of the reaction. The fluorescence intensity is inversely proportional to the number of dsDNAs present in the reaction. Information can be obtained using a real-time PNR tool, compatible with the kit (for example, StepOne Plus Real Time PCR System from ABI or lightTyper from Roche Diagnostics, Lewes, UK).

[00229] Melting peaks are obtained by plotting the temperature dependence of the negative derivative of fluorescence (-d (Fluorescence) / dT) along the y axis) versus temperature (x axis) using suitable software (eg lightTyper (Roche) or SDS Dissociation Curve, ABI). The information is analyzed in order to determine the temperature of the rapid transition of the dsDNA complex into single-stranded molecules. This temperature is called the melting point and it is directly proportional to the strength of the interaction between the two molecules. As a rule, the melting temperature exceeds 40 ° C.

Example 2: Modulation of PAR4 Polynucleotides

Treatment of HepG2 cells with antisense oligonucleotides

[00230] All antisense oligonucleotides used in Example 2 were constructed as described in Example 1. The manufacturer (IDT Inc., Coralville, IA (Coralville, Iowa)) was instructed in the preparation of synthetic phosphotioate-linked oligonucleotides and provided synthetic phosphotioate analogs, are given in table 1. An asterisk in the nucleotides indicates the presence of a phosphotioate bond. The oligonucleotides necessary for the experiment of Example 2 can be synthesized using any suitable method corresponding to the current level of technology, for example, the method used by IDT: on a solid substrate, such as 5 micron glass beads with controlled porosity (CPG), using phosphoramidite monomers (standard nucleotides with protection of all active groups with protective groups, for example, the trityl group on sugar, benzoyl on A and C and N-2-isobutyryl on G). Protecting groups prevent unwanted reactions during the synthesis of oligonucleotides. Protecting groups are removed at the end of the synthesis process. The starting nucleotide binds to the solid support through 3'-carbon and the synthesis proceeds from 3 'to 5'. The addition of a new base to the growing chain of the oligonucleotide takes place in four stages: 1) the protective group is removed from 5 'of the oxygen of the immobilized nucleotide using trichloroacetic acid; 2) the indicated immobilized nucleotide and the next nucleotide in the queue are linked using tetrazole; this reaction proceeds with the formation of an intermediate tetrazolyl derivative of phosphoramidite. 3) unreacted free nucleotides and reaction by-products are washed and unreacted immobilized oligonucleotides are capped to prevent their participation in the next synthesis cycle; capping is carried out by acetylation of free 5'-hydroxyls using acetic anhydride and N-methylimidazole; 4) to stabilize the bond between nucleotides, phosphorus is oxidized using iodine and water, if it is necessary to obtain a phosphodiester bond, or Beaucage reagent (3H-1,2-benzodithiol-3-one-1,1-dioxide), if a phosphotioate bond is required. By varying these two oxidizing agents, a hybrid skeleton can be constructed. The cycle of the four steps described above is repeated for each nucleotide in the sequence. When the sequence is fully synthesized, the oligonucleotide is cleaved from the solid support and the protecting groups are removed using ammonium hydroxide at high temperature. The protecting groups are washed with desalination, and the remaining oligonucleotides are lyophilized.

[00231] For the experiment modeled in Example 3, HepG2 cells from ATCC (cat # HB-8065) were grown on growth medium (MEM / EBSS (Hyclone cat # SH30024, or Mediatech cat # MT-10-010-CV) + 10% EBL (Mediatech cat # MT35-011-CV) + penicillin / streptomycin (Mediatech cat # MT30-002-CI)) at 37 ° C and 5% CO ₂ . The day before the experiment, the cells were reseeded with a density of 0.5 × 10 ⁴ / ml in 6-well plates and incubated at 37 ° C and 5% CO ₂ overnight. On the day of the experiment, the medium in 6-well plates was replaced with fresh growth medium.

[00232] Oligonucleotides obtained from the manufacturer in lyophilized form were diluted to a concentration of 20 μmol in deionized water that did not contain RNase and DNase. Two μl of this solution was incubated with 400 μl of Opti-MEM medium (Gibco cat # 31985-070) and 4 μl of lipofectamine 2000 (Invitrogen cat # 11668019) at room temperature for 20 minutes, then dropwise injected into one well of a 6-well plate with HepG2 cells. A similar mixture containing 2 μl of water instead of the oligonucleotide solution was used for the control "empty" transfection. After 3-18 hours of incubation at 37 ° C and 5% CO2, the medium was replaced with fresh growth medium. 48 hours after the addition of antisense oligonucleotides, the medium was removed and RNA was isolated from cells using the SV Total RNA Isolation System from Promega (cat # Z3105) or the RNeasy Total RNA Isolation kit from Qiagen (cat # 74181), following the manufacturer's instructions. 600 ng of isolated RNA was introduced into the reverse transcription reaction using the Verso cDNAkit cDNA kit from Thermo Scientific (cat # AB 1453B) or the High Capacity cDNA Reverse Transcription Kit (cat # 4368813) as described in the manufacturer's protocol. cDNA obtained from the reverse transcription reaction was used to monitor gene expression using real-time PCR using ABI Taqman Gene Expression Mix (cat # 4369510) and primers / probes designed by ABI (Applied Biosystems Taqman Gene Expression Assay: Hs01088574_ml (PAR4 ) from Applied Biosystems Inc., Foster City CA). The following PCR cycle was used: 50 ° C for 2 min, 95 ° C for 10 min, 40 cycles at (95 ° C for 15 seconds, 60 ° C for 1 min) using a StepOne real-time PCR apparatus Plus Real Time PCR Machine (Applied Biosystems). The change in the frequency of gene expression after treatment with antisense oligonucleotides was calculated based on the difference between the dCt values normalized to 18S between the treated and control samples.

Results: Real-time PCR results show that PAR4 mRNA levels in HepG2 cells significantly increase within 48 hours after treatment with two of the oligonucleotides designed for the antisense sequence of PAR4 jortybo.aApr07 (Figure 1).

Treatment of A549 cells with antisense oligonucleotides

[00233] A549 cells from ATCC (cat # CCL-185) were grown in growth medium (F-12K (ATCC 30-2004) + 10% EBL (Mediatech # 35-011-CV) + penicillin / streptomycin (Mediatech cat # MT30 -002-CI)) at 37 ° C and 5% CO ₂ . One day before the experiment, the cells were seeded with a density of 1.5 × 10 ⁵ / ml in 6-well plates and incubated at 37 ° C and 5% CO ₂ . On the day of the experiment, the medium in 6-well plates was replaced with fresh growth medium. All antisense oligonucleotides were diluted to a concentration of 20 μM. Two μl of this solution was incubated with 400 μl of Opti-MEM medium (Gibco cat # 31985-070) and 4 μl of Lipofectamine 2000 (Invitrogen cat # 11668019) at room temperature for 20 minutes and 6-well A549 cells were added to each well. . A similar mixture containing 2 μl of water instead of the oligonucleotide solution was used for the control "empty" transfection. After incubation for 3-18 hours at 37 ° C and 5% CO _{2, the} medium was replaced with fresh growth medium. 48 hours after the addition of antisense oligonucleotides, the medium was removed and RNA was isolated from cells using the SV Total RNA Isolation System from Promega (cat # Z3105) or the RNeasy Total RNA Isolation kit from Qiagen (cat # 74181) following the manufacturer's instructions. 600 ng of RNA was introduced into a reverse transcription reaction using the Verso cDNA kit from Thermo Scientific (cat # AB 1453B) or the High Capacity cDNA Reverse Transcription Kit (cat # 4368813) according to the manufacturer's protocol. The cDNA obtained from this reverse transcription reaction was used to monitor gene expression using real-time PCR using ABI Taqman Gene Expression Mix (cat # 4369510) and primers / probes designed by ABI (Applied Biosystems Taqman Gene Expression Assay: Hs01058874_ml from Applied Biosystems Inc., Foster City CA). The following PCR cycle was used: 50 ° C for 2 min, 95 ° C for 10 min, 40 cycles of (95 ° C for 15 seconds, 60 ° C for 1 min) in a MX4000 thermal cycler (Stratagene) or apparatus for Real-time PCR StepOne Plus Real Time PCR Machine (Applied Biosystems). The change in the gene expression after treatment with antisense oligonucleotides was calculated based on the difference between the 18C normalized dCt values between the treated and control samples.

Result: Real-time PCR results show that PAR4 mRNA levels in A549 cells significantly increase 48 hours after treatment with the CUR-1566 oligonucleotide aimed at the antisense sequence of the PAR4 gene jortybo.aApr07.

Treatment of HEK293 Cells with Antisense Oligonucleotides

[00234] ATCC HEK293 cells (cat # CRL-1573) were grown in growth medium (MEM / EBSS (Hyclone cat # SH30024 or Mediatech cat # MT-10-010-CV) + 10% EBL (Mediatech cat # MT-35 -011-CV) + penicillin / streptomycin (Mediatech cat # MT30-002-CI)) at 37 ° C and 5% CO ₂ . One day before the experiment, the cells were seeded with a density of 1.5 × 10 ⁵ / ml in 6-well plates and incubated at 37 ° C and 5% CO ₂ . On the day of the experiment, the medium in 6-well plates was replaced with fresh growth medium. All antisense oligonucleotides were diluted to a concentration of 20 μM. Two μl of this solution was incubated with 400 μl of Opti-MEM medium (Gibco cat # 31985-070) and 4 μl of Lipofectamine 2000 (Invitrogen cat # 11668019) at room temperature for 20 minutes and 6-well plates with HEK293 cells were added to each well . A similar mixture containing 2 μl of water instead of the oligonucleotide solution was used for the control "empty" transfection. After incubation for 3-18 hours at a temperature of 37 ° C and 5% CO2, the medium was replaced with fresh growth medium. 48 hours after the addition of antisense oligonucleotides, the medium was removed and RNA was isolated from cells using the SV Total RNA Isolation System from Promega (cat # Z3105) or the RNeasy Total RNA Isolation kit from Qiagen (cat # 74181) following the manufacturer's instructions. 600 ng of RNA was introduced into a reverse transcription reaction using the Verso cDNA kit from Thermo Scientific (cat # AB 1453B) or the High Capacity cDNA Reverse Transcription Kit (cat # 4368813) according to the manufacturer's protocol. The cDNA obtained from this reverse transcription reaction was used to monitor gene expression using real-time PCR using ABI Taqman Gene Expression Mix (cat # 4369510) and primers / probes designed by ABI (Applied Biosystems Taqman Gene Expression Assay: Hs01088574_ml from Applied Biosystems Inc., Foster City CA). The following PCR cycle was used: 50 ° C for 2 min, 95 ° C for 10 min, 40 cycles of (95 ° C for 15 seconds, 60 ° C for 1 min) in a MX4000 thermal cycler (Stratagene) or apparatus for Real-time PCR StepOne Plus Real Time PCR Machine (Applied Biosystems). The change in the gene expression after treatment with antisense oligonucleotides was calculated based on the difference between the 18C normalized dCt values between the treated and control samples.

Result: Figure 3 illustrates the increase in apoptosis in HEK293 cells 48 hours after treatment with CUR-1566 oligonucleotide introduced using Lipofectamine 2000, compared to the control.

Treatment of primary keratinocytes with antisense oligonucleotides

[00235] Primary keratinocytes (from Lifeline Technologies) were grown in growth medium (Keratinocyte culture medium, Lifeline cat # LM0004 + Lifeline growth factors! 030) at 37 ° C and 5% CO ₂ . One day before the experiment, the cells were transplanted with a density of approximately 5 × 10 ⁴ / ml (or about 1/3 dilution from 90% confluence) into 24-well collagen coated plates (Beckton Dickinson BioCoat plates cat # 35 6408) and incubated at 37 ° C and 5% CO ₂ during the night. On the day of the experiment, the medium in 24-well plates was replaced with 1 ml of fresh growth medium. The oligonucleotides supplied by the manufacturer in lyophilized form were diluted to a concentration of 20 μM in deionized water not containing RNase / DNase. Two μl of this solution was incubated with 400 μl of OptiMEM medium (Gibco cat # 31985-070) and 4 μl of TransIT®-LT1 transfection agent (Mirus cat # MIR 2300) at room temperature for 20 minutes, then added dropwise to one well 6-well plate with HepG2 cells. A similar mixture containing 2 μl of water instead of the oligonucleotide solution was used for the control "empty" transfection. After dosing, the plates were incubated overnight at 37 ° C, 5% CO ₂ . 24 hours after the addition of antisense oligonucleotides, the medium was replaced with fresh growth medium and the dosage described above was repeated. 24 hours after the second dosing, RNA was isolated from cells using the SV Total RNA Isolation System from Promega (cat # Z3105) following the manufacturer's instructions. 600 ng of total RNA was introduced into the reverse transcription reaction using the High Capacity cDNA kit cDNA kit from Applied Biosystems (cat # 4368813) according to the manufacturer's protocol. The cDNA obtained from this reverse transcription reaction was used to monitor gene expression using real-time PCR using ABI Taqman Gene Expression Mix (cat # 4369510) and primers / probes designed by ABI (Hs01088574_ml). The following PCR cycle was used: 50 ° C for 2 min, 95 ° C for 10 min, 40 cycles of (95 ° C for 15 seconds, 60 ° C for 1 min) in a StepOne real-time PCR apparatus Plus Real Time PCR Machine (Applied Biosystems). The change in the gene expression after treatment with antisense oligonucleotides was calculated based on the difference between the 18C normalized dCt values between the treated and control samples.

Result: Figure 4 illustrates the increase in the level of apoptosis in keratinocytes 48 hours after treatment with the CUR-1566 oligonucleotide introduced using Lipofectamine 2000, compared with the control (Figure 4).

Example 3: In vitro Apoptosis Studies

[00236] Apoptosis levels were determined in cells using the Titer TACS Assay kit NT following the manufacturer's protocol (Trevigen cat # 4822-96-K). Briefly, 48 hours after transfection with oligonucleotides, the cells were trypsinized, counted and plated in 96-well plates with a density of 100,000 cells / well. 96-well plates were incubated overnight at 37 ° C, 5% CO ₂ . The next morning, the medium was removed, and the cells were fixed in a 3.7% solution of formaldehyde in buffer (Sigma-252549), washed with FSB and 100% methanol (Sigma-M1775-1GA) and stored in 80% ethanol (Sigma-493511) at + 4 ° C.

[00237] To mark DNA breaks, ethanol was removed and the cells were washed with PBS and incubated with proteinase K solution at room temperature for 15 minutes, washed with water and PBS, then incubated with TACS Nuclease Solution for 10 minutes at 37 ° C. After incubation, the cells were washed with PBS and a solution of hydrogen peroxide (Sigma-MKBD1394) was added for 5 minutes at room temperature. The cells were then incubated for another 5 minutes at room temperature with a 1x TdT Labeling tag. The buffer was removed and cells were sequentially incubated with the Labeling Reaction mix labeling reaction at 37 ° C for one hour, and then a buffer was added to stop the 1x TdT Stop Buffer reaction for 5 minutes. After stopping the introduction reaction, the cells were washed with FSB, incubated with streptavidin-conjugated streptavidin Strep-HRP solution at room temperature for 10 minutes, washed with FSB / Tween mixture and incubated with TACS-Sapphire color substrate in the dark for 30 minutes. The reaction was stopped by the addition of 0.2 N HCl (Fluka-343102), and the absorbance was measured in a tablet reader at a wavelength of 450 nm.

1. Hep G2 cells (ATCC Cat # HB-8065) were transfected with 20 nM CUR-1565, CUR-1566 and CUR-1568 oligonucleotides using Lipofectamine 2000 (Lipofectamine ™ 2000) transfection agent (Invitrogen). The cell population was also transfected with 20 nM CUR-1505 (inactive oligonucleotide) and with Lipofectamine 2000 and water (false transfection).

Results: Apoptosis significantly increased in HepG2 cells 48 hours after treatment with phosphothioate oligonucleotides introduced using Lipofectamine 2000, compared with the control (Figure 5).

2. MCF-7 cells from ATCC (cat # HTB-22) were transfected with 20 mM CUR-1565, CUR-1566 and CUR-1568 oligonucleotides using Lipofectamine 2000 (Lipofectamine ™ 2000) transfection agent (Invitrogen).

Results: Apoptosis significantly increased in MCF-7 cells 48 hours after treatment with the CUR-1566 oligonucleotide introduced using Lipofectamine 2000, compared with the control (Figure 6).

[00238] Although the present invention has been illustrated and described by reference to one or more embodiments, equivalent changes and modifications will be apparent to those skilled in the art after reading and understanding the present description and the accompanying drawings. In addition, although the specific properties according to the present invention can be disclosed in only one of several embodiments, such properties can be combined with one or more other properties of other embodiments, which may be necessary and useful for any or particular application.

[00239] The abstract of the present invention will allow the reader to quickly understand the technical essence of the invention. It is understood that it will not be used for the purpose of interpreting or limiting the spirit and scope of the claims below.

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Claims (28)

1. A method of increasing the expression of a PAR4 polynucleotide in a biological system, comprising: contacting said system with at least one oligonucleotide 15 to 30 nucleotides in length, said oligonucleotide targeting a natural antisense sequence for a PAR4 polynucleotide, and wherein said natural antisense sequence has the sequence shown in SEQ ID NO: 2. 2. A method of increasing the expression of a PAR4 polynucleotide in a patient’s cells or tissues in vivo or in vitro, comprising: contacting said cells or tissues with at least one oligonucleotide of 15 to 30 nucleotides in length, said oligonucleotide targeting a natural antisense sequence for the polynucleotide PAR4, and wherein said natural antisense sequence has the sequence shown in SEQ ID NO: 2. 3. The method according to p. 2, characterized in that the at least one oligonucleotide is aimed at a nucleotide sequence containing the coding and / or non-coding nucleotide sequences of the natural antisense sequence of the PAR4 polynucleotide. 4. The method according to p. 2, characterized in that the at least one oligonucleotide is aimed at overlapping and / or non-overlapping sequences of the natural antisense sequence of the PAR4 polynucleotide. 5. The method according to p. 2, characterized in that the at least one oligonucleotide contains one or more modifications selected from: at least one modified sugar residue, at least one modified internucleoside bond, at least one modified nucleotide and their combinations. 6. The method according to p. 5, characterized in that the one or more modifications include at least one modified sugar residue selected from: 2′-O-methoxyethyl-modified sugar residue, 2′-methoxy-modified sugar residue, 2 ′ -O-alkyl-modified sugar residue, bicyclic sugar residue and combinations thereof. 7. The method according to p. 5, characterized in that the one or more modifications include at least one modified internucleoside bond selected from: phosphothioate, 2′-O-methoxyethyl (MOE), 2′-fluoro, alkylphosphonate, phosphodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, trietherphosphate, acetamidate, carboxymethyl ether, and combinations thereof. 8. The method according to p. 5, characterized in that the said one or more modifications include at least one modified nucleotide selected from: peptide nucleic acid, closed nucleic acid, arabinonucleic acid (2′-deoxy-2′-fluoro- β-D-arabinonucleic acid, FANA), their analogue, derivative and combinations. 9. The method according to p. 1, characterized in that the at least one oligonucleotide contains at least one of the oligonucleotide sequences corresponding to SEQ ID NOS: 4, 5, 7, 8 and 9. 10. A method of increasing PAR4 gene expression in mammalian cells or tissues in vivo or in vitro, comprising: contacting said cells or tissues with at least one small interfering RNA (siRNA) oligonucleotide from 15 to 30 nucleotides in length, said at least one siRNA oligonucleotide is specific for the natural antisense sequence for the PAR4 polynucleotide, and said natural antisense sequence has the sequence shown in SEQ ID NO: 2. 11. A synthetic modified oligonucleotide with a length of from 15 to 30 nucleotides containing at least one modification, wherein said at least one modification is selected from: at least one modified sugar residue; at least one modified internucleotide linkage; at least one modified nucleotide and combinations thereof; wherein said oligonucleotide is a compound that hybridizes with the natural antisense sequence of the PAR4 gene in vivo or in vitro and increases the expression of the PAR4 gene in vivo or in vitro compared to the normal control, and the specified natural antisense sequence has the sequence shown in SEQ ID NO: 2. 12. The oligonucleotide according to claim 11, wherein said at least one modification comprises an internucleotide bond selected from the group consisting of: phosphothioate, alkylphosphonate, phosphodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, trietherphosphate, acetamidate and carboxymethyl ether. their combinations. 13. The oligonucleotide according to claim 11, characterized in that said oligonucleotide contains at least one phosphotioate internucleotide bond. 14. The oligonucleotide according to claim 11, characterized in that said oligonucleotide contains a backbone of phosphatioate internucleotide bonds. 15. The oligonucleotide according to claim 11, characterized in that said oligonucleotide contains at least one modified nucleotide, wherein said modified nucleotide is selected from: a peptide nucleic acid, a closed nucleic acid, their analogue, derivative and combination. 16. The oligonucleotide according to claim 11, characterized in that said oligonucleotide contains many modifications, said modifications include modified nucleotides selected from: phosphothioate, alkylphosphonate, phosphodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, triether ether phosphate, acetamide ether, acetamide their combinations. 17. The oligonucleotide according to claim 11, characterized in that said oligonucleotide contains many modifications, said modifications including modified nucleotides selected from: peptide nucleic acids, closed nucleic acids, their analogues, derivatives and combinations. 18. The oligonucleotide according to claim 11, characterized in that said oligonucleotide contains at least one modified sugar residue selected from:
2′-O-methoxyethyl-modified sugar residue, 2′-methoxy-modified sugar residue, 2′-O-alkyl-modified sugar residue, bicyclic sugar residue, and combinations thereof. 19. The oligonucleotide according to claim 11, characterized in that said oligonucleotide contains many modifications, these modifications include modified sugar residues selected from: 2′-O-methoxyethyl-modified sugar residue, 2′-methoxy-modified sugar residue, 2′-O-alkyl-modified sugar residue, bicyclic sugar residue, and combinations thereof. 20. The oligonucleotide according to claim 11, characterized in that said oligonucleotide hybridizes with the coding and / or non-coding nucleotide sequences of the natural antisense sequence of the PAR4 polynucleotide. 21. The oligonucleotide according to claim 11, characterized in that said oligonucleotide hybridizes with the natural antisense sequence of at least one PAR4 polynucleotide and increases the expression of at least one PAR4 polynucleotide in vivo or in vitro compared to a normal control. 22. The oligonucleotide according to claim 11, characterized in that said oligonucleotide contains sequences corresponding to SEQ ID NOS: 4, 5, 7, 8 and 9. 23. A pharmaceutical composition for inducing apoptosis in a biological system expressing or acquiring the ability to express PAR4 gene products containing one or more oligonucleotides according to any one of claims. 11-22 and a pharmaceutically acceptable excipient. 24. The pharmaceutical composition according to p. 23, characterized in that said oligonucleotides contain nucleotide sequences corresponding to SEQ ID NOS: 4, 5, 7, 8 and 9. 25. The pharmaceutical composition according to p. 24, wherein the oligonucleotides corresponding to SEQ ID NOS: 4, 5, 7, 8 and 9 contain one or more modifications or substitutions. 26. The pharmaceutical composition according to p. 25, characterized in that the said one or more modifications are selected from: phosphothioate, methylphosphonate, peptide nucleic acid, closed nucleic acid, and combinations thereof. 27. A method of inducing apoptosis in a biological system expressing or acquiring the ability to express PAR4 gene products, comprising introducing an oligonucleotide of 15 to 30 nucleotides in length into the system, said oligonucleotide targeting the natural antisense sequence of SEQ ID NO: 2 PAR4 polynucleotide and increasing expression the specified polynucleotide PAR4. 28. The method according to p. 27, characterized in that said biological system is a cell or tissue of a patient.

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