TW201209163A - Treatment of BCL2 binding component 3 (BBC3) related diseases by inhibition of natural antisense transcript to BBC3 - Google Patents

Abstract

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of BCL2 binding component 3 (BBC3), in particular, by targeting natural antisense polynucleotides of BCL2 binding component 3 (BBC3). The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of BBC3.

Description

201209163 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION Embodiments of the invention include oligonucleotides and related molecules that modulate the performance and/or function of BBC3. The present application claims priority to U.S. Provisional Patent Application Serial No. 61/363,535, filed on Jan. 12, 2011, the entire disclosure of which is incorporated herein by reference. [Prior Art] 〇 DNA-RNA and RNA-RNA hybridization are important for many aspects of nucleic acid function, including DNA replication, transcription and translation. Hybridization is also important for the detection of specific nucleic acids or the various techniques that alter their performance. For example, antisense nucleotides disrupt gene expression by hybridizing to a target RNA, thereby interfering with RNA splicing, transcription, translation, and replication. Antisense DNA has the additional feature that DNA-RNA hybrids are used as substrates for ribonuclease digestion, which is present in most cell types. The antisense molecule can be delivered to a cell, such as in the case of an oxygen donating nucleotide (ODN), or it can be expressed as an RNA 0 molecule from an endogenous gene. The FDA recently approved the antisense drug VITRAVENETM (for the treatment of cytomegalovirus retinitis), indicating that the antisense drug has therapeutic utility. BRIEF DESCRIPTION OF THE DRAWINGS This summary is provided to introduce an overview of the invention This Summary is submitted with the understanding that it is not intended to be construed as limiting or limiting the scope or meaning of the scope of the application. In one embodiment, the invention provides a method for inhibiting a natural antisense transcript of 157514.doc 201209163, which is upregulated by using an antisense oligonucleotide that targets any region of a natural antisense transcript The corresponding justice gene is reached. It is also encompassed herein to inhibit natural antisense transcripts by siRNA, ribozymes and small molecules, which are considered to be within the scope of the present invention. One embodiment provides a method of modulating the function and/or expression of a BBC3 polynucleotide in a patient's cells or tissues in vivo or in vitro, comprising subjecting the cells or tissues to 5 to 3 nucleotides in length. The antisense oligonucleotide contacts 'where the oligonucleotide has a sequence identity of at least 5 〇〇/〇 with the reverse complement of the polynucleotide'. The polynucleotide comprises SEq ID NO: 2 nucleotide 1 Up to 5 to 30 contiguous nucleotides within 689, thereby modulating the function and/or performance of BBC3 polynucleotides in a patient's cells or tissues in vivo or in vitro. In one embodiment, the oligonucleotide targets a natural antisense sequence of a BBC3 polynucleotide (eg, the nucleotide set forth in SEQ ID NO: 2), any variant thereof, allele, Homologs 'mutants, derivatives, fragments and complementary sequences. An example of an antisense raised nucleotide is set forth as SEq ID NO: 3 to 8. Another embodiment provides a method of modulating the function and/or expression of a BBC3 polynucleotide in a cell or tissue of a patient in vivo or in vitro, comprising subjecting the cell or tissue to a length of 5 to 30 nucleotides The antisense oligonucleotide is contacted, wherein the oligonucleotide has a reverse complement of at least 50° with the BBC3 polynucleotide antisense transcript. Sequence identity; thereby modulating the function and/or performance of BBC3 polynucleotides in a patient's cells or tissues in vivo or in vitro. Another embodiment provides a method of modulating the function and/or expression of a BBC3 polynucleotide in a cell or tissue of a patient in vivo or in vitro, comprising subjecting the cell or tissue to a length of 5 to 30 nucleotides Antisense oligonucleotides 157514.doc 201209163 Touching wherein the oligonucleotide has at least 50% sequence identity with an antisense oligonucleotide of a BBC3 antisense polynucleotide; thereby in vivo or in vivo Externally regulates the function and/or performance of BBC3 polynucleotides in a patient's cells or tissues. In one embodiment, the 'amplifier includes one or more antisense oligonucleotides that bind to sense and/or antisense BBC3 polynucleotides. In one embodiment, the raised nucleotide comprises one or more modified or substituted nucleotides. In one embodiment, the nucleotide is raised to include one or more modified linkages.又一. ^ In yet another embodiment, the modified nucleotide comprises a modified base comprising: thiocarbonate, decyl phosphonate, peptide nucleic acid, 2, 〇 曱 thiol, fluoro- or carbon, Methylene or other locked nucleic acid (LNA) molecules. Preferably, the modified nucleotide is a nucleic acid molecule comprising a-L-LNA. In one embodiment, the nucleophilic acid is administered to the patient subcutaneously, intramuscularly, intravenously or intraperitoneally. In one embodiment, the nucleotides are administered in the form of a pharmaceutical composition. Therapeutic Q regimen comprises administering to the patient at least one antisense compound; however, the treatment can be altered to include multiple doses over a period of time. The treatment can be combined with one or more other types of therapies. * In one embodiment, the nucleotide is encapsulated in a liposome or attached to a carrier molecule (eg, cholesterol, TAT peptide). Other aspects are set forth below. [Embodiment] Several aspects of the present invention are explained below by way of example application with reference to the explanation. It should be understood that various specific details, relationships, and methods are set forth herein to provide a full understanding of the present invention: 157514.doc 201209163. However, it will be readily apparent to those skilled in the art that the present invention may be practiced without the use of the specific details or the use of other methods. The present invention is not limited to the order of the various acts or events, which may occur in a different order and/or concurrent with other acts or events.彳' does not necessarily require all riding actions or events to implement the method. All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species to which the compositions and methods disclosed herein can be applied. Thus, such terms include, but are not limited to, human and mouse genes and gene products. It will be understood that the disclosure of a gene or gene product from a particular species, unless explicitly stated in the context, is intended to be exemplary only and should not be construed as limiting. Thus, for example, for the implementation of the genes disclosed herein in the context of a mammalian nucleic acid and amino acid sequence, 5 is intended to encompass homologous and/or orthologous genes and genes from other animals. Products 'These other animals include, but are not limited to, other mammals, fish 'amphibians, reptiles, and blacks. In one embodiment the 'gene or nucleic acid sequence is human. DEFINITIONS The terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the invention. The singular forms "a", "an" and "the" are also intended to include the plural unless the context clearly dictates otherwise. When the terms "including", "includes", "having", "has", "with" or variations thereof are used in the scope, 1575l4.doc 201209163 The term is intended to mean a range of inclusions in a manner similar to the term "comprising". The term "about" or "approximately" means an acceptable range of error for a particular value determined by those skilled in the art, which depends in part on the manner in which the value is measured or measured, that is, the limits of the measurement system. Sex. For example, according to industry practice, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range that differs from a given value by up to 20%, preferably by up to 10%, more preferably by up to 5%, and even more preferably by up to 1%. Alternatively, especially for biological systems or processes, the term may mean within an order of magnitude, preferably within 5 times, and more preferably within 2 times of a numerical value. In the case of specific values stated in the scope of the application and the patent application, unless otherwise stated, the term "about" is intended to mean within the acceptable tolerance of the particular value. The term "mRNA" as used herein refers to a currently known mRNA transcript of a targeted gene, and any other transcript that can be elucidated. An "antisense oligonucleotide" or "antisense compound" means an RNA or DNA molecule that binds to another RNA or DNA (target RNA, DNA). For example, if it is an RNA oligonucleotide, it binds to another RNA target by RNA-RNA interaction and alters the activity of the target RNA. Antisense oligonucleotides can upregulate or downregulate the performance and/or function of a particular polynucleotide. This definition is intended to encompass any foreign RNA or DNA molecule that is useful from a therapeutic, diagnostic or other point of view. Such molecules include, for example, antisense RNA or DNA molecules, interfering RNA (RNAi), microRNAs, decoy RNA molecules, siRNA, enzyme RNA, therapeutically editable RNA and RNA agonists and antagonists, antisense recruitment 157514. Doc 201209163 Compounds, antisense oligonucleotides, external leader sequence (EGS) oligonucleotides, alternative splicing bodies, primers, probes, and other oligomeric compounds that hybridize to at least a portion of a target nucleic acid. Thus, such compounds may be introduced as single-stranded, double-stranded, partially single-stranded or cyclic polymeric compounds. In the context of the present invention, the term "oligonucleotide" refers to a polymer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or a mimetic thereof. The term "oligonucleotide" also encompasses natural and/or modified monomers or linked linear or cyclic oligomers comprising deoxyribonucleosides, ribonucleosides, substituted and alpha-variable Configuration forms, peptide nucleic acids (pna), locked nucleic acids (LNA), thiolates, decyl phosphonates, and the like. Oligonucleotides can specifically bind to the stem by a regular pattern of monomer-to-monomer interactions (eg, Watson-Crick-type base pairing, Ho6gsteen-type base pairing, or reverse H〇6gsteen type base pairing, or the like) Polynucleotide. The nucleotides raised may be "chimeric" nucleotides, i.e., composed of different regions. In the context of the present invention, a "chimeric" compound is a nucleotide that contains two or more chemical regions, such as a DNA region, an RNA region, a PNA region, and the like. In the case of a nucleotide compound, each chemical region is composed of at least one monomer unit (i.e., nucleotide). Such oligonucleotides typically include at least one region that is modified to exhibit one or more desired properties. Desirable properties of nucleotides include, but are not limited to, for example, increasing resistance to nuclease degradation, increasing cellular uptake, and/or increasing binding affinity for a target nucleic acid. Different regions of the raised nucleotides can thus have different properties. The chimeric oligonucleotide of the present invention can be formed into a mixed structure of two or more of the above-mentioned nucleotides, modified nucleotides, oligonucleosides and/or oligonucleosides 157514.doc 201209163 acid analogs. . Oligonucleotides can be composed of regions that can be joined in a "register", i.e., a single system of continuous linkages (e. g., in native DNA), or via a spacer. The spacers are intended to form a covalent "bridge" between the regions and, in the preferred case, have a length of no more than about one carbon atom. The spacers can have different functions, such as having a positive or negative charge, having specific nucleic acid binding properties (chimeric agents, groove binders, toxins, fluorophores, etc.), having lipophilic properties, and inducing specific secondary structures (eg, Inducing a peptide of alanine containing a helix. As used herein, "BBC3" and "BCL2 binding part 3" encompass all family members, mutants, alleles, fragments, species, coding and non-coding sequences, sense and antisense polynucleotide chains, and the like. As used herein, the terms "BCL2 binding moiety 3", "3 sense 2_binding moiety 3", BBC3, FLJ42994, JFY1, jfy], p53 up-regulators of cell death, PUMA are considered identical in the literature and are in this application Used interchangeably in the case. The term "nucleotide specific for" or "targeted oligonucleotide" as used herein refers to an oligonucleotide having the following sequence capable of forming a stable complex with a portion of a targeted gene, or (8) It is possible to form a stable duplex with a part of the mRNA transcript of the gene. The stability of the complexes and duplexes can be determined by theoretical calculations and/or in vitro analysis. An example analysis for determining the stability of hybridization complexes and duplexes is set forth in the Examples below. The term "target nucleic acid" as used herein encompasses DNA, 157514.doc 201209163 RNA (including mRNA precursors and mRNA) transcribed from the DNA, and cDNA, coding sequences, non-coding sequences, sense or antisense aggregates derived from the RNA. Nucleotide. The specific hybridization of an oligomeric compound to its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of the function of the target nucleic acid by a compound that specifically hybridizes to the target nucleic acid is commonly referred to as "antisense." The DNA function to be interfered with, for example, replication and transcription. The RNA function to be interfered with encompasses all important functions, such as translocation of RNA to protein translation sites, translation of proteins from RNA, splicing of RNA to produce one or more mRNA species, and catalytic activity in which RNA can participate or promote. The overall effect of this interference on the function of the target nucleic acid is to modulate the expression of the encoded product or oligonucleotide. RNA interference ("RNAi") is mediated by double-stranded RNA (dsRNA) molecules with sequence-specific homology to the "target" nucleic acid sequence. In certain embodiments of the invention, the mediator system has a "small interference" RNA duplex (siRNA) of 5 to 25 nucleotides. siRNA is derived from the treatment of dsRNA by an RNase enzyme called Dicer. The siRNA duplex product is recruited into a polyprotein siRNA complex called RISC (RNA-induced silencing complex). Without wishing to be bound by any particular theory, it is believed that RISC can be directed to a target nucleic acid (mRNA is preferred) in which the siRNA duplex interacts in a sequence-specific manner to catalyze cleavage in a catalytic manner. Small interfering RNAs useful in the present invention can be synthesized and used according to procedures well known in the art and well known to those skilled in the art. Small interfering RNAs for use in the methods of the invention suitably comprise from about 1 to about 50 nucleotides (nt). In an example of a non-limiting embodiment, the siRNA can comprise 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 from about 20 to 25 Nucleotides. 157514.doc -10- 201209163 Promote the selection of appropriate oligonucleotides by using a computer program that automatically aligns nucleic acid sequences and indicates regions of identity or homology. These programs are used to compare the obtained nucleic acid sequences by, for example, searching a database such as GenBank or by sequencing the PCR products. Comparison of nucleic acid sequences from various species allows selection of nucleic acid sequences that show the degree of agreement between appropriate species. In the case of unsequenced genes, Southern blots were performed to determine the degree of agreement between genes of the target species and other species. Consistency can be roughly measured by performing Southern blots (as is well known in the industry) with varying degrees of stringency. Such procedures allow for the selection of oligonucleotides that exhibit a high degree of complementarity to the target nucleic acid sequence in the individual to be controlled and a lower degree of complementarity to the corresponding nucleic acid sequences in other species. Those skilled in the art will recognize that a wide range of gene regions suitable for use in the present invention can be selected. "Enzyme RNA" means an RNA molecule having enzymatic activity (〇6(:11,(1988)]·American. Med. Assoc. 260, 3030-3035). Enzymatic nucleic acid (ribozyme) by first binding to none RNA acts. The binding is carried out via a target binding portion of an enzymatic nucleic acid that is immediately adjacent to the enzymatic portion of the molecule used to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes the target RNA and then via base pairing Binding to a target RNA and acting in an enzymatic manner to cleave the target RNA after binding to the exact site. "Bee RNA" means an RNA molecule that mimics the natural binding domain of the ligand. The decoy RNA is thereby bound to the natural target. Competition for specific ligands. For example, overexpression of HIV transactivation (TAR) RNA has been shown to be useful as a "bait" and to effectively bind HIV tat protein, thereby preventing its binding. 157514.doc • 11 - 201209163 To TAR sequences encoded in HIV RNA. This is intended to be a specific example of β. Those skilled in the art will recognize that this is only an example, and that other embodiments can be readily made using techniques generally known in the art. This article The term "monomer" as used herein generally means attached by a diester bond or an analog thereof to form a nucleus having a size ranging from a plurality of monomer units (for example, about 3-4) to about several hundreds of early aging. The monomer of the acid is composed of: thioglycolic acid vinegar, dithiophosphoric acid vinegar, phosphonic acid methyl vinegar, benzoic acid ester, amino squarate, and the like, as follows The term "nucleotide" encompasses both natural and non-natural nucleotides. It should be understood by those skilled in the art that various nucleotides previously considered "unnatural" have subsequently been found in nature. Therefore, "nucleotide" includes not only molecules containing known oxime and ringing heterocyclic rings but also heterocyclic analogs and tautomers. Interpretive examples of other types of nucleotides contain the following Molecules: adenine, guanine, thymine, cytosine, uracil, guanidine, xanthine, diamino guanidine, 8-oxo-purine 6-methyl adenine, 7-azepine, 7 - Degassing birds. Tickets, Ν4, Ν4-ethanol squirting, Ν6, Ν6-ethanol-2,6-two Base, 5_mercaptocytosine, 5_(C3_C6)_ alkynyl cytosine, 5-fluorouracil, 5 · bromoacetidine, pseudo-isocytosine, 2_hydroxy-5-methyl-4 - "Non-natural" nucleotides as described in U.S. Patent No. 5,432,272 to Benner et al. The term "nucleotide" is intended to cover each and every Such examples, as well as analogs and tautomers thereof, are of particular interest in nucleotides containing adenine, guanine, thymine, cytosine, and uracil, which are considered therapeutic and diagnostic in humans. Apply the relevant natural nucleotides. Nucleus 157514.doc -12- 201209163 Bucic acid contains natural 2'-deoxy and 2'-transglycosides (for example, as described in Kornb erg and Baker 'DNA Replication' 2nd Edition (Freeman, San Francisco, 1992) ) and its analogues. An "analog" involving a nucleotide comprises a synthetic nucleotide having a modified base moiety and/or a modified sugar moiety (see, for example, as outlined below: Scheit, Nucleotide Analogs, John Wiley, New York, 1980) Freier & Altmann, (1997) Nucl. Acid. Res., 25(22), 4429-4443, Toulme, JJ, (2001) Nature Biotechnology 19:17-〇18; Manoharan M., (1999) Biochemica et Biophysica Acta 1489: 117-139 » Freier SM, (1997) Nucl. Acid Research, 25: 4429-4443, Uhlman, E., (2000) Drug Discovery & Development, 3: 203-213, Herdewin P., ( 2000) Antisense & iVwc/eic dc/c? Drug Dev., 10:297-310); 2'-0, 3'-C linked [3.2.0] bicyclic arabinose. Such analogs comprise synthetic nucleotides designed to enhance binding properties (e.g., duplex or triplex stability, specificity, or the like). "Hybridization" as used herein means the pairing of substantially complementary strands of an oligomeric compound. A pairing mechanism involves hydrogen bonding between complementary nucleosides or nucleoside-acid bases (nucleotides) of an oligomeric compound chain, which can be Watson-Crick,

HoSgsteen or reverse Hodgsteen hydrogen bonding. For example, adenine and thymine are complementary nucleotides that form a hydrogen bond pair. Hybridization can occur in different situations. An antisense compound is "specifically hybridizable" when the binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to modulate function and / 157514.doc -13 - 201209163 or activity, and is sufficiently complementary to the extent Avoiding the occurrence of antisense compounds and _ nucleic acid sequences under conditions where it is desired to specifically bind (ie, under physiological conditions in the context of internal analysis or therapeutic treatment, and under conditions of analysis in an in vitro analytic situation) Non-specific binding. 77. The phrase "stringent hybridization conditions" & "stringent conditions" as used herein refers to conditions under which a compound of the invention hybridizes to its dry sequence but hybridizes to a minimum number of sequences. Stringent conditions are sequence dependent and differ in the context of the present invention and the context of the present invention, and the "stringent conditions" for hybridization of oligomeric compounds to dry sequences are determined by the analysis of the nature of the oligomeric compounds. In general, stringent hybridization conditions include low concentrations (<0.15 Å) of salts containing inorganic cations (e.g., Na+ or κ+) (i.e., low separation: strength). The temperature is higher than the capture but lower than the oligomerization. Compound: Tm of the dry sequence conjugate, and the presence of a denaturant (such as guanamine, dimethylformamide, bis-F-based hard) or a detergent 12-sodium sulfate (SDS). For example, the '- exchange rate was reduced by 1.1% for each 1% metformin. An example of high stringency hybridization conditions is 〇.1 χ gasified sodium _ sodium citrate buffer (ssc) / o.i% (w / v) SDS (at 60 Torr, maintained for 3 〇 minutes). As used herein, "complementary" refers to the ability to precisely pair between two nucleotides on one or both of the clusters. For example, if the nucleobase at the position of the antisense compound is capable of hydrogen bonding to a nucleobase at a position of the target nucleic acid (the target nucleic acid DNA, RNA, or oligonucleotide molecule), Then, the position at which hydrogen bonding occurs between the nutrient nucleotide and the target nucleic acid is regarded as a complementary position. The oligomeric compound and other DNA, RNA, or nucleoside molecules are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleotides that are hydrogen-bondable to each other. Thus, "specifically hybridizable" and "complementary" are used to indicate a term for a sufficient number of nucleotides to have a sufficient degree of exact pairing or complementarity to provide stability and specificity between the oligomeric compound and the target nucleic acid. Sexual union. It is understood in the art that the oligomeric compound sequence need not be 100% complementary to its target nucleic acid sequence to be specifically hybridized. In addition, oligonucleotides can hybridize in one or more regions such that insertions or adjacent segments do not participate in hybridization events (e.g., loop structures, mismatches, or hairpin structures). The oligomeric compound of the invention comprises 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, of the target region within the target nucleic acid sequence to which it is targeted. Approximately 95%, or at least about 99%, of sequence complementarity. For example, 18 of the 20 nucleotides of the antisense compound are complementary to the target region and the antisense compound that specifically hybridizes will represent 90% complementarity. In this example, the remaining non-complementary nucleotides can be clustered or dispersed with complementary nucleotides and need not be adjacent to each other or adjacent to the complementary nucleotides. Thus, an antisense compound having a length of 18 nucleotides and having 4 (four) non-complementary nucleotides (which are flanked by two regions complementary to the target nucleic acid) has 77.8% overall complementarity to the target nucleic acid. And thus fall within the scope of the invention. The percent complementarity of antisense compounds having a target nucleic acid region can generally be measured using the BLAST program (base local alignment search tool) and the PowerBLAST program known in the art. The homology, sequence identity, or percent complementarity can be used by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings (which use Smith and Waterman algorithm) measured (Adv. 157514.doc -15- 201209163

Appl. Math., (1981) 2, 482-489). The term "thermal melting point" as used herein refers to the temperature at which 5 % of the oligonucleotides complementary to the target sequence hybridize to the target sequence under equilibrium at a defined ionic strength, pH and nucleic acid concentration. Generally, for short oligonucleotides (for example, having 10 to 50 nucleotides), stringent conditions are the following: What is the salt concentration? 1 ^ 7.0 to 8.3 is at least about 0.01 to 1 _ 〇 之 ^ 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子 离子As used herein, "modulating" means increasing (stimulating) or reducing (inhibiting) the expression of a gene. When used in the context of a polynucleotide sequence, the term "variant" may encompass a gene associated with a wild-type gene. Polynucleotide sequence. This definition may also include, for example, "allele", "splicing", "species" or "polymorphism" variants. Splice variants may be significantly consistent with a reference molecule, but There is usually a larger or smaller number of polynucleotides during alternate processing due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may have additional functional domains or no domains. Species change systems vary from species to species. Polynucleotide sequences. Particularly useful in the present invention are variants of a wild-type gene product. Variants may be derived from at least one mutation in a nucleic acid sequence and may produce altered 2 mRNA or produce a structure or function that may or may not be altered. Altered polypeptide Any given natural or recombinant gene may have no allelic form, with one or more allelic forms. Common mutational changes that result in variants are usually attributed to natural deletions, additions or substitutions of nucleotides. Each of the polypeptides may be singly or in combination with others one or more times. 157514.doc -16 - 201209163 The resulting polypeptides generally have significant amino acid identity with each other. Changes in the nucleotide sequence of a particular gene between individuals in a given species. Polymorphic variants may also encompass "single nucleotide polymorphisms" (SNp) or single-sequence mutations, where the nucleotide sequence The presence of a SNP may indicate, for example, that a population has a predisposition to disease status (ie, susceptibility to resistance). Derived polynucleotides are subjected to chemical modification (eg, hydrogen from an alkane) A nucleic acid, such as a derivatized oligonucleotide, may comprise a non-natural moiety, such as an altered sugar moiety or an intersaccharide linkage. Such exemplary street biology systems Phosphoroesters and other sulfur-containing substances known in the art. Derivative nucleic acids may also contain labels, including radionucleotides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, receptors, cofactors, inhibitors, magnetics. Particles and the like. "Derivative" polypeptides or peptides (for example) are modified by: glycosylation, pegylation, phosphonium, sulfation, reduction/alkylation, saccharification, chemical coupling Or mild formalin treatment. Derivatives may also be modified to contain detectable labels (directly or indirectly), including but not limited to radioisotopes, fluorescent and enzymatic labels. The term "animal" or "patient" as used herein. It is intended to include, for example, humans, sheep, elk, deer, mule deer, shoal, scorpion animals, monkeys, horses, cows, pigs, goats, dogs, cats, rats, mice, birds, chickens, Reptiles, fish, insects and spiders. Mammals encompass warm-blooded mammals (eg, humans and domestic animals) that are usually under medical care. Examples include cats, dogs, horses, cattle, and 157514.doc -17- 201209163 humans, and only humans. "Trating or treatment" encompasses the treatment of a disease state in a mammal and comprises: (4) in a mammal, in particular when the mammal is susceptible to a disease state but has not yet been diagnosed with the disease state Preventing the occurrence of the disease state; (b) inhibiting the disease state, for example, preventing its development; and/or (c) slowing the disease state, for example, reducing the disease state until the desired endpoint is reached. Treatment also includes amelioration of the symptoms of the disease (e. g., pain relief or discomfort), wherein the improvement may or may not directly affect the disease (e.g., cause, infection, performance, etc.). As used herein, "cancer" refers to all types of cancer or neoplasms or malignancies found in mammals, including but not limited to leukemia, lymphoma, melanoma, carcinoma, and sarcoma. Cancer itself manifests itself as a "tumor" or tissue that includes malignant cells of cancer. Examples of tumors include sarcomas and carcinomas such as, but not limited to, fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovium Tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma 'sweat adenocarcinoma, Sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial ductal carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, ville Wilms, tumor, cervical cancer, sputum tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma , pineal tumor, hemangioblastoma, 157514.doc -18- 201209163 neuroma, oligodendrocyte glioma, meningioma, melanoma, neuroblastoma and retinoblastoma. Other cancers that may be treated by the compositions disclosed herein include, but are not limited to, for example, Hodgkin's DiSeaSe, non-Hodgkin's lymphoma (N〇nH〇dgkin, s Lymphoma), and steep Monthly tumor, neuroblastoma, breast cancer, Indian cancer, lung cancer, transverse, 'muscle 0 primary thrombocytosis, primary macroglobulinemia, small cell lung tumor, primary brain tumor, Gastric cancer, colon cancer, malignant pancreatic islet tumor, malignant carcinoid, bladder cancer, gastric cancer 'pre-deterioration skin damage, testicular cancer lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant high calcium Blood, cervical cancer, endometrial cancer, adrenal cortical cancer and prostate cancer. As used herein, "neurological disease or condition" refers to any disease or condition of the nervous system and/or the visual system. "Nervous diseases or conditions" include those involving the central nervous system (brain, brainstem and cerebellum), the peripheral nervous system (including cranial nerves), and the autonomic nervous system (located in the ten pivots and peripheral nervous systems). A disease or condition. Neurological diseases or conditions include, but are not limited to, acquired epilepsy aphasia; acute disseminated encephalomyelitis; adrenal leukodystrophy; age-related macular degeneration; corpus callosum dysplasia; aphasia; Aicardi syndrome; Aiexander disease, Aipers' disease; crossed limb paralysis, Alzheimer's disease; vascular dementia; amyotrophic lateral sclerosis; no brain malformation; angel syndrome (Angelrnan) Syndrome); angiomatosis; hypoxia; aphasia; apraxia; arachnoid cyst; arachnoiditis; A-Cai's malformation (Anr〇nl_Chiari 157514.doc -19- 201209163 malf〇rmati〇n); Pulse malformation; Asperger syndrome (Asperger synd_e); ataxia telangiectasia; attention deficit with hyperactivity disorder 'autism' autonomic dysfunction; back pain; Batten disease Becht's disease resembles ❹); Bells palsy, benign essential sputum; benign local muscle atrophy; benign Gu internal pressure, Binswanger's disease Binswanger, shua (10) called; eye 睑痉 ' 'Bloch Sulzberger syndrome, brachial plexus injury; brain abscess; brain injury; brain tumor (including glioblastoma multiforme); spinal cord tumor Brown-Sequard Syndrome; Canavan disease; Carpal tunnel syndrome; Burning neuralgia; Central pain syndrome; Central medullary cerebral lysis; Head disorders; Cerebral aneurysms; Cerebral arteriosclerosis; brain atrophy, cerebral giant disease; cerebral palsy; Charcot-Made-Tooth disease; chemotherapy-induced neuropathy and neuropathic pain 'Chiari malformation (Chiari Malformation; chorea; chronic inflammatory demyelinating polyneuropathy; chronic pain; chronic regional pain syndrome; c-ffin L〇wry syndrome; coma, including persistent vegetative state Congenital facial paralysis; cortical basal degeneration; cranial arteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulative traumatic disease, · Cushing's syndrome (Cushing's syndrome); giant cell inclusion disease; cytomegalovirus infection; dance eye-dance foot syndrome; DandyWalker syndrome; Dawson disease; De Morsier's syndrome ;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;; Abnormal; early childhood epilepsy encephalopathy; empty sella syndrome; encephalitis; cerebral palsy; brain trigeminal angiomatosis; epilepsy; Erb's palsy; idiopathic tremor; Fabry's disease Fahr's syndrome; fainting; familial spastic paralysis; febrile seizures; Fisher syndrome; Friedreich's ataxia; frontotemporal dementia and Other "tau lesions"; Gaucher's disease; Gerstmann's syndrome; giant cell arteritis; giant cell inclusion disease ; globular white matter dystrophy; Guillain-Barre syndrome; HTLV-1 related to myelopathy; Hallervorden-Spatz disease; head injury; headache; Hereditary spastic paraplegia; genetic polyneuritis-like ataxia; ear herpes zoster; herpes zoster; Hirayama syndrome; HIV-related dementia and neuropathy (and neurological manifestations of AIDS) Forebrain non-cracking malformation; Huntington's disease and other poly- glutamine repeat disease; water-free brain-free malformation; hydrocephalus; hypercortisolism; hypoxia; immune-mediated encephalomyelitis; inclusion bodies Myositis; pigment disorders; infant phytanic acid storage disease; infant refsum disease; infantile spasm; inflammatory myopathy; intracranial cyst; intracranial hypertension; Joubert syndrome Keams-Sayre syndrome; Kennedy disease; Kingsbrinna disease 157514.doc -21 - 201209163 Kinsboume syndrome; Ke-Fei's syndrome (Klippel Feil syndrome); Krabbe disease; Kugelberg-Welander disease; Kuru disease; Lafora disease; Lang-Ai Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome; Yan Zhao lateral (Wallenberg) syndrome; learning disability; Leigh's disease , Lennox-Gustaut syndrome; Lesch-Nyhan syndrome; leukodystrophy; Lewy body dementia; no brain return, atresia Syndrome; Lou Gehrig's disease (ie, motor neuron disease or amyotrophic lateral sclerosis); lumbar disc disease; Lyme disease - neurological sequelae; horse-cure disease (Machado) -Joseph disease); brain hypertrophy; giant brain; Melkersson-Rosenthal syndrome; Menieres disease; meningitis; Menkes disease; Malnutrition of the white matter; microcephaly Migraine; Miller Fisher syndrome; small stroke; mitochondrial myopathy; Mobius syndrome; single limb muscle atrophy; motor neuron disease; abnormal vascular network Disease; mucopolysaccharidosis; multiple infarct dementia; multifocal motor neuropathy; multiple sclerosis and other demyelinating disorders; multiple systemic atrophy with positional hypotension; muscular dystrophy; myasthenia gravis; Diffuse sclerosis; infant myoclonic encephalopathy; myoclonus; myopathy; myotonia; narcolepsy; neurofibromatosis; nerve blocker 157514.doc -22- 201209163 malignant syndrome; neurological manifestations of AIDS; Neurological sequelae; neuromuscular rigidity, neuron-like lipofuscin; abnormal brain neuron migration; Niemann-Pick disease; O'Sullivan_McLow's disease (O'Sulhvan- McLeod syndrome; occipital neuralgia; recessive spinal canal insufficiency sequence; Daejeon syndrome (〇htahara syndrome); olivine pons cerebellar atrophy; strabismic cerebral palsy; optic neuritis; Standing hypotension; overuse syndrome; paresthesia; neurodegenerative diseases or conditions (Parkins〇n, s disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALs) , dementia 'multiple sclerosis and other diseases and conditions associated with neuronal cell death), congenital accessory myotonia; paraneoplastic disease; paroxysmal seizure; parry Romberg syndrome; - Pelizaeus-Merzbacher disease; periodic paralysis; peripheral neuropathy; painful neuropathy and neuropathic pain; persistent vegetative state; systemic developmental delay; optical sneezing reflex; phytanic acid storage disease ;Pick's disease; nerve kneading; pituitary tumor; polymyositis; brain penetrating malformation' pediatric numbness syndrome late syndrome; post-constrained neuralgia, · post-infectious encephalomyelitis; orthostatic hypotension; - Prader_ Willi syndrome; primary lateral sclerosis; prion disease; progressive one-sided atrophy; progressive multifocal leukoencephalopathy; progressive sclerosis Atrophic gray matter atrophy; progressive nuclear paralysis; pseudocephaloma; Ramsay-Hunt syndrome (types I and II); Rasmussen's encephalitis; reflex sympathetic nutrition Adverse syndrome; Refsum disease; repetitive motor disorder; repetition 157514.doc -23· 201209163 sexual compression injury; restless leg syndrome; retrovirus-associated myelopathy; Rett syndrome ); Reye, s syndrome, Saint Vitus dance; Sandhoff disease, Sehilder's disease; cerebral palsy; clear septum - optic nerve dysplasia Stunning infant syndrome; vesicular therapy; Shy-Drager syndrome; Sjogren's syndrome; sleep apnea; Soto's syndrome; sputum state, · spine Splitting; spinal cord injury; spinal cord tumor; spinal muscular atrophy; Stiff_Pers〇n syndrome; stroke; Sturge-Weber syndrome; Sclerotherapy encephalitis; subcortical arteriosclerotic encephalopathy, Sydenham chorea; syncope; syringomyelia; tardive dyskinesia; Thai-sax disease (Tay_Sachs diseas uniform; Gu artery k' Spinal cord involvement syndrome; Thomsen disease; thoracic outlet syndrome; Tic Douloureux; Todd's paralysis; hyperactive slang syndrome; transient brain deficiency episode; Encephalopathy; transverse myelitis; traumatic brain injury; trembling; trigeminal neuralgia; tropical spastic paraplegia; tuberous sclerosis; jk tube dementia (multiple infarct dementia); vasculitis 'including arteritis; V〇n Hippel-Lindau disease, Wallenberg's syndrome; Werdnig-Hoffman disease; West syndrome; cervical vertebra Wounds; Williams syndrome; Wilson's disease; and Zweig's disease 157514.doc •24- 201209163 Group (Zellweger syndrome) 〇 "Proliferative A disease or condition includes, but is not limited to, a neoplastic neoplastic disorder involving a cell derived from a proliferative/neoplastic cell of a bone marrow-like, lymphoid or red blood-globular lineage, or a precursor cell thereof. Such diseases include, but are not limited to, erythroblastic leukemia, acute anterior bone marrow leukemia (APML), chronic myelogenous leukemia (CmL), lymphoid malignancies (including but not limited to acute lymphoblastic leukemia (ALL), which includes B _ lineage ALL and T-lineage ALL), chronic lymphocytic leukemia (CLL), lymphoblastic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM) ). Other forms of malignant lymphoma include, but are not limited to, non-Hodgkin's lymphoma and its variants, peripheral T-cell lymphoma, adult tau cell leukemia/lymphoma (ATL), skin tau cell lymphoma (CTCL), giant granules Lymphocytic leukemia (LGF), Hodgkin's disease, and Reed_Stowe's disease disease) ° "Inflammation" refers to systemic inflammatory conditions and local and monocyte, white blood cell and / or sputum neutral ball migration And attracting the disease. Examples of inflammation include, but are not limited to, infection with pathogenic organisms (including gram-negative bacteria, gram-negative bacteria, viruses, fungi, and parasites such as protozoa and snails), transplant rejection (including solid organs) For example, rejection of kidney, liver, heart lung or cornea, and bone marrow transplantation (including rejection of graft versus host ', disease (HD)), or local chronic or acute autoimmunity or allergies should cause fire. Autoimmune diseases include glomerulonephritis; wind-like W-responsive arthritis; chronic glomerulonephritis; inflammatory bowel disease, 157514.doc -25- 201209163 such as Crows disease, canceration Colitis and necrotizing enterocolitis, hepatitis, sepsis, alcoholic liver disease; nonalcoholic steatosis; syndrome associated with granule ball infusion; inflammatory skin, for example, contact! 4 skin fire, cowhide癖; systemic lupus erythematosus (Team E), autoimmune thyroiditis, multiple sclerosis, and some forms of diabetes, or any other autoimmune state of lesion tissue damage caused by an individual's own immune system attack. Allergic reactions include allergic asthma, chronic bronchitis, acute and delayed allergies. Systemic inflammatory conditions include traumatic events following trauma, burns, reperfusion (eg, thrombotic events in the heart, brain, intestine, or peripheral blood vessels, including myocardial infarction and stroke), sepsis, ARDS, or multi-function Inflammation associated with the disorder syndrome. Inflammatory cell recruitment also occurs in atherosclerotic plaques. Inflammation includes, but is not limited to, non-Hodgkin's lymphoma, Wegener's granulomatosis, Hashim〇t〇s thyroiditis, hepatocellular carcinoma, thymic atrophy, chronic pancreatitis, rheumatoid Arthritis, reactive lymphoproliferative, osteoarthritis, ulcerative colitis, papillary carcinoma, Crohn's disease, ulcerative colitis, acute cholecystitis, chronic cholecystitis, cirrhosis, chronic mumps, peritonitis, acute pancreas Inflammation, chronic pancreatitis, chronic gastritis, uterine adenomyosis, endometriosis, acute cervicitis, chronic cervicitis, lymphatic hyperplasia, multiple sclerosis, acute idiopathic thrombocytopenic purpura secondary hypertrophy, primary IgA nephropathy, systemic lupus erythematosus, psoriasis, emphysema, chronic pyelonephritis, and chronic cystitis. Polynucleotide and Nucleotide Compositions and Molecular Targets: In one embodiment, the target comprises a 157514.doc •26-201209163 nucleic acid sequence of BCL2 binding portion 3 (bbc3), including (without limitation) associated with BBC3 Justice and/or antisense non-coding and/or editing > 5 horse sequences. BCL2 binding moiety 3 (BBC3) or recognition 4 is a p53 regulatory gene induced after DNA damage. It was subsequently found that purna is responsible for many p53-induced pro-apoptotic effects, but Puma can also cause cell apoptosis in a p53-independent manner. Designating Puma as a sensitizer or activator has been somewhat controversial. Only the BH3 domain of the BH3 protein is required and sufficient to interact with Bcl-2 family members and appears to reproduce the function of the intact protein to a large extent. For example, 〇

The BH3 domain of Bid and Blm activates Bax and Bak ° in liposomes or mitochondria. In some studies, the Puma BH3 domain does not have this function, leading to the classification of Puma as a sensitizer by many people. However, experiments using ubiquitin translated in vitro showed an ability to activate Bax comparable to Bim and Bid. Puma has traditionally been considered a p53-inducible modulator of cell death, but it also plays a key role in the p53-independent cell death pathway, such as those in which growth factors are absent or induced by glucocorticoid therapy. The mechanism by which Puma promotes Q apoptosis is attributed to its ability to bind with high affinity and thus inhibit all Bcl-2 family members (including Bcl_2, bcl-XL, Bcl_w, Mcl-1 and A1). It has also been reported that puma can activate the intrinsic apoptotic pathway by direct binding to B ax . In the case, antisense oligonucleotides are used to prevent or treat diseases or conditions associated with members of the BBC3 family. Exemplary diseases and conditions mediated by the binding moiety 3 (BBC3), which can be used for stem cell regeneration from stem cells obtained using antisense compounds, include: diseases associated with abnormal function and/or performance of BBC3 or A disease, a cancer, a proliferative disease, or a disease or condition characterized by cell proliferation, a disease or condition associated with cell proliferation, or a disease or condition associated with abnormal or abnormal performance or function of BBC3. , 'two diseases or conditions, inflammation, with inflammation of the joint become part of the disease (" such as, osteoarthritis, arthritis, psoriatic arthritis, juvenile arthritis, Lytel's syndrome (Reiter, s syndr 〇me), arthritis associated with ulcerative colitis, Whipple's disease, arthritis associated with granulomatous ileitis, Behcet's se, lupus erythematosus, dry syndrome (Sj〇gren, s-Ο and mixed connective tissue diseases, etc.), diseases or conditions associated with mitochondrial apoptosis pathway damage, autoimmune diseases or disorders, and neurological A disease or condition associated with cell death, aging, or other condition characterized by undesirable cell loss. In embodiments of the invention, treatment and or cosmetic treatment is provided for an individual in need of skin treatment or at risk of developing a condition that may require skin treatment. The protocol and related personalized treatments can be diagnosed based on, for example, the individual's BBC3 status. The amount of BBC3 expression in a given tissue (eg, alpha skin) of a patient can be known by those skilled in the art and are herein The method set forth elsewhere, for example, by analyzing tissue using PCR or antibody-based detection methods. In one embodiment, a patient in need is treated with one or more antisense oligonucleotides to mMBC3 for prevention or treatment. Any disease or condition associated with abnormal expression, function, activity (compared to a normal control group) of BBC3. In one embodiment, the oligonucleotide is specific for a polynucleotide of BBC3, including (but not limited to) Non-coding regions. BBC3 targets include variants; mutants of BBC3, including SNp; non-coding sequences of 3; 157514.doc •28- 201209163 genes, fragments and Preferably, the oligonucleotide is an antisense rNa molecule. According to an embodiment of the invention, the target nucleic acid molecule is not limited to the BBC3 polynucleotide but extends to any isoform, receptor, homologue of BBC3. , non-coding regions, and the like. In one embodiment, the oligonucleotide targets a natural antisense sequence of a BBC3 target (a natural sequence encoding a non-coding region) comprising, without limitation, variants, alleles thereof , homologues, mutants, derivatives, fragments and complementary sequences. Preferably, the oligonucleotide is an antisense RNA or DNA molecule. In one embodiment, the oligomeric compound of the invention is also included in the compound. Variants of different bases are present at one or more nucleotide positions. For example, if the first nucleotide is adenine, a variant containing a chestnut, guanosine, cytidine or other natural or non-natural nucleotide at this position can be produced. This can occur anywhere in the antisense compound. The compounds are then tested using the methods described herein to determine their ability to inhibit the performance of the target nucleic acid. 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 6% to about 7%. In some embodiments the 'homology, sequence identity or complementarity is from about 7% 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 angstroms/inch. The antisense compound can specifically hybridize in the following cases: the compound and the target nucleus 157514.doc •29·201209163 cause loss of activity, and the binding of the acid can interfere with the normal function of the target nucleic acid. Such conditions occur at a level of sufficient complementarity to avoid non-specific binding under conditions in which the antisense compound specifically binds to the non-targeted receptor (ie, physiological conditions in the case of in vivo or therapeutic treatment and in vivo) Conditions for performing the analysis in the case of external analysis. An antisense compound (whether DNA, RNA, chimeric compound, or substituted compound, etc.) can specifically hybridize under the following conditions (the binding of the compound to the target DNA or RNA molecule can interfere with the normal function of the target DNA or RNA) Loss of utility, and sufficient degree of complementarity to avoid antisense compounds and non-target sequences under conditions that are expected to specifically bind (ie, under physiological conditions in vivo or in therapeutic treatment or in vitro) Non-specific binding occurs under conditions in which the analysis is performed under analysis. In one embodiment, the BBC3 is targeted (including, without limitation, an antisense sequence identified and expanded using, for example, PCR, hybridization, etc., one or more of the sequences set forth as seq N〇: 2, and the like) The performance or function of the BBC3. In one embodiment, the performance or function is up-regulated compared to a control group. In a consistent example, performance or function was down-regulated compared to the control group. In one embodiment, the oligonucleotide comprises a nucleic acid sequence set forth as SEQ ID NOS: 3 to 8, comprising an antisense sequence identified and expanded using, for example, PCR, hybridization, and the like. Such nutrient acids may include one or more modified nucleocapnias, shorter or longer fragments, modified linkages, and the like. Examples of the modified bond or the internucleoside linkage include a phosphorothioate, a dithiophosphate or the like. In one embodiment, the nucleotide comprises a phosphorus derivative. A phosphorus derivative (or 1575l 4.doc • 30-201209163 modified phosphate group) that can be attached to a sugar or a sugar analog moiety of a modified oligonucleotide of the invention can be a monophosphate, a diphosphate, Triphosphates, alkyl phosphates, alkyl phosphates, phosphorothioates, and the like. The preparation of the above-described phosphate vinegar analogs, and their incorporation in nucleotides, modified nucleotides and nutrient acid are also known per se and need not be set forth herein. • Those skilled in the art also utilize the specificity and sensitivity of antisense oligonucleotides in therapeutic applications. Antisense oligonucleotides are used as therapeutic moieties to treat disease states in animals and humans. Antisense oligonucleotides have been safely and efficiently administered to humans and many clinical trials are currently being performed. It has thus been determined that the recruitment of nucleoside niacin can be a useful therapeutic modality that can be used in the treatment of cells, tissues and animals, especially humans. In an embodiment of the invention, a recruitment antisense compound, particularly an oligonucleotide, binds to a target nucleic acid molecule and modulates the expression and/or function of the molecule encoded by the target gene. DNA functions to be interfered with include, for example, replication and transcription. The RNA function to be interfered with includes all important functions, such as translocation of RNA to protein translation sites, translation of proteins from RNA, generation of one or more splicing of rNA, and catalytic activity in which RNA can participate or promote. The function can be adjusted or suppressed depending on the desired function. Antisense compounds comprise an antisense oligomeric compound, an antisense oligonucleotide, an external leader sequence (EGS) nucleotide, an alternative splicing, a primer, a probe, and other oligomeric compounds that hybridize to at least a portion of the dry nucleic acid. Thus, such compounds can be introduced as single-stranded, double-stranded, partially single-stranded or cyclic oligomeric compounds. In the context of the present invention, the drying of an antisense compound to a particular nucleic acid molecule can be a multi-step process. This process usually begins with the identification of the dry core to be regulated. 157514.doc • 31 201209163 Acid. For example, the '(iv) nucleic acid can be a cellular gene (or mRNA transcribed from the gene) associated with a particular disorder or disease state or a nuclear I knife from an infectious agent. In the present invention, the target nucleic acid encodes a BCL2 binding moiety 3 (BBC3) ). The targeting process also typically involves determining at least one stem region, region or site within which the antisense interaction occurs within the target nucleic acid to produce a desired effect (e. g., to modulate performance). In the context of the present invention, the term "region" is defined as a moiety having at least one identifiable structure, function or property in a dry nucleic acid. There is a segment within the region of the target nucleic acid. The "segment" $ is the smaller or sub-portion of the region within the target nucleic acid. The "site" used in the present invention is defined as the position within the dry nucleic acid. In one embodiment, the antisense raised nucleotide binds to the natural antisense sequence of BCL2 binding portion 3 (BBC3) and regulates the expression and/or function of BBC3 (SEQ 〇 n〇: 1). Examples of antisense sequences include Eq ID N〇: 2 to 8. In one embodiment, the antisense oligonucleotide binds to one or more segments of a BcL2 binding portion 3 (BBC3) polynucleotide and modulates BBC3i expression and/or function. The segment includes at least 5 contiguous nucleotides of a BBC3 sense or antisense polynucleotide. In one embodiment, the antisense oligonucleotide is specific for the natural antisense sequence of BBC3, wherein binding of the raised nucleotide to the natural antisense sequence of BBC3 modulates the expression and/or function of BBC3. In one embodiment, an 'oligonucleotide compound comprises an antisense sequence characterized as SEq ID N〇: 3 to 8, which is identified and expanded using, for example, pcR, hybridization, and the like. The ## oligonucleotide may comprise one or more modified nucleotides, shorter 157514.doc •32· 201209163 Ο

Or longer fragments, modified keys, and the like. Examples of modified bonds or internucleotide linkages include thiolates, dithiophosphates, and the like. In one embodiment, the nucleotide comprises a fill derivative. The filled derivative (or modified phosphate group) that can be attached to the sugar or sugar analog moiety of the modified nucleotide of the present invention can be monophosphate, diphosphate, triphosphate, tartaric acid. Base esters, alkyl phosphates, phosphorothioates, and the like. The preparation of the above-described acid-filled analogs, and their incorporation in nucleotides, modified nucleotides and oligonucleotides are also known per se and need not be set forth herein. As is known in the art, 'the translation initiation codon is usually 5, _AUG (in the transcription of mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also called "AUG codon", Start codon or rAUG start codon. A few genes have a translation initiation codon with an RNA sequence of 5'-GUG, 5I-UUG or 5I-CUG; and it has been shown that 5, _aua, 5, _acg and 5, -CUG can function in vivo. Thus, even if the starting amino acid is in each case usually thiol amide (in eukaryotes) or methionine (in prokaryotes), the terms "translation start codon" and "start" The code "" can also cover a variety of codon sequences. Eukaryotic and prokaryotic genes can have two or more alternative initiation codons, either of which can be preferentially used for translational initiation in a particular cell type or tissue or under a particular set of conditions. In the context of the present invention, the "start codon" and "translating start codon" are in vivo (4) from the transcription of the gene encoding the binding member 2 (BBC3) to the one of $ & Multiple codons, regardless of the sequence of such codons. The translation stop codon (or "stop codon") of US ^ ^ ^丞 can have two loyalty; Wei-species sequence (ie, 5'-UAA, 51-157514.doc •33· 201209163 5'- TAG and UAG and 51-UGA 'the corresponding DNA sequences are one of 5, _taa 5'-TGA, respectively. The terms "start codon region" and "translation initiation codon region" refer to about -25 to about 5 contiguous regions in the mRNA- or gene-directed direction (ie, 5 or 3) from the translation initiation codon. The bitter acid - part. Class m also #f 5 I "stop codon region" and "translation stop codon region I" refers to this 1RNA or gene in the ^: direction from the translation stop codon (ie, 5' or 3,) About 25 to about 5 adjacent nucleotides - 4 knives, the 'start codon region' (or "translation start codon region J" and "stop codon region" (or "transfer stop codon region" The antisense compound of the present invention is effective to dry to all regions. Open reading frames (〇RF) or "encoding regions" are known in the art to refer to the region between the sub-translating stop codon and the region that can be effectively targeted. In the context of the present invention The targeting region is the intra-gene region of the translation start or stop codon of the open reading frame (0RF) of the gene. The other target region contains 5, non-translated regions (5, utr), which are known in the art. In the region of the mRNA from the direction 5 of the translation initiation codon, and thus the mRNA 5, the nucleotide between the capping site and the translation initiation codon (or another target region of the corresponding nucleotide on the gene) Contains the 3, non-translated region (3'UTR) known in the art, which refers to the portion of the mRNA in the direction from the translation stop codon 3, and thus contains the translation stop codon of the mRNA and the 3' end Nucleotide (or the corresponding nucleotide on the gene). mRNA25, the capping site includes 5, 5, -3, triphosphate linkage to the mRNA 5, _ the most residual 157514.doc • 34· 201209163 N7-T-based bird bitter residue. mRNAi5, capped area is considered to contain the capping structure itself and The cap site is adjacent to the previous 5 nucleotides. Another dry region of the invention is the 5' capped region. Although some eukaryotic mRNA transcripts can be directly translated, many true nuclear organisms 111 ft 1 transcript Contains one or more regions that are cleaved from the transcript prior to translation and are referred to as "introns." The thorny (and thus translated) regions are referred to as "exons" and spliced together to form a contiguous mRNA sequence. In one embodiment, a targeted splice site (ie, an intron-exon node or an exon-intron node) is particularly useful for aberrant splicing associated with disease or overproduction of a particular splicing product In a disease-related situation, another embodiment of an abnormal fusion node that is due to rearrangement or deletion is a target site. mRNA transcripts generated by splicing two (or more) mRNAs from different gene sources For "fusion transcripts", antisense compounds that target, for example, 1) or 111} 前^ precursors can be used to effectively target introns. In one embodiment, the antisense oligonucleotide binds to the Q and/or non-coding region of the target polynucleotide and modulates the expression and/or function of the target molecule. In one embodiment, the antisense oligonucleotide binds to a natural antisense polynucleotide and modulates the expression and/or function of the target molecule. In one embodiment, the antisense raised nucleotide binds to the sense polynucleotide and modulates the expression and/or function of the molecule. Alternative RNA transcripts can be produced from the same genomic region of DNA. Such alternative transcripts are commonly referred to as "variants." More specifically, the "1111^^8 precursor variant" is a transcript produced from the same genomic DNA, which has a starting or stopping position with respect to other transcripts generated from the same genomic DNA 157514.doc •35 · 201209163 is different and contains intron and exon sequences. When one or more exons or intron regions, or portions thereof, are cleaved during splicing, the mRNA precursor variants produce smaller "mRNA variants". Thus, mRNA variants of treated mRNA precursor variants and each unique mRNA precursor variant must always produce a unique mRNA variant by splicing. Such mRNA variants are also referred to as "alternate splice variants". If the mRNA precursor variant is not spliced, the mRNA precursor variant is identical to the mRNA variant. Variants can be generated via the use of alternating signals that initiate or stop transcription. The mRNA steroid and mRNA may have more than one start codon or stop post code. Variants derived from mRNA precursors or mRNAs using alternative initiation codons are referred to as "alternative initiation variants" of mRNA precursors or mRNA. Their use of alternative stop codon transcripts is referred to as an "alternative stop variant" of the mRNA precursor or mRNA. One specific type of alternative stop variant is a "polyA variant" in which multiple transcripts are derived from alternatives to one of the "polyA stop signals" of the transcriptional machinery, resulting in a unique polyA position. Point-terminated transcript. Above the invention, the types of variants described herein are also examples of target nucleic acids. The position on the target nucleic acid that hybridizes to the antisense compound is defined as the portion of the target region that is 5 nucleotides long that is targeted by the active antisense compound. Although specific sequences of certain exemplary target segments are set forth herein, those skilled in the art will recognize that such sequences are used to illustrate and exemplify particular embodiments within the scope of the invention. Other target segments can be readily identified by those skilled in the art in light of this disclosure. A target segment of 5 to 100 nucleotides in length and comprising at least five (5) 157514.doc - 36 - 201209163 contiguous nucleotides selected from the preferred interpretive target segments is also considered suitable for dryness. The target segment may comprise a DNA or RNA sequence comprising at least 5 contiguous nucleotides from the 5'-end of one of the preferred interpretable target segments (remaining nucleotide acid is contiguous in the same DNA or RNA) A segment that begins immediately upstream of the 5'-end of the target segment and continues until the DNA or RNA contains from about 5 to about 1 nucleotide. Similarly, a preferred target segment is represented as a DNA or RNA sequence comprising at least 5 contiguous nucleotides from the 3, _ terminus of one of the preferred interpretable target segments (the remaining nucleotide is the same DNA) Or a continuous segment of the RNA 'which is immediately downstream of the 3, _ terminal of the target segment and continues until the DNA or RNA contains about 5 to about 1 nucleotide.) Those skilled in the art can identify other preferred dry segments without undue experimentation. Once one or more target regions, segments or sites have been identified, an antisense compound that is sufficiently complementary to the target (i.e., hybridized sufficiently and sufficiently specific) can be selected to achieve the desired effect. In an embodiment of the invention, the raised nucleotide binds to the antisense strand of a particular target. The nucleotides are raised to a length of at least 5 nucleotides and can be synthesized such that each nucleus is directed to an overlapping sequence' whereby the oligo-acid is synthesized to cover the entire length of the dry polynucleotide. The target also contains both coding and non-coding regions. In one embodiment, the antisense oligonucleotide preferably targets a specific nucleic acid. The targeting of antisense compounds to specific nucleic acids is a multi-step process. This process typically begins with the identification of the nucleic acid sequence for which the function is to be modulated. This may, for example, be a cellular gene (or 157514.doc-37-201209163 mRNA from gene transcription) or a non-coding polynucleotide (eg, non-coding RNA (ncRNA)) that is associated with a particular condition or disease state. . RNA can be classified into (1) messenger RNA (mRNA), which is translated into protein; and (2) non-protein-encoding RNA (ncRNA). ncRNAs include microRNAs, antisense transcripts, and other transcription units (TUs) that contain high-density stop codons and lack any extensive "open reading frame". Many ncRNAs appear to start at the promoter site in the 3' non-translated region (3'UTR) of the locus encoding the protein. ncRNAs are generally rare and at least half of the ncRNAs sequenced by the FANTOM Alliance appear to be unpolyadenylated. Most researchers pay attention to the polyadenosylated mRNA that is processed and exported to the cytoplasm for obvious reasons. Recently, the group showing non-polyadenylated nuclear RNA may be extremely large, and many of these transcripts are derived from so-called intergenic regions. The mechanism by which ncRNA regulates gene expression is base pairing with the target transcript. RNAs that function by base pairing can be grouped into: (1) cis-encoded RNA, which encodes at the same genetic position at which it functions, but encodes at the relative bond of the RNA and thus shows complete complementarity with its stem And (2) trans-encoded RNA, which encodes at a different chromosomal location than the RNA it functions with and typically does not exhibit full base pairing potential with its target. Without wishing to be bound by theory, the interference of antisense oligonucleotides described herein with antisense polynucleotides may alter the performance of the corresponding sense messenger RNA. However, this regulation can be uncoordinated (increased messenger RNA caused by knockdown) or coordinated (antisense weakening causes concomitant messenger RNA reduction). In such cases, the antisense oligonucleotide can target overlapping or non-overlapping portions of the antisense transcript to create a weak or sequestration. The encoding of 157514.doc -38-201209163 and non-coded antisense transcripts can be performed in the same manner, and any species can regulate the corresponding sense transcripts in a coordinated or uncoordinated manner. The strategy used to identify new oligo (tetra) acids against the stem can be based on any other way of weakening antisense transcripts or modulating the target by antisense oligo-acids. Strategy 1: In the case of uncoordinated regulation, weakening antisense transcripts increases the performance of the (just) gene. If the post-gene coding is known or assumed to be dry, it is conceivable to weaken its antisense counterpart to mimic the effects of a receptor agonist or an enzyme stimulator. Strategy 2: In the case of coordinated regulation, both antisense and justice can be weakened at the same time and thus synergistically reduce the expression of the (just) gene. For example, if the money is used to achieve a weakening by antisense (4) acid, then this strategy can be applied to the antisense oligodeoxynucleotide of the antisense oligos of the sense transcript to the antisense oligo of the corresponding antisense transcript. A single energy symmetric antisense raised nucleotide that is acid, or simultaneously dry, overlapping overlapping sense and antisense transcripts. According to the present invention, antisense compounds include antisense oligonucleotides, ribozymes, exo-P-directing sequence (EGS) oligonucleotides, siRNA compounds, and mono-哎 double-stranded interference (RNAi) compounds (eg, Xiancai) Compounds), and other oligomeric compounds that hybridize to at least a portion of a nucleic acid and modulate its function. Thus, it may be DNA, RNA, a flank, a class ship, or a mixture thereof, or may be a mimetic of one or more of substances such as 5 hai. Such compounds may be mono-double-stranded, %-like or hairpin-type oligomeric compounds' and may contain structural elements such as internal or terminal expansion, mismatch or loop. Antisense compounds are typically made in a linear form, but may be joined or otherwise formed into a cyclic and/or branched form. Antisense compounds can include, for example, the following: hybridization with I57514.doc-39-201209163, Chengyuanwang or. The two strands of the double bond compound are split, or have a single autoligand sufficient to complement the hybridization and form a single strand of the fully or partially double stranded compound. The two chains can be internally connected to create a free 3, or 5, end or connectable to form a continuous cooling structure or loop. The hairpin structure can be at 5 or 3, and the end has a pendant single XL long single chain feature. The double-stranded compound may contain an overhanging moiety at the end as needed. Other modifications may include attachment to one end, a selected nucleotide position, or a coupling group attached to an internucleoside linkage. Alternatively, the two strands can be joined via a non-nucleic acid moiety or a linker group. When the field is formed only by a strand, the dsRNA may be in the form of an auto-complementary hairpin-type molecule that folds itself to form a duplex. Thus, the dsRNA can be fully or partially double stranded. Gene expression can be specifically regulated by the stable expression of dsRNA hairpins in transgenic cell lines, however, in some embodiments, the genes are shown or stressed up. In the formation of a single strand from two strands, or an auto-complementary hairpin-type molecule (folded to form a duplex), the two strands (or regions that form a duplex in a single strand) are Wats〇nCrick Method of pairing complementary RNA strands. Following introduction into the system, the compounds of the invention may elicit - or a plurality of enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid or may act via a mechanism based on occupancy. In general, nucleic acids (including oligonucleotides) can be described as "class jobs" (ie, usually have one or more 2, deoxy sugars and (usually) τ instead of u-test) or "RNA-like" (i.e., usually one or more 2,-trans-based or 2,-zinc-plated sugar and (usually) 1; not a τ-test group). Nucleic acid snails: More than one type of structure can be used, most commonly Α type and Β type. As a general rule, the nucleotide type "DNA" of the B-type structure is 157514.doc -40-201209163 and those having the A-type structure are "like RNA". In some (chimeric) embodiments, the antisense compound can contain both A- and B-type regions. In one embodiment, the desired oligonucleotide or antisense compound comprises at least one of the following: an antisense RNA, an antisense DNA, a chimeric antisense oligonucleotide, an antisense oligonucleotide comprising a modified linkage Glycosidic acid, interfering RNA (RNAi), short interfering RNA (siRNA); small interfering RNA (miRNA); small temporal RNA (stRNA); or short hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small Activated RNA (saRNA), or a combination thereof. dsRNA also activates gene expression, a mechanism known as "small RNA-induced gene activation" or RNAa. A dsRNA targeting a gene promoter can induce efficient transcriptional activation of the gene. RNAa is displayed in human cells using synthetic dsRNA (referred to as "small active RNA" (saRNA). It is not yet known whether RNAa is conserved in other organisms. Small double-stranded RNA (dsRNA), such as small interfering RNA (siRNA) and microRNA (miRNA), have been found to be triggers of an evolutionarily conserved mechanism of RNA interference (RNAi). RNAi typically causes gene silencing by remodeling chromatin to thereby suppress transcription, thereby degrading complementary mRNA, or blocking protein translation. However, in the context of the detailed description of the Examples section below, the display oligonucleotides enhance the performance and/or function of the BCL2 binding portion 3 (BBC3) polynucleotide and its encoded product. dsRNA can also be used as a small activating RNA (saRNA). Without wishing to be bound by theory, saRNA can induce target gene expression by targeting sequences in gene promoters, a phenomenon known as dsRNA-induced transcriptional activation (RNAa). In another embodiment, the "preferred target segment" identified herein can be used to screen for additional compounds that modulate the expression of a BCL2 binding moiety 3 (BBC3) polynucleotide. "Modulators" are compounds which reduce or increase the performance of a nucleic acid molecule encoding BBC3 and include at least a 5-nucleotide portion that is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding a sense or natural antisense polynucleotide of BBC3 with one or more candidate modulators, and selecting one or more to reduce or increase the encoding of the BBC3 polynucleus Candidate modulators of the expression of nucleic acid molecules of glucosinolates (e.g., SEQ ID NOS: 3 to 8). If one or more candidate modulators are shown to be capable of modulating (eg, reducing or increasing) the performance of a nucleic acid molecule encoding a BBC3 polynucleotide, the modulator can be used in other investigative studies of BBC3 polynucleotide function, or as a A research, diagnostic, or therapeutic agent of the invention. The function of the gene is preferably adjusted to the natural antisense sequence. For example, the BBC3 gene (for example, accession number is NM_001127242). In one embodiment, the target is an antisense polynucleotide of the BBC3 gene. In one embodiment, the antisense oligonucleotide targets a sense and/or natural antisense sequence of a BBC3 polynucleotide (eg, accession number NM_001127242), variants, alleles, isoforms, homologs thereof , mutants, derivatives, fragments and complementary sequences. Preferably, the oligonucleotide is an antisense molecule and the target comprises the coding and non-coding regions of the antisense and/or sense BBC3 polynucleotide. Preferred target segments of the invention may also be combined with their corresponding complementary antisense compounds of the invention to form stable double-stranded (duplex) oligonucleotides. It has been shown in the art that these double-stranded oligonucleotide moieties can modulate target expression and regulate translation and RNA processing via antisense mechanisms. In addition, the double-stranded moiety can be chemically modified. For example, it has been shown that the double-stranded portions can inhibit the target by typical hybridization of the antisense strand of the double stranded 157514.doc-42-201209163 to the target, thereby triggering the enzymatic degradation of the target. In one embodiment, the antisense oligonucleotide targets a BCL2 binding portion 3 (BBC3) polynucleotide (eg, accession number NM_001127242), variants thereof, alleles, isoforms, homologs, mutants , derivatives, fragments and complementary sequences. Preferably, the oligonucleotide is an antisense molecule. According to an embodiment of the invention, the target nucleic acid molecule is not limited to BBC3 but extends to any isoform, receptor, homologue, and the like of the BBC3 molecule.

In one embodiment, the oligonucleotide targets a native antisense sequence of a BBC3 polynucleotide (eg, a polynucleotide set forth as SEQ ID NO: 2), any variant thereof, allele, Homologs, mutants, derivatives, fragments and complementary sequences. An example of an antisense oligonucleotide is set forth as SEQ ID NO: 3 to 8 °. In one embodiment, the nucleic acid sequence of the oligonucleotide and the BBC3 antisense molecule (including, without limitation, is associated with a BBC3 polynucleotide) The non-coding sense and/or antisense sequences complement or bind to and modulate the expression and/or function of the BBC3 molecule. In one embodiment, the oligonucleotide complements or binds to the nucleic acid sequence of BBC3 natural antisense molecule (described as SEQ ID NO: 2) and modulates the expression and/or function of the BBC3 molecule. In one embodiment, the oligonucleotide comprises the sequence of at least 5 contiguous nucleotides of SEQ ID NOs: 3 to 8 and modulates the expression and/or function of the BBC3 molecule. Polynucleotide targets include BBC3 (including members of its family), variants of BBC3; mutants of BBC3, including SNP; non-coding sequences of BBC3; 157514.doc-43-201209163 alleles of BBC3; species variants, Fragments and the like. Preferably, the oligonucleotide is an antisense molecule. In one embodiment, the nucleotides that target the BBC3 polynucleotide include: antisense RNA, interfering RNA (RNAi), short interfering RNA (siRNA); micro interfering RNA (miRNA); small temporal rnA (stRNA) Or short hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); or small activating RNA (saRNA). In one embodiment, 'targeting BCL2 binding moiety 3 (BBC3) polynucleotides (e. g., SEQ ID NOs: 2 to 8) can modulate the performance or function of such targets. In one embodiment, the performance or function is up-regulated compared to a control group. In one embodiment, the performance or function is downregulated as compared to the control group. In one embodiment, the antisense compound comprises the sequence set forth as SEQ ID NOs: 3 to 8. Such oligonucleotides can include one or more modified nucleotides, shorter or longer fragments, modified linkages, and the like. In one embodiment, SEQ ID NOs. 3 to 8 include one or more LNA nucleotides. Table 1 shows exemplary antisense nucleotides for use in the methods of the invention. Table 1 :

Sequence number antisense sequence name sequence SEQ ID NO: 3 CUR-1675 C*C*A*C*C*A*A*G*C*C*A*G*A*T*T*C*C*C*A *C SEQ ID NO: 4 CUR-1676 A*T*C*Cs|tA*C*Csf:C:iiA*T*C*Ti!!C*G*G*C*Cs,eT*T*C SEQ ID NO: 5 CUR-1677 A*G*G*C*T*C*A*A*G*C*G*T*T*C*C*T*C*C*C*A SEQ ID NO : 6 CUR-1678 C*C*C*A5,!G*T*T*A*G*G*T*T*T!|:C*C*G*C*A*T!i!G* T SEQ ID NO: 7 CUR-1679 T*C*T*G*Ti|iC*G*C*Cs|eC*A*A*Gs|!C*T*G*G*A*G*T SEQ ID NO: 8 CUR-1505 T*C*T*G*T*C*G*C*C*C*C*A*A*G*C*T*G*G*A*G*T 157514.doc - 44 - 201209163 The desired target nucleic acid can be modulated in a number of ways known in the art. For example, antisense oligonucleotides, siRNA, and the like are used. An enzymatic nucleic acid molecule (e.g., a ribozyme) is a nucleic acid molecule capable of catalyzing two or more different reactions, including the ability to repeatedly cleave other individual nucleic acid molecules in a nucleotide base sequence-specific manner. The enzymatic nucleic acid molecule can be used, for example, to target substantially any RNA transcript. Due to their sequence specificity, trans-cleaved enzymatic nucleic acid molecules can be used as a therapeutic agent for human diseases. Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the cellular RNA background. This cleavage event causes the mRNA to lose functionality and remove protein expression from the RNA. In this way, the synthesis of proteins associated with disease states can be selectively inhibited. In general, an enzymatic nucleic acid having an RNA cleavage activity functions by first binding to a target RNA. The binding is carried out via a target binding portion of an enzymatic nucleic acid that is immediately adjacent to the enzymatic portion of the molecule used to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes the target RNA and then binds to the target RNA via a complementary base pair and acts enzymatically to cleave the target RNA upon binding to the exact site. This cleavage strategy for a target RNA will disrupt its ability to direct synthesis of the encoded protein. After the enzymatic nucleic acid has bound and cleaves its RNA target, it is released from the RNA to find another target and can repeatedly bind and cleave the new target. Several methods such as in vitro selection (evolution) strategies (Orgel, (1979) Proc_R. Soc. London, B 205, 43 5) have been used to generate various reactions (eg phosphodiester linkages and guanamine linkages) New nucleic acid catalyst for cleavage and ligation). 157514.doc -45- 201209163 The development of ribozymes with optimal catalytic activity will significantly contribute to any strategy that uses RNA lytic ribozymes to regulate gene expression. For example, hammerhead ribozymes act at a catalytic rate (kcat) of about 1 min-1 in the presence of a saturated (1 mM) concentration of Mg2+ cofactor. Artificial "rna ligase" ribozymes have been shown to catalyze the corresponding self-modification reactions at a rate of about 00 min. In addition, it is known that certain modified hammerhead ribozymes have a binding arm composed of DNA that can modulate tRNA cleavage by a multiple of the turnover rate of 1 〇〇 min. Finally, the use of certain nucleotide analogs in place of hammerhead ribozymes to catalyze specific residues within the core produces modified ribozymes that exhibit up to a 10-fold increase in catalytic rate. These findings indicate that ribozymes can significantly promote chemical conversion by significantly greater than the catalytic rate of the catalytic rate exhibited by most naturally occurring cleavage ribozymes in vitro. The structure of some self-cleaving ribozymes can then be optimized for maximum catalytic activity, or a novel RNA motif that exhibits a significantly faster rate of RNA phosphodiester cleavage can be prepared. The intermolecular cleavage of RNA receptors catalyzed by RNA catalysts in accordance with the "hammerhead" model was first shown in 1987 (Uhlenbeck, 〇 c 〇 987) _(10), 328: 596-600). The RNA catalyst is recovered and reacted with a plurality of rna molecules to indicate that it is indeed catalytic. Catalytic RNA based on the "hammerhead" motif design has been used to cleave specific stem sequences' which is achieved by making appropriate base changes in the catalytic RNA to maintain the necessary sequence with the stem sequence. This allows the (iv) rna to cleave the specific sequence and demonstrates that the catalytic RNA designed according to the "hammerhead" model can cleave the specific receptor Rna in vivo. RNA interference (RNAi) has become an effective tool for regulating gene expression in mammalian and mammalian cells 157514.doc -46- 201209163. This approach requires the delivery of small interfering RNA (siRNA) in the form of RNA itself or DNA using expression plasmids or viruses and coding sequences for small hairpin RNAs that are processed into siRNA. This system enables efficient delivery of siRNA precursors to their active cytoplasm and allows the use of regulated and tissue-specific promoters for gene expression. In one embodiment, the oligonucleotide or antisense compound comprises an oligo or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA), or a mimetic, chimera, analog or homolog thereof Things. This term encompasses nucleotides consisting of natural nucleotides, sugars, and covalent internucleoside (backbone) linkages; and nucleotides that have non-natural portions and function in a similar manner. Such modified or substituted oligonucleotides are generally preferred over native forms due to desirable properties such as enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases. According to the invention, an oligonucleotide or "antisense compound" comprises an antisense oligonucleotide (eg RNA, DNA, mimetic, chimera, analog or homolog thereof), ribozyme, external leader sequence (EGS) Oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds (eg, siRNA compounds), saRNAs, aRNAs, and other oligomeric compounds that hybridize to and modulate at least one portion of a dry nucleic acid. Thus, it can be DNA, RNA, DNA-like, RNA-like, or a mixture thereof, or can be a mimetic of one or more of such substances. The compounds may be single chain, double stranded, cyclic or hairpin polymeric compounds and may contain structural elements such as internal or terminal expansion, mismatch or loop. Antisense compounds are typically made in a linear form, but may be joined or otherwise formed into a cyclic and/or branched form. Antisense 157514.doc 47· 201209163 may comprise a construct such as two strands that are hybridized to form a fully or partially double-stranded compound, or have sufficient auto-complementation to hybridize and form a fully or partially double-stranded compound. Single chain. The two chains can be internally connected to create a free 1 or 5, and the ends can be joined to form a continuous hairpin structure or loop. The hairpin structure may have an overhang at the end of 5, or 3 to extend the single-strand feature. The double-stranded compound may contain an overhang at the end as needed. Other modifications may include attachment to one end, a selected nucleotide position, a sugar position, or a coupling group attached to an internucleoside linkage. Alternatively, the two strands can be joined via a non-nucleic acid moiety or a linker group. Upon formation from only one strand, the dsRNA can be in the form of an auto-complementary hairpin-type molecule that folds in itself to form a duplex. Thus, the dsRNA can be fully or partially double stranded. The gene expression can be specifically regulated by stably expressing the dsRNA hairpin in the transgenic cell line. When formed from a single strand of two strands, or in the form of an auto-complementary hairpin-type molecule (self-folding to form a duplex), the two strands (or regions forming a duplex in a single strand) are in Watson_Crick mode. The complementary strand of base pairing. Following introduction into the system, the compounds of the invention may elicit - or a plurality of enzymes or structural proteins to effect cleavage or other modification of the target (d) or may act via a mechanism based on occupancy. In general, nucleic acids (including oligonucleic acids) can be described as "brain-like" (ie, usually having one or more 2,_deoxy sugars and (usually) Τ instead of U-killers) or "class ships" (ie, usually with one or more 2'-trans-based or 2|-modified sugars and (usually Chuanchuan instead of τ illusion. Nucleic acid helix can be used in more than one type of structure, most commonly Α type and _ According to 仏 slave, the oligonucleotides with B-like structure are "DNA-like, 157514.doc -48- 201209163 且 and they have _-like structure "Rna" h, antisense compounds The VIII and 6 regions may be included. - The antisense compound of this month may comprise an antisense portion of from about 5 to about rib nucleotides (i.e., from about 5 to about 80 linked nucleops). Means the length of the antisense strand or portion of the antisense compound. In other words, the single bond antisense compound of the invention comprises 5 to a fine nucleotide acid, and the double-stranded antisense compound of the invention (eg, dsRNA) comprises a length of 5 to about 8 正义 sense and antisense strands or parts of nuclear phytic acid. Those skilled in the art should understand that this covers a length of 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 6, 7, 19, 20 31 43 55 67 32 44 56 68 :, 9 '21 33 45 57 69, 22 34 46 58 23 35 47 59 24 36 48 60 25 37 49 61 26 38 50 62 27 39 51 63 28 40 52 64 29 41 53 65 30 42 54 66 Ο 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 or 80 nucleotides , or The antisense portion of any of the ranges. In a pertinent example, the antisense compound of the invention has an antisense portion of from i 〇 to 50 nucleosides in length. 35 Applicants should understand that this exhibits antisense Partial lengths are 1〇, u, 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 any range of oligonucleotides thereof In some embodiments, the oligonucleotide is 15 nucleotides in length. In one embodiment, the antisense or nucleotide compound of the invention has 157514.doc •49·201209163 length 12 or 13 Up to 30 nucleotides of the antisense portion. Those skilled in the art should Understand that 'this shows that the antisense part is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleosides An acid, or any range of antisense compounds thereof. In one embodiment, the oligomeric compounds of the invention also comprise variants in which different bases are present at one or more nucleotide positions in the compound. For example, if the first nucleotide is adenine, a variant containing chest pain, bird or bitterness at this position can be produced. This can occur anywhere in the antisense or dsRNA compound. The compounds are then tested using the methods described herein to determine their ability to inhibit the performance of dry nucleic acids. In some embodiments, the homology, sequence identity or complementarity between the antisense compound and the target is from about 4% to about 6%. In some embodiments, homology, sequence identity or complementarity is from about 6% to about 7%. In some embodiments, homology, sequence identity or complementarity is from about 7% 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 1%. In one embodiment, the antisense raised nucleotides (e.g., the nucleic acid knives set forth in SEQ ID NOS: 3 to 8) include - or a plurality of substitutions or modifications. In one embodiment, the nucleotide is replaced by a locked nucleic acid (LNA).

In one embodiment, the oligonucleotide targets a BBC3-related coding and/or non-coding sequence and one or more regions of the sense and/or antisense nucleic acid molecule set forth as the sequences of SEQ ID NOs: 1 and 2. . Oligonucleotide is also dried to the overlapping region of SEQ ID 157514.doc, 50-201209163 NO: 1 and 2.

Certain preferred oligonucleotides of the invention are chimeric oligonucleotides. The "chimeric oligonucleotide" or "chimera" in the context of the present invention is an oligonucleotide comprising two or more chemically distinct regions, each region consisting of at least one nucleotide acid. The oligonucleotides typically contain at least one modified nucleotide region that confers one or more beneficial properties (eg, increased nuclease resistance, increased cellular uptake, increased binding affinity to dryness); and is capable of cleaving RNA : DNA or RNA: the region of the RNA heterozygote that is regulated by the enzyme. For example, the RNase tether can cleave RNA: an endonuclease of the rNA chain of a DNA duplex. Thus, activated RNase H can lyse dry, thereby greatly increasing the efficiency of antisense regulation of gene expression. Thus, comparable results can be obtained with shorter oligonucleotides when using chimeric oligo(4) compared to phosphorothioate deoxyoligonucleotides that hybridize to the same target region. It is believed that the cleavage of RNA & can be detected by gel electrophoresis and, if desired, nucleic acid hybridization techniques known in the art. In one embodiment, the chimeric nucleocapsid acid comprises at least one region modified to increase target binding affinity, and (usually) used as a region for RNAse Η receptor. The affinity of the nucleus acid and its dry (in this case, one of the codes (1)), the temperature at which the Tm-type oligonucleotide dissociates from the target, is usually determined by measuring the Tm of the oligonucleotide/dry pair; Photometric method to detect dissociation. The higher the Tm, the affinity of the oligonucleotide with the dry one. A chimeric antisense compound of the invention may be two or more as described above: nuclear, modified nucleotides, nucleosides and/or recruitment The structural form of the nucleotide simulation is formed. Therefore, the compounds are also known in the art as heterozygous 157514.doc -51 · 201209163 bodies or combinations. Representative U.S. patents which teach the preparation of such hybrid structures include, but are not limited to, U.S. Patent Nos. 5,013,830, 5,149,797, 5,220,007, 5,256,775, 5,366,878, 5,403,711, 5,491,133, 5, 565, 35, 5, 623 065, 5, 652, 355, 5, 652, 356, and 5, 7, 0, 922, each of which is incorporated herein by reference. In one embodiment, the modified region of the nucleotide comprises at least one nucleotide modified at the 2' position of the sugar, preferably via 2,-0 alkyl, 2,-indole-alkyl _ - an alkyl or 2'-fluoro modified nucleotide. In another embodiment, the RNA modification comprises a ribose for a pyrimidine, an abasic residue or a 21-fluoro, a 2-amino group and a 2, 〇-mercapto group modification of the 3, terminal reverse base. These modifications are typically incorporated into the raised nucleotides and have been shown to have a higher Tm (i.e., higher target binding affinity) for a given target relative to the 2,-deoxyoligonucleotide. This increased affinity effect greatly enhances the inhibition of gene expression by RNAi-raised nucleotides. RNAse Η cleavage RNA: An endonuclease of an RNA strand in a DNA duplex; therefore, activation of this enzyme cleaves an RNA target, and thereby greatly enhances the efficiency of RNAi inhibition. Lysis of the RNA target can usually be visualized by gel electrophoresis. In one embodiment, the chimeric oligonucleotide is also modified to enhance nuclease resistance. The cells contain a variety of degradable nucleic acid exonuclease and endonuclease. A variety of nucleotide and nucleoside modifications have been shown to allow the oligonucleotides incorporating such modifications to be more resistant to nuclease digestion than native oligodeoxynucleotides. Nuclease resistance is usually measured by incubating the oligonucleotide with a cell extract or a knife-free core fee enzyme solution and measuring the amount of remaining intact oligonucleotide by gel electrophoresis after a period of time. . Oligonucleotides that have been modified to enhance their nuclease resistance at 1575l4.doc -52-201209163 remain intact longer than unmodified oligonucleotides. A variety of oligonucleotide modifications have been shown to enhance or confer nuclease resistance. Currently, oligonucleotides containing at least one phosphorothioate modification are preferred. In some cases, oligonucleotides that enhance target binding affinity are also modified to independently enhance nuclease resistance. Specific examples of some preferred oligonucleotides to be used in the present invention include those comprising a modified backbone, such as phosphorothioate, phosphotriester, methyl phosphonate, Short chain alkyl or cycloalkyl sugar linkages or short chain heteroatoms or heterocyclic sugar linkages. The most preferred are oligonucleotides having a phosphorothioate backbone and those having a hetero atom backbone, such as CH2-NH--0--CH2, CH, -N (CH3)--0--CH2 [referred to as methylene (methylimido) or MMI backbone], (:112--0--:^((:^13)--CH2, CH2 - N (CH3)--N (CH3)--CH2 and 0--N (CH3)--CH2 -CH2 backbone, wherein the natural phosphodiester backbone is represented by O--P-O--CH). The guanamine backbone disclosed by De Mesmaeker et al. (1995) Acc. Chem. Res. 28:366-374 is also preferred. Also preferred are oligonucleotides having a morpholino backbone structure (Summerton and Weller, U.S. Patent No. 5,034,506. In another embodiment (e.g., peptide nucleic acid (PNA) backbone), the phosphodiester backbone of the oligonucleotide is replaced by a polyamine backbone, which is directly or indirectly Binding to the aza nitrogen atom of the polyamine backbone. The nucleotide may also include one or more substituted sugar moieties. Preferred oligonucleotides include one of the following groups at the 1-position: OH, SH , SCH3, F, OCN, OCH3 OCH3, OCH3 0(CH2)n CH3, 0(CH2)n NH2 or 0(CH2)n CH3 (n is from 1 to about 10); Cl to CIO Carboalkyl, alkoxyalkoxy, substituted lower alkane 157514.doc •53·201209163 base, aryl or aryl group; Cl; Br; CN; CF3; OCF3; Ο--, S-- Or N-alkyl; 0--, S--, or N-alkenyl; SOCH3; S02 CH3; 0N02; N02; N3; NH2; heterocycloalkyl; heterocycloalkylaryl; Polyalkylamino group; substituted decyl group; RNA cleavage group; reporter group; immigration agent; group for improving the pharmacokinetic properties of the oligonucleotide; or for modifying the oligo a pharmacodynamic property of a glycoside group and other substituents having similar properties. A preferred modification comprises 2'-methoxyethoxy [2LO-CH2 CH2 OCH3, also known as 2'-0-(2) - methoxyethyl)]. Other preferred modifications include 2'-decyloxy (2'-0--CH3), 2'-propoxy (2I-OCH2 CH2CH3) and 2'-fluoro (2' -F). Similar modifications can be made at other positions on the oligonucleotide, especially at the 3' position of the sugar at the 3' terminal nucleotide and at the 5' position of the 5' terminal nucleotide. It may have a sugar mimetic, for example using cyclobutyl instead of pentofuran. It may additionally or alternatively include nucleobase (often abbreviated as the industry's "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleotides include adenine (A), guanine (G), and thymus. D D (T), cells. dense. Set (C) and urine ° density (U). Modified nucleocapnia includes nucleotides found only occasionally or transiently in natural nucleic acids, such as hypoxanthine, 6-mercapto adenine, 5-Me pyrimidine, especially 5-methylcytosine (also known as 5- Methyl-2'-deoxycytosine and commonly referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentiolysyl HMC; and synthetic nucleotides, For example, 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(alkylalkylamino)adenine or other hetero-substituted alkyl glands Bismuth, 2-thiouracil, 2-thiothymidine, 5-bromouracil, 5-157514.doc -54- 201209163 hydroxymethyluracil, 8-indene guanine, 7-deazaguanine, N6 (6-Aminohexyl) adenine and 2,6-diaminopurine. "Common" bases known in the art (e.g., inosine) can be included. The 5_Me_c substitution has been shown to increase the stability of the nucleic acid duplex by 0.6 ° C to 1.2 ° C and is currently the preferred base substitution. Another modification to the oligonucleotides of the invention involves chemically linking one or more of the conjugates that enhance the activity or cellular uptake of the nucleotide to the nucleotide. Such moieties include, but are not limited to, lipid moieties (eg, cholesterol moieties), cholesteryl moieties, aliphatic chains (eg, dodecanediol or undecyl residues), polyamines or polyethylene glycol chains, or diamonds Alkanoic acid. Oligonucleotides comprising a lipophilic moiety, and methods of making such oligonucleotides are known in the art, for example, U.S. Patent No. 5,138, No. 5, No. 5,218, No. 5, and No. 5,459,255. All positions in a given oligonucleotide need not be uniformly modified, and in fact one or more of the above modifications may be incorporated into a single oligonucleotide or even incorporated into a single nucleoside within a nucleotide. The invention also encompasses oligonucleotides which are chimeric oligonucleotides as defined above. In another embodiment, a nucleic acid molecule of the invention is conjugated to another moiety, including but not limited to abasic nucleotides, polyethers, polyamines, polyamines, peptides, carbohydrates, lipids, or Polyhydrocarbon compound. Those skilled in the art will recognize that such molecules can be attached to one or more of any of the nucleic acid molecules at several positions on the sugar, base or phosphofccSa group. Oligonucleotides for use in the present invention can be prepared in a convenient conventional manner via well known solid phase sigma techniques. A number of 157514.doc -55- 201209163 suppliers including Applied Biosystems sell equipment for the implementation of this synthesis. Any other means for this synthesis may also be employed, and the current synthesis of oligonucleotides is well known to those skilled in the art. It is also well known to use similar techniques to prepare other oligonucleotides such as phosphorothioates and alkylated derivatives. Similar techniques and commercially available modified DNA synthetic nucleotides (amidhe) and fixed pore glass (CPG) products (eg, DNA synthesis nuclei modified with biotin, luciferin, acridine or psoralen) are also well known in the art. Glycosylate and/or CPG (available from Glen Research, Sterling, VA)) to synthesize fluorescently labeled, biotinylated or otherwise modified oligonucleotides, such as cholesterol-modified oligonucleotides. In accordance with the present invention, the use of modifications (e.g., using LNA monomers) to enhance the efficacy, specificity, and duration of action and broaden the route of administration of oligonucleotides includes current chemical modalities such as MOE, ANA, FANA, PS, and the like. This can be achieved by replacing some of the monomers in the current oligonucleotide with LNA monomers. The LNA modified oligonucleotide may have a size similar to the parent compound or may be larger or preferably smaller. Preferably, the LNA-modified oligonucleotide contains less than about 70%, more preferably less than about 60%, optimally less than about 50% of the LNA monomer, and is between about 5 and 25 nucleotides in size, More preferably between about 12 and 20 nucleotides. Preferred modified oligonucleotide backbones include, but are not limited to, phosphorothioates, palmitic phosphorothioates, dithiophosphates, phosphotriesters, aminoalkyl phosphates, methyl phosphonates, and Other alkyl phosphonates (including 3'-alkyl esters of phosphonic acids and palmitic phosphonates), phosphinates, amino phosphates (including 3'-amino phosphates of aminophosphates and aminophosphoric acid) Aminoalkyl esters, thiocarbonylamino phosphates, thiocarbonylalkyl phosphonates, thiocarbonylalkyl phosphates, and boron having a 157514.doc -56-201209163 see 3'-5' linkage An alkanoate, a 2,-5' linked analog of the backbone, and those having a reverse polarity (wherein adjacent nucleoside units are linked by 3, -5 to 5'-3' or Change from 2, -5' to 5'-2.). Also included are various salts, mixed salts and free acid forms. Representative U.S. patents including the above-described bonding include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476, 3, 1, 5,023,243, 5,177,196, 5,188,897 , Nos. 5,264,423, 5,276,019, 5,278,3,2, 0,5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925 No. 5,519,126, 5,536,821, 5,541,306, 5,550,111., 5,563,253, 5,571,799, 5,587,361 and 5,625,050, each of which is incorporated by reference. Into this article. Preferred modified oligonucleotide backbones containing no phosphorus atoms have a backbone formed by the following form: short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms, and alkyl or cycloalkyl groups Internucleoside linkages, or one or more short chain heteroatoms or heterocyclic internucleoside linkages. The backbones include those having a morpholino linkage (partially formed from a nucleoside sugar moiety); a siloxane backbone; a sulfide, an alkali, and a sulfone backbone; an oxime group and a thioformate The main chain of methylene sulfonyl group and the main chain of thioformamidine; the main chain containing olefin; the main chain of amino sulfonate; methylene imine and methylene The base of the thiol group; the main chain of the acid and the main chain of the lanthanum: Lai domain; and others with mixed N, 〇, Sach2 components. 157514.doc -57- 201209163 teaches that the representative U.S. patents of the above-mentioned nucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033. , Nos. 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,610,289 , Nos. 5,602,240, 5,608, 046, 5, 610, 289, 5, 618, 704, 5, 623, 070, 5, 663, 312, 5, 633, 360, 5, 677, 437, and 5, 677, 439 each incorporated herein by reference. In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e., the backbone) in the nucleotide unit are replaced by a new group. The base unit is retained to hybridize to a suitable nucleic acid target compound. One such polycondensation compound (an oligonucleotide mimetic that has been shown to have excellent hybridization properties) is called a peptide nucleic acid (pNA). In the PN A sigma, the sugar-main chain in the oligo-nucleotide is replaced by a guanamine-containing main chain, a thiol-ethylaminoglycine backbone. The nucleobase is retained and it is t-amine with the backbone. The aza nitrogen atom of the file is directly or indirectly bonded. Representative U.S. patents for the preparation of the teachings include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262 each incorporated herein by reference. Other teachings of pNA compounds can be found in > Milk 613611, science 254, 1497-1500. "Shenzhizhi 1 case is an oligonucleotide with a thiophene sulphuric acid backbone and an oligonucleotide with a heteroatom backbone and, in particular, the above mentioned US 157514.doc -58- 201209163 Lithium No. 5,489,677 -CH2-NH-0-CH2-, -CH2-N (CH3)-0-CH2-[referred to as methylene (methylimido) or MMI backbone], -CH2 -0-N(CH3)-CH2-'-CH2N(CH3)-N(CH3)CH2- and -0-N(CH3)-CH2-CH2-[where the natural phosphodiester backbone is represented by -0-PO -CH2-], and the guanamine backbone of U.S. Patent No. 5,602,240, incorporated herein by reference. Also preferred are the nucleotides having the morpholino backbone structure of U.S. Patent No. 5,034,506, which is incorporated herein by reference. The modified oligonucleotide may also contain one or more substituted sugar moieties. Preferred nucleotides include one of the following in the 2' position: OH; F; Ο-, S- or N-alkyl, Ο-, S- or N-alkenyl; Ο-, S- or N - alkynyl; or decyl-0-alkyl, wherein the alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl groups. Particularly preferred are O(CH2)n OmCH3, 0(CH2)n, OCH3, 0(CH2)nNH2, 0(CH2)nCH3, 0(CH2)nONH2, and 0(CH2n0N(CH2)nCH3)2, where n And m can be from 1 to about 10. Other preferred oligonucleotides include one of the following in the 2' position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, 0-alkylaryl or anthracene-aryl Alkyl, SH, SCH3, OCN, CM, Br, CN, CF3, OCF3, SOCH3, S02CH3, 0Ν02, Ν02, Ν3, ΝΗ2, heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, poly Alkylamino, substituted decyl, RNA cleavage group, acceptor group, intercalator, pharmacokinetic properties of modified oligonucleotides, or pharmacodynamics of modified nucleotides a group of properties, and other substituents of similar nature. Preferred modifications include 2'-methoxyethoxyethoxy (2'-0-CH2CH20CH3, also known as 2'-0-(2-decyloxyethyl) or 2,-oxime), 157514.doc-59 - 201209163 Also known as 'alkoxy alkoxy. Other preferred modifications include 2,-dimethylaminooxyethoxy (i.e., 0(CH2)20N(CH3)2 groups, also known as 2ι_DMAOE, as described in the Examples below), and 2,_Dimethylaminoethoxyethoxy (also known in the industry as 2,_〇_didecylaminoethoxyethyl or 2ι_DMAEOE'), ie, 2i_〇CH2_〇_CH2 N (CH2) 2). Other preferred modifications include 2,-decyloxy (2'-〇CH3), 2,-aminopropoxy (2'-0 CH2CH2CH2NH2) and 2,-fluoro(2,-F). Similar modifications can be made at other positions on the nucleotide, especially 3, terminal nucleotides or 2, _5, 3, 5 and 5, terminal nucleotides in the oligo. Bit. The oligonucleotide may also have a sugar mimetic, for example, using a cyclobutyl moiety in place of the pentofuranosyl sugar. Representative U.S. patents which teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359, 〇44, 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, Nos. 5,658,873, 5,670,633, and 5,700,920 each incorporated herein by reference. Oligonucleotides may also include nucleobases (often abbreviated as "bases" in the art) for modification or substitution. As used herein, "unmodified" or "native" nucleotides include purines (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-hydroxydecyl 157514.doc -60- 201209163 Cellular bite, yellow, yellow „Ticket, 2_Aminogland (4), 6-methyl and other alkyl derivatives of adenine and guanine, 2_propyl and other alkyl derivatives of adenine and guanine, 2_thio Uracil, 2_thiothymidine and 2-thiocytosine, 5_dentate uracil and cytosine, 5-propynyl uracil and cytosine, 6-azouracil, cytosine and thymine , 5- uracil (pseudouracil), 4-thiouracil, generation, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5_halogenated, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosine, 7-methylguanine and 7-methyl adenine, 8-azaguanine and 8 Azadenine, 7-deazaguanine, and 7-deaza adenine, and 3-deazaguanine and 3-deaza adenine. Additional 'nucleotides including those disclosed in U.S. Patent No. 3,687 , in the 8th and 8th, they are revealed in 'The Concise Encyclopedia o f P〇lymer

Science And Engineering', pp. 858-859, Kroschwitz, JI Editor, John Wiley & Sons, 1990, by Englisch et al. 'Angewandle Chemie, International Edition', 1991, 30, p. 613 And their disclosure by Sanghvi, YS, Chapter 15, 'Antisense Research and Applications', pp. 289-302, Crooke, ST and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleotides are particularly useful for increasing the binding affinity of the polymeric compounds of the present invention. Such nucleotides include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted indole, including 2-aminopropyl adenine, 5-propynyl uracil and 5 - Propynyl cytosine. 5-methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6 ° C to 1.2 ° C (Sanghvi, YS, Crooke, 157514.doc -61 - 201209163 ST and Lebleu, B. Edit ' 'Antisense Research And Applications', CRC Press, Boca Raton, 1993 > 276-278) and is currently the preferred substituent substitution, even more particularly when combined with the 2'-methoxyethyl saccharide modification. Representative U.S. patents which teach the preparation of the above-described modified nucleotides and other modified nucleotides include, but are not limited to, U.S. Patent Nos. 3,687,8,8, and 4,845,205, 5,130,302, 5,134,066. Nos. 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,9,8, 5,502,177, 5,525,711, 5,552,54, Nos. 5,587,469, 5,596,091, 5, 614, 617, 5, 750, 692, and 5, 681, 941, each incorporated herein by reference. Another modification to the oligonucleotides of the invention involves chemically linking one or more conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide to the oligonucleotide. Such moieties include, but are not limited to, lipid moieties (eg, cholesterol moieties), cholic acid, thioethers (eg, hexyl-s-triphenylsulfonyl mercaptan), thiosteroids, aliphatic chains (eg, 'dodecane a diol or an undecyl residue), a phospholipid (for example, di-hexadecyl-racemic-glycerol or triethylammonium 1,2-di-indole-hexadecyl)-racemic mH_ Phosphonic acid vinegar), polyamine or polyethylene glycol chain, or diamond, acetic acid, palmity moiety, or octadecylamine or hexylaminocarbonyl-oxycholesterol moiety. Representative U.S. patents which teach the preparation of such nucleophilic acid conjugates include 157514.doc-62-201209163 but are not limited to U.S. Patent Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313. No. 5.545.730, 5,552,538, 5,578,717, 5.580.731, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, Nos. 5,414,077, 5,486,603, 5,512,439, 5,578,718, 5,608,046, 4,587,044, 4,605,735, 4,667,025, 4,762,779, 4,789,737, 4,824,941, 4,835,263, 4,876,335 No. 4,904,582, 4,958,013, 5.082.830, 5,112,963, 5,214,136, 5.082.830, 5,112,963, 5,214,136, 5,245,022, 5,254,469, 5,258,506 No. 5,262,536, 5,272,250, 5,292,873, 5,317,098, 5,371,241, 5,391 , 723, 5,416,203, 5,451,463, 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 No. 5, 5 ", 923, 5, 599, S > 28 and 5,688, 941, each of which is incorporated herein by reference.濞# 兮: The compounds of the invention may also be added to the area of drug research and target validation. The present invention encompasses the use of the compounds identified herein and preferred target segments in the drug study 9, which attempts to elucidate the bcl2 binding moiety 157514.doc •63·201209163 Polymeric acid and disease state, phenotype, or condition The relationship between the two. The methods comprise detecting or modulating BBC3 polynucleic acid comprising contacting a sample, tissue, cell, or organism with a compound of the invention, and measuring the nucleic acid or protein content of BBC3 polyphosphate at a time after treatment and / or related phenotype or chemical endpoint, and if necessary, compare the measured value to an untreated sample or a sample treated with another compound of the present invention. Such methods can also be performed in parallel or in combination with other experiments to determine the function of an unknown gene for use in a dry validation process, or to determine the effectiveness of a particular gene product (4) (d) in treating or preventing a particular disease, condition or phenotype. Evaluation of gene expression up-regulation or inhibition: The transfer of exogenous nucleic acid into a host cell or organism can be assessed by directly detecting the presence of nucleic acid in the cell or organism. This detection can be achieved by a number of methods known in the industry. For example, the presence of an exogenous nucleic acid can be detected by Southern blotting or by a poly-enzyme chain reaction (PCR) technique using primers that specifically amplify a nucleic acid-related nucleotide sequence. The performance of exogenous nucleic acids can also be measured using conventional methods including gene expression analysis. For example, Northern blotting and reverse transcription PCR (RT_pCR) can be used to detect and quantify mRNA produced from exogenous nucleic acids. The performance of purines in exogenous nucleic acids can also be detected by measuring enzymatic activity or reporter protein activity. For example, the expression of the exogenous nucleic acid in the effector RNA can be indicated by the reduction or increase in the expression of the nucleic acid. Based on sequence conservation, primers can be designed and used to amplify the coding region of the target gene. Initially, the control gene model can be created using the highest coding region from each gene, but any coding or non-coding region can be used. Each control gene was assembled by inserting each coding region between the reporter coding region and its poly(A) signal. Such plasmids will produce mRNAs having a reporter gene in the upstream portion of the gene and a potential RNAi target in the 1 non-coding region. The effectiveness of individual antisense oligonucleotides is analyzed by adjusting the reporter gene. The reporter gene used in the method of the present invention comprises acetaminolate synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucuronidase (GUS), chlorine Acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP) ), luciferase (Luc), saponin synthase (NOS), octopine carnitine synthase (0CS) and its derivatives. A variety of selectable markers that confer resistance to: ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin can be used. , kanamycin, lincomycin, methotrexate, phosphinothricin, pi purmycin, and tetracycline. Methods for determining modulation of reporter genes are well known in the art and include, but are not limited to, fluorescent methods (eg, fluorescence spectroscopy, fluorescence activated cell sorting (FACS), fluorescence microscopy), antibiotic resistance assays . BBC3 protein and mRNA expression can be analyzed using methods known to those skilled in the art and described elsewhere herein. For example, immunological assays such as ELISA can be used to measure protein content. The BBC3 ELISA assay kits are commercially available, for example, from R&D Systems (Minneapolis, 157514.doc-65-201209163 MN). In the examples, the sample treated with the antisense oligonucleotide of the present invention (for example, cells or tissues in vivo or in vitro) is evaluated by comparison with the BBC3 expression in the control sample. BBC3 performance (eg, mRNA or protein). For example, methods known to those skilled in the art can be used to compare the performance of a protein or nucleic acid to a simulated or untreated sample. Alternatively, the desired profile can be used to compare the alpha pattern with the control antisense oligonucleotide (e.g., with altered or different sequences). In another embodiment, the treated sample can be compared to the untreated sample using a difference in the performance of the different nucleic acids in the untreated sample (including any criteria deemed suitable by the investigator, eg, the ruthenium gene). The difference in the expression of BBC3 protein or nucleic acid in the sample. The observed difference / can be expressed, for example, in ratio or fractional form as needed for comparison with the pair, the group. In the examples, the amount of BBC3 mRNA or protein in the sample treated with the antisense oligonucleotide of the present invention is increased or decreased by about 1.25 times to about 1 Å relative to the untreated «sample or sample treated with the control nucleic acid. Multiple or higher. In embodiments, the BBC3 mRNA or protein is increased or decreased by at least about 1.25 fold, at least about 13 fold, at least about 1.4 fold, at least about 10 fold, at least about h6 fold, at least about 17 fold, at least about 1.8 fold. At least about 2 times, at least about 2-5 times, at least about 3 times, at least about 35 times, at least about 4 times, at least about 4 to 5 times, at least about 5 times, at least about 5 · 5 times, at least about 6 times At least about 6.5 times, at least about gamma fold, at least about 7.5 fold, at least about 8 fold, at least about 8.5 fold, at least about 9 fold, at least about 9.5 fold, or at least about 1 fold or greater. 157514.doc -66 - 201209163 Sets of 'Groups, Research Reagents, Diagnosis and Treatment The compounds of the present invention are useful in the diagnosis, treatment, and prophylaxis, and can be used as kit 2 research reagents and components. In addition, those skilled in the art typically use the antisense nucleotides that strongly inhibit gene expression to explain the function of a particular gene or to distinguish the function of each member of a biological pathway. For use in kits and diagnostics and in a variety of biological systems, this topical compound (alone or in combination with other compounds or therapeutic agents) can be used as a tool in differential zero and/or combinatorial analysis to elucidate cells and tissues. The pattern of expression of the gene or part of the entire complement. The term "biological system" or "plant system" as used herein is defined to mean any organism, cell, cell culture or tissue that exhibits, or is sufficient to represent, a BCL2 binding portion 3 (BBC3) gene product. Such systems include, but are not limited to, transgenic animals, cells, cell cultures, tissues, xenografts, grafts, and combinations thereof. According to one non-limiting example, a pattern of expression in a sputum cell or tissue treated with one or more antisense compounds is compared to a control cell or tissue that has not been treated with an antisense compound, and the resulting pattern is analyzed for, for example, a disease Correlation, nickname transmission pathway, cell localization, degree of expression, degree of difference in gene expression of the size, structure or function of the gene being tested. Such analysis can be performed in stimulated or unstimulated cells and in the presence or absence of other compounds that affect performance patterns. Examples of methods for gene expression analysis known in the art include DNA arrays or microarrays, SAGE (series analysis of gene expression), restriction enzyme amplification), TOGA (general gene expression analysis), protein array 157514.doc •67· 201209163 and proteomics, the sequence of the performance of the standard ST) sequencing, subtraction of rna fingerprint technology (surf), subtraction selection, differential display _, comparative genomic hybridization, FISH (fluorescence in situ hybridization) and mass spectrometry. The compounds of the invention are useful in research and diagnostics because such compounds hybridize to nucleic acids encoding BCL2 binding moiety 3 (BBC3). For example, the efficiency of the #effective BBC3 modulator as disclosed herein and the nucleotides that hybridize under such conditions are effective primers or probes under conditions conducive for gene amplification or detection, respectively. Such primers and probes can be used in methods that require specific detection of nucleic acid molecules encoding BBC3 and amplification of such nucleic acid molecules for use in purine detection or in other BBC3 studies. Hybridization of the antisense oligonucleotides (especially primers and probes) of the invention with nucleic acids encoding BBC3 can be detected by methods known in the art. Such means may involve coupling the enzyme to the oligoacid, radiolabeled oligonucleotide, or any other suitable means of detection. It is also possible to manufacture a kit that uses these assays to detect the B B C 3 content of the sample. Those skilled in the art also utilize the specificity and sensitivity of the antisense compound in therapeutic applications. Antisense compounds have been used as therapeutic moieties to treat disease states in animals (匕3 humans). Antisense oligonuclear (IV) drugs have been safe and effective. Many clinical trials have been implemented. It was thus determined that antisense can be a useful treatment for the treatment of cells, tissues and animals (especially humans). ', 〇 #therapy > 胄 胄 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投 投For example, in a non-limiting embodiment 157514.doc-68 - 201209163, the methods comprise the step of administering to a subject in need of treatment a therapeutically effective amount of a BBC3 modulator. The BBC3 modulator of the present invention is effective for regulating the activity of BBC3 or regulating the expression of BBC3 protein. In one embodiment, the activity or performance of BBC3 in the animal is inhibited by about 10% compared to the control group. Preferably, the activity or performance of BBC3 in the animal is inhibited by about 30%. More preferably, the activity or performance of BBC3 in an animal is inhibited by 50% or more. Therefore, the oligomeric compound can modulate the expression of BCL2 binding moiety 3 (BBC3) mRNA by at least 10°/ compared to the control group. 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%. In one embodiment, the activity or performance of BCL2 binding moiety 3 (BBC3) and/or in the animal is increased by about 10% compared to the control group. Preferably, the activity or performance of BBC3 in the animal is increased by about 30%. More preferably, the activity or performance of BBC3 in the animal is increased by 50% or more. Thus, the oligomeric compound can modulate the performance of BBC3 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%, compared to the control group. At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%. For example, the increase or decrease in the expression of BCL2 binding moiety 3 (BBC3) in serum, blood, adipose tissue, liver or any other body fluid, tissue or organ of an animal can be measured. Preferably, the cells contained in the fluid, tissue or organ analyzed comprise a 157514.doc •69·201209163 nucleic acid molecule encoding the BBC3 peptide and/or the BBC3 protein itself. The compound can be used in a pharmaceutical composition by adding an effective amount of a compound of the invention to a suitable pharmaceutically acceptable diluent or carrier. The uses and methods of the compounds of the invention may also be used for prophylactic purposes. Conjugates Additional modification of the oligonucleotides of the present invention involves chemically linking one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the nucleus acid to the oligonucleotide. The moieties or conjugates may comprise a coupling group covalently bonded to a functional group such as a - or a secondary radical. The present invention: a coupling group comprising an intercalating agent, a reporter molecule, a polyamine, a polyamine, a polyethylene glycol, a group for enhancing the pharmacodynamic properties of the oligomer, and a drug for strengthening the polymer A group of metabolic kinetic properties. Typical coupling base: Contains cholesterol, lipids, stone scorpion, biotin, cockroach, folic acid, phenanthrene, phenanthrene, luciferin, rhodamine (-), coumarin and dye: in this In the context of the invention, the group 3 which enhances the pharmacodynamic properties can improve uptake, enhance degradation resistance, and/or enhance the sequence-specific hybridization with the (tetra) acid. In the context of the present invention, Xiao Qiang The group of the pharmacokinetic properties of the drug comprises a group which can improve the uptake, distribution, metabolism or secretion of the compound of the present invention. Representative coupling groups are disclosed in (4) (4) on the 23rd of the international patent application in the request for the pcT /us92/()9(9), and U.S. Patent No. 6,287,86, incorporated herein by reference, which is incorporated herein by reference in its entirety in its entirety in the the the the the the the the -5-diphenylmethyl mercaptan), thiocholesterol, aliphatic chain (for example, dodecyl diol or decyl residue), dish grease (for example, 157514.doc -70· 201209163 two-ten Hexacyclo-racemic glycerol or triethylammonium hydride, 2_di-〇-hexadecyl _ Racemic-glycerol-3-Hphosphonate), polyamine or polyethylene glycol chain, or adamantane acetic acid, palmitoyl moiety, or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides can also be coupled to active drug substances such as 'aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen ( Fenbufen), _ 洛芬 (ket0pr0fen), (S)- (+)-praprofen ((S)-(+)-pranoprofen), carprofen (carprofen), dansylsarcosine, 2 , 3,5-three angstromic acid, flufenamic acid, leucovorin, benzothiadiazine, chlorothiazide, diazepam, indomethacin, barbiturate , cephalosporin, sulfa drugs, antidiabetic agents, antibacterial agents or antibiotics. Representative US patents for the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979, 4,948,882 , Nos. 5,218,105, 5,525,465, 5,541,313, 5.545.730, 5,552,538 Nos. 5,578,717, 5.580.731, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,077, 5,486,603, 5,512,439 No. 5,578,718, 5,608,046, 4,587,044, 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 , 157514.doc •71-201209163 5.082.830, 5.082.830, 5,245,022 '5,262,536 '5,317,098, 5,416,203, 5,512,667, 5,567,810, 5,112,963, 5,214,136, Nos. 5,112,963, 5,214,136, 5,254,469, 5,258,506, 5,272,250, 5,292,873, 5,371,241, 5,391,723, 5,451,463, Nos. 5,510,475, 5,514,785, 5,565,552, 5,574,142, 5,585,481, 5,587,371 , Nos. 5,595,726, 5,597,696, 5,599,923, 5,599,928 and 5,688,941. Formulations The compounds of the invention may also be mixed, encapsulated, coupled or otherwise with other molecules, molecular structures or mixtures of compounds (eg, liposomes, molecules that target receptors, oral, rectal, topical or other formulations). The combination is combined to aid in ingestion, distribution and/or absorption. Representative U.S. patents which teach the preparation of such formulations which facilitate ingestion, distribution and/or absorption include, but are not limited to, U.S. Patent Nos. 5,1,8,921, 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, 5,013,556, 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, 157514.doc •72·201209163 5,534,259, 5,543,152, Nos. 5,556,948, 5,580,575, and 5,595,756 each incorporated herein by reference. Although it is not necessary to administer antisense nucleotides in the context of a vector to modulate target expression and/or function, embodiments of the invention relate to expression vector constructs for expressing antisense oligonucleotides, which include a promoter , a hybrid promoter gene sequence and having a strong constitutive promoter activity, or a promoter activity that can be induced under the desired conditions. In one embodiment, the practice of the invention involves the administration of at least one of the above antisense oligonucleotides using a suitable nucleic acid delivery system. In one embodiment, the system comprises a non-viral vector operably linked to a polynucleotide. Examples of such non-viral vectors include only oligonucleotides (e.g., any one or more of SEQ ID NOS: 3 to 8) or a combination with a suitable protein, polysaccharide or lipid formulation. Further, a suitable nucleic acid delivery system comprises a viral vector, typically from a Q adenovirus, an adeno-associated virus (AAV), a helper cell-dependent adenosis, a retrovirus, or a Japanese liposome-coagulated virus (HVJ) complex. A sequence of at least one of them. Preferably, the viral vector comprises a strong eukaryotic promoter operably linked to an I nucleotide (e.g., a 'cytomegalovirus (Cmv) promoter). Further, the preferred vector comprises a viral vector, a fusion protein and a chemical conjugate. The retroviral vector comprises M〇i〇ney murine white virus and an HIV-based virus. A preferred HIV-based viral vector comprises at least two vectors wherein the gag and P〇1 genes are from the HIV genome and the env gene is derived from 157514.doc-73-201209163 additional-virus. The dNA viral vector is preferred. Such vectors include a ρ 〇χ vector (e.g., orthopoxvirus or fowlpox virus vector), a herpes virus vector (e.g., herpes simplex virus (HSV) vector), an adenoviral vector, and an adeno-associated viral vector. The antisense compound of the invention encompasses any pharmaceutically acceptable salt, ester, or salt of such vinegar, or is capable of providing a biologically active metabolite or residue thereof after administration to an animal (including humans) Any other compound. The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of a compound of the present invention: that is, a salt which retains the desired biological activity of the parent compound and which does not impart an undesirable toxicological effect. Preferred examples of pharmaceutically acceptable salts and their use for oligonucleosides are further described in U.S. Patent No. 6,287,860, incorporated herein by reference. The invention also encompasses pharmaceutical compositions and formulations comprising the antisense compounds of the invention. The pharmaceutical compositions of the present invention can be administered in a variety of ways, depending on the desired local or systemic treatment and the area to be treated. Administration may be local (including ocular administration and administration to the mucosa, including vaginal and rectal delivery of the lungs (eg, by inhalation or spraying of powder or aerosol, including by nebulizer); intratracheal, nasal Internal, epidermal, and transdermal, oral or parenteral administration. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, or intracranial (eg, intralesia or brain) Intravital administration. For the treatment of tissues in the central nervous system, administration can be carried out, for example, by injection or by injection into the cerebrospinal fluid. The administration of antisense RNA in cerebrospinal fluid is described, for example. U.S. Patent Application Publication No. 2/7/117, 772 to "Methods for -g familial ALS disease pr〇gressi〇n" 157514.doc-74-201209163, the entire contents of which is incorporated herein by reference. When the antisense nucleoside acid of the present invention is intended to be administered into cells of the central nervous system, it may be administered - or a plurality of drugs capable of promoting the penetration of the antisense oligodeoxynucleotide into the blood-brain barrier. Within the stinky cortex or hippocampus area for injection. Elaborated by the muscle tissue to deliver to the motor neurons in the administration of adenoviral vectors for neurotrophic factors (for example) US Patent "No. The first M32,427 Adenoviral-vector_mediated BU coffee ⑷

In medullary motor neur〇ns, which is incorporated herein by reference. It is known in the art to deliver a carrier directly to the brain (e.g., striatum, thalamus 'hippocampus or substantia nigra) and is described, for example, in "Aden〇virus vectors f〇r plus (4) of U.S. Patent No. 7 (2). Curry f(10) as in y(10) into cells of the central nervous system particularly in bram", which is incorporated herein by reference. Administration can be by slow infusion or administration of a sustained release formulation by injection for rapid administration or Ik time. The antisense oligo (4) of the target may also be linked or coupled to an agent that provides the desired pharmaceutical or pharmacodynamic properties. For example, antisense oligonucleotides can be coupled to any of the substances known in the art to promote penetration or delivery across the blood-brain barrier (eg, antibodies to the transferrin receptor) and administered by intravenous injection. . The antisense beta can be linked to a viral vector which, for example, can render the antisense compound more effective and/or facilitate delivery of the antisense compound across the blood brain barrier. The osmotic blood-brain barrier can also be destroyed by, for example, infusion of: sugars including but not limited to meso-erythritol, xylitol, D(d)galactose, D(1) lactose, D(+) xylose, Dulcitol, myo-inositol, L(_) fructose, 157514.doc -75- 201209163 D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, fiber Disaccharide, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(one) ribose, ribitol, D(+) arabitol, L(- Alginitol, D(+) fucose, L (one) fucose, D (one) to threose, L (+) to threose and L (-) to threose; or amino acids, including but not limited to bran Aminic acid, lysine, arginine, aspartame, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, Proline, serine, threonine, tyrosine, proline and taurine. Methods and materials for enhancing blood-brain barrier penetration are described, for example, in "Method for the delivery of genetic material across the blood brain barrier", No. 6,294, 520, "Material for passage through the blood-brain". Barriers and "Parenteral delivery systems", No. 6,936,589, the entire contents of each of which are incorporated herein by reference. The antisense compounds of the present invention may be mixed, encapsulated, coupled or otherwise mixed with other molecules, molecular structures or mixtures of compounds (eg, liposomes, molecules to the receptor, oral, rectal, topical or other formulations). Other ways to combine to aid in ingestion, distribution and/or absorption. For example, a cationic lipid can be included in the formulation to facilitate oligonucleotide uptake. One composition which exhibits uptake is LIPOFECTIN (available from 018 (:0-BRL, Bethesda, MD). It is believed that oligonucleotides having at least one 2'-0-decyloxyethyl modification are particularly suitable. Oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, plugs 157514.doc -76- 201209163, mouth Aerosols, liquids and powders. Pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be required or desired. Coated condoms, gloves and the like may also be suitable. Conveniently presented in unit dosage form. The pharmaceutical formulations of the present invention can be prepared according to conventional techniques well known in the art. F 4 includes the steps of combining the active ingredient with a pharmaceutical carrier (group) or excipient (group). The preparation can be prepared by uniformly and intimately combining the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product. The compositions of the invention can be formulated into a number of possible dosage forms. Either For example, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. The liquid may further comprise a substance which increases the viscosity of the suspension, and comprises (for example, methylcellulose sodium behenitol and/or dextran. The suspension may also contain a stabilizer. Ο I The pharmaceutical composition of the invention includes but is not limited to Solutions, emulsions, foams, and formulations containing liposomes. The pharmaceutical compositions and formulations of the present invention may include or be osmotically enhanced, loaded, excipients or other active or non-active ingredients. The liquid is usually a heterogeneous system in which a liquid is dispersed in another liquid in the form of a droplet (the diameter usually exceeds 〇_ι μιη). The emulsion may contain additional group damage in addition to the dispersed phase, and may be present in the aqueous phase or the oil phase. An active drug in the form of a solution or self-dissipating as a knife. One embodiment of the invention comprises a microemulsion. The emulsion and its use are well known in the art and are further described in U.S. Patent No. 1575. 14.doc • 77· 201209163 6,287, 860. The formulations of the invention comprise liposome formulations. The term "liposome" as used in the present invention means a vesicle composed of an amphiphilic lipid arranged in one or more spherical bilayers. Lipid system monolayer or multilamellar vesicles having a membrane formed from a lipophilic material and an aqueous internal structure containing the composition to be delivered. The cationic lipid system is a positively charged liposome which is believed to be associated with a negatively charged DnA Molecular interactions to form stable complexes. pH-sensitive or negatively charged liposomes are believed to capture DNA rather than complex with it. Both cationic and non-cationic liposomes can be used to deliver DNA to cells. Liposomes also contain "space. Stable Liposomes As used herein, the term refers to liposomes comprising one or more specific lipids. When incorporated into liposomes, these particular lipids produce liposomes that have an extended cycle life relative to liposomes that lack the particular lipids. Examples of sterically stabilized liposomes are those in which the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derived from one or more hydrophilic polymers (e.g., polyethylene glycol (pEG) moieties). Liposomes and their use are further described in U.S. Patent No. 6,287,86. ~ The pharmaceutical formulations and compositions of the present invention may also comprise a surfactant. The use of surfactants in pharmaceutical products, formulations and emulsions is well known in the art. Surfactants and their use are further described in U.S. Patent No. 6,287,860, incorporated herein by reference. In a wide variety of embodiments, the present invention employs various permeation enhancers to achieve efficient delivery of the core, particularly oligonucleotide. In addition to contributing to the diffusion of non-lipophilic drugs in the cell membrane, the penetration enhancer also enhances the penetration of lipophilic drugs 157514.doc -78· 201209163. Penetration enhancers can be classified as belonging to one of five categories: that is, surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Permeation enhancers and their use are further described in the disclosure of which is incorporated herein by reference. National Studies (4) Those who are familiar with this technology should recognize that 'mixtures are usually designed according to their intended application (ie, the route of administration). Preferred formulations for topical administration comprise the oligonucleotides of the invention and oxime

The topical delivery agent is, for example, a lipid, a liposome, a fatty acid, a fatty acid vinegar, a steroid, a chelating agent, and a surfactant. Preferred lipids and liposomes comprise neutral lipids and liposomes (for example, dioleic acid base _ 曰醯 曰醯 D D D D D 乙醇 乙醇 、 、 、 、 、 、 、 一 一 一 一 一 一 一 一 一 一 一 一 一 一Choline), negative lipids and liposomes (such as di-myristylphosphatidylglycerol DMpG) and cationic lipids and liposomes (such as dioleyltetramethylaminopropyl D〇TAp and two oils) Base-Linsterylethanolamine DOTMA). ^ f is in the bureau. In the case of sputum or other administration, the oligonucleotides of the present invention may be encapsulated in or form complexes (especially cationic liposomes) with the ruthenium. Alternatively, the oligonucleoside can be complexed with a lipid, especially a cationic lipid. Preferably, the hydroxy acid and the pharmaceutically acceptable salt thereof, and their use are further described in U.S. Patent No. 6,287,86. ^ 2 = Stolen and compositions and formulations comprising powders or granules, micro-r-grain granules, suspensions or solutions in water or non-aqueous media, '^gel capsules, sachets, lozenges or Micro-tablets. It may be desirable to use a flavoring agent, diluent, emulsifier, dispersing sword or adhesive. 157514.doc -79-201209163 Good oral formulations are the same as the ones that have been combined with one or more penetration enhancers, surfactants and chelating agents. Preferably, the surfactant comprises a fatty acid and/or an ester or a salt thereof, a bile acid / τ warm and/or a salt thereof. The preferred bile acids/salts and fatty acids and their use are further described in U.S. Patent No. 6,287,860, the disclosure of which is incorporated herein by reference. And t h ^ liter and T are also preferred as a combination of penetration enhancers, for example, a combination of a fatty acid/salt and a bile acid/salt. The combination of Youjia is sodium salt of lauric acid, citric acid and UDCA. I am going to do it*. Other permeation enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene __ 铃铃纯甘乳 C" 碎20_cetyl ether. The oligonucleotides of the present invention may be orally delivered in the form of particles comprising spray dried particles or complexed to form micro or nano particles. Oligonucleotide® acid complexes and their use are further described in U.S. Patent No. 6,287, the disclosure of which is incorporated herein by reference. Compositions and formulations for parenteral, intralesional or intraventricular administration may comprise sterile aqueous solutions which may also contain buffering agents, diluents and other suitable additives such as, but not limited to, permeation. Enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. Certain embodiments of the invention provide pharmaceutical compositions comprising one or more oligomeric compounds and one or more other chemotherapeutic agents that function by non-antisense mechanisms. Examples of such chemotherapeutic agents include, but are not limited to, cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactjn〇mycin, doxorubicin, and Epirubicjn, idarubicin, es〇rubicin, bleomycin, mafosfamide, if〇sfamide, cytosine Arabinoside 157514.doc 201209163

(cytosine arabinoside), bischloroethyl-nitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, Prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, Wujia Pent ame thy lmel amine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen Nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, arabinose Cytarabine), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-peroxy-cyclohexylamine, 5-fluorourine bite (5- Fluorouracil) (5-FU), 5-deoxyuridine (5-fluorodeoxyuridine) (5-FUdR), guanamine pterin (MTX), colchicines, taxol, vincristine, vinblastine, etoposide (VP-16) ), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin, and diethylstilbestrol ) (DES). When used with a compound of the invention, the chemotherapeutic agents can be isolated (eg, 5-FU and oligonucleotide), sequentially (eg, 5-FU and oligonucleotide, followed by a period of time 157514. Doc -81 - 201209163 is a combination of MTX and nucleotides, or in combination with one or more other such chemotherapeutic agents (eg, 5-FU, MTX and oligonucleotides, or 5_Fu, radiation therapy, and oligonucleotides) )use. Anti-inflammatory drugs (including but not limited to non-steroidal anti-inflammatory drugs and corticosteroids, as well as antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir (acycl vir) And ganciclovir may also be combined in the compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of the invention. Compounds of two or more combinations may be used together or sequentially. In another related embodiment, the compositions of the invention may contain one or more antisense compounds (especially raised nucleotides) that target the first nucleic acid and one or more additional antisense compounds that target the second nucleic acid target. For example, the first target can be a specific antisense sequence of BCL2 binding portion 3 (BBC3) and the second target can be region $ from the other-nucleotide sequence. Alternatively, the inventive composition may contain two or more antisense compounds which are dried to different regions of the same BCL2 binding moiety 3(10)c3) nucleic acid. Many examples of antisense compounds are disclosed herein and other examples may be selected from suitable compounds known in the art. Compounds of two or more combinations may be used together or sequentially. Dosing: It is believed that those skilled in the art should be aware of the formulation of the therapeutic composition and its subsequent administration (administration). Administration depends on the severity and responsiveness of the condition to be treated, where the course of treatment can last from days to months, or until a cure or amelioration of the disease is achieved. The optimal dosing regimen can be calculated by measuring the accumulation of music in the patient's body.孰 π Staying hot with this technology can easily determine the appropriate dosage, method of administration and re-sucking. Dare doses can vary depending on the relative efficacy of the 1575l4.doc -82- 201209163 oligo-nucleotide, and can usually be estimated based on the £(:5〇) found to be effective in in vitro and in vivo animal models. Generally, the dose is from 0.01 call/kg body weight to 100 mg/kg body weight, and can be given once or more per week, maternal week, monthly or yearly, or even every 2 to 20 years. The skilled artisan can easily estimate the repetition rate of administration based on the measured residence time and concentration of the drug in the body fluid or tissue. After successful treatment, it may be desirable to have the patient maintain the therapy to prevent recurrence of the disease state, wherein A maintenance dose between pg/kg body weight to 100 mg/kg body weight is administered to the oligonucleotide and administered once or more times per day to every 20 years. In the examples, the following doses of the drug are used. To treat a patient: at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg Weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, Less than about 9 mg/kg body weight, at least about 1 mg/kg body weight, at least about μ mg/kg body weight, Q at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 3 mg/kg body weight At least about 35 mg/kg body weight, at least about 40 mg/kg body weight, at least about 45 mg/kg body weight, at least about 50 mg/kg body weight, at least about 6 mg/kg body weight, at least about 70 mg/kg body weight, At least about 80 mg/kg body weight, at least about 90 mg/kg body weight, or at least about 1 mg/kg body weight. Certain injection doses of antisense oligonucleotides are described, for example, in "Antisense", U.S. Patent No. 7,563,884. Modulation of PTP1B expression, the entire contents of which are incorporated herein by reference. Although the various embodiments of the present invention have been described above, it is understood that 157514.doc -83 - 201209163 these embodiments are merely examples The present invention is not limited by the spirit and scope of the present invention. The breadth and scope of the present invention should not be limited by the above embodiments. One limit. All the documents mentioned in this article are In this way, all publications and patent documents cited in this application are hereby incorporated by reference inso- In general, the applicant does not recognize that any particular reference is a "prior art" of the invention of the invention. Examples of compositions and methods of the invention are illustrated in the following examples. EXAMPLES The following non-limiting examples are illustrative of selected embodiments of the invention. It is to be understood that the change in the ratio of the elements of the components shown and the alternatives are known to those skilled in the art and are within the scope of the embodiments of the invention. Example 1: Design of an antisense nucleic acid molecule that binds to an antisense nucleic acid molecule of BCL2 binding moiety 3 (BBC3) and, or a sense strand of a BBC3 polynucleotide, as described above, the term "specific to "Nuclear nucleotide" or "undirected oligonucleotide" refers to a nucleotide having the following sequence: (i) capable of forming a stable complex with a part of the dry gene, or (ii) capable of A portion of the mRNA transcript of the dry gene forms a stable duplex.

By using a computer program (eg IDT AntiSense Design, IDT)

OligoAnalyzer) to facilitate the selection of appropriate nucleotides that automatically identify sub-sequences of 19-25 nucleotides in each given sequence, which form a hybrid with the target polynucleotide sequence and It has the desired melting temperature of 157514.doc • 84 - 201209163 (usually 50 ° C to 60 ° C) and does not form self-dimers or other complex secondary structures. The selection of appropriate oligonucleotides is further facilitated by the use of computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Use these

The program compares the obtained nucleic acid sequences by, for example, searching a database such as GenBank or by sequencing the PCR products. Comparing nucleic acid sequences from a range of genes and intergenic regions in a given genome makes optional

A nucleic acid sequence that exhibits an appropriate degree of specificity for the target gene. Such procedures allow for the selection of a degree of high complementarity for a target nucleic acid sequence in a given genome and a lower degree of complementarity to other nucleic acid sequences. Cooked

Those skilled in the art will recognize that a wide range of gene regions suitable for use in the present invention can be selected. X can specifically hybridize": a compound can bind to a dry nucleic acid when it binds to a dry nucleic acid to interfere with the normal function of the dry nucleic acid to regulate function and/or activity' and to have sufficient complementarity to avoid antisense compound plaque The nucleic acid sequence undergoes non-specific binding under conditions where it is desired to undergo specific binding (i.e., under physiological conditions in the case of in vivo analysis or therapeutic treatment, and under conditions in which the assay is performed in the context of in vitro analysis). ^ The hybridity f of the oligoacid described herein can be measured by an in vitro assay known in the art. For example, VIII, & 丨-wax is obtained by using a smelting curve to determine the binding strength of an innocent molecule to a potential drug molecule to obtain the properties of the oligonucleotides described herein. And = any of the established methods of measuring the strength of the intermolecular interaction (eg '(four) curve analysis) to estimate the combination of natural antisense molecules with potential 157514.doc •85· 201209163 drug molecules (molecules) strength. Melting curve analysis measures the temperature at which a natural antisense molecule/molecular complex rapidly transitions from a double strand to a single stranded form. This temperature has been widely accepted as a reliable measure of the strength of the interaction between two molecules. Melt curve analysis can be performed using a cDNA copy of the actual native antisense RNA molecule or a synthetic DNA or RNA nucleotide corresponding to the molecular binding site. Multiple kits containing all necessary reagents to perform this assay (e.g., Applied Biosystems, MeltDoctor kit) can be used. Such kits comprise a suitable buffer solution containing one of a double stranded DNA (dsDNA) binding dye (e.g., ABI HRM dye, SYBR Green, SYTO, etc.). The nature of the dsDNA dye is that it emits almost no fluorescence in free form, but has high fluorescence when bound to dsDNA. To perform the analysis, the cDNA or corresponding oligonucleotide is mixed with the molecule at a concentration defined by a particular manufacturer's protocol. The mixture was heated to 95 °C to dissociate all preformed dsDNA complexes and then slowly cooled to room temperature or other lower temperatures as defined by the kit manufacturer to anneal the DNA molecules. The newly formed composite was then slowly heated to 95 ° C while continuing to collect data on the amount of fluorescence produced in the reaction. The intensity of the fluorescence is inversely proportional to the amount of dsDNA present in the reaction. Data can be collected using a real-time PCR instrument compatible with the kit (eg ABI’s StepOne Plus Real-Time PCR System or lightTyper instrument, Roche Diagnostics, Lewes, UK). Draw a plot of the negative derivative of fluorescence relative to temperature (-d (fluorescence) / dT), y-axis) and temperature (X-axis) by using appropriate software (eg lightTyper (Roche) or SDS Dissociation Curve, ABI) To build a melting peak. 157514.doc -86- 201209163 Analyze the data to determine the field when the dsDNA complex is rapidly converted to a single-stranded molecule. This temperature is called Tm and is proportional to the intensity of interaction between the two molecules. Typically, the Tm is above 40. (Example 2. Example 2: Regulation of BBC3 Polynucleotide Treatment of MCF-7 Cells Using Antisense Nucleotide Nucleotide All antisense priming acids used in Example 2 were performed as described in the Examples. Commercial (IDT, Coralville, IA) to manufacture the designed phosphorothioate-bonded oligonucleotides and provide the designed phosphorothioate analogs shown in the table. The inter-nucleotide star symbol indicates the presence Phosphorothioate linkages. The oligonucleotides required for the experiments in Example 2 can be synthesized using any suitable state of the art method, such as the method used for IDT: in a solid support (eg, 5 micron fixed glass beads (CPG)) The use of a phosphoramidite monomer (common nucleotides, all of which are protected by a protecting group, such as a trityl group on a sugar, a benzoquinone on A and C, and a N on G) 2-Isobutyl-based. The protecting group prevents undesired reactions during oligonucleotide synthesis. The protecting group is removed at the end of the synthesis process. Via 3, the carbon is attached to the initial nucleotide. Solid carrier and synthesis in the direction of 3' to 5, in the following 4 steps Adding a new base to the nucleotide chain: 1) using tri-acetic acid to remove the protecting group from the 5' oxygen of the fixed nucleotide; 2) using tetrazole to immobilize the nucleotide with the next nucleotide in the sequence Coupling together; the reaction is continued via the tetrazolylphosphinamide intermediate; 3) washing removes unreacted free nucleotides and reaction by-products and capping unreacted fixed nucleotides to prevent their participation The next round of synthesis; by using acetic anhydride and N-mercaptopimid for free 5, acetylation of the base to achieve end-capping; 4) to stabilize the bond between nucleotides, so that 157514.doc -87-201209163姨 and water (if you want to produce acid diester bonds), or Beaucage reagent (3H-1,2-benzodithiol-3-one-1,1-dioxide) (if desired phosphorothioate linkage) ) to phosphorus oxide. The chimeric backbone can be constructed by alternately using two oxidizing agents. The above 4-step cycle is repeated for each nucleotide in the sequence. When the complete sequence is synthesized, the oligonucleotide is cleaved from the solid support at a high temperature using ammonium hydroxide and deprotected. The protecting group is removed by washing with desalting and the remaining oligonucleotide is lyophilized. To carry out the experiment designed in Example 2, the growth medium (MEM/EBSS (Hyclone catalog number: SH30024, or Mediatech catalog number: MT-10-010-CV) + 10% FBS at 37 ° C and 5% CO 2 ) (Mediatech catalog number: MT35-011-CV) + penicillin/streptomycin (Mediatech catalog number: MT30-002-CI)) MCF-7 cells grown from ATCC (Catalog No.: HB- twenty two). On the day before the experiment, the cells were plated again in a 6-well plate at a density of 1.5 χ 105 cells/ml and incubated overnight at 37 ° C and 5% CO 2 . On the day of the experiment, the medium in the 6-well plate was changed to fresh growth medium. Oligonucleotides delivered by the manufacturer in lyophilized form were diluted to a concentration of 20 μM in deionized water without RNAse/DNAse. 2 μΐ of this solution was incubated with 400 μΐ OptiMEM medium (Gibco catalog number: 31985-070) and 4 μL Lipofectamine 2000 (Invitrogen catalog number: 11668019) for 20 min at room temperature, and then applied dropwise to a 6-well plate. One well of MCF-7 cells. A similar mixture containing 2 μM water instead of the oligonucleotide solution was used as a mock transfection control group. After incubation for 3 to 18 h at 37 ° C and 5% CO 2 , the medium was changed to fresh growth medium. After adding antisense oligonucleotides for 48 h, the medium was removed and used to 157514.doc •88· 201209163 from Promega's SV Total RNA Isolation System (Catalog No.: Z3105) or RNeasy Total RNA Isolation Kit from Qiagen (catalog) No. 74181) RNA was extracted from cells according to the manufacturer's instructions. 6 ng of extracted RNA was added to the reverse transcription reaction using the Verso cDNA kit from Thermo Scientific (Catalog No.: AB1453B) or the high-capacity cDNA reverse transcription kit (Catalog No.: 4368813), such as the manufacturer's protocol. Said in the middle. The cDNA from this reverse transcription reaction was used by real-time PCR using ABI Taqman Gene Expression Mix (catalog number: 4369510) and primers/probes designed by ABI (Applied Biosystems Taqman Gene Expression Assay: Hs00248075_m 1 > Applied Biosystems, Foster City CA) to monitor gene expression. The following PCR cycles were performed using a stepOne Plus real-time PCR machine (Applied Biosystems): 2 min at 50 °C, 10 min at 95 °C, 40 cycles (15 seconds at 95 C, at 60 °) Keep 1 min under C). The fold change in gene expression after treatment with antisense oligonucleotides was calculated based on the difference in 18S normalized dCt values between treated and mock transfected samples. The real-time PCR results showed that the content of BBC3 mRNA in MCF-7 cells was significantly increased after 48 h treatment with oligonucleotides designed to be BBC3 antisense mRNA CA306306 (Fig. 1). Example 3: For 耙Increased degree of apoptosis in primary human fibroblasts following antisense nucleoside treatment of PUMA-specific natural antisense transcripts in Example 3 'in final concentration of 20 nM in primary human fibroblasts Antisense oligonucleotides targeting PUMA-specific natural antisense transcripts were tested. The following data confirms that up-regulation of PUMA mRNA by modulating the function of PUMA-specific natural antisense transcription 157514.doc •89·201209163 is associated with an increased degree of apoptosis as determined by TUNEL analysis. Materials and methods Use the dry direction #异磔天资 and 4 #录勿之和J Raised nucleotides to deal with the second sub-Yu Yong. Iron 襻 mother care. At 37. (: and 5% C02 of primary human skin fibroblasts introduced into the medium by Dr. N-Kenyon (University of Miami) by a-MEM (Gibco, catalog number: 12561-056) + 10% FBS ( Mediatech, catalog number: 35-015 CV) + l〇/〇Antimycotic-Antibiotic (Gibco, catalog number: 15240-062) was grown in growth medium. Cells were treated with antisense nucleotides using the Next Day method. One day before the experiment, the cells were plated at a density of about 2×1 〇 5 cells/well in 6-well plates in growth medium and incubated overnight at 37° C. and 5% CO 2 . The next day, 6-well plates were placed. The medium was changed to fresh medium (1.5 ml/well) and the cells were administered antisense nucleotides. All antisense nucleoside acids were manufactured by IDT (Coralville, IA). Sequence of all oligonucleotides Listed in Table 1. The oligonucleotide storage solution was diluted to a concentration of 20 uM in sterile water without DNAse/RNAse. 2 ul of this solution was administered to one well with 400 ul of 〇pti_MEM medium (Gibc〇 catalog No.: 31985-070) and 4 ul Lip〇fectamine 2000 (Invitrogen catalog number: 11668019), which will Incubate for 2 〇 min at room temperature and apply dropwise to one well of the 6-well plate with cells. A similar mixture containing 2 ul of water instead of the oligonucleotide solution was used as a mock transfection control group. Inactive nucleotide CUR-1 505 was used as an inactive control group. After incubation for 18 hours at 37 C and 5% C〇2, the medium was changed to fresh 157514.doc •90- 201209163 growth medium. Add antisense After 48 hours of oligonucleotide removal, the medium was removed and the cells were trypsinized and re-plated in 96-well plates at a density of approximately 70,000 cells/well. After incubation at 37 ° C and 5% C 〇 2 for 24 hours Cells were used for TUNEL apoptosis analysis. rt/see the source of death. The cells were assayed using the HT Titer TACS assay kit according to the manufacturer's instructions (Trevigen catalog number: 4822-96-K). In short, the cells were fixed in 3.7% buffered furfural solution (Sigma-252549), rinsed with PBS and 100% methanol (Sigma_ 1775-1〇8) and used at +4 before analysis. °(: stored in 80% ethanol (Sigma-493511). The labeled DNA was cleaved, the ethanol was removed, and the cells were washed with PB S and incubated with proteinase K solution for 15 minutes at room temperature, rinsed in water and PBS, and then incubated with TACS nuclease solution for 10 minutes at 37 °C. . After the incubation, the cells were washed with PBS, and a hydrogen peroxide solution (Sigma-MKBD1394) was added. After incubation for 5 minutes at room temperature, the cells were incubated with 1X TdT labeling buffer for 5 minutes at room temperature, then q was discarded. The cells were then reacted with the labeling reaction mixture at 37. (: Incubate for one hour, then add lx TdT to stop the buffer for 5 minutes. After the labeling reaction is stopped, wash the cells with PBS and incubate with Strep-HRP solution for 10 minutes at room temperature using PBS/Tween Wash and incubate for 30 minutes with TACS sapphire in the dark. Stop the reaction by adding 0.2 N HCl (Fluka-343102) and read the absorbance at 450 nm in a plate reader unless otherwise stated. All of the above solutions were supplied in the HT Titer TACS assay kit. . As shown in Figure 2, antisense oligos targeting PUMA-specific natural 157514.doc •91-201209163 antisense transcripts at 20 nM After nucleotide treatment, the degree of apoptosis in primary human fibroblasts increased more than 5-fold, and the antisense oligonucleotide was previously shown to upregulate PUMA mRNA content (Fig. 1, Example 2). This up-regulation was significant compared to the chemically inactive oligonucleotide-treated control samples. The results indicate that up-regulation of PUMA mRNA can up-regulate PUMA protein using oligonucleotides targeting PUMA-specific natural antisense transcripts and Increase cell > week Degree 4. Example 4: Reduced Proliferative Capacity in Primary Human Fibroblasts Treated with Antisense Oligonucleotides Targeting PUMA-Specific Natural Antisense Transcripts In Example 4 'Test Targeted PUMA Specific Natural Antisense Antisense oligonucleotides of transcripts inhibit the ability of colonies of primary human fibroblasts. The following data confirm that up-regulation of PUMA mRNA by modulating the function of PUMA-specific natural antisense transcripts allows oligonucleotides to be processed Proliferation in cells is reduced. Materials and Methods Selection #立分#. The primary human skin fibroblasts introduced into the medium by Dr. N. Kenyon (University of Miami) at 37 ° C and 5% C02 a-MEM (Gibco, catalog number: 12561-056) + 10% FBS (Nlediatech ' catalog. 35-015 CV) + 1% Antimycotic-

Growth was carried out in a growth medium consisting of Antibiotic (Gibco, catalog number: 1 5240-062). Cells were treated with antisense oligonucleotides using the Next Day method. On the day before the experiment, cells were plated at a density of about 2 x 105 cells/well in 6-well plates in growth medium and incubated overnight at 37 ° C and 5% CO 2 . Day - Day 'Change the medium in the 6-well plate to fresh medium (1.5 ml/well) 157514.doc -92- 201209163 and administer the antisense oligo to the cells. All antisense oligonucleotides are manufactured by IDT (C〇ralviUe, IA). The sequences of all oligonucleotides are listed in Table 1. The stock solution of the oligonucleotide was diluted to a concentration of 20 uM in sterile water without DNAse/RNAse. 2 ul of this solution was administered to a well with 400 ul of Opu-MEM medium (Gibco catalog number: 31985-070) and 4 ui Lipofectamine 2000 (invitrogeng number: ι1668〇19), which were incubated together at room temperature 2 〇min, and applied dropwise to one well of a 6-well plate with cells. A similar mixture containing 2 ul of water instead of the nucleotide solution was used as a mock transfection control group. The same concentration of the inactive conjugate nucleotide CUR-1505 was used as a control group. After incubation for about 18 h at 37 ° C and 5% CO 2 , the medium was changed to fresh growth medium. After 48 hours of addition of antisense oligonucleotide, the medium was removed and the cells were trypsinized and re-plated into 6-well plates at a density of about 5000 cells/well. The plate was incubated at 37 ° C, 5% C 〇 2 for 5 to 7 days until colonies of 3 to 10 cells were formed. Fixed colonies and used for 0.25 % in 95% ethanol for better visibility 1 9-Dimercapto-indenyl blue (sigma Aldrich, Cat. No. 34, 108.8) was stained and rinsed with PBS. Colonies were counted and compared to control wells treated with inactive nucleotides and mock transfected controls. The results, as shown in Figure 3, were treated with antisense raised nucleotides targeting PUMA-specific natural antisense transcripts compared to cells treated with inactive oligonucleotides and mock-transfected controls. The number of colonies formed by the primary fibroblasts is reduced. The results indicate that PUMA mRNA upregulated by antisense raised nucleotides produces a functional protein and reduces in vitro cell proliferation. 157514.doc • 93- 201209163 Although the present invention has been illustrated and described in accordance with one or more embodiments of the present invention, it will In addition, although specific features of the invention may be disclosed in accordance with one of the various embodiments, the features may be combined with one or more other features of other embodiments, which may be possible for any given or particular application. It is desirable and advantageous. The Abstract of the Disclosure allows the reader to quickly ascertain the nature of the technical disclosure. The Abstract is submitted with the understanding that it is not intended to limit or limit the scope or meaning of the following claims. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the up-regulation of PUMA mRNA by antisense oligonucleotides targeting PUMA-specific natural antisense transcripts with MCF7 cells. The MCF7 breast cancer cell line was administered 20 nM of different antisense oligonucleotides targeting CUMMA-specific natural antisense transcripts (CUR-1675, CUR-1676, CUR-1677, CUR-1678 and CUR-1679). After 48 h of administration, PUMA mRNA was quantified by reverse transcription (RT) real-time polymerase chain reaction (PCR). The relative nucleotide processing of PUMA mRNA was calculated based on the 1 8S normalized difference between the administered cells (adding CUR nucleotides to the cells) and the mock cells (no oligonucleotide added to the cells). Performance changes. The bars designated CUR-1675, CUR-1676, CUR_1677, CUR-1678, and CUR-1679 correspond to the samples treated with SEQ ID NOS: 3 to 7, respectively. Figure 2 shows the increase in the degree of apoptosis in primary human fibroblasts treated with antisense oligonucleotides targeting PUMA-specific natural antisense transcripts as determined by TUNEL analysis. CUR-1675 - previously shown upregulated 157514.doc -94 · 201209163 PUMA mRNA targeting oligonucleotides of PUMA natural antisense transcript; CUR-1505 - inactive oligonucleotide with similar chemical properties. The bars indicated as CUR-1675 and CUR-1505 correspond to the samples treated with SEQ ID NOS: 3 and 8, respectively. ***Indication Ρ<0·001 Figure 3 shows the ability to reduce proliferation in primary human fibroblasts treated with antisense oligonucleotides targeting PUMA-specific natural antisense transcripts, as analyzed by selection Measured. CUR-1675 - an oligonucleotide previously shown to up-regulate PUMA mRNA targeting PUMA natural antisense transcript; CUR-1505 - an inactive oligonucleotide with similar chemical properties. The bars denoted as CUR-1675 and CUR-1509 correspond to the samples treated with SEQ ID NOS: 3 and 8, respectively. *Indication Ρ<0·05. BRIEF DESCRIPTION OF THE SEQUENCE LISTING - SEQ ID NO: 1 : Homo sapiens BCL2 binding part 3 (BBC3), transcript variant 3, mRNA (NCBI accession number: NM_001127242); SEQ ID NO: 2: natural BBC3 antisense sequence (CA3 06306); SEQ ID NOS: 3 to 8: antisense oligonucleotides. * indicates a phosphorothioate bond. 157514.doc 95- 201209163 Sequence Listing <110> American Okokena Co., Ltd. <120> By treating BCL2 binding part 3 (BBC3) natural antisense transcript to treat BBC3 related diseases <130> BBC3 <140> 100124653 <141> 2011-07-12 <150> 61/363,535 <151> 2010-07-12 <160> 8 <170> Patentln version 3.5 <210> 1 <211> 1347 <212> m <213 > Homo sapiens <400> 1 gaggegattg cgattgggtg agacccagta aggatggaaa gtgtagagga gacaggaatc 60

Ο cacggctttg gaaaaaggaa ggacaaaact caccaaacca gageagggea ggaagtaaca 120 atgagaaact gaaaaagaaa cggaatggaa agetatgaga caggatgaaa tttggcatgg 180 ggtctgccca ggcatgtcca tgccaggtgc ccagggctgc ttccacgacg tgggtcccct 240 gccagatttg tgagacaaga ggagcagcag cggcaccgcc cctcaccctg gagggtcctg 300 tacaatctca tcat £ ggact cctgccctta cccaggggcc acagagcccc egagatggag 360 cccaattagg tgcctgcacc cgcccggtgg acgtcaggga ctcggggggc aggcccctcc 420 cacctcctga caccctggcc agcgcggggg actttctctg caccatgtag catactggac 480 tcccagccct gcctgtcccg ggggcgggcc ggggcagcca ctccagcccc agcccagcct 540 ggggtgcact gaeggagatg cggactcctg ggtccctggc caagaagcca ggagagggac 600 ggctgatgga ctcagcatcg gaaggtggcg gtgaccgagg gggtggggac tgagccgccc 660 gcctctgccg cccaccacca tctcaggaaa ggctgttgtg ctggtgcccg ttccagctgc 720 aggggtgaca ctgggagggg ggggctctcc tctcggtgct ccttcactct gggcctggcc 780 tcaggcccct ggtgcttccc cccctcctcc tgggaggggg cccgtgaaga gcaaatgagc 840 caaacgtgac cactagcctc ctggagccag agagtggggc tcgtttgccg gttgctccag 900 cccggcgccc agcca tcttc cctgagccag ccggcgggtg gtgggcatgc ctgcctcacc 960 ttcatcaggg ggtggccagg aggggcccag actgtgaatc ctgtgctctg cccgtgaccg 1020 ccccccgccc catcaatccc attgeatagg tttagagaga gcacgtgtga ccactggcat 1080 tcatttgggg ggtgggagat gccgccccag ccttagtccc cagggccaag 1140 cgctgggggg aagacgggga gtcagggagg gggggaaatc teggaagagg gaggagtetg 1200 ggagtgggga gggatggccc agcctgtaag atactgtata tgcgctgctg tagataccgg 1260 aatgaatttt ctgtacatgt ttggttaatt ttttttgtac atgatttttg tatgtttcct 1320 tttcaataaa atcagattgg tttggctgaa Aacagtg 1347 <210> 2 <211> 689 <212> DNA <213> Homo sapiens <400> 2 cttttctttt ctttttttag taaagatggg gtctcattat gttgcccagg ttggtctcaa 60 actcctgggc tcaa£tgatc ctccctcctt ggcctcccaa agtgcctgga ttatggtcat 120 157514-sequence table.doc 201209163 gaggcaccat gcccagcccc ggaatgttct tatttstttg tttgtttgta gagacagggt 180 ctcactatgt tgcccaggct ggtctcaaat tccagacctc aagcaatcgt tcatccttga 240 cctcccaaaa tgctgggatt acaggtgtga gccaccacac cggcctataa tgttcttttg 300 agaagaaaat gagtgtgaag gata gtg ££ a atctggcttg gtggctggag gtagaaaaga 360 tggggasagg ccaagagtct atttggccat aagcgttatc actagtcaaa atcctcccca 420 cagctgggcg tggtggctca cgtctgcaat cccagtatca cggaaggccg agatgggtgg 480 atcacctgag cccaggagtt ccagaccagc ctgggcaaca tgcggaaacc taactgggga 540 ggctggggtg ggaggaacgc ttgagcctgt gaggtcgagg ctgcagtagg ctgtgatcgc 600 acattgcact ccagcttggg cgacagagcg agaccctttc tcaaaaaaaa aaaaaacggc 660 atggccgcgg ccctcgtgcc gaattcttg 689 < 210 & gt 3 <211> 20 <212> DNA <213>Artificial sequence<220><223> Antisense nucleoside <400> 3 ccaccaagcc agattcccac 20 <210> 4 <211> 20 <;212> DNA <213>Artificial sequence<220><223> Antisense nucleoside <400> 4 atccacccat ctcggccttc 20 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220><223> antisense oligonucleoside <400> 5 aggctcaagc gttcctccca 20 <210> 6 <211> 21 <212> DNA <213> artificial sequence <220>223>antisense oligonucleoside <400> 6 cccag Ttagg tttccgcatg t 21 <210> 7 <211> 20 <212> DNA <213>Artificial sequence <220><223> Antisense nucleoside-2 - 157514 - Sequence Listing.doc 201209163 <4〇0> 7 tctgtcgccc aagctggagt <210> 8 <211> 21 <212> DNA <213> artificial sequence <220><223> antisense oligonucleoside <400> 8 cctctccacg cgcagtacat

157514 - Sequence Listing. doc

Claims (1)

201209163 VII. Patent Application Range 1. A method for regulating the function and/or performance of a BCL2 binding portion 3 (BBC3) polynucleotide in a biological system in vitro, comprising: making the system with at least one length of 5 Up to 30 nucleotide antisense oligonucleotide contacts, wherein the at least one oligonucleotide has at least 50% of the reverse complement of the native antisense transcript of the BCL2 binding portion 3 (BBC3) polynucleotide Sequence identity; thereby modulating the function and/or performance of the BCL2 binding moiety 3 (BBC3) polynucleotide. Ο 2. A method of modulating the function and/or expression of a BCL2 binding portion 3 (BBC3) polynucleotide in a biological system according to claim 1, comprising: bringing the biological system to at least one nucleoside of 5 to 30 in length An acid antisense oligonucleotide contact, wherein the at least one oligonucleotide has at least 50% sequence identity to the reverse complement of the polynucleotide, the polynucleotide comprising the natural inverse of SEQ ID NO: 5 to 30 contiguous nucleotides within the transcript nucleotides 1 to 689; thereby modulating the function and/or expression of the BCL2 binding portion 3 (BBC3) polynucleotide. 〇3. A method of modulating the function and/or expression of a BCL2 binding portion 3 (BBC3) polynucleotide in a patient cell or tissue in vitro, comprising: causing the cells or tissues to have at least one length of 5 Contacting to a 30 nucleotide antisense oligonucleotide having at least 50% sequence identity to the antisense oligonucleotide of the BCL2 binding portion 3 (BBC3) polynucleotide; The function and/or expression of the BCL2 binding portion 3 (BBC3) polynucleotide in the patient's cells or tissues is thereby regulated in vitro. 4. A method of modifying the function and/or expression of a BCL2 binding portion 157514.doc 201209163 3 (BBC3) polynucleotide in a patient cell or tissue according to claim 3, comprising: bringing the biological system to at least one length of 5 Contacting to a 30 nucleotide antisense oligonucleotide, wherein the at least one oligonucleotide has at least 50% sequence identity to the reverse complement of the polynucleotide, the polynucleotide comprising SEQ ID NO : 5 to 30 contiguous nucleotides within nucleotides 1 to 689 of the natural antisense transcript; thereby modulating the function and/or expression of the BCL2 binding portion 3 (BBC3) polynucleotide. 5 _ A method for modulating the function and/or expression of a BCL2 binding portion 3 (BBC3) polynucleotide in a biological system in vitro, comprising: polymerizing the system with at least one stem to the BCL2 binding portion 3 (BBC3) The antisense raised nucleotides of the region of the nucleotide's natural antisense oligonucleotide are contacted; thereby modulating the function and/or expression of the BCL2 binding portion 3 (BBC3) polynucleotide. 6. The method of claim 5, wherein the function and/or performance of the BCL2 binding moiety 3 (BBC3) is increased in vitro compared to the control group. 7. The method of claim 5, wherein the at least one antisense oligonucleotide targets a natural antisense sequence of a BCL2 binding portion 3 (BBC3) polynucleotide. 8. The method of claim 5, wherein the at least one antisense oligonucleotide targets a nuclear I sequence, the nucleic acid sequence comprising a BCL2 binding portion 3 (BBC3) polynucleotide encoding and/or non-coding nucleic acid sequence. 9. The method of claim 5, wherein the at least one antisense oligonucleotide targets σ. The overlap and/or non-weight of the 4 points 3 (BBC3) polynucleotides. And 々 '1 0. such as claim 5 $ f, 土 ^ . _ 〆 方法 method, wherein the at least one antisense nucleotide comprises 5 k from the following modifications: at least one modified sugar moiety, to 157514 .doc 201209163 One less modified internucleoside linkage, at least one modified nucleotide, and combinations thereof. 11. The method of claim 10, wherein the modified sugar moiety is selected from the group consisting of: or a plurality of modifications comprising modifying at least one sugar moiety, 2, methoxyl via 2'-fluorene-methoxyethyl Modified sugar moiety, sugar moiety modified by 2,_q_(tetra), bicyclic sugar moiety, and combinations thereof. 12. The method of claim 1 , wherein the one or more modifications comprise at least one modified internuclear linkage selected from the group consisting of: phosphorothioate, 2,-methoxyethyl (ΜΟΕ), 2'-fluoro, alkyl phosphonate, dithiophosphate, thiophosphonic acid alkyl vinegar, amino citrate, amino carboxylic acid brewing, carbonated vinegar, phosphate triester, amino acetate, carboxymethyl Base esters and combinations thereof. 13. The method of claim 10, wherein the one or more modifications comprise at least one modified nucleotide acid selected from the group consisting of peptide nucleic acid (ρΝΑ), locked nucleic acid (LNA), arabinic acid (FANA), and the like Things, derivatives and combinations. 14. The method of claim 1, wherein the at least one oligonucleotide comprises at least one oligonucleotide sequence set forth as SEQ ID NOs: 3 to 8. 15. A method of modulating the function and/or expression of a BCL2 binding portion 3 (BBC3) gene in a mammalian cell or tissue in vitro, comprising: bringing the cells or tissues to at least one length of 5 to 3 a short interfering RNA (siRNA) nucleotide contact, the at least one siRNA oligonucleotide specific for an antisense polynucleotide of a BCL2 binding portion 3 (BBC3) polynucleotide, wherein the at least one An siRNA oligonucleotide having at least 5% sequence identity to an antisense of the BCL2 binding portion 3 (BBC3) polynucleotide and/or a complement of at least about 5 contiguous nucleic acids in a sense nucleic acid molecule; 157514. Doc 201209163 and in vitro regulation of the function and/or performance of binding moiety 3 (BBC3) in mammalian cells or tissues. 16. The method of claim 15, wherein the oligonucleotide has at least 8% sequence identity to a sequence having at least about $ contiguous nucleic acid, the sequence and the BCL2 binding portion 3 (BBC3) polynuclear Acid antisense and/or sense nucleic acid molecules are complementary. 17. Seeding, 3⁄4• in vitro 6 weeks_mammalian cells or tissue squadrons [2 binding part 3 (BBC3) functions and/or methods of seeing, including: making such cells or tissues with at least An antisense oligodeoxynucleotide conjugate having a length of about 5 to 30 nucleotides, and a sigma antisense oligonucleotide pair 8 (:] 2 binding moiety 3 (BBC3) polynucleotide for justice and/or The non-coding and/or coding sequence of the natural antisense strand is specific in that it aligns the at least one antisense oligonucleotide with at least one sequence of at least 5 % of the nucleic acid sequences set forth as SEQ ID NOS: 1 and 2 And the function and/or expression of the bcL2 binding moiety 3 (BBC3) in mammalian cells or tissues in vitro. 18. Use of an antisense nucleus for the manufacture of a biological system for regulation An agent for the function and/or expression of a BCL2 binding moiety 3 (BBC3) polynucleotide wherein the nucleotide is 5 to 3 nucleotides in length and binds to the BCL2 binding moiety 3 (BBC3) The reverse complement of the acid natural antisense transcript has at least 50% sequence identity. 19. The use of the term 18 The reverse complement of a polynucleotide comprising 5 to 30 contiguous nucleotides of nucleotides 1 to 689 of the natural antisense transcripts of SEq id 具有 2 has a sequence identity of at least 5%. An use of an antisense oligonucleotide for the manufacture of a medicament for modulating the function and/or expression of a BCL2 binding moiety 3 (BBC3) polynucleotide in a cell or tissue of a patient 157514.doc 201209163, wherein The antisense oligonucleotide is 5 to 30 nucleotides in length and has at least 50% sequence identity to the antisense oligonucleotide of the BCL2 binding portion 3 (BBC3) polynucleotide. The use of claim 20, wherein the oligonucleotide is inverted complement to a polynucleotide comprising 5 to 30 contiguous nucleotides within nucleotides 1 to 689 of the natural antisense transcript of SEQ ID NO: 2. Having at least 50% sequence identity. 22. Use of an antisense oligonucleotide for the manufacture of a function and/or expression for modulating a BCL2 binding portion 3 (BBC3) polynucleotide in a biological system An agent, wherein the antisense oligonucleotide targets a region of the natural antisense oligonucleotide of the BCL2 binding portion 3 (BBC3) polynucleotide. The use of claim 22, wherein the function and/or performance of the BCL2 binding member 3 (BBC3) is increased in vivo compared to the control group. 24. The use of claim 22, wherein the antisense oligonucleotide targets BCL2 binding The natural antisense sequence of a portion 3 (BBC3) polynucleotide. 25. The use of claim 22, wherein the antisense oligonucleotide targets a coding comprising a BCL2 binding portion 3 (BBC3) polynucleotide and/ Or a nucleic acid sequence that does not encode a nucleic acid sequence. 26. The use of claim 22, wherein the antisense oligonucleotide targets overlapping and/or non-overlapping sequences of a BCL2 binding portion 3 (BBC3) polynucleotide. 27. The use of claim 22, wherein the antisense oligonucleotide comprises one or more modifications selected from the group consisting of at least one modified sugar moiety, at least one modified internucleoside linkage, at least one modified core Glycosylates and combinations thereof. 28. The use of claim 27, wherein the one or more modifications comprise at least one 157514.doc 201209163 modified sugar moiety selected from the group consisting of 2,-〇-methoxyethyl modified sugar moiety, via 2 , a ft-modified sugar moiety, a sugar moiety modified by 2, _ 〇 炫, a bicyclic sugar moiety, and combinations thereof. 29. The use of claim 27, wherein the one or more modifications comprise at least one modified internuclear linkage selected from the group consisting of: thiophosphoric acid vinegar, 2, 〇 methoxyethyl (ΜΟΕ), 2 ,- said, phosphonic acid ester, dithiophosphoric acid vinegar, thiophosphonic acid alkyl vinegar, amino phosphoric acid, amino carboxylic acid vinegar, carbonated vinegar, triglyceride, aminoacetic acid vinegar, carboxymethyl ester And their combinations. 30. The use of claim 27, wherein the one or more modifications comprise at least one modified nucleotide selected from the group consisting of a peptide nucleic acid (ρνα), a locked nucleic acid (LNA), an arabinic acid (FANA), and the like Things, derivatives and combinations. 31. The use of claim 18, wherein the oligonucleotide comprises at least one oligonucleotide sequence set forth as SEQ ID NOs: 3 to 8. 32. Use of a short interfering RNA (siRNA) to recruit a bitter acid for the manufacture of a medicament for modulating the function and/or expression of a BCL2 binding portion 3 (BBC3) gene in a mammalian cell or tissue The nucleus acid is 5 to 30 nucleotide acids in length and specific for the antisense polynucleotide of the BCL2 binding portion 3 (BBC3) polynucleotide, wherein the siRNA raises the binding moiety of the bitter acid to the BCL2 The antisense of the 3 (BBC3) polynucleotide and/or the complement of at least about 5 contiguous nucleic acids in the sense nucleic acid molecule has a sequence identity of at least 50%. 33. The use of claim 32, wherein the nutrient acid has at least 80% sequence identity to a sequence having at least about $ contiguous nucleic acid, the sequence and the BCL2 binding portion 3 (BBC3) polynucleotide Antisense and/or Justice Nuclear 157514.doc 201209163 Molecular complementarity. 34. Use of an antisense oligonucleotide for the manufacture of an agent for modulating the function and/or expression of BCL2 binding portion 3 (BBC3) in a mammalian cell or tissue, wherein the antisense nucleotide a length of about 5 to 30 nucleotides and specific for a non-coding and/or coding sequence of a sense and/or natural antisense strand of a BCL2 binding portion 3 (BBC3) polynucleotide, wherein the antisense oligo The nucleotide has at least 50% sequence identity to at least one of the nucleic acid sequences set forth as SEQ ID N:. 35. A modified oligonucleotide comprising at least one modified synthesis, wherein the at least one modification is selected from the group consisting of: at least one modified sugar moiety, at least one modified internucleotide linkage, at least one modified Nucleotide and its group, wherein the β-coupon nucleotide line hybridizes with the bcl2 binding part 3 (BBC3) gene in vivo or in vitro and can regulate the BCL2 binding part 3 (BBC3) compared with the normal control group. An antisense compound of the function and/or expression of a gene and wherein the nucleotide of the nucleotide has at least 50°/° sequence identity with a sequence of at least about 5 contiguous nucleic acids, the sequence and the BCL2 binding moiety 3 ( BBC3) Antisense and/or sense nucleic acid molecules of a polynucleotide and their alleles, homologs, isoforms, variants, derivatives, mutants, fragments or combinations are complementary. 36. The nucleotide of claim 35, wherein the nucleotide is 5 to 30 nucleotides in length and 5 to 30 contiguous nucleotides in the natural antisense transcript of the BBC3 gene. The reverse complement has a sequence identity of at least 5%. 37. The nucleotide of claim 36, wherein the at least one modification comprises an internucleotide linkage selected from the group consisting of 157514.doc 201209163: phosphorothioate, phosphonic acid ester, disulfide Phosphate, alkyl thiophosphonate, amino phosphate, amino phthalate, carbonate, phosphate, amino acetate, carboxymethyl ester, and combinations thereof. 3. The nucleotide of claim 36, wherein the oligonucleotide comprises at least one phosphorothioate internucleotide linkage. 39. The nucleotide of claim 36, wherein the nucleotide comprises a backbone of a phosphorothioate internucleotide linkage. 40. The nucleotide of claim 36, wherein the nucleotide comprises at least one modified nucleotide selected from the group consisting of: a peptide nucleic acid, a locked nucleic acid (LNA), an analog thereof, and a derivative. Things and combinations. 41. The nucleotide of claim 3, wherein the oligonucleotide comprises a plurality of modifications wherein the modifications comprise modified nucleotides selected from the group consisting of phosphorothioates, phosphonic acid esters, Dithiophosphates, alkyl thiophosphonates, amino phosphates, urethanes, carbonates, phosphotriesters, aminoacetic acid vinegars, and slow methyl groups are intended to be combined. 42. The nucleotide of claim 36, wherein the oligonucleotide comprises a plurality of modifications, wherein the modifications comprise modified nucleotides selected from the group consisting of peptide nucleic acids, locked nucleic acids (LNA), analogs thereof , derivatives and combinations. 43. The nucleotide of claim 36, wherein the oligonucleotide comprises at least one modified sugar moiety selected from the group consisting of a 2,_〇-methoxyethyl modified sugar moiety, via 2'- a methoxy-modified sugar moiety, a 2,_〇-alkyl-modified sugar moiety, a bicyclic sugar moiety, and combinations thereof. 44. The oligonucleotide according to claim 36, wherein the oligonucleotide comprises a plurality of 157514.doc 201209163 ornaments wherein the modifications comprise a modified sugar moiety selected from the group consisting of 2, _ 〇-曱 oxygen a thioethyl-modified sugar moiety, a 2, decyloxy-modified sugar moiety, a 2,-fluorene-alkyl modified sugar moiety, a bicyclic sugar moiety, and combinations thereof. 45. The oligonucleotide of claim 36, wherein the oligonucleotide is at least about 5 to 30 nucleotides in length and is antisense and/or sensed with a BCL2 binding portion 3 (BBC3) polynucleotide. Chain hybridization wherein the oligonucleotide has at least about an antisense to the BCL2 binding portion 3 (BBC3) polynucleotide and/or a complementary sequence of at least about 5 contiguous nucleic acids in a sense encoding and/or non-coding nucleic acid sequence. 60% sequence identity. 46. The oligonucleotide of claim 36, wherein the oligonucleotide is antisense and/or sense encoding and/or non-coding nucleic acid sequence of the 8 (:1^ binding moiety 3 (BBC3) polynucleotide The complement of at least about 5 contiguous nucleic acids has a sequence identity of at least about 80%. 47. The oligonucleotide of claim 36, wherein the nucleotide is conjugated to at least one BCL2 in vivo or ex vivo A portion 3 (BBC3) polynucleotide hybridizes and modulates the expression and/or function of the BCL2 binding portion 3 (BBC3) polynucleotide relative to a normal control group. 48. The oligonucleotide of claim 36 Wherein the oligonucleotide comprises the sequence SEQ ID NOs: 3 to 8. 49. A pharmaceutical composition comprising one or more of one or more BCL2 binding moiety 3 (BBC3) as claimed in claim 35 Nucleotide-specific oligonucleotides and pharmaceutically acceptable excipients. 50. The composition of claim 49, wherein the oligonucleotides are nucleated as seq ID NO: 3 to 8 Alignment of any of the nucleotide sequences, having a sequence identity of at least about 157514.doc 201209163 40%. 51. The oligonucleotides include the nucleotide sequences set forth as SEQ ID NOS: 3 to 8. 52. The composition of claim 51, wherein the exemplified are SEQ ID NOs: 3 to 8 The nucleotide includes one or more modifications or substitutions. 53. The composition of claim 52, wherein the one or more modifications are selected from the group consisting of phosphorothioate, methyl phosphonate, peptide nucleic acid, and locked nucleic acid ( LNA) Molecules and combinations thereof 54. Use of an antisense nucleotide to produce or prevent at least one of BCL2 binding moiety 3 (BBC3) polynucleotide and/or at least one thereof An agent encoding a product-related disease, wherein the antisense oligonucleotide binds to a natural antisense sequence of at least one BCL2 binding portion 3 (BBC3) polynucleotide and modulates the at least one BCL2 binding portion 3(7)BC3) polynuclei The performance of glycosides. 55. The use of claim 54, wherein the disease associated with the at least one 8 〇 2 binding portion 3 (BBC3) polynucleotide is selected from the group consisting of: a disease or condition associated with abnormal function and/or performance of BBC3, cancer , a proliferative disease or condition 'a disease or condition characterized by cell proliferation or characterized by cell proliferation, a disease or condition associated with a mutation or abnormal expression or function of BBC3, a neurological disease or condition, inflammation, an inflammation of the joint becoming a disease — · Part of the condition (for example, osteoarthritis, arthritis, psoriasis joints, inflammation, juvenile arthritis, Lyttle syndrome syndrome), arthritis associated with ulcerative colitis, and Whirlple's disease (Wlnpple's Disease), joints associated with granulomatous ileitis 157514.doc -10- 201209163 56. Ο 57. 58. 59. 〇60. 61. Behcet's disease, systemic lupus erythematosus, dry Symptoms (Sjogrenis syndr〇me) and mixed connective tissue diseases, etc., diseases or conditions associated with mitochondrial apoptosis pathway damage, autoimmune diseases or conditions, and with God A disease or condition associated with death of a cell, aging, or other condition characterized by undesired cell loss. A method for inducing apoptosis in a biological system in vitro, comprising administering to the system a nucleotide that is 5 to 30 nucleotides in length and is naturally associated with a BBC3 polynucleotide At least 50 of 5 to 30 contiguous nucleotides in the reverse complement of the antisense transcript. /. Consistent. The method of claim 56, wherein the natural antisense transcript has a method of 〇 IDNO·· 2 〇, such as β, 56, wherein the biological system is a patient cell or tissue. A method for inducing apoptosis in a biological system in vitro, comprising administering to the system a nucleotide of about 5 to 30 nucleotides in length, wherein the oligonucleotide is targeted by a target gene The natural antisense transcript up-regulates the delta gene' and wherein the nucleotide is at least 5% identical to the reverse complement of the native antisense transcript of the up-regulated gene. A use of a nucleotide for the manufacture of a medicament for inducing a 'cell' in a biological system, wherein the nucleotide is 5 to 30 nucleotides in length and BBC3 At least 5% of the 5 to 3 contiguous nucleotides in the reverse complement of the natural antisense transcript of the polynucleotide are at least 5%. For example, the use of beta 60 wherein the natural antisense transcript has SEq IDNO: 2. 157514.doc -11 - 201209163 Μ. The use of claim 6G, wherein the biological system is a patient cell or tissue. 63. The use of an oligo-nucleic acid for the manufacture of a sputum for inducing a cytoplasmic/peripheral death in a biological system, wherein the nucleocapnic acid is about 5 to W nucleotides in length And upregulating the gene by targeting a natural antisense transcript of the target gene and wherein the nucleotide is at least 5% identical to the reverse complement of the natural antisense transcript of the upregulated gene. 64. A method of identifying and selecting at least one oligonucleotide that is selective for a natural antisense transcript of a gene of a selected target polynucleotide for in vivo administration, the method comprising: identifying at least one of Oligonucleotides of at least 5 contiguous nucleotides, at least 5 contiguous nucleotides at least partially complementary to an antisense poly(picoic acid) of "Hypergic acid; and under stringent hybridization conditions Measuring the thermal melting point of the antisense raised nucleotide and the hybrid of the target polynucleotide or the antisense polynucleotide of the selected dry polynucleotide; and selecting at least one oligonucleotide based on the obtained information Invested in the body. 157514.doc 12-