WO2011146674A2 - Treatment of bcl2-like 11 (bcl2l11) related diseases by inhibition of natural antisense transcript to bcl2l11 - Google Patents

Abstract

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of BCL2-like 11 (BCL2L11), in particular, by targeting natural antisense polynucleotides of BCL2-like 11 (BCL2L11). The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of BCL2L11.

Description

TREATMENT OF BCL2-L1 E 1 1 (BCL2L11) RELATED DISEASES BY INHIBITION OF NATURAL

ANTISENSE TRANSCRIPT TO BCL2L11

FIELD OF THE INVENTION

[00011 The present application claims the priority of U.S. provisional patent application No. 61/346252 filed May 1 , 2010 which is incorporated herein by reference in its entirety.

[0002] Embodiments of the invention comprise oligonucleotides modulating expression and/or function of BCL2L1 1 and associated molecules.

BACKGROUND

[0003 ) DNA-RNA and RNA-RNA hybridization arc important to many aspects of nucleic acid function including DNA replication, transcription, and translation. Hybridization is also central to a variety of technologies that either detect a particular nucleic acid or alter its expression. Antisensc nucleotides, for example, disrupt gene expression by hybridizing to target RNA, thereby interfering with RNA splicing, transcription, translation, and replication. Antisense DNA has the added feature that DNA-RNA hybrids serve as a substrate for digestion by ribonuclease H, an activity that is present in most cell types. Antisense molecules can be delivered into cells, as is the case for oligodeoxynucleotides (ODNs), or they can be expressed from endogenous genes as RNA molecules. The FDA recently approved an antisense drug, VITRAVENE™ (for treatment of cytomegalovirus retinitis), reflecting that antisense has therapeutic utility.

SUMMARY

[0004] This Summary is provided to present a summary of the invention to briefly indicate the nature and substance of the invention. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.

[0005] In one embodiment, the invention provides methods for inhibiting the action of a natural antisense transcript by using antisense oligonuclcotide(s) targeted to any region of the natural antisense transcript resulting in up-regulation of the corresponding sense gene. It is also contemplated herein that inhibition of the natural antisensc transcript can be achieved by siRNA, ribozymcs and small molecules, which are considered to be within the scope of the present invention.

[0006] One embodiment provides a method of modulating function and/or expression of an BCL2L1 1 polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antisensc oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within nucleotides I to 3026 of SEQ ID NO: 2 and I to 1512 of SEQ ID NO: 3 thereby modulating function and/or expression of the BCL2L1 1 polynucleotide in patient cells or tissues in vivo or in vitro. (0007] In an embodiment, an oligonucleotide targets a natural antisense sequence of BCL2L1 1 polynucleotides, for example, nucleotides set forth in SEQ ID NOS: 2 and 3, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Examples of antisense oligonucleotides arc set forth as SEQ ID NOS: 4 to 13.

[0008] Another embodiment provides a method of modulating function and/or expression of an BCL2L1 1 polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to a reverse complement of the an antisense of the BCL2L1 1 polynucleotide; thereby modulating function and/or expression of the BCL2LI 1 polynucleotide in patient cells or tissues in vivo or in vitro.

[000 1 Another embodiment provides a method of modulating function and/or expression of an BCL2LI I polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to an antisense oligonucleotide to an BCL2LI 1 antisense polynucleotide; thereby modulating function and/or expression of the BCL2L 1 1 polynucleotide in patient cells or tissues in vivo or in vitro.

[0010] In an embodiment, a composition comprises one or more antisense oligonucleotides which bind to sense and/or antisense BCL2L1 1 polynucleotides.

10 1 11 In an embodiment, the oligonucleotides comprise one or more modified or substituted nucleotides.

10 121 In an embodiment, the oligonucleotides comprise one or more modified bonds.

[0013] In yet another embodiment, the modified nucleotides comprise modified bases comprising phosphorothioatc, mcthylphosphonatc, peptide nucleic acids, 2'-0-mcthyl, iluoro- or carbon, methylene or other locked nucleic acid (LNA) molecules. Preferably, the modified nucleotides are locked nucleic acid molecules, including a-L-LNA.

[0014] In an embodiment, the oligonucleotides arc administered to a patient subcutaneously, intramuscularly, intravenously or intrapcritoncally.

[0015] In an embodiment, the oligonucleotides are administered in a pharmaceutical composition. A treatment regimen comprises administering the antisense compounds at least once to patient; however, this treatment can be modified to include multiple doses over a period of time. The treatment can be combined with one or more other types of therapies.

[0016] In an embodiment, the oligonucleotides arc encapsulated in a liposome or attached to a carrier molecule (e.g. cholesterol, TAT peptide).

100171 Other aspects arc described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

[0 1 ] Figure 1 is a graph of real time PCR results showing the fold change + standard deviation in BCL2L 1 1 mRN A after treatment of HepG2 cells with phosphorothioatc oligonucleotides introduced using Lipofectaminc 2000, as compared to control. Real time PCR results show that the levels of the BIM mRNA in MCF-7 cells arc significantly increased 48 h after treatment with two of the oligos designed to BIM antisense Hs.652337. Bars denoted as CUR- 1521 and CUR- 1522 correspond to samples treated with SEQ ID NOS: 10 and 1 1 respectively.

[001 ] Figure 2 is a graph of real time PCR results showing the fold change + standard deviation in BCL2L 1 1 mRNA after treatment of HcpG2 cells with phosphorothioatc oligonucleotides introduced using Lipofcctaniine 2000, as compared to control. Real time PCR results show that the levels of the BIM mRNA in HcpG2 cells arc significantly increased 48 h after treatment with one of the oligos designed to BIM antisense Hs.652337. Bars denoted as CUR- 1519, CUR- 1520, CUR- 1523, CUR- 1525, CUR- 1524, CUR- 1526, CUR- 1521 and CUR- 1522 correspond to samples treated with SEQ ID NOS: 4 to 13 respectively.

[0020] Figure 3 shows fold change in BCL2L1 1 mRNA expression in primary keratinocytes treated with phosphorothioatc oligonucleotides. CUR- 1.522 significantly uprcgulatcd BIM mRNA in Keratinocytes LL. Bar denoted as CUR- 1522 correspond to sample treated with SEQ ID NO: 1 1.

[0021 J Figure 4 is a graph of Apoptosis Assay Results showing percent increase in Apoptosis in A549 cells treated with phophorothioate oligonucleotides introduced using lipofectamine 2000 as compared to control. Apoptosis results show that CUR- significantly increased apoptosis in A549 cells compared to control. Bars denoted as CUR-1 174 and CUR-1522 correspond to samples treated with SEQ ID NO: 1 1 and 12

[00221 Figure 5 shows percent increase in Apoptosis in cells treated with phophorothioate oligonucleotides. CUR- 1522 significantly increased apoptosis in cells compared to control. Bars denoted as CUR- 151 , CUR-1521 CUR-1 522 and CUR- 1505 correspond to samples treated with SEQ ID NO: SEQ ID NO: 4, 10, 1 1 and 13

[0023] Figure 6 shows percent increase in Apoptosis in cells treated with phophorothioate oligonucleotides. CUR- 1522 significantly increased apoptosis in cells compared to control. Bar denoted as CUR- 1519, CUR- 1521 CUR-1522 and CUR- 1505 correspond to samples treated with SEQ ID NO: 4, 10, 1 1 and 13

|0024] Sequence Listing Description- SEQ ID NO: 1 : Homo sapiens BCL2-like 1 1 (apoptosis facilitator) (BCL2L1 1 ), transcript variant 9, mRNA (NCBI Accession No.: NM_2()7002); SEQ ID NO: 2: Natural BCL2L 1 1 antisense sequence (Hs.652337); SEQ ID NO: 3: Natural BCL2L1 1 antisense sequence (Hs.6 l 8461 ); SEQ ID NOs: 4 to 13: Antisense oligonucleotides. * indicates phosphothioate bond.

DETAILED DESCRIPTION

[0025] Several aspects of the invention arc described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in die relevant art, however, will readily recognize tliat the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

[0026] All genes, gene names, and gene products disclosed herein arc intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but arc not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In an embodiment, the genes or nucleic acid sequences are human. Definitions

10027 J The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0028] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than I standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1 % of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0029] As used herein, the term "mRNA" means the presently known mRNA transcript(s) of a targeted gene, and any further transcripts which may be elucidated.

[0030] By "antisense oligonucleotides" or "antisense compound" is meant an RNA or DNA molecule that binds to anotlicr RNA or DNA (target RNA, DNA). For example, if it is an RNA oligonucleotide it binds to another RNA target by means of RNA-RNA interactions and alters the activity of the target RNA. An antisense oligonucleotide can Liprcgulatc or downrcgulatc expression and/or function of a particular polynucleotide. The definition is meant to include any foreign RNA or DNA molecule which is useful from a therapeutic, diagnostic, or other viewpoint. Such molecules include, for example, antisense RNA or DNA molecules, interference RNA (RNAi), micro RNA, decoy RNA molecules, siRNA, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA, antisense oligomeric compounds, antiscnsc oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomcric compounds that hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomcric compounds.

[00311 In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimctics thereof. The term "oligonucleotide", also includes linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomcric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioatc, mcthylphosphonatc, and the like. Oligonucleotides arc capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Hoogstccn or reverse Hoogstccn types of base pairing, or the like.

[ 0032 J The oligonucleotide may be "chimeric", that is, composed of different regions. In the context of this invention "chimeric" compounds are oligonucleotides, which contain two or more chemical regions, for example, DNA region(s), RNA region(s), PNA region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotides compound. These oligonucleotides typically comprise at least one region wherein the oligonucleotide is modified in order to exhibit one or more desired properties. The desired properties of the oligonucleotide include, but are not limited, for example, to increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. Different regions of the oligonucleotide may therefore have different properties. The chimeric oligonucleotides of die present invention can be formed as mixed structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide analogs as described above.

[0033] The oligonucleotide can be composed of regions that can be linked in "register", that is, when the monomers are linked consecutively, as in native DNA, or linked via spacers. The spacers are intended to constitute a covalent "bridge" between the regions and have in preferred cases a length not exceeding about l(K) carbon atoms. The spacers may carry different functionalities, for example, having positive or negative charge, carry special nucleic acid binding properties (intercalators, groove binders, toxins, fluorophors etc.), being lipophilic, inducing special secondary structures like, for example, alanine containing peptides that induce alpha-helices.

[0034] As used herein "BCL2LI 1 " and "BCL2-likc 1 1" arc inclusive of all family members, mutants, alleles, fragments, species, coding and noncoding sequences, sense and antisense polynucleotide strands, etc.

[0035] As used herein, the words BCL2-likc 1 1 , BCL2L1 1, BAM, Bcl2-intcracting mediator of cell death, Bcl2-L-1 1 , BIM, BIM-alpha6, BIM-beta6, BIM-beta7, BimEL, BitnL, BOD, are considered the same in the literature and are used interchangeably in the present application. [0036] As used herein, the term "oligonucleotide specific for" or "oligonucleotide which targets" refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (ii) capable of forming a stable duplex with a portion of a mRNA transcript of the targeted gene. Stability of the complexes and duplexes can be determined by theoretical calculations and/or in vitro assays. Exemplary assays for determining stabi lity of hybridization complexes and duplexes arc described in the Examples below.

10037] As used herein, the temi "target nucleic acid" encompasses D A, RNA (comprising prcmR A and mRNA) transcribed from such DNA, and also cDNA derived from such RNA, coding, noncoding sequences, sense or antisense polynucleotides. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds, which specifically hybridize to it, is generally referred to as "antisense". The functions of DNA to be interfered include, for example, replication and transcription. The functions of RNA to be interfered, include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of an encoded product or oligonucleotides.

[0038] RNA interference "RNAi" is mediated by double stranded RNA (dsRNA) molecules that have sequence- specific homology to their "target" nucleic acid sequences. In certain embodiments of the present invention, the mediators are 5-25 nucleotide "small interfering" RNA duplexes (siRNAs). The siRNAs are derived from the processing of dsRN A by an RNase enzyme known as Dicer. siRNA duplex products are recruited into a multi-protein siRNA complex termed RISC (RNA Induced Silencing Complex). Without wishing to be bound by any particular theory, a RISC is then believed to be guided to a target nucleic acid (suitably mRNA), where the siRNA duplex interacts in a sequence-specific way to mediate cleavage in a catalytic fashion. Small interfering RN As that can be used in accordance with the present invention can be synthesized and used according to procedures that arc well known in the art and that will be familiar to the ordinarily skilled artisan. Small interfering RNAs for use in the methods of the present invention suitably comprise between about 1 to about 50 nucleotides (nt). In examples of non limiting embodiments, siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25 nucleotides.

[0039] Selection of appropriate oligonucleotides is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species. In the case of genes that have not been sequenced, Southern blots arc performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, as is well known in the art, it is possible to obtain an approximate measure of identity. These procedures allow the selection of oligonucleotides that exhibit a high degree of complementarity to target nucleic acid sequences in a subject to be controlled and a lower degree of complementarity to corresponding nucleic acid sequences in other species. One skilled in the art will realize that there is considerable latitude in selecting appropriate regions of genes for use in the present invention.

[0040] By "enzymatic RNA" is meant an R A molecule with enzymatic activity (Cech, (1988) J. American. Med. Assoc. 260, 3030-3035). Enzymatic nucleic acids (ribozymes) act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. 10041 J By "decoy RNA" is meant an RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a "decoy" and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA. This is meant to be a specific example. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art.

[0042] As used herein, the term "monomers" typically indicates monomers linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomelic units, e.g., from about 3-4, to about several hundreds of monomelic units. Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, mcthylphosphornatcs, phosphoroselenoate, phosphoramidate, and die like, as more fully described below.

[00431 The term "nucleotide" covers naturally occurring nucleotides as well as nonnaturally occurring nucleotides. It should be clear to the person skilled in the art that various nucleotides which previously have been considered "nonnaturally occurring" have subsequently been found in nature. Thus, "nucleotides" includes not only the known purine and pyrimidinc hctcrocycles-containing molecules, but also heterocyclic analogues and tautomers thereof. Illustrative examples of other types of nucleotides are molecules containing adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopiirine, 8-oxo- N6-mcthyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin, N6.N6- cthano-2,6- diaminopurinc, 5-mcthylcytosinc, 5-(C3-C6)-alkynylcytosine, 5-fiuorouracil, 5-bromouracil, pscudoisocytosine, 2-hydroxy-5-mcthyl-4-triazolopyridin, isocytosinc, isoguanin, inosinc and the "non-naturally occurring" nucleotides described in Bcnncr el «/., U.S. Pat No. 5,432,272. The term "nucleotide" is intended to cover every and all of these examples as well as analogues and tautomers thereof. Especially interesting nucleotides arc those containing adenine, guanine, tliymine, cytosine, and uracil, which are considered as the naturally occurring nucleotides in relation to therapeutic and diagnostic application in humans. Nucleotides include the natural 2'-deoxy and 2'- hydroxyl sugars, e.g., as described in Kombcrg and Baker, DNA Replication, 2nd Ed. CFrceman, San Francisco, 1992) as well as their analogs.

[0044] "Analogs" in reference to nucleotides includes synthetic nucleotides having modified base moieties and/or modified sugar moieties (sec e.g., described generally by Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Frcicr & Altmann, ( 1 97) Nticl. Acid. Res., 25(22), 4429- 4443, Toulme, J.J., (2001) Nature Biotechnology 19: 17-18; anoharan M., (1999) Biachemica el Biophysica Acta 1489: 1 17-139; Freier S. M., (1997) Nucleic Acid Research, 25:4429-4443, Uhlman, E., (2000) Drug Discovery <$ Development, 3: 203-213, Herdewin P., (2000) Antisensc c6 Nucleic Acid Drug Dev., 10:297-310); 2'-0, 3^'-C-linked [3.2.0] bicycloarabinonuclcosides. Such analogs include synthetic nucleotides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.

[0045] As used herein, "hybridization" means the pairing of substantially complementary strands of oligomeric compounds. One mechanism of pairing involves hydrogen bonding, which may be Watson-Criek, Hoogstccn or reversed Hoogstecn hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleotides) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleotides which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

[0046^'] An antisensc compound is "specifically hybridizable" when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a modulation of function and/or activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

[0047] As used herein, the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions arc sequence-dependent and will be different in different circumstances and in the context of this invention, "stringent conditions" under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they arc being investigated. In general, stringent hybridization conditions comprise low concentrations (<0.15M) of salts with inorganic cations such as Na++ or K.++ (i.e., low ionic strength), temperature higher than 20°C - 25° C. below the Tm of the oligomeric conipound.target sequence complex, and the presence of dcnaturants such as fomiamidc, dimcthylformamide, dimcdiyl sulfoxide, or the detergent sodium dodecyl sulfate (SDS). For example, the hybridization rate decreases 1.1 % for each 1% formamide. An example of a high stringency hybridization condition is 0.1X sodium chloride-sodium citrate buffer (SSC)/0.1% (w/v) SDS at 60° C. for 30 minutes.

100481 "Complementary," as used herein, refers to the capacity for precise pairing between two nucleotides on one or two oligomeric strands. For example, if a nucleobasc at a certain position of an antisensc compound is capable of hydrogen bonding with a nuclcobasc at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonicric compound and the further DNA, RNA, or oligonucleotide molecule arc complementary to each other when a sufficient number of complementary positions in each molecule arc occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizablc" and "complementary" are terms which arc used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleotides such that stable and specific binding occurs between the oligomeric compound and a target nucleic acid.

[004 1 It is understood in the art diat the sequence of an oligomeric compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizablc. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments arc not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure). The oligomeric compounds of the present invention comprise at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining non-complementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. As such, an antisense compound which is 18 nucleotides in length having 4 (four) non-complementary nucleotides which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowcrBLAST programs known in the art. Percent homology, sequence identity or complementarity, can be determined 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 uses the algorithm of Smith and Waterman (Adv. Appl. Math., (1981) 2, 482-489).

[0050] As used herein, the term "Thermal Melting Point (Tm)" refers to the temperature, under defined ionic strength, pH, and nucleic acid concentration, at which 50% of the oligonucleotides complementary to the target sequence hybridize to the target sequence at equilibrium. Typically, stringent conditions will be those in which the salt concentration is at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and die temperature is at least about 30°C for short oligonucleotides (e.g., 10 to 50 nucleotide). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. [00511 As used herein, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a genc.

[00521 The term "variant", when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, "allelic," "splice," "species," or "polymorphic" variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during m NA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. Of particular utility in the invention arc variants of wild type genc products. Variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant genc may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

|(K)53 | The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs,) or single base mutations in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population with a propensity for a disease state, that is susceptibility versus resistance.

[0054] Derivative polynucleotides include nucleic acids subjected to chemical modification, for example, replacement of hydrogen by an alkyl, acyl, or amino group. Derivatives, e.g., derivative oligonucleotides, may comprise non- naturally-occurring portions, such as altered sugar moieties or inter-sugar linkages. Exemplary among these are phosphorothioatc and other sulfur containing species which are known in the art. Derivative nucleic acids may also contain labels, including radionuclcotidcs, enzymes, fluorescent agents, chemiluminescent agents, chromogcnic agents, substrates, cofactors, inhibitors, magnetic particles, and the like.

[0055] A "derivative" polypeptide or peptide is one that is modified, for example, by glycosylation, pcgylation, phosphorylation, sulfation, reduction/alkylation, acylation, chemical coupling, or mild formalin treatment. A derivative may also be modified to contain a detectable label, cither directly or indirectly, including, but not limited to. a radioisotope, fluorescent, and enzyme label.

[0056] As used herein, the term "animal" or "patient" is meant to include, for example, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys, horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken, reptiles, fish, insects and arachnids.

[0057] "Mammal" covers warm blooded mammals that are typically under medical care (e.g., humans and domesticated animals). Examples include feline, canine, equine, bovine, and human, as well as just human. [005 1 "Treating" or "treatment" covers the treatment of a disease-state in a mammal, and includes: (a) preventing the discasc-statc from occurring in a mammal, in particular, when such mammal is predisposed to the disease-state but has not yet been diagnosed as having it; (b) inhibiting the discasc-statc, e.g., arresting it development; and/or (c) relieving the discasc-statc, e.g., causing regression of the disease state until a desired endpoint is reached. Treating also includes the amelioration of a symptom of a disease (e.g., lessen the pain or discomfort), wherein such amelioration may or may not be directly affecting the disease (e.g., cause, transmission, expression, etc.).

[ 0059] As used herein, "cancer" refers to all types of cancer or neoplasm or malignant tumors found in mammals, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas. The cancer manifests itself as a "tumor" or tissue comprising malignant cells of the cancer. Examples of tumors include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, cndothcliosarcoma, lymphangiosarcoma, lymphangiocndothcliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma. medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' rumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Additional cancers which can be treated by the disclosed composition according to the invention include but not limited to, for example, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulincmia, small-cell lung rumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, gastric cancer, prcmalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and prostate cancer. The term "apoptosis" means programmed cell death. It also means "cell death" or "targeted cell death" which occurs in the context of administration of an antisensc oligonucleotide recited herein to treat or mitigate diseases or disorders associated with uncontrolled cell growth and/or the malfunction of normal apoptosis and/or apoptotic pathways.

|()060| As used herein a "Neurological disease or disorder" refers to any disease or disorder of the nervous system and/or visual system. "Neurological disease or disorder" include disease or disorders that involve the central nervous system (brain, brainstem and cerebellum), the peripheral nervous system (including cranial nerves), and the autonomic nervous system (parts of which are located in both central and peripheral nervous system). A Neurological disease or disorder includes but is not limited to acquired epileptiform aphasia; acute disseminated encephalomyelitis; adrenolcukodystrophy; agc-rclatcd macular degeneration; agenesis of the corpus callosum; agnosia; Aicardi syndrome; Alexander disease; Alpcrs' disease; alternating hemiplegia; Alzheimer's disease; Vascular dementia; amyotrophic lateral sclerosis; ancnccphaly; Angclman syndrome; angiomatosis; anoxia; aphasia; apraxia; arachnoid cysts; arachnoiditis; Anronl-Chiari malformation; arteriovenous malformation; Asperger syndrome; ataxia tclcgicctasia; attention deficit hyperactivity disorder; autism; autonomic dysfunction; back pain; Batten disease; Behcet's disease; Bell's palsy; benign essential blepharospasm; benign focal; amyotrophy; benign intracranial hypertension; Binswanger's disease; blepharospasm; Bloch Sulzberger syndrome; brachial plexus injury; brain abscess; brain injury; brain tumors (including glioblastoma multiforme); spinal tumor; Brown- Sequard syndrome; Canavan disease; carpal tunnel syndrome; causalgia; central pain syndrome; central pontine myelinolysis; cephalic disorder; cerebral aneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism; cerebral palsy; Charcot-Maric-Tooth disease; chemotherapy-induced neuropathy and neuropathic pain; Chiari malformation; chorea; chronic inflammatory demyelinating polyneuropathy; chronic pain; chronic regional pain syndrome; Coffin Lowry syndrome; coma, including persistent vegetative state: congenital facial diplegia; corticobasal degeneration; cranial arteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing's syndrome; cytomegalic inclusion body disease; cytomegalovirus infection; dancing eyes-dancing rect syndrome; DandyWalker syndrome; Dawson disease; De Morsier's syndrome; Dcjerine- lumke palsy; dementia; dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia; dysgraphia; dyslexia; dystonias; early infantile epileptic encephalopathy; empty sella syndrome; encephalitis; encephaloceles; encephalotrigeminal angiomatosis; epilepsy; Erb's palsy; essential tremor; Fabry's disease; Fahr's syndrome; fainting; familial spastic paralysis; febrile seizures; Fisher syndrome; Friedreich's ataxia: fronto-temporal dementia and other "tauopathics"; Gauchcr's disease; Gerstmann's syndrome; giant cell arteritis; giant cell inclusion disease; globoid cell leukodystrophy; Guillain-Barre syndrome; HTLV- 1 -associated myelopathy; Hallcrvordcn-Spatz disease; head injury; headache; hemifacial spasm; hereditary spastic paraplegia; hcredopathia atactic a polyncuritiformis; herpes zoster oticus; herpes zoster; Hirayama syndrome; HIVassociated dementia and neuropathy (also neurological manifestations of AIDS); holoprosencephaly; Huntington's disease and other polyglutaminc repeat diseases; hydranencephaly; hydrocephalus; hypercortisolism; hypoxia; immune- mediated encephalomyelitis: inclusion body myositis; incontinentia pigmcnti; infantile phytanic acid storage disease; infantile refsum disease: infantile spasms; inflammatory myopathy; intracranial cyst; intracranial hypertension; Joubcrt syndrome; Kcams-Sayrc syndrome; Kennedy disease Kinsboumc syndrome; Klippcl Fcil syndrome: Krabbe disease; Kugelbcrg-Welandcr disease; kuru; Lafora disease; Lambert-Eaton myasthenic syndrome; Landau-KIeffner syndrome; lateral medullary (Wallenberg) syndrome; learning disabilities; Leigh's disease; Lcnnox-Gustaut syndrome; Lesch-Nyhan syndrome; leukodystrophy; Lcwy body dementia; Lissencephaly; locked-in syndrome; Lou Gehrig's disease (i.e., motor neuron disease or amyotrophic lateral sclerosis); lumbar disc disease; Lyme disease-neurological sequelae; Machado-Joscph disease; macrcnccphaly; nicgalcnccphaly; Melkersson-Rosenthal syndrome; Mcnicrcs disease; meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly; migraine; Miller Fisher syndrome; mini-strokes; mitochondrial myopathies; Mobius syndrome; monomelic amyotrophy; motor neuron disease; Moyamoya disease; mucopolysaccharidoses; milti-infarct dementia; multifocal motor neuropathy; multiple sclerosis and other dcmyclinating disorders; multiple system atrophy with postural hypotension; muscular dystrophy; myasthenia gravis; myclinoclastic diffuse sclerosis; myoclonic encephalopathy of infants; myoclonus; myopathy; myotonia congenital; narcolepsy; neurofibromatosis; neuroleptic malignant syndrome; neurological manifestations of AIDS; neurological sequelae of lupus; ncuromyotonia; neuronal ceroid lipofuscinosis; neuronal migration disorders; Nicmann-Pick disease; O'Sullivan-McLeod syndrome; occipital neuralgia; occult spinal dysraphism sequence; Ohtahara syndrome; olivopontocerebellar atrophy; opsoclonus myoclonus; optic neuritis; orthostatic hypotension; overuse syndrome: paresthesia; a neurodegenerative disease or disorder (Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), dementia, multiple sclerosis and other diseases and disorders associated with neuronal cell death); paramyotonia congenital; paraneoplastic diseases; paroxysmal attacks; Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodic paralyses; peripheral neuropathy; painful neuropathy and neuropathic pain; persistent vegetative state; pervasive developmental disorders; photic sneeze reflex; phytanic acid storage disease; Pick's disease; pinched nerve; pituitary rumors; polymyositis; porencephaly; post- polio syndrome; postherpetic neuralgia; postinfectious encephalomyelitis; postural hypotension; Prader- Willi syndrome; primary lateral sclerosis; prion diseases; progressive hemifacial atrophy; progressive multifocalleukoencephalopathy; progressive sclerosing poliodystrophy; progressive supranuclear palsy; pseudotumor cerebri; Ramsay-Hunt syndrome (types I and 1 1 ); Rasmussen's encephalitis; reflex sympathetic dystrophy syndrome; Rcfsum disease; repetitive motion disorders; repetitive stress injuries; restless legs syndrome rctrovirus-associatcd myelopathy; Rett syndrome; Rcyc's syndrome; Saint Vitus dance; Sandhoff disease; Schildcr's disease; schizencephaly; septo-optic dysplasia; shaken baby syndrome; shingles; Shy-Drager syndrome: Sjogren's syndrome; sleep apnea; Soto's syndrome; spasticity: spina bifida; spinal cord injury; spinal cord tumors; spinal muscular atrophy; Stiff-Person syndrome; stroke; Sturge-Weber syndrome; subacute sclerosing panencephalitis; subcortical arteriosclerotic encephalopathy; Sydenham chorea; syncope; syringomyelia; tardive dyskinesia; Tay-Sachs disease; temporal arteritis; tethered spinal cord syndrome; Thomsen disease; thoracic outlet syndrome; Tic Douloureux; Todd's paralysis; Tourettc syndrome; transient ischemic attack; transmissible spongiform encephalopathies; transverse myelitis; traumatic brain injury; tremor; trigeminal neuralgia; tropical spastic paraparesis; tuberous sclerosis; vascular dementia (multi-infarct dementia); vasculitis including temporal arteritis; Von Hippel-Lindau disease; Wallenberg's syndrome; Werdnig-Hoffman disease; West syndrome; whiplash; Williams syndrome; Wildon's disease; and Zellweger syndrome. [0061 1 A "proliferative disease or disorder" includes, but is not limited to, hematopoietic neoplastic disorders involving hyperplastic/neoplastic cells of hematopoietic origin arising from myeloid, lymphoid or crythroid lineages, or precursor cells thereof. These include, but arc not limited to erythroblastic leukemia, acute promycloid leukemia (APML), chronic myelogenous leukemia (CML), lymphoid malignancies, including, but not limited to, acute lymphoblastic leukemia (ALL), which includes B-lineagc ALL and T-lincagc ALL, chronic lymphocytic leukemia (CLL), prolymphocyte leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to, non- Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia lymphoma (ATL), cutaneous T-ccll lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Rced- Sternberg disease.

[0062 J A "hematological disease or disorder" includes a disease, disorder, or condition which affects a hematopoietic cell or tissue. Hematological disorders include diseases, disorders, or conditions associated with aberrant hematological content or function. Examples of hematological disorders include disorders resulting from bone marrow irradiation or chemotherapy treatments for cancer, disorders such as pernicious anemia, hemorrhagic anemia, hemolytic anemia, aplastic anemia, sickle cell anemia, sideroblastic anemia, anemia associated with chronic infections such as malaria, trypanosomiasis, HIV, hepatitis virus or other viruses, myelophthisic anemias caused by marrow deficiencies, renal failure resulting from anemia, anemia, polycethemia, infectious mononucleosis (IM), acute non-lymphocytic leukemia (ANLL), acute Myeloid Leukemia (A L), acute promyelocyte leukemia (APL), acute myelomonocytic leukemia (AMMoL), polycethemia vera, lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia, Wilm's tumor, Ewing's sarcoma, retinoblastoma, hemophilia, disorders associated with an increased risk of thrombosis, herpes, thalesscmia, antibody-mediated disorders such as transfusion reactions and erythroblastosis, mechanical trauma to red blood cells such as micro-angiopathic hemolytic anemias, thrombotic thrombocytopenic purpura and disseminated intravascular coagulation, infections by parasites such as Plasmodium, chemical injuries from, e.g., lead poisoning, and hypcrsplcnisni. Lymphatic diseases include, but arc not limited to, lymphadenitis, lymphagicctasis, lymphangitis, lymphedema, lymphocclc, lymphoproliferative disorders, mucocutaneous lymph node syndrome, reticuloendotheliosis, splenic diseases, thymus hyperplasia, thymus neoplasms, tuberculosis, lymph node, pseudolymphoma, and lymphatic abnormalities. Disorders of hcmatolymphoid system include, but arc not limited to. non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and reactive lymphoid hyperplasia.

Polynucleotide and Oligonucleotide Compositions and Molecules

[00631 Targets: In one embodiment, the targets comprise nucleic acid sequences of BCL2-likc 1 1 (BCL2L 1 1 ), including without limitation sense and/or antisense noncoding and/or coding sequences associated with BCL2L 1 I . [0064] Members of the Bcl-2 family of proteins arc structurally similar, but have divergent activities. This family of proteins is divided into the "pro-apoptotic" family members (i.e., Bax, Bak, Bok), and the "pro-survival" family members (i.e., Bcl-2, Bel-XL, Bcl-w). The former can be further subdivided into those proteins containing the BH3 domain and those lacking it. The BH3 domain is thought to facilitate binding of these proteins directly to members of the "pro-survival" proteins, thereby inhibiting the "pro-survival" activity and promoting cell death or apoptosis.

[0065] The human BCL2L1 1 locus, located on chromosome 2q 13, encodes a protein of 198 amino acids structurally and functionally related to the Bi-D-only group of pro-apoptotic BCL2 family members. The gene is frequently mutated in diverse human tumors leading to loss of BCL2LI 1 activity. Expression of BCL2L 1 1 is induced by a diverse range of apoptotic stimuli such as deprivation of growth factors/cytokines, ionizing radiation, and cytotoxic peptides.

[0066] In an embodiment, antisense oligonucleotides are used to prevent or treat diseases or disorders associated with BCL2L1 1 family members. Exemplary BCL2-likc I I (BCL2LU) mediated diseases and disorders which can be treated with cell/tissues regenerated from stem cells obtained using the antisense compounds comprise: cancer, aberrant apoptosis, a proliferative disease or disorder, a disease or disorder associated with mitochondrial apoptosis pathway, leukemia, an autoimmune disease or disorder, a disease or disorder associated with impaired immunity, infection, hyperproliferative disorders, hyperproliferation of reproductive tissue (such as uterine, testicular and ovarian carcinomas, endometriosis, and squamous, glandular epithelial carcinomas of the cervix etc.), a neurological disease or disorder, a developmental disorder associated with the developing embryo, a hematological disease or disorder, impaired tissue homeostatis, rheumatoid arthritis, sepsis, retinal ganglion cell death, a hepatic disease and a renal disease.

[0067] -In an embodiment, modulation of BCL2L1 1 by one or more antisense oligonucleotides is administered to a patient in need thereof, to prevent or treat any disease or disorder related to BCL2L 1 1 abnormal expression, function, activity as compared to a normal control.

[0068] In an embodiment, the oligonucleotides are specific for polynucleotides of BCL2L1 1 , which includes, without limitation noncoding regions. The BCL2LI 1 targets comprise variants of BCL2L 1 1 ; mutants of BCL2LI 1, including SNPs; noncoding sequences of BCL2L1 1 ; alleles, fragments and the like. Preferably the oligonucleotide is an antisense RNA molecule.

[0069] In accordance with embodiments of the invention, the target nucleic acid molecule is not limited to BCL2L1 1 polynucleotides alone but extends to any of the isoforms, receptors, homologs, non-coding regions and the like of BCL2L1 1.

[0070] In an embodiment, an oligonucleotide targets a natural antisensc sequence (natural antisense to the coding and non-coding regions) of BCL2L 1 1 targets, including, without limitation, variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Preferably the oligonucleotide is an antisense RNA or DNA molecule. [0071 1 In an embodiment, the oligomcric compounds of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenine, variants may be produced which contain thymidine, guanosinc, cytidine or other natural or unnatural nucleotides at this position. This may be done at any of the positions of the antisense compound. These compounds arc then tested using the methods described herein to determine their ability to inhibit expression of a target nucleic acid. 100721 In some embodiments, homology, sequence identity or complementarity, between the antisense compound and target is from about 50% to about 60%. In some embodiments, homology, sequence identity or complementarity, is from about 60% to about 70%. In some embodiments, homology, sequence identity or complementarity, is from about 70% to about 80%. In some embodiments, homology, sequence identity or complementarity, is from about 80% to about 90%. In some embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.

[0073 J An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired. Such conditions include, i.e., physiological conditions in the case of in vivo assays or therapeutic treatment, and conditions in which assays are performed in the case of in vitro assays. 100741 An antisense compound, whether DNA, NA, chimeric, substituted etc, is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complcmcntarily to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays arc performed.

[00751 In an embodiment, targeting of BCL2L 1 1 including without limitation, antisense sequences which arc identified and expanded, using for example, PCR, hybridization etc., one or more of the sequences set forth as SEQ ID NOS: 2 and 3, and the like, modulate the expression or function of BCL2L1 1. In one embodiment, expression or function is up-regulatcd as compared to a control. In an embodiment, expression or function is down-regulated as compared to a control.

[00761 In an embodiment, oligonucleotides comprise nucleic acid sequences set forth as SEQ ID NOS: 4 to 13 including antisense sequences which arc identified and expanded, using for example, PCR, hybridization etc. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like. Examples of modified bonds or intemucleotide linkages comprise phosphorothioate, phosphorodithioatc or the like. In an embodiment, the nucleotides comprise a phosphorus derivative. The phosphorus derivative (or modified phosphate group) which may be attached to the sugar or sugar analog moiety in the modified oligonucleotides of the present invention may be a monophosphate, diphosphate, triphosphate, alkylphosphatc, alkancphosphatc, phosphorothioatc and the like. The preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, per sc, is also known and need not be described here.

[0077] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.

[0078] Jn embodiments of the present invention oligomeric antisense compounds, particularly oligonucleotides, bind to target nucleic acid molecules and modulate the expression and/or function of molecules encoded by a target gene. The functions of DNA to be interfered comprise, for example, replication and transcription. The functions of RNA to be interfered comprise all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The functions may be up-regulated or inhibited depending on the functions desired.

[0079] The antisense compounds, include, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.

[0080] Targeting an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target nucleic acid encodes BCL2-like 1 1 (BCL2L11).

[0081 ] The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. Within the context of the present invention, the term "region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids arc segments. "Segments" arc defined as smaller or sub-portions of regions within a target nucleic acid. "Sites," as used in the present invention, arc defined as positions within a target nucleic acid.

|0082] In an embodiment, the antisense oligonucleotides bind to the natural antisense sequences of BCL2-like 1 1 (BCL2L1 1) and modulate the expression and/or function of BCL2LI I (SEQ ID NO: 1). Examples of antisense sequences include SEQ ID NOS: 2 to 13. [0083] [n an embodiment, the antisense oligonucleotides bind to one or more segments of BCL2-likc 1 1 (BCL2L 1 1 ) polynucleotides and modulate the expression and/or function of BCL2L1 1. The segments comprise at least five consecutive nucleotides of the BCL2L 11 sense or antisense polynucleotides.

[0084] In an embodiment, the antisense oligonucleotides are specific for natural antisense sequences of BCL2L1 1 wherein binding of the oligonucleotides to the natural antisense sequences of BCL2L1 1 modulate expression and/or function of BCL2Ll l .

[0085] In an embodiment, oligonucleotide compounds comprise sequences set forth as SEQ ID NOS: 4 to 13, antisense sequences which are identified and expanded, using for example, PCR, hybridization etc These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like. Examples of modified bonds or intcmucleotide linkages comprise phosphorothioate, phosphorodithioate or the like. In an embodiment, the nucleotides comprise a phosphorus derivative. The phosphorus derivative (or modified phosphate group) which may be attached to the sugar or sugar analog moiety in the modified oligonucleotides of the present invention may be a monophosphate, diphosphate, triphosphate, alkylphosphate. alkanephosphatc, phosphorothioate and the like. The preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, perse, is also known and need not be described here.

[0086] Since, as is known in the art, the translation initiation codon is typically 5 -AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon". A minority of genes has a translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG; and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in cukaryotcs) or fonnylmcthionine (in prokaryotcs). Eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, "start codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding BCL2-likc 1 1 (BCL2LI I ), regardless of the sequcnce(s) of such codons. A translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5 -UAA, 5 -UAG and 5 -UGA (the corresponding DNA sequences arc 5'- TAA, 5'- TAG and 5'-TGA, respectively).

[0087] The terms "start codon region" and "translation initiation codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in cither direction (i.e., 5' or 3') from a translation termination codon. Consequently, the "start codon region" (or "translation initiation codon region") and the "stop codon region" (or 'translation termination codon region") arc all regions that may be targeted effectively with the antiscnsc compounds of the present invention.

[0088] The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between the translation initiation codon and die translation termination codon, is also a region which may be targeted effectively. Within the context of die present invention, a targeted region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

10089 ] Another target region includes the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene). Still another target region includes the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA (or corresponding nucleotides on the gene). The 5' cap site of an mRNA comprises an N7-methylatcd guanosinc residue joined to the 5'-most residue of the mRNA via a 5'-5^* triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap site. Another target region for this invention is the 5' cap region.

10090] Although some cukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," which arc excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and arc spliced together to form a continuous mRNA sequence. In one embodiment, targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, is particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. An aberrant fusion junction due to rearrangement or deletion is another embodiment of a target site. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources arc known as "fusion transcripts". Introns can be effectively targeted using antisense compounds targeted to, for example, DNA or prc-mRNA.

[0091 1 In an embodiment, the antisense oligonucleotides bind to coding and/or non-coding regions of a target polynucleotide and modulate the expression and/or function of the target molecule.

[0092] In an embodiment, the antiscnse oligonucleotides bind to natural antiscnsc polynucleotides and modulate the expression and/or function of the target molecule.

[00931 In an embodiment, the antiscnsc oligonucleotides bind to sense polynucleotides and modulate the expression and/or function of the target molecule.

100941 Alternative R A transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as "variants". More specifically, "pre-mRNA variants" are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in cither their start or stop position and contain both intronic and exonic sequence.

0095J Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA variants". Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as "alternative splice variants". If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

1 0961 Variants can be produced through the use of alternative signals to start or stop transcription. Prc-mRNAs and inRNAs can possess more than one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon arc known as "alternative stop variants" of that pre-mRNA or mRNA. One specific type of alternative stop variant is the "polyA variant" in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also embodiments of target nucleic acids.

[00971 The locations on the target nucleic acid to which the antisense compounds hybridize pre defined as at least a 5-nucleotide long portion of a target region to which an active antisense compound is targeted.

[0098] While the specific sequences of certain exemplary target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional target segments are readily identifiable by one having ordinary skill in the art in view of this disclosure.

[0099] Target segments 5-100 nucleotides in length comprising a stretch of at least five (5) consecutive nucleotides selected from within the illustrative preferred target segments arc considered to be suitable for targeting as well.

[00100] Target segments can include DNA or RNA sequences that comprise at least the 5 consecutive nucleotides from the 5'-tcrminus of one of the illustrative preferred target segments (the remaining nucleotides being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5'-terminus of the target segment and continuing until the DNA or RNA contains about 5 to about 100 nucleotides). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 5 consecutive nucleotides from the 3'-terminus of one of the illustrative preferred target segments (.the remaining nucleotides being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3'-tcrminus of the target segment and continuing until the DNA or RNA contains about 5 to about 100 nucleotides). One having skill in the art armed with the target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments. [00101 ] Once one or more target regions, segments or sites have been identified, antiscnsc compounds arc chosen which arc sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[00102] In embodiments of the invention the oligonucleotides bind to an antisense strand of a particular target. The oligonucleotides arc at least 5 nucleotides in length and can be synthesized so each oligonucleotide targets overlapping sequences such that oligonucleotides arc synthesized to cover the entire length of the target polynucleotide. The targets also include coding as well as non coding regions.

[00103] In one embodiment, it is preferred to target specific nucleic acids by antisense oligonucleotides. Targeting an antiscnsc compound to a particular nucleic acid is a multistcp process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a non coding polynucleotide such as for example, non coding RNA (ncR A).

[00104] RNAs can be classified into (1 ) messenger RNAs (mRNAs), which are translated into proteins, and (2) non- protein-coding R As (ncRNAs). ncRNAs comprise microRNAs, antisense transcripts and other Transcriptional Units (TU) containing a high density of stop codons and lacking any extensive "Open Reading Frame". Many ncRNAs appear to start from initiation sites in 3' untranslated regions (3'UTRs) of protein-coding loci. ncRNAs arc often rare and at least half of the ncRNAs that have been sequenced by the FANTOM consortium seem not to be polyadenylated. Most researchers have for obvious reasons focused on polyadenylated mRNAs that are processed and exported to the cytoplasm. Recently, it was shown that the set of non-polyadenylated nuclear RNAs may be very large, and that many such transcripts arise from so-called intergenic regions. The mechanism by which ncRNAs may regulate gene expression is by base pairing with target transcripts. The RNAs that function by base pairing can be grouped into (1) cis encoded RNAs that arc encoded at the same genetic location, but on the opposite strand to the RNAs they act upon and therefore display perfect complementarity to their target, and (2) trans-encoded RNAs that arc encoded at a chromosomal location distinct from the RNAs they act upon and generally do not exhibit perfect base-pairing potential with their targets.

[00105] Without wishing to be bound by theory, perturbation of an antisense polynucleotide by the antisense oligonucleotides described herein can alter the expression of the corresponding sense messenger RNAs. However, this regulation can cither be discordant (antisense knockdown results in messenger RNA elevation) or concordant (antiscnsc knockdown results in concomitant messenger RNA reduction). In these cases, antiscnsc oligonucleotides can be targeted to overlapping or non-overlapping parts of the antisense transcript resulting in its knockdown or sequestration. Coding as well as non-coding antisense can be targeted in an identical manner and that eitlier category is capable of regulating the corresponding sense transcripts - either in a concordant or disconcordant manner. The strategics that arc employed in identifying new oligonucleotides for use against a target can be based on the knockdown of antisense RNA transcripts by antisense oligonucleotides or any other means of modulating the desired target.

[00106] Strategy 1: In the case of discordant regulation, knocking down the antisense transcript elevates the expression of the conventional (sense) gene. Should that latter gene encode for a known or putative drug target, then knockdown of its antisense counterpart could conceivably mimic the action of a receptor agonist or an enzyme stimulant.

1 01071 Strategy' 2: In the case of concordant regulation, one could concomitantly knock down both antisense and sense transcripts and thereby achieve synergistic reduction of the conventional (sense) gene expression. If, for example, an antisense oligonucleotide is used to achieve knockdown, then this strategy can be used to apply one antisense oligonucleotide targeted to the sense transcript and another antisense oligonucleotide to the corresponding antisense transcript, or a single energetically symmetric antisense oligonucleotide that simultaneously targets overlapping sense and antisense transcripts.

[00108 ] According to the present invention, antisense compounds include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleorides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid and modulate its function. As such, they may be DNA, RNA, DNA-like, RNA-!ike, or mixtures thereof, or may be mimctics of one or more of these. These compounds may be single-stranded, doublcstranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges, mismatches or loops. Antisense compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and/or branched. Antisense compounds can include constructs such as, for example, two strands hybridized to form a wholly or partially doub!c-stranded compound or a single strand with sufficient sclf- complcmcntarity to allow for hybridization and formation of a fully or partially double-stranded compound. The two strands can be linked internally leaving free 3' or 5' termini or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on cither the 5' or 3' terminus producing an extension of single stranded character. The double stranded compounds optionally can include overhangs on the ends. Further modifications can include conjugate groups attached to one of the termini, selected nucleotide positions, sugar positions or to one of the intemucleosidc linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group. When formed from only one strand, dsRNA can take the fonn of a self-complementary hairpin-rypc molecule that doubles back on itself to form a duplex. Thus, the dsRNAs can be fully or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines, however, in some embodiments, the gene expression or function is up regulated. When formed from two strands, or a single strand that takes the form of a self-complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) arc complementary RNA strands that base pair in Watson-Crick fashion.

[00109] Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid or may work via occupancy-based mechanisms. In general, nucleic acids (including oligonucleotides) may be described as "DNA-likc" (i.e., generally having one or more 2'-deoxy sugars and, generally, T rather than U bases) or "RNA-likc" (i.e., generally having one or more 2'- hydroxyl or 2'-modified sugars and, generally U rather than T bases). Nucleic acid helices can adopt more than one type of stmcture, most commonly the A- and B-forms. It is believed that, in general, oligonucleotides which have B-form-likc structure are "DNA-likc" and those which have A-formlikc structure are "RNA-Iike." In some (chimeric) embodiments, an antisense compound may contain both A- and B-form regions.

[001. 10] In an embodiment, the desired oligonucleotides or antisense compounds, comprise at least one of: antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRN A); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof

[001 1 1 1 dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNAa. dsRNAs targeting gene promoters induce potent transcriptional activation of associated genes. RNAa was demonstrated in human cells using synthetic dsRNAs, termed "small activating RNAs" (saRNAs). It is currently not known whether RNAa is conserved in other organisms.

[001 12] Small double-stranded RNA (dsRNA), such as small interfering RNA (siRNA) and microRNA (miRNA), have been found to be the trigger of an evolutionary conserved mechanism known as RNA interference (RNAi). RNAi invariably leads to gene silencing via remodeling chromatin to thereby suppress transcription, degrading complementary mRNA, or blocking protein translation. However, in instances described in detail in the examples section which follows, oligonucleotides arc shown to increase the expression and/or function of the BCL2-likc 1 1 (BCL2L 1 1) polynucleotides and encoded products thereof. dsRNAs may also act as small activating RNAs (saRNA). Without wishing to be bound by theory, by targeting sequences in gene promoters, saRNAs would induce target gene expression in a phenomenon referred to as dsRNA-induccd transcriptional activation (RNAa).

[001 13] In a further embodiment, the "preferred target segments" identified herein may be employed in a screen for additional compounds that modulate the expression of BCL2-like 1 1 (BCL2L1 1) polynucleotides. "Modulators" arc those compounds tliat decrease or increase the expression of a nucleic acid molecule encoding BCL2L 11 and which comprise at least a 5-nucleotidc portion that is complemeatary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding sense or natural antisense polynucleotides of BCL2L1 1 with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding BCL2L1 1 polynucleotides, e.g. SEQ ID NOS: 4 to 13. Once it is shown that the candidate modulator or modulators arc capable of modulating (e.g. cither decreasing or increasing) the expression of a nucleic acid molecule encoding BCL2LI 1 polynucleotides, the modulator may then be employed in further investigative studies of the function of BCL2L1 1 polynucleotides, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

1001 14_.1 Targeting the natural antisense sequence preferably modulates the function of the target gene. For example, the BCL2L1 1 gene (e.g. accession number NM_207002). in an embodiment, the target is an antisense polynucleotide of the BCL2L 1 1 gene. In an embodiment, an antisense oligonucleotide targets sense and/or natural antisense sequences of BCL2LI I polynucleotides (e.g. accession number NM_207002), variants, alleles, isoforms, homologs, mutants, derivatives, fragments and complementary sequences thereto. Preferably the oligonucleotide is an antisense molecule and the targets include coding and noncoding regions of antisense and/or sense BCL2L11 polynucleotides.

[001 .15] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.

[001 16] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications. For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target.

[001 17] In an embodiment, an antisense oligonucleotide targets BCL2-like 1 1 (BCL2L1 1 ) polynucleotides (e.g. accession number NM_207002), variants, alleles, isoforms, homologs, mutants, derivatives, fragments and complementary sequences thereto. Preferably the oligonucleotide is an antisense molecule.

(001 18 ) In accordance with embodiments of the invention, the target nucleic acid molecule is not limited to BCL2L 1 1 alone but extends to any of the isoforms, receptors, homologs and the like of BCL2L1 1 molecules.

[001 1 | In an embodiment, an oligonucleotide targets a natural antisense sequence of BCL2L1 1 polynucleotides, for example, polynucleotides set forth as SEQ ID NOS: 2 and 3, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Examples of antisense oligonucleotides arc set forth as SEQ ID NOS. 4 to 13.

[00120] In one embodiment, the oligonucleotides arc complementary to or bind to nucleic acid sequences of BCL2L1 1 antisense, including without limitation noncoding sense and/or antisense sequences associated with BCL2L1 1 polynucleotides and modulate expression and/or function of BCL2L1 1 molecules.

[00121 1 In an embodiment, the oligonucleotides are complementary to or bind to nucleic acid sequences of BCL2L 1 1 natural antisense. set forth as SEQ ID NOS: 2 and 3, and modulate expression and/or function of BCL2L1 1 molecules. (00122] In an embodiment, oligonucleotides comprise sequences of at least 5 consecutive nucleotides of SEQ ID NOS: 4 to 13 and modulate expression and/or function of BCL2L 1 1 molecules.

|0()123 | The polynucleotide targets comprise BCL2L 1 1 , including family members thereof, variants of BCL2L11 ; mutants of BCL2L 1 1 , including SNPs; noncoding sequences of BCL2L1 1 ; alleles of BCL2L 1 1 ; species variants, fragments and the like. Preferably the oligonucleotide is an antisense molecule.

[00124] In an embodiment, the oligonucleotide targeting BCL2LI 1 polynucleotides, comprise: antisense RNA, interference RNA (R Ai), short interfering RNA (siRNA); micro interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); or, small activating RNA (saRNA).

[00125] In an embodiment, targeting of BCL2-like I I (BCL2LI 1 ) polynucleotides, e.g. SEQ ID NOS: 2 to 13 modulate the expression or function of these targets. In one embodiment, expression or function is up-rcgulatcd as compared to a control. In an embodiment, expression or function is down-regulated as compared to a control.

[00126] In an embodiment, antisense compounds comprise sequences set forth as SEQ ID NOS: 4 to 13. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like.

[00127] In an embodiment, SEQ ID NOS: 4 to 13 comprise one or more LNA nucleotides. Table I shows exemplary antisense oligonucleotides useful in the methods of the invention.

Table 1:

[00128] The modulation of a desired target nucleic acid can be carried out in several ways known in the art. For example, antisense oligonucleotides, siRNA etc. Enzymatic nucleic acid molecules (e.g., ribozymcs) arc nucleic acid molecules capable of catalyzing one or more of a variety of reactions, including the ability to repeatedly cleave other separate nucleic acid molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be used, for example, to target virtually any RNA transcript. [00129] Because of their sequence-specificity, trans-cleaving enzymatic nucleic acid molecules show promise as therapeutic agents for human disease. Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the mRNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited.

[00130] In general, enzymatic nucleic acids with RNA cleaving activity act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

[00131 1 Several approaches such as in vitro selection (evolution) strategies (Orgel, (1979) Proc. R. Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing a variety of reactions, such as cleavage and ligation of phosphodiester linkages and amide linkages.

[00132] The development of ribozymes that are optimal for catalytic activity would contribute significantly to any strategy that employs RNA-cleaving ribozymes for the purpose of regulating gene expression. The hammerhead ribozyme, for example, functions with a catalytic rate (kcat) of about 1 min-1 in the presence of saturating (10 mM) concentrations of Mg2+ cofactor. An artificial "RNA ligase" ribozyme has been shown to catalyze the corresponding self-modification reaction with a rate of about 100 min-1 . In addition, it is known that certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turn-over rates that approach 100 min- 1. Finally, replacement of a specific residue within the catalytic core of the hammerhead with certain nucleotide analogues gives modified ribozymes that show as much as a 10-fold improvement in catalytic rate. These findings demonstrate that ribozymes can promote chemical transformations with catalytic rates that arc significantly greater than those displayed in vitro by most natural self-cleaving ribozymes. It is then possible that the structures of certain selfcleaving ribozymes may be optimized to give maximal catalytic activity, or that entirely new RNA motifs can be made that display significantly faster rates for RNA phosphodiester cleavage.

[00133] Intermolccular cleav age of an RNA substrate by an RNA catalyst that fits the "hammerhead" model was first shown in 1 87 (Uhlcnbcck, O. C. ( 1987) Nature, 328: 596-600). The RNA catalyst was recovered and reacted with multiple RNA molecules, demonstrating that it was truly catalytic.

10013 1 Catalytic RNAs designed based on the "hammerhead" motif have been used to cleave specific target sequences by making appropriate base changes in the catalytic RNA to maintain necessary base pairing with the target sequences. This has allowed use of the catalytic RNA to cleave specific target sequences and indicates that catalytic RNAs designed according to the "hammerhead" model may possibly cleave specific substrate RNAs in vivo.

|()()135| RNA interference (RNAi) has become a powerful tool for modulating gene expression in mammals and mammalian cells. This approach requires the delivery of small interfering RNA (siRNA) cither as RNA itself or as DNA, using an expression plasmid or virus and the coding sequence for small hairpin RNAs that arc processed to siRNAs. This system enables efficient transport of the prc-siRNAs to the cytoplasm where they arc active and permit the use of regulated and tissue specific promoters for gene expression.

[00136] In an embodiment, an oligonucleotide or antisense compound comprises an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA), or a mimetic, chimera, analog or homolog thereof. This term includes oligonucleotides composed of naturally occurring nucleotides, sugars and covalent intemucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often desired over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

[00137] According to the present invention, the oligonucleotides or "antisense compounds" include antisense oligonucleotides (e.g. RNA, DNA, mimetic, chimera, analog or homolog thereof), ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, saR A, aRNA, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid and modulate its function. As such, they may be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimctics of one or more of these. These compounds may be single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges, mismatches or loops. Antisense compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and/or branched. Antisense compounds can include constructs such as, for example, two strands hybridized to form a wholly or partially double-stranded compound or a single strand with sufficient sclf-complcmcntarity to allow for hybridization and formation of a fully or partially double-stranded compound. The two strands can be linked internally leaving free 3' or 5' termini or can be linked to form a continuous hairpin structure or loop. The hairpin staicture may contain an overhang on cither the 5' or 3' terminus producing an extension of single stranded character. The double stranded compounds optionally can include overhangs on the ends. Further modifications can include conjugate groups attached to one of the termini, selected nucleotide positions, sugar positions or to one of the intemucleoside linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group. When formed from only one strand, dsRNA can take the fonn of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the dsRNAs can be fully or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines. When formed from two strands, or a single strand that takes the form of a self-complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) arc complementary RNA strands that base pair in Watson- Crick fashion.

100138] Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid or may work via occupancy-based mechanisms. In general, nucleic acids (including oligonucleotides) may be described as "DNA-likc" (i.e., generally having one or more 2'-deoxy sugars and, generally, T rather than U bases) or " NA-like" (i.e., generally having one or more 2'- hydroxyl or 2'-modificd sugars and, generally U rather than T bases). Nucleic acid helices can adopt more than one type of structure, most commonly the A- and B-forms. It is believed that, in general, oligonucleotides which have B-form-like structure arc "DNA-like" and those which have A-formlike structure are "RNA-like." In some (chimeric) embodiments, an antisensc compound may contain both A- and B-form regions.

[00139] The antisensc compounds in accordance with this invention can comprise an antisensc portion from about 5 to about 80 nucleotides (i.e. from about 5 to about 80 linked nucleosides) in length. This refers to the length of the antisense strand or portion of the antisense compound. In other words, a single-stranded antisense compound of the invention comprises from 5 to about 80 nucleotides, and a double-stranded antisensc compound of the invention (such as a dsRNA, for example) comprises a sense and an antisense strand or portion of 5 to about 80 nucleotides in length. One of ordinary skill in the art will appreciate that this comprehends antisense portions of 5, 6, 7,8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18. 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44. 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 2, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length, or any range thercwithin.

[00140] In one embodiment, the antisensc compounds of the invention have antisense portions of 10 to 50 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies oligonucleotides having antisensc portions of 10, 1 1. 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 in length, or any range thercwithin. In some embodiments, the oligonucleotides arc 15 nucleotides in length.

[001411 In one embodiment, the antisense or oligonucleotide compounds of the invention have antisense portions of 12 or 13 to 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies antisensc compounds having antisensc portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any range thercwithin.

|0(⁾142 | In an embodiment, the oligomenc compounds of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenosine, variants may be produced which contain thymidine, guanosine or cytidine at this position. This may be done at any of the positions of the antisense or dsRNA compounds. These compounds arc then tested using the methods described herein to determine their ability to inhibit expression of a target nucleic acid.

[00143] In some embodiments, homology, sequence identity or complementarity, between the antisense compound and target is from about 40% to about 60%. In some embodiments, homology, sequence identity or complementarity, is from about 60% to about 70%. In some embodiments, homology, sequence identity or complementarity, is from about 70% to about 80%. In some embodiments, homology, sequence identity or complementarity, is from about 80% to about 90%. In some embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about I (X)%.

|(⁾0144| In an embodiment, the antisense oligonucleotides, such as for example, nucleic acid molecules set forth in SEQ ID NOS: 4 to 13 comprise one or more substitutions or modifications. In one embodiment, the nucleotides arc substituted with locked nucleic acids (LNA).

[00145] In an embodiment, the oligonucleotides target one or more regions of the nucleic acid molecules sense and/or antisense of coding and/or non-coding sequences associated with BCL2L 1 1 and the sequences set forth as SEQ ID NOS: 1 to 3. The oligonucleotides are also targeted to overlapping regions of SEQ ID NOS: I to 3.

[00146| Certain preferred oligonucleotides of this invention are chimeric oligonucleotides. "Chimeric oligonucleotides" or "chimeras," in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular cndonuclcase which cleaves the RNA strand of an RNA.DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense modulation of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides arc used, compared to phosphorothioate dcoxyoligonuclcotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. In one an embodiment, a chimeric oligonucleotide comprises at least one region modified to increase target binding affinity, and, usually, a region that acts as a substrate for RNAsc H. Affinity of an oligonucleotide for its target (in this case, a nucleic acid encoding ras) is routinely determined by measuring the Tm of an oligonuclcotidc/targct pair, which is the temperature at which the oligonucleotide and target dissociate; dissociation is detected spcctrophotomctrically. The higher the Tm, the greater is the affinity of the oligonucleotide for the target.

100147] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotides mimetics as described above. Such; compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures comprise, but arc not limited to, US patent nos. 5,013,830; 5, 149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,71 1 ; 5,491 , 133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference.

[00148] In an embodiment, the region of the oligonucleotide which is modified comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-Oalkyl, 2'-0-alkyl-0-alkyl or 2'-fluoro-modificd nucleotide. In other an embodiment, RNA modifications include 2'-fluoro, 2'-amino and 2' O-mcthyl modifications on the ribose of pyrimidiues, abasic residues or an inverted base at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than; 2'-deoxyoligonucleotides against a given target. The effect of such increased affinity is to greatly enhance RNAi oligonucleotide inhibition of gene expression. RNAse H is a cellular endonuclease that cleaves the RNA strand of RN A:DNA duplexes; activation of this enzyme therefore results in cleavage of the RNA target, and thus can greatly enhance the efficiency of RNAi inhibition. Cleavage of the RNA target can be routinely demonstrated by gel electrophoresis. In an embodiment, the chimeric oligonucleotide is also modified to enhance nuclease resistance. Cells contain a variety of exo- and endo-nuc leases which can degrade nucleic acids. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide. Nuclease resistance is routinely measured by inaibating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified oligonucleotides. A variety of oligonucleotide modifications have been demonstrated to enhance or confer nuclease resistance. Oligonucleotides which contain at least one phosphorothioatc modification arc presently more preferred. In some cases, oligonucleotide modifications which enhance target binding affinity arc also, independently, able to enhance nuclease resistance.

[00149] Specific examples of some preferred oligonucleotides envisioned for this invention include those comprising modified backbones, for example, phosphorothioatcs, phosphotriestcrs, methyl phosphonatcs, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with phosphorothioatc backbones and those with heteroatom backbones, particularly CH2 -NH--0-- CH2, CH,~N(CH3)-0--CH2 [known as a mcthy!cncimcthylimino) or MM] backbone], CH2 -0~N (CH3>-CH2, CH2 -N (CH3)-N (CH3)-CH2 and O-N (CH3)-CH2 --CH2 backbones, wherein the native phosphodicstcr backbone is represented as 0--P--0-CH,). The amide backbones disclosed by De Mcsmaekcr ct al. (1995) Acc. Chcm. Res. 28:366-374 arc also preferred. Also preferred arc oligonucleotides having morpholino backbone structures (Summerton and Weller, U.S. Pat. No. 5,034,506). In other an embodiment, such as the peptide nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide is replaced with a po!yamidc backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamidc backbone. Oligonucleotides may also comprise one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 0(CH2)n CH3, 0(CH2)n NH2 or 0(CH2)n CH3 where n is from I to about 10; CI to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF3 ; OCF3; O--, S--, or N-alkyl; 0-, S-, or N-alkcnyl; SOCH3; S02 CH3; ON02; N02; N3; NH2; hctcrocycloalkyl; hctcrocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy |2'-0-CH2 CH2 OCH3, also known as 2'-0-(2-mcthoxyethyl)]. Other preferred modifications include 2'-mcthoxy (2'-0--CH3), 2'- propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimctics such as cyclobutyls in place of the pentofuranosyl group.

[00150] Oligonucleotides may also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleotides include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleotides include nucleotides found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5- hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleotides, e.g., 2- aminoadenine, 2-(mcthylamino)adenine, 2-(imidazolylalkyl)adenine, 2- (aminoalklyamino)adenine or other hcterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5- bromouracil, 5-hydroxymethyluracil, 8-azaguaninc, 7- dcazaguanine, N6 (6-aminohexyl)adcnine and 2,6-diaminopurinc. A "universal" base known in the art, e.g., inosinc. may be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C. and arc presently preferred base substitutions.

|00151 ) Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undccyl residues, a polyaminc or a polyethylene glycol chain, or Adamanfane acetic acid. Oligonucleotides comprising lipophilic moieties, and methods for preparing such oligonucleotides arc known in the art, for example, U.S. Pat Nos. 5, 138,045, 5,218, 105 and 5,459,255.

[001521 It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of tlic aforementioned modifications may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide. The present invention also includes oligonucleotides which arc chimeric oligonucleotides as hereinbefore defined. [0015 J In another embodiment, the nucleic acid molecule of the present invention is conjugated with another moiety including but not limited to abasic nucleotides, polycthcr, polyaminc, polyamidcs, peptides, carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in the art will recognize that these molecules can be linked to one or more of any nucleotides comprising the nucleic acid molecule at several positions on the sugar, base or phosphate group.

[00154 J The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of one of ordinary skill in the art. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioatcs and alkylated derivatives. It is also well known to use similar techniques and commercially available modified amidites and controUed-pore glass (CPG) products such as biotin, fluorescein, acridinc or psora len-modified amidites and/or CPG (available from Glen Research, Sterling VA) to synthesize fluorcscciitly labeled, biotinylated or other modified oligonucleotides such as cholesterol- modified oligonucleotides.

[00155] In accordance with the invention, use of modifications such as the use of LNA monomers to enhance the potency, specificity and duration of action and broaden the routes of administration of oligonucleotides comprised of current chemistries such as OE, ANA, FANA, PS etc. This can be achieved by substituting some of the monomers in the current oligonucleotides by LNA monomers. The LNA modified oligonucleotide may have a size similar to the parent compound or may be larger or preferably smaller. It is preferred that such LNA-modified oligonucleotides contain less than about 70%, more preferably less than about 60%, most preferably less than about 50% LNA monomers and that their sizes are between about 5 and 25 nucleotides, more preferably between about 12 and 20 nucleotides.

[00156] Preferred modified oligonucleotide backbones comprise, but not limited to, phosphorothioatcs, chiral phosphorothioatcs, phosphorodithioates, phosphotricstcrs, aminoalkylphosphotricstcrs, methyl and other alkyl phosphonatcs comprising 3'alkylcnc phosphonatcs and chiral phosphonatcs, phosphinatcs, phosphoramidatcs comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2 -5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units arc linked 3'-5' to 5'- 3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms arc also included.

[00157] Representative United States patents that teach the preparation of the above phosphorus containing linkages comprise, but are not limited to, US patent nos. 3,687,808; 4,469,863; 4,476,301 ; 5,023,243; 5, 177, 196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321 ,131 ; 5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925; 5,519, 126; 5,536,821 ; 5,541 ,306; 5,550, 1 1 1 ; 5,563, 253; 5,571 ,799; 5.587.361 ; and 5,625.050, each of which is herein incorporated by reference. [001.58] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that arc formed by short chain alkyl or cycloalkyl intcmuclcosidc linkages, mixed hcteroatom and alkyl or cycloalkyl intcmuclcosidc linkages, or one or more short chain hcteroatomic or heterocyclic intcmuclcosidc linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxanc backbones; sulfide, sulfoxide and sulfonc backbones; formacctyl and thioformacctyl backbones; methylene fonnacctyl and thioformacctyl backbones; alkene containing backbones; sulfamate backbones; mcthylcncimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and odiers having mixed N, 0, S and CH2 component pans.

[00159] Representative United States patents that teach the preparation of the above oligonucleosides comprise, but are not limited to, US patent nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216, 141 ; 5,235,033; 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; 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 of which is herein incorporated by reference.

[00160] In other preferred oligonucleotide mimetics, both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nuclcobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds comprise, but are not limited to, US patent nos. 5.539,082; 5,714,331 : and 5,719,262, each of which is herein incorporated by reference . Further teaching of PNA compounds can be found in Nielsen, et al. ( 1991) Science 254, 1497-1500.

|()()161 | In an embodiment of the invention the oligonucleotides with phosphorothioate backbones and oligonucleosides with hcteroatom backbones, and in panicular- CH2-NH-0-CH2-,-CH2-N (CH3 0-CH2-known as a methylene (methylimino) or MMI backbone,- CH2-0-N (CH3)-CH2-,-CH2N(CH3)-N(CH3) CH2-and-0-N(CH3)- CH2-CH2- wherein the native phosphodicster backbone is represented as-0-P-0-CH2- of the above referenced US patent no. 5,489,677, and the amide backbones of the above referenced US patent no. 5,602,240. Also prcfcircd are oligonucleotides having morpholino backbone structures of the above-referenced US patent no. 5,034,506.

[00162] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C to CO alkyl or C2 to CO alkenyl and alkynyl. Particularly preferred are O (CH2)n OmCH3, 0(CH2)n,OCH3, 0(CH2)nNH2, 0(CH2)nCH3, 0(CH2)nONH2, and 0(CH2nON(CH2)nCH3)2 where n and m can be from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C to CO, (lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3, H2, hcterocyc!oalky], hetcrocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification comprises 2'-methoxyethoxy (2'-0-CH2CH20CH3, also known as 2'-0-(2- methoxyethyl) or 2'-M0E) i.e., an alkoxyalkoxy group. A further preferred modification comprises 2'-dimethylaminooxycthoxy, i.e. , a 0(CH2)20N(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'- dimethylaminoethoxyethoxy (also known in the art as 2'O-dimethylaminoethoxyethyl or 2'- DMAEOE), i.e., 2'-0-CH2-0-CH2-N (CH2)2.

|⁽⁾0163 | Other preferred modifications comprise 2'-methoxy (2'-0 CH3), 2 -aminopropoxy (2'-0 CH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2 -5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures comprise, but are not limited to, US patent nos. 4,981 ,957; 5, 1 18,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514, 785; 5,519, 134; 5,567,81 1 ; 5,576,427; 5,591 ,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5.646, 265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference.

[00164) Oligonucleotides may also comprise nuclcobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleotides comprise the purine bases adenine (A) and guanine (G), and the pyrimidinc bases thymine (T), cytosine (C) and uracil (U). Modified nucleotides comprise other synthetic and natural nucleotides such as 5-methylcytosinc (5-inc-C), 5-hydroxymcthyl cytosine, xanthine, hypoxanthinc, 2- aminoadeninc, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutcd adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromcthyl and other 5-substitutcd uracils and cytosincs, 7-methylquaninc and 7-mcthyladcninc, 8-azaguanine and 8-azaadcninc, 7-dcazaguaninc and 7-dcazaadcninc and 3-deazaguaninc and 3-dcazaadcninc.

|00165| Further, nucleotides comprise those disclosed in United States Patent No. 3,687,808, those disclosed in The Concise Encyclopedia of Polymer Science And Engineering', pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., 'Angcwandle Chcmie, International Edition', 19 1 , 30, page 613, and those disclosed by Sanghvi, Y.S., Chapter 15, 'Antisense Research and Applications', pages 289-302, Crookc, S . and Lcblcu. B. ca., CRC Press, 1993. Certain of these nucleotides arc particularly useful for increasing the binding affinity of the oligomcric compounds of the invention. These comprise 5-substitutcd pyrimidincs, 6- azapyrimidincs and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladeninc, 5- propynyluracil and 5-propynylcytosine. 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crookc, S.T. and Lcblcu, B., eds, 'Antiscnse Research and Applications', CRC Press, Boca Raton, 1 93, pp. 276-278) and arc presently preferred base substitutions, even more particularly when combined with 2'-Omethoxycthyl sugar modifications.

[00166] Representative United States patents that teach the preparation of the above noted modified nucleotides as we^'ll as other modified nucleotides comprise, but are not limited to, US patent nos. 3,687,808, as well as 4,845,205; 5, 130,302; 5,134,066; 5,175, 273; 5, 367,066; 5,432,272; 5,457, 187; 5,459,255; 5,484,908; 5,502,177; 5,525,71 1 ; 5,552,540; 5,587,469; 5,596,091 ; 5,614,617; 5,750,692, and 5,681 ,941 , each of which is herein incorporated by reference.

[00167] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.

[00168] Such moieties comprise but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l ,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or Adamantane acetic acid, a palmityl moiety, or an octadccylamine or hcxylamino-carbonyl-t oxycholesterol moiety.

[00169] Representative United States patents that teach the preparation of such oligonucleotides conjugates comprise. but are not limited to, US patent nos. 4,828,979; 4,948,882; 5,218, 105; 5,525,465; 5,541 ,313; 5,545,730; 5,552, 538; 5,578,717, 5,580,731 ; 5,580,731 ; 5,591 ,584; 5,109,124; 5, 1 18,802; 5,138,045; 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; 4,904,582: 4,958,013; 5,082, 830; 5,1 12,963; 5,214, 136; 5,082,830; 5, 1 12,963; 5,214, 136; 5, 245,022; 5,254,469;

5,258,506; 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; 5,599,923;

5,599, 928 and 5,688,941 , each of which is herein incorporated by reference.

[00170 J Drag dismveiy: The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between BCL2-like 1 1 (BCL2L1 1 ) polynucleotides and a disease state, phenotype, or condition. These methods include detecting or modulating BCL2L 1 1 polynucleotides comprising contacting a sample, tissue, cell, or organism with die compounds of the present invention, measuring the nucleic acid or protein level of BCL2L1 1 polynucleotides and or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotypc.

Assessing Up-regulaiion or Inh ibition of Gene Expression:

[00171 ] Transfer of an exogenous nucleic acid into a host cell or organism can be assessed by directly detecting the presence of the nucleic acid in the cell or organism. Such detection can be achieved by several methods well known in the art. For example, the presence of the exogenous nucleic acid can be detected by Southern blot or by a polymerase chain reaction (PGR) technique using primers that specifically amplify nucleotide sequences associated with the nucleic acid. Expression of the exogenous nucleic acids can also be measured using conventional methods including gene expression analysis. For instance, mRNA produced from an exogenous nucleic acid can be detected and quantified using a Northern blot and reverse transcription PCR (RT-PCR).

[001721 Expression of RN A from the exogenous nucleic acid can also be detected by measuring an enzymatic activity or a reporter protein activity. For example, antisense modulatory activity can be measured indirectly as a decrease or increase in target nucleic acid expression as an indication that the exogenous nucleic acid is producing the effector RNA. Based on sequence conservation, primers can be designed and used to amplify coding regions of the target genes. Initially, the most highly expressed coding region from each gene can be used to build a model control gene, although any coding or non coding region can be used. Each control gene is assembled by inserting each coding region between a reporter coding region and its poly(A) signal. These plasmids would produce an mRNA with a reporter gene in the upstream portion of the gene and a potential R Ai target in the 3' non-coding region. The effectiveness of individual antisense oligonucleotides would be assayed by modulation of the reporter gene. Reporter genes useful in the methods of the present invention include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), lucifcrasc (Luc), nopalinc synthase (NOS), octopinc synthase (OCS), and derivatives thereof. Multiple selectable markers arc available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline. Methods to determine modulation of a reporter gene arc well known in the art, and include, but arc not limited to, fluoromctric methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), antibiotic resistance determination. [001731 BCL2L 1 1 protein and mRNA expression can be assayed using methods known to those of skill in the art and described elsewhere herein. For example, immunoassays such as the EL1SA can be used to measure protein levels. BCL2L 1 1 ELISA assay kits arc available commercially, e.g., from R&D Systems (Minneapolis, MN).

100174 J In embodiments, BCL2L1 1 expression (e.g., mRNA or protein) in a sample (e.g.. cells or tissues in vivo or in vitro) treated using an antisense oligonucleotide of the invention is evaluated by comparison with BCL2L1 1 expression in a control sample. For example, expression of the protein or nucleic acid can be compared using methods known to those of skill in the art with that in a mock-treated or untreated sample. Alternatively, comparison with a sample treated with a control antisense oligonucleotide (e.g., one having an altered or different sequence) can be made depending on the information desired. In another embodiment, a difference in the expression of the BCL2L1 1 protein or nucleic acid in a treated vs. an untreated sample can be compared with the difference in expression of a different nucleic acid (including any standard deemed appropriate by the researcher, e.g., a housekeeping gene) in a treated sample vs. an untreated sample.

[001751 Observed differences can be expressed as desired, e.g., in the form of a ratio or fraction, for use in a comparison with control. In embodiments, the level of BCL2LI 1 mRNA or protein, in a sample treated with an antisense oligonucleotide of the present invention, is increased or decreased by about 1.25-fold to about 10-fold or more relative to an untreated sample or a sample treated with a control nucleic acid. In embodiments, the level of BCL2L1 1 mRNA or protein is increased or decreased by at least about 1.25-fold, at least about 1.3-fold, at least about

1.4- fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1 .7-fold, at least about 1.8-fold, at least about 2- fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-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 10- fold or more.

Kits. Research Reagents. Diagnostics, and Therapeutics

| ()0176 | The compounds of the present invention can be utilized for diagnostics, therapeutics, and prophylaxis, and as research reagents and components of kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.

[001 7] For use in kits and diagnostics and in various biological systems, the compounds of the present invention, cither alone or in combination with other compounds or therapeutics, are useful as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues. [0017 Ί As used herein the term "biological system" or "system" is defined as any organism, cell, cell culture or tissue that expresses, or is made competent to express products of the BCL2-like 1 1 (BCL2L 1 1) genes. These include, but arc not limited to, humans, transgenic animals, cells, cell cultures, tissues, xenografts, transplants and combinations thereof.

[00179] As one non limiting example, expression patterns within cells or tissues treated with one or more antisense compounds arc compared to control cells or tissues not treated with antisense compounds and the patterns produced arc analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds that affect expression patterns.

[0 1801 Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays, SAGE (serial analysis of gene expression), READS (restriction enzyme amplification of digested cDNAs), TOGA (total gene expression analysts), protein arrays and proteomics, expressed sequence tag (EST) sequencing, subtractive RNA fingerprinting (SuRF), subtractive cloning, differential display (DD), comparative genomic hybridization, FISH (fluorescent in situ hybridization) techniques and mass spectrometry methods.

100 I S 1 1 The compounds of the invention arc useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding BCL2-like 11 (BCL2L1 1). For example, oligonucleotides that hybridize with such efficiency and under such conditions as disclosed herein as to be effective BCL2L1 1 modulators are effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding BCL2L1 1 and in the amplification of said nucleic acid molecules for detection or for use in further studies of BCL2L1 I . Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding BCL2LI 1 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabcling of the oligonucleotide, or any other suitable detection means. Kits using such detection means for detecting the level of BCL2L1 1 in a sample may also be prepared.

[001 2] The specificity and sensitivity of antisense are also harnessed by those of skill in the an for therapeutic uses: Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense . compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

1001831 For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of BCL2L1 1 polynucleotides is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of BCL2L1 1 modulator. The BCL2L1 1 modulators of the present invention effectively modulate the activity of the BCL2L1 1 or modulate the expression of the BCL2L 1 1 protein. In one embodiment, the activity or expression of BCL2L1 1 in an animal is inhibited by about 10% as compared to a control. Preferably, the activity or expression of BCL2L1 1 in an animal is inhibited by about 30%. More preferably, the activity or expression of BCL2L1 1 in an animal is inhibited by 50% or more. Thus, the oligomcric compounds modulate expression of BCL2-likc 1 1 (BCL2L1 1 ) mRNA by at least 10%, by at least 50%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100% as compared to a control.

[001 1 In one embodiment, the activity or expression of BCL2-Iike 1 1 (BCL2LI I) and/or in an animal is increased by about 10% as compared to a control. Preferably, the activity or expression of BCL2L11 in an animal is increased by about 30% More preferably, the activity or expression of BCL2L1 1 in an animal is increased by 50% or more. Thus, the oligomcric compounds modulate expression of BCL2L I 1 mRNA by at least 10%, by at least 50%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100% as compared to a control.

[00185] For example, the reduction of the expression of BCL2-like 1 1 (BCL2L 1 1 ) may be measured in serum, blood, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding BCL2L11 peptides and/or the BCL2L 1 1 protein itself.

[00186] The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.

Conjugates

[00187] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethcrs, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typicalconjugatc groups include cholcstcrols, lipids, phospholipids, biotin, phenazinc, folate, phenanthridine, anthraquinone, acridinc, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence- specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups arc disclosed in International Patent Application No.

PCT/US92/0 1 6, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference.

Conjugate moieties include, but arc not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thiocthcr, e.g., hcxyl-5- rrityl thiol, a thiocholcstcrol, an aliphatic chain, e.g., dodecandiol or undccyl residues, a phospholipid, e.g., di-hcxadccyl-rac -glycerol or tricthylammonium l ,2-di-0-hcxadccyl-rac-glyccro-3-Hphosphonatc, a po!yaminc or a polyethylene glycol chain, or Adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino- carbonyl-oxycholcsterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, kctoprofen, (SM+^pranoprofcn, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazidc, chlorothiazide, a diazepinc, indomcthicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

|00188 J Representative United States patents that teach die preparation of such oligonucleotides conjugates include. but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541 ,313; 5,545,730; 5,552,538;

5,578,717, 5,580.731 ; 5,580,731 ; 5,591 ,584; 5, 109,124; 5,1 18,802; 5,138,045; 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;

4,904,582; 4,958,013; 5,082,830; 5,1 12,963; 5,214,136; 5,082,830; 5, 1 12,963; 5,214,136; 5,245,022; 5,254,469;

5,258,506; 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; 5,599,923;

5,599,928 and 5,688,941 .

Formulations

|00189] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as forcxample, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.

Representative United States patents that teach the preparation of such uptake, distribution and or absorption-assisting fonnulations include, but are not limited to, U.S. Pat. Nos. 5, 108,921; 5,354,844; 5,416,016; 5,459, 127; 5,521 ,291 ;

5,543, 165; 5,547,932; 5,583,020; 5,591 ,721 ; 4,426,330; 4,534,899; 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; 5,534.259;

5,543, 152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[001 0] Although, the antisense oligonucleotides do not need to be administered in the context of a vector in order to modulate a target expression and/or function, embodiments of the invention relates to expression vector constructs for the expression of antisense oligonucleotides, comprising promoters, hybrid promoter gene sequences and possess a strong constitutive promoter activity, or a promoter activity which can be induced in the desired case. [00191 ] In an embodiment, invention practice involves administering at least one of the foregoing antisensc oligonucleotides with a suitable nucleic acid delivery system. In one embodiment, that system includes a non-viral vector opcrably linked to the polynucleotide. Examples of such nonviral vectors include the oligonucleotide alone (e.g. any one or more of SEQ ID NOS: 4 to 13) or in combination with a suitable protein, polysaccharide or lipid formulation.

1001921 Additionally suitable nucleic acid delivery systems include viral vector, typically sequence from at least one of an adenovirus, adenovirus-associated virus (AAV), helper-dependent adenovirus, retrovirus, or hemagglutinatin virus of Japan-liposome (HVJ) complex. Preferably, the viral vector comprises a strong eukaryotic promoter opcrably linked to the polynucleotide e.g., a cytomegalovirus (CM V) promoter.

[00193] Additionally preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia viruses and HIV-bascd viruses. One preferred HIV-bascd viral vector comprises at least two vectors wherein the gag and pol genes are from an HIV genome and the env gene is from another virus. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex 1 virus (HSV) vector, Adenovirus Vectors and Adeno-associated Virus Vectors.

[001 4] The antisensc compounds of the invention encompass any pharmaceutical ly acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.

[001 5 ] The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesircd toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses arc further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.

[001 6] The present invention also includes pharmaceutical compositions and formulations that include the antisensc compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

[001 7] For treating tissues in the central nervous system, administration can be made by, e.g., injection or infusion into the cerebrospinal fluid. Administration of antisensc RNA into cerebrospinal fluid is described, e.g., in U.S. Pat. App. Pub. No. 2007/01 17772, "Methods for slowing familial ALS disease progression," incorporated herein by reference in its entirety.

| 0()198 | When it is intended that the antisensc oligonucleotide of the present invention be administered to cells in the central nervous system, administration can be with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier. Injection can be made, e.g., in the cntorhinal cortex or hippocampus. Delivery of neurotrophic factors by administration of an adenovirus vector to motor neurons in muscle tissue is described in, e.g., U.S. Pat. No. 6,632,427, "Adenoviral-vector-mediated gene transfer into medullary motor neurons," incorporated herein by reference. Delivery of vectors directly to the brain, e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra, is known in the art and described, e.g., in U.S. Pat. No. 6,756,523, ''Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain,^" incorporated herein by reference. Administration can be rapid as by injection or made over a period of time as by slow infusion or administration of slow release formulations.

[00199] The subject antisense oligonucleotides can also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. For example, the antisense oligonucleotide can be coupled to any substance, known in the art to promote penetration or transport across the blood-brain barrier, such as an antibody to the transferrin receptor, and administered by intravenous injection. The antisense compound can be linked with a viral vector, for example, that makes the antisense compound more effective and/or increases the transport of the antisense compound across the blood-brain barrier. Osmotic blood brain barrier disruption can also be accomplished by, e.g., infusion of sugars including, but not limited to, meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffmosc. L(+) rhamnose, D(+) melibiosc. D(-) ribosc, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, U-) fucose, D{-) lyxosc, L(+) lyxose, and L(-) lyxosc, or amino acids including, but not limited to, glutaminc, lysine, argininc, asparaginc, aspaitic acid, cysteine, glutamic acid, glycine, histidinc, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine. Methods and materials for enhancing blood brain barrier penetration arc described, e.g., in U. S. Patent No. 4,866,042, "Method for the delivery of genetic material across the blood brain barrier," 6,294,520, "Material for passage through the blood-brain barrier," and 6,936,589, "Parenteral delivery systems," all incorporated herein by reference in their entirety.

[00200] The subject antisense compounds may be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. For example, cationic lipids may be included in the formulation to facilitate oligonucleotide uptake. One such composition shown to facilitate uptake is L1POFECTIN (available from GIBCO-BRL, Bethesda, MD). [00201 1 Oligonucleotides with at least one 2'-0-mcthoxycthyl modification arc believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[00202] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipicnt(s). In general, the formulations arc prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[002031 The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulosc, sorbitol and/or dextran. The suspension may also contain stabilizers.

[00204] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.

[00205] Emulsions are typically heterogeneous systems of one liquid dispersed in another in the fonn of droplets usually exceeding 0.1 μιη in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active ding that may be present as a solution in cither the aqueous phase, oily phase or itself as a separate phase. Microcmulsions arc included as an embodiment of the present invention. Emulsions and their uses arc well known in the ait and arc further described in U.S. Pat. No. 6,287,860. ·

[00206] Formulations of the present invention include liposomal formulations. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes arc positively charged liposomes that arc believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that arc pH-scnsitivc or negatively-charged arc believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[00207] Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomeslacking such specialized lipids. Examples of stcrically stabilized liposomes arc those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses arc further described in U.S. Pat. No. 6,287,860.

[002081 The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.

[00209] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non- chclating nonsurfactants. Penetration enhancers and their uses arc further described in U S. Pat. No. 6,287,860, which is incorporated herein by reference.

[00210] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0021 11 Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoyl-phosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphospharidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramcthylaminopropyl DOTAP and dioleoyl-phosphatidyl cdianolaminc DOTMA).

[00212] For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexcd to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses arc further described in U.S. Pat. No. 6,287,860.

100213 f Compositions and formulations for oral administration include powders or granules, microparticulatcs, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablcts. Thickeners, flavoring agents, diluents, cmulsificrs, dispersing aids or binders may be desirable. Preferred oral formulations arc those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses arc further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxycthylcnc-20-ccty] ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complcxcd to form micro or nanoparticlcs. Oligonucleotide complcxing agents and their uses arc further described in U.S. Pat. No. 6,287.860, which is incorporated herein by reference.

100214] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[00215] Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chcmotherapcutic agents that function by a non-antisense mechanism. Examples of such chcmotherapcutic agents include but are not limited to cancer chemothcrapcutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamidc, cytosine arabinosidc, bischloroethyl- nitrosurca, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamcthylmclamine, pentamethylmelamine, mitoxantrone, amsacrinc, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- azacytidine, hydroxyurea, dcoxycoformycin, 4-hydroxypcroxycyclo-phosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP- 1 ), trimetrexate, irinotecan, topotecan, getneitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chcmotherapcutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chcmotherapcutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Antiinflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabinc, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisensc drugs arc also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0021 ] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. For example, the first target may be a particular antisense sequence of BCL2-likc 1 1 (BCL2L 1 1 ), and the second target may be a region from another nucleotide sequence. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same BCL2-Iike 1 1 (BCL2L 1 1) nucleic acid target. Numerous examples of antiscnsc compounds are illustrated herein and others may be selected from among suitable compounds known in the art. Two or more combined compounds may be used together or sequentially.

Dosing: 100217] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within die skill of those in the art. Dosing is dependent on seventy and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in vitro and in vivo animal models. In general, dosage is from 0.01 ng to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years.

100218 J In embodiments, a patient is treated with a dosage of drug that is at least about 1 , at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20. at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 mg/kg body weight. Certain injected dosages of antisense oligonucleotides are described, e.g., in U.S. Pat. No. 7,563,884, "Antisense modulation of PTPI B expression," incorporated herein by reference in its entirety.

[00219 J While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above described embodiments.

[00220] All documents mentioned herein arc incorporated herein by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is "prior art" to their invention. Embodiments of inventive compositions and methods arc illustrated in the following examples.

EXAMPLES

[00221 1 The following non-limiting Examples serve to illustrate selected embodiments of the invention. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and arc within the scope of embodiments of the present invention. Example I : Design of antisense oligonucleotides specific for a nucleic acid molecule antisense to a BCL2-like II (HC 2L 11 ) and/or a sense strand of BCL2U I polynucleotide

[00222] As indicated above the term "oligonucleotide specific for" or "oligonucleotide targets" refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (ii) capable of forming a stable duplex with a portion of an mRNA transcript of the targeted gene.

100223] Selection of appropriate oligonucleotides is facilitated by using computer programs (e.g. IDT AntiSensc Design, IDT OligoAnalyzer) that automatically identify in each given sequence subsequences of 19-25 nucleotides that will form hybrids with a target polynucleotide sequence with a desired melting temperature (usually 50-60°C) and will not form sclf-dimers or other complex secondary structures.

[00224] Selection of appropriate oligonucleotides is further facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs arc used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of genes and intergenic regions of a given genome allows the selection of nucleic acid sequences that display an appropriate degree of specificity to the gene of interest. These procedures allow the selection of oligonucleotides that exhibit a high degree of complementarity to target nucleic acid sequences and a lower degree of complementarity to other nucleic acid sequences in a given genome. One skilled in the art will realize that there is considerable latitude in selecting appropriate regions of genes for use in the present invention.

100225 j An antisense compound is "specifically hybridizablc" when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a modulation of function and/or activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays arc performed in the case of in vitro assays.

[00226] The hybridization properties of the oligonucleotides described herein can be determined by one or more in vitro assays as known in the art. For example, the propenies of the oligonucleotides described herein can be obtained by determination of binding strength between the target natural antisense and a potential drug molecules using melting curve assay.

[00227] The binding strength between the target natural antiscnsc and a potential drug molecule (Molecule) can be estimated using any of the established methods of measuring the strength of intcrmolccular interactions, for example, a melting curve assay. 100228] Melting curve assay determines the temperature at which a rapid transition from double-stranded to singlc- stranded conformation occurs for the natural antiscnsc/ olcculc complex. This temperature is widely accepted as a reliable measure of the interaction strength between the two molecules.

[00229] A melting curve assay can be performed using a cDNA copy of the actual natural antisensc RNA molecule or a synthetic DNA or RNA nucleotide corresponding to the binding site of the Molecule. Multiple kits containing all necessary reagents to perform this assay are available (e.g. Applied Biosystcms Inc. McltDoctor kit). These kits include a suitable buffer solution containing one of the double strand DNA (dsDNA) binding dyes (such as ABI HRM dyes, SYBR Green, SYTO, etc.). The properties of the dsDNA dyes are such that they emit almost no fluorescence in free form, but arc highly fluorescent when bound to dsDNA.

[00230] To perform the assay the cDNA or a corresponding oligonucleotide are mixed with Molecule in concentrations defined by the particular manufacturer's protocols. The mixture is heated to 95 °C to dissociate all preformed dsDNA complexes, then slowly cooled to room temperature or other lower temperature defined by the kit manufacturer to allow the DNA molecules to anneal. The newly formed complexes are then slowly heated to 95 °C with simultaneous continuous collection of data on the amount of fluorescence that is produced by the reaction. The fluorescence intensity is inversely proportional to the amounts of dsDNA present in the reaction. The data can be collected using a real time PCR instrument compatible with the kit (e.g.ABl's StepOne Plus Real Time PCR System or lightTyper instrument, Roche Diagnostics, Lewes, UK).

[00231 1 Melting peaks are constructed by plotting the negative derivative of fluorescence with respect to temperature (-d(Fluorescence)/dT) on the y-axis) against temperature (x-axis) using appropriate software (for example lightTyper ( Roche) or SDS Dissociation Curve, ABI). The data is analyzed to identify the temperature of the rapid transition from dsDNA complex to single strand molecules. This temperature is called Tm and is directly proportional to the strength of interaction between the two molecules. Typically, Tm will exceed 40 °C.

Example 2: Modulation ofBCLlLI 1 polynucleotides

Treatment o/^'MCF-7 cells with antisen.se oligonucleotides

[00232] All antisense oligonucleotides used in Example 2 were designed as described in Example 1. The manufacturer (IDT Inc. of Coralville, 1A) was instructed to manufacture the designed phosphothioate bond oligonucleotides and provided the designed phosphothioate analogs shown in Table 1. The asterisk designation between nucleotides indicates the presence of phosphothioate bond. The oligonucleotides required for the experiment in Example 2 can be synthesized using any appropriate state of the art method, for example the method used by IDT: on solid support, such as a 5 micron controlled pore glass bead (CPG), using phosphoramiditc monomers (normal nucleotides with all active groups protected with protection groups, e.g. trityl group on sugar, benzoyl on A and C and N-2-isobutyryl on G). Protection groups prevent the unwanted reactions during oligonucleotide synthesis. Protection groups arc removed at the end of the synthesis process. The initial nucleotide is linked to the solid support through the .Vcarbon and the synthesis proceeds in the 3' to 5'dircction. The addition of a new base to a growing oligonucleotide chain takes place in four steps: 1 ) the protection group is removed from the 5' oxygen of the immobilized nucleotide using trichloroacetic acid; 2) the immobilized and the ncxt-in-scquencc nucleotides arc coupled together using tctrazole: the reaction proceeds through a tetrazolyl phosphoramidite intermediate; 3) the unreactcd free nucleotides and reaction byproducts arc washed away and the unreactcd immobilized oligonucleotides are capped to prevent their participation in the next round of synthesis; capping is achieved by acetylating the free 5' hydroxy! using acetic anhydride and N-methyl imidazole; 4) to stabilize the bond between the nucleotides the phosphorus is oxidized using iodine and water, if a phosphodiestcr bond is to be produced, or Beaucage reagent (3H- l ,2-benzodithiol-3-one-l , l- dioxidc), if a phosphothioatc bond is desired. By alternating the two oxidizing agents, a chimeric backbone can be constructed. The four step cycle described above is repeated for every nucleotide in the sequence. When the complete sequence is synthesized, the oligonucleotide is cleaved from the solid support and dcprotcctcd using ammonium hydroxide at high temperature. Protection groups are washed away by desalting and the remaining oligonucleotides are lyophilized. To perform the experiment designed in Example 2, MCF-7 cells from ATCC (cat# HTB-22) were grown in growth media (MEM/EBSS (Hyclone cat #SH30024, or Mediatech cat # MT- 10-010-CV) + 10% FBS (Mediatech cat# MT35- 01 1 -CV)+ penicillin/streptomycin (Mediatech cat# MT30-002-CI)) at 37°C and 5% CCk One day before the experiment the cells were replated at the density of 1.5 * l Vml into 6 well plates and incubated at 37°C and 5% CO_: overnight. On the day of the experiment the media in the 6 well plates was changed to fresh growth media.

Oligonucleotides shipped by the manufacturer in lyophilized form were diluted to the concentration of 20 uM in deionized RNAsc/DNAse-free water. Two μΐ of this solution was incubated with 400 μΙ of OptiMEM media (Gibco cat#31 85-070) and 4 μΐ of Lipofcctaminc 2000 (Invitrogen cat# 1 1668019) at room temperature for 20 min, then applied dropwisc to one well of the 6 well plate with MCF-7 cells. Similar mixture including 2 μΐ of water instead of the oligonucleotide solution was used for the mock-transfcctcd controls. After 3-18 h of incubation at 37°C and 5% CO₂ the media was changed to fresh growth media. 48 h after addition of antisense oligonucleotides the media was removed and RNA was extracted from the cells using SV Total RNA Isolation System from Promcga (cat # Z3105) or RNcasy Total RNA Isolation kit from Qiagen (cat# 74181 ) following the manufacturers' instructions. 600 ng of extracted RNA was added to the reverse transcription reaction performed using Verso cDNA kit from Thermo Scientific (cat#AB 1453B) or High Capacity cDNA Reverse Transcription Kit (cat# 4368813) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real time PCR using ABI Taqman Gene Expression Mix (cat#4369510) and primers probes designed by ABI (Applied Biosystems Taqman Gene Expression Assay: Hs00l 97982_ml by Applied Biosystems Inc., Foster City CA). The following PCR cycle was used: 50°C for 2 min, 95°C for 10 min, 40 cycles of (95°C for 15 seconds, 60°C for 1 min) using StcpOnc Plus Real Time PCR Machine (Applied Biosystems). Fold change in gene expression after treatment with antiscnsc oligonucleotides was calculated based on the difference in 18S-normalized dCt values between treated and mock-transfcctcd samples.

Results: Real time PGR results show that the levels of the BIM mRNA in MCF7 cells arc significantly increased 48 h after treatment with one of the oligos designed to BIM antisense Hs.652337.

Treatment ofHepG2 cells with antisense oligonucleotides

[00233] All antisense oligonucleotides used in Example 2 were designed as described in Example 1. The manufacturer (IDT Inc. of Coralville, 1A) was instructed to manufacture the designed phosphothioate bond oligonucleotides and provided the designed phosphothioate analogs shown in Table 1. The asterisk designation between nucleotides indicates the presence of phosphothioate bond. The oligonucleotides required for the experiment in Example 2 can be synthesized using any appropriate state of the art method, for example the method used by IDT: on solid support, such as a 5 micron controlled pore glass bead (CPG), using phosphoramiditc monomers (normal nucleotides with all active groups protected with protection groups, e.g. trit l group on sugar, benzoyl on A and C and N-2-isobutyryl on G). Protection groups prevent the unwanted reactions during oligonucleotide synthesis. Protection groups are removed at the end of the synthesis process. The initial nucleotide is linked to the solid support through the 3'carbon and the synthesis proceeds in the 3' to i'direction. The addition of a new base to a growing oligonucleotide chain takes place in four steps: 1 ) the protection group is removed from the 5' oxygen of the immobilized nucleotide using trichloroacetic acid; 2) the immobilized and the next-in-sequence nucleotides arc coupled together using tetrazole: the reaction proceeds through a tetrazolyl phosphoramidite intermediate; 3) the unrcacted free nucleotides and reaction byproducts arc washed away and the unrcacted immobilized oligonucleotides are capped to prevent their participation in the next round of synthesis; capping is achieved by acetylating the free 5' hydroxyl using acetic anhydride and N-mcthyl imidazole; 4) to stabilize the bond between the nucleotides the phosphorus is oxidized using iodine and water, if a phosphodiestcr bond is to be produced, or Bcaucagc reagent (3H-l ,2-benzodithiol-3-onc-l , l - dioxidc), if a phosphothioate bond is desired. By alternating the two oxidizing agents, a chimeric backbone can be constructed. The four step cycle described above is repeated for every nucleotide in the sequence. When the complete sequence is synthesized, the oligonucleotide is cleaved from the solid support and deprotectcd using ammonium hydroxide at high temperature. Protection groups are washed away by desalting and the remaining oligonucleotides are lyophilized.

[00234] To perform the experiment designed in Example 2, HcpG2 cells from ATCC (cat# HB-8065) were grown in growth media (M EM/EBSS (Hyclonc cat #SH30024, or Mcdiatcch cat # MT-10-010-CV) +1.0% FBS (Mcdiatcch cat# MT35- 01 l -CV)+ penicillin/streptomycin (Mcdiatcch cat# MT30-O02-CI)) at 37°C and 5% C02. One day before the experiment the cells were rcplatcd at the density of 0.5 lO4/mI into 6 well plates and incubated at 37°C and 5% C02 overnight. On the day of the experiment the media in the 6 well plates was changed to fresh growth media. [00235] Oligonucleotides shipped by the manufacturer in lyophilized form were diluted to the concentration of 20 uM in deionized RNAsc/DNAsc-frcc water. Two μΐ of this solution was incubated with 400 ul of OptiMEM media (Gibco cat#31985-070) and 4 μΐ of Lipofcctaminc 2000 (Invitrogcn cat# 1 1668019) at room temperature for 20 min, then applied dropwisc to one well of the 6 well plate with HcpG2 cells. Similar mixture including 2 μΐ of water instead of the oligonucleotide solution was used for the mock-transfcctcd controls. After 3-18 h of incubation at 37°C and 5% C02 the media was changed to fresh growth media. 48 h after addition of antisense oligonucleotides the media was removed and RNA was, extracted from the cells using SV Total RNA Isolation System from Promega (cat # Z3105) or Ncasy Total RNA Isolation kit from Qiagen (cat# 74181 ) following the manufacturers' instructions. 600 ng of extracted RNA was added to the reverse transcription reaction performed using Verso cDNA kit from Thermo Scientific (cat#AB I453B) or High Capacity cDNA Reverse Transcription Kit (cat# 4368813) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real time PGR using ABI Taqman Gene Expression Mix (cat#4369510) and primers probes designed by AB1 (Applied Biosystcms Taqman Gene Expression Assay: Hs00197982_ml (BCL2L1 1 ) by Applied Biosystems Inc., Foster City CA). The following PCR cycle was used: 50°C for 2 min, 95°C for 10 min, 40 cycles of (95°C for 15 seconds, 60°C for 1 min) using StepOne Plus Real Time PCR Machine (Applied Biosystems). 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.

[00236| Resufts: Real time PCR results show that the levels of the BIM mRNA in HepG2 cells are significantly increased 48 h after treatment with one of the oligos designed to BIM antisense Hs.652337 (Fig 2).

Treatment of primary keratinocytes with antisense oligonucleotides

[00237] Primary keratinocytes (from Lifeline Technologies) were grown in a growth media (Keratinocyte Growth Media, Lifeline cat# LM0004 + Lifeline growth factorsl030) at 37°C with 5% C02. One day before the experiment the cells were rcplatcd at approximately 5xlO^A4/ml (or about 1/3 dilution from 90% conflucncy) into 24-wcll collagen- coated plates (Bcckton Dickinson BioCoat plates cat #35 6408) and incubated at 37oC and 5% C02 overnight. On the day of the experiment the media in the 24-wcll plates was changed to 1 ml fresh growth media. Oligonucleotides shipped by the manufacturer in lyophilized form were diluted to the concentration of 20 uM in deionized RNAsc/DNAsc-frcc water. Two μΙ of this solution was incubated with 400 μ) of OptiMEM media (Gibco cat#31 85- 070) and 4 μΙ of TransIT®-LTl Transfection Reagent (Minis cat # MIR 2300) at room temperature for 20 min, then applied dropwisc to one well of the 6 well plate with HcpG2 cells. Similar mixture including 2 μ] of water instead of the oligonucleotide solution was used for the mock-transfcctcd controls.Aftcr dosing die plates were incubated overnight at 37oC, 5% C02. 24 h after addition of antisense oligonucleotides the media was replaced with fresh growth media and the dosing was repeated as described above. 24 h after second dosing RNA was extracted from the cells using SV Total RNA Isolation System from Promega (cat # Z3 I05) following the manufacturers' instructions. 600 ng of total RNA was added to the reverse transcription reaction performed using High Capacity cDNA kit from Applied Biosystcms (cat#4368813) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real time PCR using ABI Taqman Gene Expression Mix (cat#4369510) and primers/probes designed by ABI (HsOOI97982_m l). The following PCR cycle was used: 50°C for 2 min, 95°C for 10 min, 40 cycles of (95°C for 15 seconds, 60°C for 1 min) using StcpOnc Plus Real Time PCR Machine (Applied Biosystcms). 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.

[00238 J Result: Real time PCR results show that the levels of the BIM mRNA in keratinocyte cells are significantly increased 48 h after treatment with one of the oligos designed to BIM antisense Hs.652337 (Fig 3).

Example 3: In Vitro Apoptosis Assays

[00239] The level of apoptosis was detected in cells using the HT Titer TACS Assay kit following the manufacturer's protocol (Trcvigcn cat# 4822-96-K). Briefly, 48 hours post-oligonucleotide transfection cells were trypsinized, counted and replated in 96-well plates at a density of 100,000 cells/well. The 96-well plates were then incubated overnight at 37oC, 5% C02. The following morning the media was removed and the cells were fixed in 3.7% Buffered Formaldehyde Solution (Sigma-252549) rinsed with PBS and 100% methanol (Sigma-M 1775- 1 GA) and stored in 80% ethano! (Sigma-4935 1 1 ) at +4oC,

[00240] To label DNA breaks, ethanol was removed, and the cells were washed with PBS and incubated with Proteinase K solution at room temperature for 1 minutes, rinsed in water and PBS, then incubated with TACS Nuclease Solution for 10 minutes at 37°C. After incubation the cells were rinsed with PBS, and hydrogen peroxide solution (Sigma-MKBD I 394) was added for 5 minutes at room temperature. The cells were then incubated 5 more minutes at room temperature with l TdT Labeling. Buffer was discarded and cells were subsequently incubated with Labeling Reaction mix at 37°C for one hour and then lx TdT Stop Buffer was added for 5 minutes. After the labeling reaction was stopped, cells were washed with PBS, incubated with Strcp-HRP solution at room temperature for 10 minutes, washed with PBS Tween and incubated with TACS-Sapphirc in the dark for 30 minutes. The reaction was stopped by addition of 0.2N HCI (Fluka-343102) and the absorbance was read in a plate reader at 450nm.

1. A549 cells (ATCC # CCL- 185™) were transfected with 20nM CUR- 1 174, a p63 antisense oligonucleotide, and CUR- 1522, a BIM antisense oligonucleotide using Lipofectamine™ 2000 Transfection Reagent (Invitrogcn).

Results: Apoptosis is significantly increased in A549 cells transfected with CUR-1 174 and CUR- 1522 (Fig 4).

2. Hep G2 cells (ATCC Cat# HB-8065) were transfected with CUR-1519, CUR- 1 21 and CUR-I522 at 20nM using Lipofectamine™ 2000 Transfection Reagent (Invitrogcn). A subset of cells was also tranfectcd with 20nM CUR- 1505

(inactive oligonucleotide) and with Lipofectamine™ 2000 and water (mock transfection).

Results: Apoptosis is significantly increased in HepG2 cells transfected with CUR-1522. Other oligonucleotides did not have a significant effect on apoptosis (Fig. 5 and 6). [00241] Although the invention has been illustrated and described with respect to one or more implementations, equivalent alterations and modifications will occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In addition, while a particular feature of the invention may have been disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application.

| 00242 | The Abstract of the disclosure will allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the following claims.

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Claims

CLAIMS What is claimed is:

1. A method of modulating a function of and/or the expression of a BCL2-like 1 1 (BCL2L 1 1 ) polynucleotide in a biological system comprising: contacting said system with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a natural antisense of a BCL2-likc 11 (BCL2L1 I) polynucleotide; thereby modulating a function of and/or the expression of the BCL2-likc 1 1 (BCL2L11) polynucleotide.

2. A method of modulating a function of and/or the expression of a BCL2-like 1 1 (BCL2L1 1 ) polynucleotide in a biological system according to claim 1 comprising: contacting said biological system with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within the natural antisense transcript nucleotides 1 to 3026 of SEQ ID NO: 2 and 1 to 1512 of SEQ ID NO: 3; thereby modulating a function of and/or the expression of the BCL2-like 1 1 (BCL2L1 1 ) polynucleotide.

3. A method of modulating a function of and/or the expression of a BCL2-like 1 1 (BCL2L1 1) polynucleotide in patient cells or tissues in vivo or in vitro comprising: contacting said cells or tissues with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to an antisense oligonucleotide to the BCL2-likc 1 1 (BCL2L1 1) polynucleotide; thereby modulating a function of and/or the expression of the BCL2-likc 1 1 (BCL2L 1 1 ) polynucleotide in patient cells or tissues in vivo or in vitro.

4. A method of modulating a function of and/or the expression of a BCL2-like 1 1 (BCL2L1 1) polynucleotide in patient cells or tissues according to claim 3 comprising: contacting said biological system with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within the natural antisense transcript nucleotides 1 to 3026 of SEQ ID NO: 2 and I to 1512 of SEQ ID NO: 3; thereby modulating a function of and/or the expression of the BCL2-likc 1 1 (BCL2L 1 1 ) polynucleotide.

5. A method of modulating a function of and/or the expression of a BCL2-likc 1 1 (BCL2L1 1 ) polynucleotide in a biological system comprising: contacting said system with at least one antisense oligonucleotide that targets a region of a natural antisense oligonucleotide of the BCL2-like 1 1 (BCL2L1 1 ) polynucleotide; thereby modulating a function of and/or the expression of the BCL2-like 1 1 (BCL2L 1 1) polynucleotide.

6. The method of claim 5, wherein a function of and/or the expression of the BCL2-like 1 1 (BCL2L I 1) is increased in v vo or in virrrj with respect to a control.

7. The method of claim 5, wherein the at least one antisense oligonucleotide targets a natural antisense sequence of a BCL2-likc 1 1 (BCL2L1 1 ) polynucleotide.

8. The metliod of claim 5, wherein the at least one antisense oligonucleotide targets a nucleic acid sequence comprising coding and/or non-coding nucleic acid sequences of a BCL2-likc 1 1 (BCL2L1 1) polynucleotide.

9. The metliod of claim 5, wherein the at least one antisense oligonucleotide targets overlapping and/or non- overlapping sequences of a BCL2-likc 1 1 (BCL2LI I) polynucleotide.

10. The method of claim 5, wherein the at least one antisense oligonucleotide comprises one or more modifications selected from: at least one modified sugar moiety, at least one modified intcmuclcoside linkage, at least one modified nucleotide, and combinations thereof.

1 1 . The metliod of claim 10, wherein the one or more modifications comprise at least one modified sugar moiety selected from: a 2'-0-mcthoxycthyl modified sugar moiety, a 2'-mcthoxy modified sugar moiety, a 2'-0-alkyl modified sugar moiety, a bicyclic sugar moiety, and combinations thereof.

12. The method of claim 10, wherein the one or more modifications comprise at least one modified intemucleoside linkage selected from: a phosphorothioate, 2'- Omethoxyethyl ( OE), 2'-fluoro. alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.

1 3. The method of claim 10, wherein the one or more modifications comprise at least one modified nucleotide selected from: a peptide nucleic acid (PNA), a locked nucleic acid (LNA), an arabino-nucleic acid (FANA), an analogue, a derivative, and combinations thereof.

14. The method of claim I , wherein the at least one oligonucleotide comprises at least one oligonucleotide sequences set forth as SEQ ID NOS: 4 to 13.

1 5. A method of modulating a function of and/or the expression of a BCL2-likc 1 1 (BCL2L1 1 ) gene in mammalian cells or tissues in vivo or in vitro comprising: contacting said cells or tissues with at least one short interfering RNA (siRNA) oligonucleotide 5 to 30 nucleotides in length, said at least one siRNA oligonucleotide being specific for an antisense polynucleotide of a BCL2-like 1 1 (BCL2LI 1) polynucleotide, wherein said at least one siRNA oligonucleotide has at least 50% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense nucleic acid molecule of the BCL2-likc 1 1 (BCL2LI 1) polynucleotide; and, modulating a function of and or the expression of BCL2- like 1 1 (BCL2L1 1 ) in mammalian cells or tissues in vivo or in vitro.

1 . The method of claim 15, wherein said oligonucleotide has at least 80% sequence identity to a sequence of at least about five consecutive nucleic acids that is complementary to the antisense and/or sense nucleic acid molecule of the BCL2-like 1 1 (BCL2L1 1) polynucleotide.

17. A method of modulating a function of and/or the expression of BCL2-likc 1 1 (BCL2L11) in mammalian cells or tissues in vivo or in vitro comprising: contacting said cells or tissues with at least one antisense oligonucleotide of about 5 to 30 nucleotides in length specific for noncoding and/or coding sequences of a sense and/or natural antisense strand of a BCL2-like 11 (BCL2L1 1) polynucleotide wherein said at least one antisense oligonucleotide lias at least 50% sequence identity to at least one nucleic acid sequence set forth as SEQ ID NOS: 1 to 3; and, modulating the function and/or expression of the BCL2-likc 1 1 (BC,L2L1 1 ) in mammalian cells or tissues in vivo or in vitro.

18. A synthetic, modified oligonucleotide comprising at least one modification wherein the at least one modification is selected from: at least one modified sugar moiety; at least one modified internuclcotide linkage; at least one modified nucleotide, and combinations thereof; wherein said oligonucleotide is an antisense compound which hybridizes to and modulates the function and/or expression of a BCL2-likc 1 1 (BCL2L1 1 ) gene in vivo or in vitro as compared to a normal control and wherein said oligonucleotide has at least 50% sequence identity to a sequence of at least about five consecutive nucleic acids that is complementary to the antisense and/or sense nucleic acid molecule of the BCL2-like 1 1 (BCL2L 1 1 ) polynucleotide and alleles, homologs, isoforms, variants, derivatives, mutants, fragments, or combinations thereof.

1 . The oligonucleotide according to claim 18 wherein said oligonucleotide is 5 to 30 nucleotides in length and has at least 50% sequence identity to the reverse complement of 5-30 consecutive nucleotides within a natural antisense transcript of the BCL2L1 1 gene.

20. The oligonucleotide of claim 1 , wherein the at least one modification comprises an intemucleotide linkage selected from the group consisting of: phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioatc, phosphoramidatc, carbamate, carbonate, phosphate tricster, acctamidatc, carboxymcthyl ester, and combinations thereof.

21. The oligonucleotide of claim 19, wherein said oligonucleotide comprises at least one phosphorothioate intemucleotide linkage.

22. Tlie oligonucleotide of claim 1 , wherein said oligonucleotide comprises a backbone of phosphorothioate intemucleotide linkages.

23. The oligonucleotide of claim 19, wherein the oligonucleotide comprises at least one modified nucleotide, said modified nucleotide selected from: a peptide nucleic acid, a locked nucleic acid (LNA), analogue, derivative, and a combination thereof.

24. The oligonucleotide of claim 19, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from: phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidatc, carbamate, carbonate, phosphate tricstcr, acctamidatc, carboxymcthyl ester, and a combination thereof.

25. The oligonucleotide of claim 19, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from: peptide nucleic acids, locked nucleic acids (LN A), analogues, derivatives, and a combination thereof.

26. The oligonucleotide of claim 19, wherein the oligonucleotide comprises at least one modified sugar moiety selected from: a 2'-0-methoxycthyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-0-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.

27. The oligonucleotide of claim 1 , wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified sugar moieties selected from: a 2'-0-methoxyethyl modified sugar moiety, a 2'-mcthoxy modified sugar moiety, a 2'-0-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.

28. The oligonucleotide of claim 19, wherein the oligonucleotide is of at least about 5 to 30 nucleotides in length and hybridizes to an antisense and/or sense strand of a BCL2-like 1 1 (BCL2L1 1 ) polynucleotide wherein said oligonucleotide has at least about 60% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense coding and/or noncoding nucleic acid sequences of the BCL2-!ikc 1 1 (BCL2LI I ) polynucleotide.

29. The oligonucleotide of claim 1 , wherein the oligonucleotide has at least about 80% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense coding and/or noncoding nucleic acid sequence of the BCL2-like 1 1 (BCL2L1 1 ) polynucleotide.

30. The oligonucleotide of claim 1 , wherein said oligonucleotide hybridizes to and modulates expression and/or function of at least one BCL2-likc 1 1 (BCL2L1 1 ) polynucleotide in vivo or in vitro, as compared to a normal control.

31. The oligonucleotide of claim 19, wherein the oligonucleotide comprises the sequences set forth as SEQ ID NOS: 4 to 13.

32. A pharmaceutical composition comprising one or more oligonucleotides specific for one or more BCL2-likc 1 1 (BCL2LI I ) polynucleotides according to claim 18 and a pharmaceutically acceptable excipicnt.

33. The composition of claim 32, wherein the oligonucleotides have at least about 40% sequence identity as compared to any one of the nucleotide sequences set forth as SEQ I D NOS: 4 to 13.

34. The composition of claim 32, wherein the oligonucleotides comprise nucleotide sequences set forth as SEQ ID NOS: 4 to 13.

35. The composition of claim 34, wherein the oligonucleotides set forth as SEQ ID NOS: 4 to 13 comprise one or more modifications or substitutions.

36. The composition of claim 35. wherein the one or more modifications arc selected from: phosphorothioatc, mcthylphosphonatc, peptide nucleic acid, locked nucleic acid (LNA) molecules, and combinations thereof.

37. A method of preventing or treating a disease associated with at least one BCL2-like 11 (BCL2L1 1) polynucleotide and/or at least one encoded product thereof, comprising: administering to a patient a therapeutically effective dose of at least one antiscnsc oligonucleotide that binds to a natural antiscnsc sequence of said at least one BCL2-likc 1 1 (BCL2L1 1) polynucleotide and modulates expression of said at least one BCL2-like 11 (BCL2L1 1) polynucleotide; thereby preventing or treating the disease associated with the at least one BCL2-likc 1 1 (BCL2L 1 1 ) polynucleotide and/or at least one encoded product thereof.

38. The method of claim 37, wherein a disease associated with the at least one BCL2-like I I (BCL2L1 I ) polynucleotide is selected from: a disease or disorder associated with abnormal function and/or expression of BCL2L1 1 , cancer, aberrant apoptosis, a proliferative disease or disorder, a disease or disorder associated with mitochondrial apoptosis pathway, leukemia, an autoimmune disease or disorder, a disease or disorder associated with impaired immunity, infection, hyperproliferative disorders, hyperproliferation of reproductive tissue (such as uterine, testicular and ovarian carcinomas, endometriosis, and squamous, glandular epithelial carcinomas of the cervix etc.), a neurological disease or disorder, a developmental disorder associated with the developing embryo, a hematological disease or disorder, impaired tissue homeostatis, rheumatoid arthritis, sepsis, retinal ganglion cell death, a hepatic disease and a renal disease.

39. A method of inducing apoptosis in a biological system comprising administration of an oligonucleotide of 5 to 30 nucleotides in length and which is at least 50% identical to 5 to 30 consecutive nucleotides within a reverse complement of a natural antiscnsc transcript to a BCL2L1 1 polynucleotide to said system.

40. The method according to claim 39 wherein the natural antisense transcript has SEQ ID NOS: 2 or 3.

41. The method according to claim 39 wherein the biological system is a patient cell or tissue.

42. A method of inducing apoptosis in a biological system comprising administration of an oligonucleotide of about 5 to 30 nucleotides in length to said system wherein said oligonucleotide uprcgulates a target gene by targeting to a natural antiscnsc transcript of the gene and wherein said oligonucleotide is at least 50% identical to a reverse complement of the natural antisense transcript of the upregulated gene.

43. A method of identifying and selecting at least one oligonucleotide selective for a natural antisense transcript of a BC.L2L1 1 gene as a selected target polynucleotide for in vivo administration comprising: identifying at least one oligonucleotide comprising at least five consecutive nucleotides which arc at least partially complementary to a polynucleotide that is antiscnsc to the selected target polynucleotide; measuring the thermal melting point of a hybrid of an antisense oligonucleotide and the target polynucleotide or the polynucleotide that is antisense to the selected target polynucleotide under stringent hybridization conditions; and selecting at least one oligonucleotide for in vivo administration based on the information obtained.