CA2762987A1 - Treatment of transcription factor e3 (tfe3) and insulin receptor substrate 2 (irs2) related diseases by inhibition of natural antisense transcript to tfe3 - Google Patents

Abstract

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotides, in particular, by targeting natural antisense polynucleotides of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2(IRS2). The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of TFE3 and/or IRS2.

Description

TREATMENT OF TRANSCRIPTION FACTOR E3 (TFE3) and INSULIN RECEPTOR SUBSTRATE 2 (IRS2) RELATED DISEASES BY INHIBITION OF NATURAL ANTISENSE TRANSCRIPT TO TFE3 FIELD OF THE INVENTION
100011 The present application claims the priority of U.S. provisional patent application No. 61/180,515 filed May 22, 2009 and U.S. provisional patent application No. 61/291,419 filed Dec 31, 2(x)9 which is incorporated herein by reference in its entirety.
100021 Embodiments of the invention comprise oligonucleotides modulating expression and/or function of TFE3 and/or IRS2 and associated molecules.
BACKGROUND
100031 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. Antisense 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 oligodcoxynueicotides (ODNs), or they can be expressed from endogenous genes as RNA molecules. The FDA
recently approved an antiscnsc drug, VITRAVENE''"1 (for treatment of cytomcgalovirus retinitis), reflecting that antiscnsc has therapeutic utility.
SUMMARY
100041 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.
100051 In one embodiment, the invention provides methods for inhibiting the action of a natural antisense transcript by using antisense oligonucleotide(s) targeted to any region of the natural antiscnsc transcript resulting in up-regulation of the corresponding sense gene. It is also contemplated herein that inhibition of the natural antisense transcript can be achieved by siRNA, ribozymcs and small molecules, which are considered to be within the scope of the present invention.
100061 One embodiment provides a method of modulating function and/or expression of a TFE3 and/or IRS2 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 a polynucleotide comprising 5 to 30 consecutive nucleotides within nucleotides I to 497 of SEQ ID NO: 5 thereby modulating function and/or expression of the Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (1RS2) polynucleotide in patient cells or tissues in vivo or in vitro.

100071 In another preferred embodiment, an oligonucleotide targets a natural antiscnse sequence of TFE3 polynucleotidcs, for example, nuclcotides set forth in SEQ ID NO: 3, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Examples of antiscnsc oligonucleotides arc set forth as SEQ ID NOS: 4 to 9.
100081 Another embodiment provides a method of modulating function and/or expression of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotides in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antiscnsc oligonuclcotidc 5 to 30 nuclcotides in length wherein said oligonuclcotidc has at least 50% sequence identity to a reverse complement of the an antiscnsc of the Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide; thereby modulating function and/or expression of the TFE3 and/or IRS2 polynucleotide in patient cells or tissues in-vivo or in vitro.
100091 In a preferred embodiment, a composition comprises one or more antiscnsc oligonucleotides which bind to sense and/or antiscnsc TFE3 and/or I RS2 polynucleotides.
100101 In another preferred embodiment, the oligonucleotides comprise one or more modified or substituted nucleotides.
100111 In another preferred embodiment, the oligonucleotides comprise one or more modified bonds.
100121 In yet another embodiment, the modified nuclcotides comprise. modified bases comprising phosphorothioate, methylphosphonate, peptide nucleic acids, 2'-O-methyl, fluoro- or carbon, methylene or other locked nucleic acid (LNA) molecules. Preferably, the modified nuclcotides arc locked nucleic acid molecules, including a-L-LNA.
100131 In another preferred embodiment, the oligonucleotides are administered to a patient subcutaneously, intramuscularly, intravenously or intrapcritoncally.
100141 In another preferred embodiment, the oligonucleotides are administered in a pharmaceutical composition.
A treatment regimen comprises administering the antiscnsc 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.
(0015] In another preferred embodiment, the oligonucleotides arc encapsulated in a liposomc or attached to a carrier molecule (e.g. cholesterol, TAT peptide).
100161 Other aspects are described inf =a.
BRIEF DESCRIPTION OF THE DRAWINGS
100171 Figure I
Figure I shows a graph of real time PCR results showing the fold change +
standard deviation in IRS2 mRNA
after treatment of HepG2 cells and 518A2 cells with siRNA oligonucleotides introduced using Lipofectarninc 2000, as compared to control. Bars denoted as 518A2 CUR-0603, 5I8A2 CUR-0605 correspond to 5I8A2 cells' samples treated with SEQ ID NOS 4 and 5, respectively. And Bars denoted as HcpG2 CUR-0603, HcpG2 CUR-0605, correspond to HcpG2 cells' samples treated with SEQ ID NOS 4 and 5, respectively.
Figure 2 shows a graph of real time PCR results showing the fold change +
standard deviation in TFE3 mRNA
after treatment of HcpG2 cells with siRNA oligonucleotides introduced using Lipofectamine 2000, as compared to control. Bars denoted as TFE3 CUR-0603 and CUR-0605 correspond to SEQ ID NOS 4 and 5 respectively.
Figure 3 shows a graph of real time PCR results showing the fold change +
standard deviation in TFE3 mRNA
after treatment of HcpG2 cells with siRNA oligonuclcotidcs introduced using Lipofectaminc 2000, as compared to control. Real time PCR results show that the levels of TFE3 mRNA in HcpG2 cells is significantly increased 48h after treatment with one of the siRNAs designed to TFE3 antisense Hs.708291.Bars denoted as CUR-0603, CUR-0605, CUR-0607, CUR-0599, CUR-0601, and CUR-0609 correspond to SEQ ID NOS 4 to 9.
100181. Sequence Listing Description SEQ ID NO: 1: Homo sapiens insulin Receptor Substrate 2 (IRS2), mRNA
(accession number: NM_003749), SEQ ID NO: 2: Homo sapiens transcription factor binding to IGHM enhancer 3 (TFE3), mRNA (accession number: NM_00652 1) SEQ ID NO: 3: TFE3 Natural antisense sequence (Hs.708291) SEQ ID NOs: 4 to.9: Hs.70829I antisense oligonuclcotidcs, `r' indicates RNA.
SEQ ID NO: 10 to 15: Hs.708291 sense oligonuclcotidcs. These are the reverse complements of the antisense oligonuclcotidcs SEQ ID NO: 4 to 9 respectively. `r' indicates RNA.
DETAILED DESCRIPTION
100191 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 the relevant art, however, will readily recognize that 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.
100201 All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is 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 preferred embodiments, the genes or nucleic acid sequences are human.
Definitions 100211 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."
100221 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 I 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.
100231 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.
100241 By "antisensc oligonucleotides" or "antisense compound" is meant an RNA
or DNA molecule that binds to another RNA or DNA (target.RNA, DNA). For example, if it is an RNA
oligonucleotide it binds to another RNA target by means of RNA-RNA interactions and alters the activity of the target RNA (Eguchi el a!., (1991) Ann. Rev. Biochem. 60, 631-652). An antisense oligonucleotide can upregulate or downregulate 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, 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.
100251 In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. The term "oligonucleotide", also includes linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like. Oligonucleotides are 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, Hoogstecn or reverse Hoogstcen types of base pairing, or the like.
100261 The oligonucleotide may be "chimeric", that is, composed of different regions. In the context of this invention "chimeric" compounds arc oligonucleotidcs, 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 oligonucleotidcs compound. These oligonucleotidcs typically comprise at least one region wherein the oligonuclcotide 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 oligonuclcotidc may therefore have different properties. The chimeric oligonucleotidcs of the present invention can be formed as mixed structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide analogs as described above.
100271 The oligonuclcotidc 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 100 carbon atoms. The spacers may carry different functional i ties, 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, alaninc containing peptides that induce alpha-helices.
100281 As used herein "TFE3" and "Transcription factor E3" arc inclusive of all family members, mutants, alleles, fragments, species, coding and noncoding sequences, sense and antisense polynuclcotidc strands, etc.
100291 As used herein, the words Transcription factor E3, TFE3, bHLHe33, RCCP2; and TFEA are considered the same in the literature and arc used interchangeably in the present application.
100301 As used herein, the words Insulin Receptor Substrate 2, IRS2, are considered the same in the literature and are used interchangeably in the present application.
100311 As used herein, the term "oligonuclcotidc specific for" or "oligonuclcotidc which targets" refers to an oligonuclcotidc 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 stability of hybridization complexes and duplexes are described in the Examples below.
100321 As used herein, the term "target nucleic acid" encompasses DNA, RNA
(comprising prcmRNA and mRNA) transcribed from such DNA, and also eDNA 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 oligonuclcotides.
100331 RNA interference "RNAi" is mediated by double stranded RNA (dsRNA) molecules that have sequence-specific homology to their "target" nucleic acid sequences (Caplen, N. J., el al. (2001) Proc. Nail. Acad. Sc!. USA
98:9742-9747). 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 dsRNA by an RNasc enzyme known as Dicer (Bernstein, E.. ei al. (2001) Manure 409:363-366). ssRNA
duplex products are recruited into a multi-protein ssRNA 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 (Bernstein, E., ei al. (2001) Nature 409:363-366; Boutla, A., et a!. (2001) Curr. Biol.
11:1776-1780). Small interfering RNAs that can be used in accordance with the present invention can be synthesized and used according to procedures that are 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 I 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.
100341 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 GcnBank 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 are 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 oligonuclcotides that exhibit a high degree of coin plementari ty to target nucleic acid sequences in a subject to be controlled and a lower degree of complcmentarity 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.

100351 By "enzymatic RNA" is meant an RNA molecule with enzymatic activity (Cech, (1989).J. American.
Med. Assoc. 260, 3030-3035). Enzymatic nucleic acids (ribozymcs) 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.
100361 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 (Sullcnger ei al. (1990) Cell, 63, 601- 608). 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.
100371 As used herein, the term "monomers" typically indicates monomers linked by phosphodicstcr bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., from about 3-4, to about several hundreds of monomeric units. Analogs of phosphodicstcr linkages include: phosphorothioate, phosphorodithioate. mcthylphosphomates, phosphoroselenoate, phosphoramidate, and the like, as more fully described below.
100381 The term "nucleotide" covers naturally occurring nucleotides as well as nonnaturally occurring nucleotides. It should be clear to the person skilled in the an that various nucleotides which previously have been considered "non-naturally occurring" have subsequently been found in nature.
Thus, "nucleotides" includes not only the known purinc and pyrimidine heterocycles-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, xanthinc, diaminopurine, 8-oxo- N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin, N6,N6-ethano-2,6- diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pscudoisocytosinc, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, inosine and the "non-naturally occurring" nucleotides described in Benner et al., 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 are those containing adenine, guanine, thymine, cytosine, and uracil, which are considered as the naturally occurring nuclcotidcs 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. (Freeman, San Francisco, 1992) as well as their analogs.
100391 "Analogs" in reference to nuclcotidcs includes synthetic nuclcotidcs having modified base moieties and/or modified sugar moieties (see e.g., described generally by Scheit, Nucleotide Analogs, John Wiley, New York, 1980: Frcier & Altmann, (1997) Nrrcl. Acid. Res., 25(22), 4429- 4443, Toulme, J.J., (2001) Nature Biotechnology 19:17-18: Manoharan M., (1999) Hiochemica el Biophyycica Acia 1499:117-139, Frcicr S. M., (1997) Nucleic Acid Research, 25:4429-4443, Uhlman, E., (2(x10) Drug Discovery & Dcnvelopmeni, 3:
203-213, Herdewin P., (2000) Aniicense & Nucleic Acid Drug Derv., 10:297-310); 2'-O, 3'-C-linked 13.2.01 bicycloarabinonucleosides (see e.g.
N.K Christiensen., ei al, (1998).1. Am. ('hem. Soc., 120: 5458-5463; Prakash TP. Bhat B. (2007) C'urr Top Med ('hem. 7(7):641-9; Cho EJ, el al. (2009) Annual Review ofAnalylical C'hemisiry, 2, 241-264). Such analogs include synthetic nucleotides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.
100401 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-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleotides) of the strands of oligomeric compounds. For example, adenine and thyminc are complementary nucleotides which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
10041 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.
100421 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 are 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 compound:target sequence complex, and the presence of denaturants such as formamide, dimethylformamide, dirtiethyl 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 0.1 X sodium chloride-sodium citrate buffer (SSC)/0.1 % (w/v) SDS
at 60 C. for 30 minutes.
100431 "Complementary," as used herein, refers to the capacity for precise pairing between two nucleotides on one or two oligomeric strands. For example, if a nuclcobasc at a certain position of an antisense 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 oligomeric compound and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizablc" and "complementary"
are terms which are 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.
100441 It is understood in the art that the sequence of an oligomeric compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonuclcotide 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 complementariry. In this example, the remaining noncomplementary 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) noncomplcmentary, nucleotides which are flanked by two regions of complete complcmentarity 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 PowerBLAST
programs known in the art (Altschul et al., (1990) J. Mot. Biol., 215, 403-410; Zhang and Madden, (1997) Genorne Res., 7, (A9-656). 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. App!. Math., (1981) 2, 482-489).
100451 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 the 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.
100461 As used herein, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.
100471 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 mRNA 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 are variants of wild type gene 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 gene 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.
(0048( The resulting polypeptides generally will have significant amino acid identity relative to each other. A
polymorphic variant is a variation in the polynucleotidc 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.
(0049( 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 arc phosphorothioate and other sulfur containing species which are known in the art. Derivative nucleic acids may also contain labels, including radionucleotides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles, and the like.
(0050( A "derivative" polypcptide 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, either directly or indirectly, including, but not limited to, a radioisotope, fluorescent, and enzyme label.
(0051( 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.
(0052( "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.
(0053( "Treating" or "treatment" covers the treatment of a disease-state in a mammal, and includes: (a) preventing the disease-state 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 disease-state, e.g., arresting it development; and/or (c) relieving the disease-state, 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.). papillary carcinoma, , alveolar soft-part sarcoma papillary 100541 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, lymphangioendotheliosarcoma, 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 renal cell carcinoma, alveolar soft-part sarcoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma. Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pincaloma, hemangioblastoma, acoustic neuroma, oligodcndroglioma, 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 macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endonictrial cancer, adrenal cortical cancer, and prostate cancer.
100551 "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). Examples of neurological disorders include but are not limited to, headache, stupor and coma, dementia, seizure, sleep disorders, trauma, infections, neoplasms, neuroopthalmology, movement disorders, demyclinating diseases, spinal cord disorders, and disorders of peripheral nerves, muscle and neuromuscular junctions. Addiction and mental illness, include, but are not limited to, bipolar disorder and schizophrenia, are also included in the definition of neurological disorder. The following is a list of several neurological disorders, symptoms, signs and syndromes that can be treated using compositions and methods according to the present invention: acquired epileptiform aphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy; age-related macular degeneration; agenesis it of the corpus callosum: agnosia; Aicardi syndrome; Alexander disease; Alpers' disease; alternating hemiplegia;
Vascular dementia; amyotrophic lateral sclerosis; anencephaly; Angelman syndrome; angiomatosis, anoxia;
aphasia: apraxia: arachnoid cysts; arachnoiditis; Anronl-Chiari malformation;
artcriovenous malformation;
Aspcrgcr syndrome; ataxia tclegicctasia; attention deficit hyperactivity disorder; autism; autonomic dysfunction;
back pain: Batten disease: Bchcct's disease, Bell's palsy; benign essential blcpharospasm; benign focal;
amyotrophy; benign intracranial hypertension; Binswangcr's disease;
blepharospasm; Bloch Sulzberger syndrome:
brachial plexus injury; brain abscess; brain injury; brain tumors (including glioblastoma multiformc); spinal humor;
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-Marie-Tooth disease; chemotherapy-induced neuropathy and neuropathic pain;
Chian 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 artcritis; craniosynostosis; Creutzfeldt-Jakob disease, cumulative trauma disorders; Cushing 's syndrome; cytomegalic inclusion body disease;
cytomegalovirus infection, dancing eyes-dancing feet syndrome; DandyWalker syndrome; Dawson disease; De Morsier's syndrome, Dejerine-Klumke palsy; dementia; dermatomyositis; diabetic neuropathy; diffuse sclerosis;
dysautonomia; dysgraphia; dyslexia;
dystonias; early infantile epileptic encephalopathy, empty sclla syndrome, encephalitis, encephaloceles;
encephalotngeminal 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": Gaucher's disease; Gerstmann's syndrome; giant cell arteritis; giant cell inclusion disease;
globoid cell lcukodystrophy; Guillain-Barn syndrome; HTLV-1-associated myclopathy; Hallcrvorden-Spatz disease; head injury: headache; hemifacial spasm; hereditary spastic paraplegia; hcredopathia atactic a polyncuritifonnis; herpes zoster oticus; herpes zostcr; Hirayama syndrome;
HiVassociated dementia and ncuropathy (also neurological manifestations of AIDS); holoproscncephaly;
Huntington's disease and other polyglutamine repeat diseases; hydranenccphaly; hydrocephalus;
hypcrcortisolism, hypoxia; immune-mediated encephalomyelitis; inclusion body myositis; incontinentia pigmenti, infantile phytanic acid storage disease, infantile refsum disease; infantile spasms; inflammatory myopathy;
intracranial cyst; intracranial hypertension, Joubcrt syndrome; Kcams-Sayre syndrome; Kennedy disease Kinsboume syndrome;
Klippcl Feil syndrome;
Krabbc disease; Kugclbcrg-Wclandcr disease; kuru; Lafora disease; Lambert-Eaton myasthenic syndrome, Landau-Klcffner syndrome; lateral medullary (Wallenberg) syndrome; learning disabilities; Leigh's disease;
Lennox-Gustaut syndrome; Lesch-Nyhan syndrome: leukodystrophy, Lewy body dementia; Lissenccphaly:
locked-in syndrome; Lou Gehri3 s disease (i.e., motor neuron disease or amyotrophic lateral sclerosis); lumbar disc disease; Lyme disease--neurological sequelae; Machado-Joseph disease;
macrencephaly; megalenccphaly;
Melkersson-Rosenthal syndrome; Menicres disease; meningitis; Menkcs disease;
metachrornatic leukodystrophy;

microcephaly, migraine: Miller Fisher syndrome; mini-strokes: mitochondrial myopathics; Mobius syndrome:
monomelic amyotrophy; motor neuron disease; Moyamoya disease:
mucopolysaccharidoses; milti-infarct dementia; multifocal motor neuropathy; multiple sclerosis and other demyclinating disorders; multiple system atrophy with postural hypotension; p muscular dystrophy; myasthenia gravis:
myclinoclastic diffuse sclerosis:
myoclonic enccphalopathy of infants; myoclonus; myopathy; myotonia congenital;
narcolepsy, neurofibrornatosis-, ncuroleptic malignant syndrome; neurological manifestations of AIDS;
neurological sequelae oflupus;
ncuromyotonia: neuronal ccroid lipofuscinosis; neuronal migration disorders;
Nieman-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: Neurodegcncrative 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; parancoplastic diseases;
paroxysmal attacks; Parry Romberg syndrome; Pelizaeus-Merzbachcr 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 tumors;
polymyositis; porencephaly; post-polio syndrome; postherpetic neuralgia; postinfectious encephalomyelitis; postural hypotension; Pradcr- Willi syndrome:
primary lateral sclerosis; prion diseases: progressive hemifacial atrophy;
progressive multi focalleukoencephalopathy: progressive sclerosing poliodystrophy;
progressive supranuclcar palsy;
pseudotumor cerebri; Ramsay-Hunt syndrome (types I and 11); Rasmussen's encephalitis: reflex sympathetic dystrophy syndrome: Rcfsum disease; repetitive motion disorders; repetitive stress injuries; restless legs syndrome;
retrovirus-associated nryclopathy; Rett syndrome: Reye's syndrome; Saint Vitus dance; Sandhoff disease;
Schildcr's disease: schizencephaly; septo-optic dysplasia; shaken baby syndrome; shingles; Shy-Drager syndrome:
Sjogrcn's syndrome; sleep apnea: Soto's syndrome: spasticity; spina bifida;
spinal cord injury; spinal cord tumors;
spinal muscular atrophy; Stiff-Person syndrome: stroke: Sturgc-Weber syndrome;
subacute sclerosing pancncephalitis; subcortical arteriosclerotic eneephalopathy; Sydcnham chorea;
syncope; syringomyelia: tardivc dyskinesia: Tay-Sachs disease: temporal arteritis: tethered spinal cord syndrome: Thomsen disease: thoracic outlet syndrome; Tic Douloureux; Todd's paralysis: Tourette syndrome; transient ischemic attack; transmissible spongiform cnccphalopathics; 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 Zellwegcr syndrome.
100561 An "Inflammation" refers to systemic inflammatory conditions and conditions associated locally with migration and attraction of monocytes, leukocytes and/or neutrophils. Examples of inflammation include, but are not limited to. Inflammation resulting from infection with pathogenic organisms (including gram-positive bacteria, gram-negative bacteria, viruses, fungi, and parasites such as protozoa and helminths), transplant rejection (including rejection of solid organs such as kidney, liver, heart, lung or cornea, as well as rejection of bone marrow transplants including graft-versus-host disease (GVHD)), or from localized chronic or acute autoimmune or allergic reactions. Autoimmune diseases include acute glomeruloncphritis;
rheumatoid or reactive arthritis; chronic glomcruloncphritis, inflammatory bowel diseases such as Crohn's disease, ulcerative colitis and necrotizing cntcrocolitis, granulocyte transfusion associated syndromes; inflammatory dermatoses such as contact dermatitis, atopic dermatitis, psoriasis; systemic lupus crythcmatosus (SLE), autoimmune thyroiditis, multiple sclerosis, and some forms of diabetes, or any other autoimmune state where attack by the subject's own immune system results in pathologic tissue destruction. Allergic reactions include allergic asthma, chronic bronchitis, acute and delayed hypersensitivity. Systemic inflammatory disease states include inflammation associated with trauma, burns, repcrfusion following ischemic events (e.g. thrombotic events in heart, brain, intestines or peripheral vasculature, including myocardial infarction and stroke), sepsis, ARDS or multiple organ dysfunction syndrome. Inflammatory cell recruitment also occurs in atherosclerotic plaques. Inflammation includes, but is not limited to, Non-Hodgkin's lymphoma, Wegener's granulomatosis, Hashimoto's thyroiditis, hepatocellular carcinoma, thymus atrophy, chronic pancreatitis, rheumatoid arthritis, reactive lymphoid hyperplasia, osteoarthritis, ulcerative colitis, papillary carcinoma, Crohn's disease, ulcerative colitis, acute cholecystitis, chronic cholecystitis, cirrhosis, chronic sialadenitis, peritonitis, acute pancreatitis, chronic pancreatitis, chronic Gastritis, adenomyosis, endometriosis, acute cervicitis, chronic cervicitis, lymphoid hyperplasia, multiple sclerosis, hypertrophy secondary to idiopathic thrombocytopenic purpura, primary IgA nephropathy, systemic lupus erythematosus, psoriasis, pulmonary emphysema, chronic pyelonephritis, and chronic cystitis.
(0057) A cardiovascular disease or disorder includes those disorders that can either cause ischcmia or are caused by reperfusion of the heart. Examples include, but are not limited to, atherosclerosis, coronary artery disease, granulomatous myocarditis, chronic myocarditis (non-granulomatous), primary hypcrtrophic cardiomyopathy, peripheral artery disease (PAD), stroke, angina pectoris, myocardial infarction, cardiovascular tissue damage caused by cardiac arrest, cardiovascular tissue damage caused by cardiac bypass, cardiogenic shock, and related conditions that would be known by those of ordinary skill in the art or which involve dysfunction of or tissue damage to the heart or vasculature, especially, but not limited to, tissue damage related to ADAM activation. CVS
diseases include, but are not limited to, atherosclerosis, granulomatous myocarditis, myocardial infarction, myocardial fibrosis secondary to valvular heart disease, myocardial fibrosis without infarction, primary hypertrophic cardiomyopathy, and chronic myocarditis (non-granulomatous).
100581 A 'Metabolic disease or disorder" refers to a wide range of diseases and disorders of the endocrine system including, for example, insulin resistance, diabetes, obesity, impaired glucose tolerance, high blood cholesterol, hyperglycemia, hyperinsulinemia, dyslipidemia and hyperlipidemia.

100591 A connective tissue disease or disorder includes, but is not limited to, cystic fibrosis, myocardial fibrosis, myclofibrosis, hepatic fibrosis, interstitial lung fibrosis. ncoplastic fibrosis, pancreatic fibrosis, pulmonary fibrosis, subcpidcrnial fibrosis, panmural fibrosis of the bladder, proliferative fibrosis, replacement fibrosis, retroperitoncal fibrosis and root sleeve fibrosis, osteogenesis imperfecta, Ehlcrs-Danlos syndrome, chondrodysplasias, Marfan syndrome, Alport syndrome, familial aortic aneurysm, achondroplasia, mucopolysaccharidoses, osteoporosis, ostcopetrosis, Paget's disease, rickets, ostcomalacia, hypcrparathyroidism, renal osteodystrophy, osteonecrosis, osteomyelitis, ostcoma, osteoid ostcoma, osteoblastoma, osteosarcoma, osteochondroma, chondroma, chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous cortical defect, nonossifying fibroma, fibrous dysplasia, fibrosarcoma, malignant fibrous histiocytoma, Ewing's sarcoma, primitive neuroectodennal tumor, giant.
cell tumor, osteoarthritis, rheumatoid arthritis, ankylosing spondyloarthritis, Reiter's syndrome, psoriatic arthritis, enteropathic arthritis, infectious arthritis, gout, gouty arthritis, calcium pyrophosphate crystal deposition disease, ganglion, synovial cyst, villonodular synovitis, systemic sclerosis, Dupuytren's contracture, lupus crythematosus, mixed connective tissue disease, epidermolysis bullosa simplex, bullous congenital ichthyosiform erythroderma (cpidermolytic hyperkeratosis), non-epidermolytic and epidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens, pachyonychia congenita, and white sponge nevus.
100601 A cell proliferative disease or disorder includes, but is not limited to, actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis. mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hcmoglobinuria, polycythemia vcra, psoriasis, primary thrombocythcmia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, mycloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
Polynucleolide and Oligonucleolide Compositions and Molecules 100611 targets: In one embodiment, the targets comprise nucleic acid sequences of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2), including without limitation sense and/or antisense noncoding and/or coding sequences associated with TFE3 and/or IRS2.
100621 TFE3, a basic helix-loop-helix (bHLH) protein, as a transactivator of metabolic genes that are regulated through an E-box in their promoters. Adcnovirus-mcdiatcd expression of TFE3 in hcpatocytcs in culture and in vivo strongly activated expression of IRS-2 and Akt and enhanced phosphorylation of insulin-signaling kinases such as Akt, glycogen synthase kinasc 3(3 and p70S6 kinase. TFE3 is a bHLH
transcription factor that strongly activates various insulin signaling molecules, protecting against the development of insulin resistance and the metabolic syndrome (Nakagawa Y., el a!. (2005) Nature Medicine 12, 107 - 113).

100631 Transcription factor binding to IGHM enhancer 3, also known as TFE3; is a human gene. TFE3, a member of the helix-loop-helix family of transcription factors, binds to the mu-E3 motif of the immunoglobulin heavy-chain enhancer and is expressed in many cell types.
(0064) Regulation of IRS-2 is the primary site where TFE3 in synergy with Foxol, and SREBP-lc converge.
Taken together, TFE3/Foxol andSREBP-lc reciprocally regulate IRS-2 expression and insulin sensitivity in the liver (Shimano H. (2007).1 Mo! Mecl. 85(5):437-44).
100651 Members of the IRS-protein family are tyrosine phosphorylated by the receptors for insulin and IGF-I, as well as certain cytokines receptors coupled to Janus kinases (Yenush L, et al.
(1997) Bio Essays 19:491-500). At least four IRS-proteins occur in mammals: IRS-I and IRS-2 are widely expressed, IRS-3 is restricted to adipose tissue, B-cells, and possibly liver; and IRS-4 is expressed in the thymus, brain, and kidney. IRS-proteins have a conserved amino terminus composed of adjacent pleckstrin homology and phosphotyrosine-binding domains that mediate coupling to activated receptor tyrosine kinascs.
100661 Exemplary Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) mediated diseases and disorders which can be treated with cell/tissues regenerated from stem cells obtained using the antisense compounds comprise: diabetes or related disorders thereof (e.g., type I, type 11 diabetes, gestational diabetes, diabetic ketoacidosis, nonkctotic hyperosmolar coma, hypoglycemia, diabetic coma etc.), a metabolic disease or disorder (e.g., an insulin resistant non diabetic state such as obesity, impaired glucose tolerance (IGT) and Metabolic Syndrome), Polycystic Ovary Syndrome, atherosclerosis, cancer (e.g..
example papillary renal cell carcinoma, alveolar soft-part sarcoma), a disease associated with apoptosis, aging and senescence; a neurological disease or disorder (e.g. Alzhcimcrs disease, Parkinson's disease, amyotrophic lateral sclerosis etc.) a disease or disorder associated with infectious organisms, autoimmunity, a disease or disorder associated with immune system, an inflammation, an allergy, a cardiovascular disease or disorder (e.g.
diabetic retinopathy, diabetic neuropathy, diabetic amyotrophy, diabetic nephropathy, diabetic cardiomyopathy), a macrovascular disease or disorder, a coronary artery disease, angina, myocardial infarction ("heart attack"), stroke, a peripheral vascular disease (e.g.
including those that contribute to intermittent claudication (exertion-related leg and foot pain) as well as diabetic foot, diabetic myonecrosis ('muscle wasting'), diabetic foot etc.), a connective tissue disease or disorder, a cell proliferative disease or disorder.
100671 In a preferred embodiment, the oligonucleotides are specific for polynucleotides of TFE3 and/or IRS2, which includes, without limitation noncoding regions. The TFE3 and/or IRS2 targets comprise variants of TFE3 and/or IRS2; mutants of TFE3 and/or IRS2, including SNPs; noncoding sequences of TFE3 and/or IRS2; alleles, fragments and the like. Preferably the oligonuclcotidc is an antiscnse RNA
molecule.
100681 In accordance with embodiments of the invention, the target nucleic acid molecule is not limited to TFE3 and/or IRS2 polynucleotides alone but extends to any of the isoforms, receptors, homologs, non-coding regions and the like of TFE3 and/or IRS2.

100691 In another preferred embodiment, an oligonucleotide targets a natural antisense sequence (natural antiscnsc to the coding and non-coding regions) of TFE3 targets, including, without limitation, variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto.
Preferably the oligonucleotidc is an antisense RNA or DNA molecule.
100701 In another preferred embodiment, the oligomeric 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, guanosine, cytidinc or other natural or unnatural nucleotides at this position. This may be done at any of the positions of the antisense compound. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of a target nucleic acid.
10071 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 ./x. In some embodiments, homology, sequence identity or complementarity, is about 90%, about 92'/'0, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.
10072 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 complcmentarity to avoid non-specific binding of the antisensc 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 arc performed in the case of in vitro assays.
100731 An antisense compound, whether DNA, RNA, chimeric, substituted etc, is specifically hybridizable when binding of the compotmd 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 complementarily 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 are performed.
100741 In another preferred embodiment, targeting of TFE3 including without limitation, antisense sequences which are identified and expanded, using for example, PCR, hybridization etc., one or more of the sequences set forth as SEQ ID NOS: 5, and the like, modulate the expression or function of TFE3 and/or IRS2. In one embodiment, expression or function is up-regulated as compared to a control.
In another preferred embodiment, expression or function is down-regulated as compared to a control.

(0075( In another preferred embodiment, oligonuclcotides comprise nucleic acid sequences set forth as SEQ ID
NOS: 4 to 9 including antiscnsc sequences which are identified and expanded, using for example, PCR, hybridization etc. These oligonuclcotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like. Examples of modified bonds or internuclcotide linkages comprise phosphorothioatc, phosphorodithioatc or the like. In another preferred 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 oligonuclcotides of the present invention may be a monophosphate, diphosphate, tiphosphate, alkylphosphate, alkancphosphate, phosphorothioate and the like. The preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, per se, is also known and need not be described here.
(0076( The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonuclcotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antiscnse oligonuclcotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can he useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.
100771 In embodiments of the present invention oligomcric antiscnsc compounds, particularly oligonuclcotides, bind to target nucleic acid molecules and modulate the expression and/or function of molecules encoded by a target gene. The fitnctions 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.
100781 The antiscnsc compounds, include, antisense oligomcric compounds, antisense 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 oligomeric compounds.
(0079( 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 Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2).

(00801 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 ]cast one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. "Segments" are defined as smaller or sub-portions of regions within a target nucleic acid. "Sites," as used in the present invention, are defined as positions within a target nucleic acid.
100811 In a preferred embodiment, the antisense oligonucleotides bind to the natural antisense sequences of Insulin Receptor Substrate 2 (IRS2) and/or Transcription factor E3 (TFE3) and modulate the expression and/or function of Transcription factor E3 (TFE3) and/or insulin Receptor Substrate 2 (IRS2). Examples of antisense sequences include SEQ ID NOS: 3 to 9.
100821 In another preferred embodiment, the antisense oligonucleotides bind to one or more segments of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) and modulate the expression and/or function of TFE3 and/or IRS2. The segments comprise at least five consecutive nucleotides of the Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) sense or antisense polynueleotides.
100831 In another preferred embodiment, the antisense oligonucleotides are specific for natural antisense sequences of Transcription factor E3 (TFE3) wherein binding of the oligonucleotides to the natural antisense sequences of TFE3 modulate expression and/or function of TFE3 and/or IRS2.
100841 In another preferred embodiment, oligonucleotide compounds comprise sequences set forth as SEQ ID
NOS: 4 to 9, 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 another preferred embodiment, the nucleotides comprise a phosphonis 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 monophosphatc, diphosphate, tiphosphate, alkylphosphate, alkanephosphate, phosphorothioate and the like.
The preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, per se, is also known and need not be described here.
100851 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 methioninc (in eukaryotes) or formylmcthionine (in prokaryotes). 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 Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2), regardless of the sequence(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).
100861 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 either 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") are all regions that may be targeted effectively with the antisense compounds of the present invention.
100871 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 the translation termination codon, is also a region which may be targeted effectively. Within the context of the 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.
100881 Another target region includes the 5' untranslatcd 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' untranslatcd 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-methylated guanosinc residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphatc 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.
100891 Although some cukaryotic mRNA transcripts arc directly translated, many contain one or more regions, known as "introns," which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to fonn a continuous mRNA sequence. In one embodiment, targeting splice sites, i.e., intron-cxon 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 are known as "fusion transcripts". Introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.
100901 In another preferred embodiment, the antisensc oligonucleotides bind to coding and/or non-coding regions of a target polynucleotide and modulate the expression and/or function of the target molecule.
100911 In another preferred embodiment, the antisense oligonucleotides bind to natural antisense polynucleotides and modulate the expression and/or function of the target molecule.
100921 In another preferred embodiment, the antisense oligonucleotides bind to sense polynucleotides and modulate the expression and/or function of the target molecule.
100931 Alternative RNA 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 either their start or stop position and contain both intronic and exonic sequence.
100941 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.
100951 Variants can be produced through the use of alternative signals to start or stop transcription. Prc-mRNAs and mRNAs can possess more than one start codon or stop codon. Variants .that originate from a pre-mRNA or mRNA that use alternative start codons arc known as "alternative start variants" of that pre-mRNA or mRNA.
Those transcripts that use an alternative stop codon are 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 poly A sites. Within the context of the invention, the types of variants described herein are also embodiments of target nucleic acids.
100961 The locations on the target nucleic acid to which the antisense compounds hybridize are defined as at least a 5-nucleotide long portion of a target region to which an active antisense compound is targeted.
100971 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 arc readily identifiable by one having ordinary skill in the art in view of this disclosure.
100981 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.

100991 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 nuclcotides 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 nuclcotides). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 5 consecutive nuclcotides from the 3'-terminus of one of the illustrative preferred target segments (the remaining nuclcotides being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3'-terminus of the target segment and continuing until the DNA or RNA contains about 5 to about 100 nuclcotides). 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.
1001001 Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
11 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 are synthesized to cover the entire length of the target polynucleotide. The targets also include coding as well as non coding regions.
100102 In one embodiment, it is preferred to target specific nucleic acids by antisense oligonucleotides. Targeting an antisense compound to a particular nucleic acid, is a multistep 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 mR.NA transcribed from. the gene) whose expression is associated with a particular disorder or disease state, or a non coding polynucleotidc such as for example, non coding RNA
(ncRNA).
1001031 RNAs can be classified into (I) messenger RNAs (mRNAs), which arc translated into proteins, and (2) non-protein-coding RNAs (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 are 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 (Cheng, J. el al. (2005) Science 308 (5725), 1149-1154, Kapranov, P.
et a!. (2005). Genome Regis 15 (7), 987-997). 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 are 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 are encoded at a chromosomal location distinct from the RNAs they act upon and generally do not exhibit perfect base-pairing potential with their targets.
1001041 Without wishing to be bound by theory, perturbation of an antisense polynucleotidc by the antiscnsc oligonucleotides described herein can alter the expression of the corresponding sense messenger RNAs. However, this regulation can either be discordant (antisense knockdown results in messenger RNA elevation) or concordant (antisense knockdown results in concomitant messenger RNA reduction). In these cases, antisense 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 either category is capable of regulating the corresponding sense transcripts - either in a concordant or disconcordant manner. The strategies 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.
1001051 SYraiekn- 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.
1001061 Srraiek, 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.
1001071 According to the present invention, antisense compounds include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, 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-like, or mixtures thereof, or may be mimetics of one or more of these. These compounds may be single-stranded, doublestranded, 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. Antiscnsc 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 self-complenientarity 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 either 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 internucleoside linkages. Alternatively, the two strands can be linked via a non-nucleic, acid moicry or linker group. When formed from only one strand, dsRNA can take the form S 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 transgcnic 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) are complementary RNA strands that base pair in Watson-Crick fashion.
1001081 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-like" (i.e.. generally having one or more 2'-deoxy sugars and, generally, T
rather than U bases) or "RNA-like" (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 structure, most commonly the A- and B-forms. It is believed that, in general, oligonucleotides which have B-form-like structure are "DNA-like" and those which have A-formlikc structure are "RNA-like." In some (chimeric) embodiments, an antisense compound may contain both A- and B-form regions.
1001091 In another preferred embodiment, the desired oligonucleotides or antisensc 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 (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof.
1001101 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.
1001111 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 are shown to increase the expression and/or function of the TFE3 and/or IRS2 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-induced transcriptional activation (RNAa).
1001121 In a further embodiment, the "preferred target segments" identified herein may be employed in a screen for additional compounds that modulate the expression of TFE3 and/or IRS2 polynucleotides. "Modulators" are those compounds that decrease or increase the expression of a nucleic acid molecule encoding TFE3 and/or IRS2 and which comprise at least a 5-nucleotide portion that is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding sense or natural antisensc polynucleotides of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) 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 Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2), e.g. SEQ ID NOS: 4 to 9. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding TFE3 and/or IRS2 polynucleotides, the modulator may then be employed in further investigative studies of the function of TFE3 and/or IRS2 polynucleotides, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.
1001131 Targeting the natural antisensc Transcription factor E3- (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) sequence preferably modulates the function of the target gene TFE3 and/or IRS2 (e.g. accession number NM_003749 and/or NM 006521). In a preferred embodiment, the target is an antiscnse polynuclcotide of the Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) gene. In a preferred embodiment, an antiscnsc oligonuclcotidc targets sense and/or natural antisensc sequences of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) (e.g. accession number NM_003749 and/or NM_(0)6521) 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 antisensc and/or sense TFE3 and/or IRS2 polynucleotides.
1001141 The preferred target segments of the present invention may be also be combined with their respective complementary antisensc compounds of the present invention to form stabilized double-stranded (duplexcd) oligonucleotidcs.
1001151 Such double stranded oligonuclcotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processing via an antiscnse mechanism.
Moreover, the double-stranded moieties may be subject to chemical modifications (Fire ei al., (1998) Nature, 391, 806-811; Timmons and Fire, (1998) Nature, 395, 854; Timmons et al., (2001) Gene. 263, 103-112; Tabara el at, (1998) Science. 282, 430-43 I :
Montgomery e/ al., (1998) Proc. Nail. Acad. Sci. LISA, 95, 15502-15507; Tuschl ci al., (1999) Genes Dev., 13, 3191-3197; Elbashir et a!., (2001) Natu e, 411, 494-498; Elbashir et a!., (2001) Genes Dev. 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisensc strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman at a/., (2002) Science, 295, 694-697).
1001161 In a preferred embodiment, an antisensc oligonucleotide targets Transcription factor E3 (TFE3) and/or = Insulin Receptor Substrate 2 (IRS2) (e.g. accession number NM_003749 and/or NM_006521) variants, alleles, isoforms, homologs, mutants, derivatives, fragments and complementary sequences thereto. Preferably the oligonucleotide is an antisensc molecule.
1001171 In accordance with embodiments of the invention, the target nucleic acid molecule is not limited to Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) alone but extends to any of the isoforms, receptors, homologs and the like of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) molecules.
1001181 In another preferred embodiment, an oligonucleotide targets a natural antisense sequence of TFE3 and/or IRS2 polynucleotides, for example, polynucleotides set forth as SEQ ID NOS: 5, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto.
Examples of antisensc oligonucleotides are set forth as SEQ ID NOS: 4 to 9.
1001191 In one embodiment, the oligonucleotidcs are complementary to or bind to nucleic acid sequences of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) antisensc, including without limitation noncoding sense and/or antisensc sequences associated with Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotides and modulate expression and/or function of TFE3 and/or IRS2 molecules.
1001201 In another preferred embodiment, the oligonucleotidcs are complementary to or bind to nucleic acid sequences of TFE3 and/or IRS2 natural antisensc, set forth as SEQ ID NO:
3,.and modulate expression and/or function of TFE3 and/or IRS2 molecules.
1001211 In a preferred embodiment, oligonucleotidcs comprise sequences of at least 5 consecutive nucleotides of SEQ ID NOS: 4 to 9 and modulate expression and/or function of TFE3 and/or IRS2 molecules.
1001221 The polynucleotide targets comprise TFE3 and/or IRS2, including family members thereof, variants of TFE3 and/or IRS2; mutants of TFE3 and/or IRS2, including SNPs; noncoding sequences of TFE3 and/or IRS2;
alleles of TFE3 and/or IRS2; species variants, fragments and the like.
Preferably the oligonucleotide is an antisensc molecule.
1001231 In another preferred embodiment, the oligonucleotide targeting Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2), comprise: antisensc RNA, interference RNA
(RNAi), 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).
1001241 In another preferred embodiment, targeting of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynuclcotides, e.g. SEQ ID NOS: 5 modulates the expression or function of these targets. In one embodiment, expression or function is up-regulated as compared to a control. In another preferred embodiment, expression or function is down-regulated as compared to a control.
1001251 In another preferred embodiment, antisense compounds comprise sequences set forth as SEQ ID NOS: 4 to 9. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like.
1001261 In another preferred embodiment, SEQ ID NOS: 4 to 9 comprise one or more LNA nucleotides.
1001271 Table I shows exemplary antisense oligonucleotides useful in the methods of the invention.
Antisense Sequence ID Sequence Sequence Name SEQ ID NO:6 CUR-0603 rArUrArCrCrUrArCrArCrCrCrArArGrGrUrUrGrUrCrArGrCrUrUrU
SEQ ID NO:7 CUR-0605 rCrArGrCrArArGrGrCrArGrArArGrCrUrUrGrGrArGrGrArGrGrGrU
SEQ ID NO:8 CUR-0607 rCrUrGrUrArArUrCrArGrGrCrArArGrGrArGrGrArGrGrArGrUrCrA
SEQ ID NO:9 CUR-0599 rCrUrCrCrCrArArCrUrCrCrCrUrArCrUrUrUrCrUrGrUrCrUrCrUrU
SEQ ID NO:10 CUR-0601 rGrCrCrUrGrGrGrUrUrUrGrUrUrCrCrCrArArCrUrGrGrUrGrGrUrU
SEQ ID NO:11 CUR-0609 rCrUrGrGrGrUrArUrArCrUrUrArArGrArUrUrGrArCrGrUrGrCrUrC

The modulation of a desired target nucleic acid can be carried out in several ways known in the art. For example, antiscnsc oligonuclcotides, siRNA etc. Enzymatic nucleic acid molecules (e.g., ribozymcs) are 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 (7..aug el a/., 324, Nature 429 1986;
Ccch. 260 JAMA 3030, 1988; and Jeffcrics et a1., 17 Nucleic Acids Research 1371, 1989).
1001281 Because of their sequence-specificity, trans-cleaving enzymatic nucleic acid molecules show promise as therapeutic agents for human disease (Unman & McSwiggen, (1995) Ann. Rep. Mel.
(:'hem. 30, 285-294;
Christofferson and Marr, (1995) 1. Med. Chem. 38, 2023-2037). 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.
1001291 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 a 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 WO 2010/135695 . PCT/US2010/035842 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.
1001301 Several approaches such as, in vitro selection (evolution) strategies (Orgcl, (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 ofphosphodiestcr linkages and amide linkages, (Joyce, (1989) Gene, 82, 83-87; Bcaudry et al., (1992) Science 257, 635-641; Joyce, (1992) .Scientific American 267, 90-97; Breaker ei al., (1994) IB'1'LY'H
12, 268; Bartel et al., (1993) Science 261:1411- 1418; Szostak, (1993) SIBS
17, 89-93; Kumar ei al., (1995) /,ASLH J., 9, 1183: Breaker, (1996) Curr. Op. Biotech., 7,442).
1001311 The development of ribozmes 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 (kcal) of about I min-I in the presence of saturating (10 mM) concentrations of Mg2+ cofactor. An artificial "RNA ligase" riboryme has been shown to catalyze the corresponding self-modification reaction with a rate of about 100 min-I. In addition, it is known that certain modified hammerhead riborymes that have substrate binding arms made of DNA
catalyze RNA cleavage with multiple turn-over rates that approach 100 min-l. 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 riborymes can promote chemical transformations with catalytic rates that arc significantly greater than those displayed in vitro by most natural self-cleaving ribozmcs. 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.
1001321 Intermolecular cleavage of an RNA substrate by an RNA catalyst that fits the "hammerhead" model was first shown in 1987 (Uhlenbeck, 0. C. (1987) Nanrre, 328: 596-600). The RNA
catalyst was recovered and reacted with multiple RNA molecules, demonstrating that it was truly catalytic.
1001331 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 (Haseloff and Gerlach, (1988) Nature, 334, 585; Walbot and Bruening, (1988) Nature, 334, 196;
Uhlenbeck, 0. C. (1987) Nature, 328: 596-600; Koizumi, M., ei al. (1988) 1'EBS
Lett., 228: 228-230). 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. (sec Haseloff and Gerlach, (1988) Nature, 334, 585; Walbot and Bru ring, (1988) Nature, 334, 196; Uhlenbeck, 0. C. (1987) Nalnre, 328: 596-600).
1001341 RNA interference (RNAi) has become a povierful tool for modulating gene expression in mammals and mammalian cells. This approach requires the delivery of small interfering RNA
(siRNA) either as RNA itself or as DNA, using an expression plasmid or virus and the coding sequence for small hairpin RNAs that are processed to siRNAs. This system enables efficient transport of the pre-siRNAs to the cytoplasm where they are active and permit the use of regulated and tissue specific promoters for gene expression.
1001351 In a preferred embodiment, an oligonucleotide or antisensc 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 intemuclcosidc (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides arc 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.
1001361 According to the present invention, the oligonucleotides or "antisense compounds" include antisense oligonucleotides (e.g. RNA, DNA, mimetic, chimera, analog or homolog thereof), ribozymcs, external guide sequence (EGS) oligonuclcotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, saRNA, 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 mimetics 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. Antiscnse compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and/or branched. Antiscnse 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 self-complementanty 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 either 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 internucleoside 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 form 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 (Hammond ef a/., (1991) Nat. Rev.
Gene!., 2, 110-119-, Matzke el aL, (2001) ('urr. Opin. Genet. Dev., 11, 221-227; Sharp, (2001) Genes Dev., 15, 485-490). 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 stran d) are complementary RNA strands that base pair in Watson-Crick fashion.

1001371 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 oligonuclcotides) may be described as "DNA-like" (i.e., generally having one or more 2'-dcoxy sugars and, generally, T
rather than U bases) or "RNA-like" (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 structure, most commonly the A- and B-forms. It is believed that, in general, oligonucleotides which have B-form-like structure are "DNA-like" and those which have A-formlikc structure are "RNA-like." In some (chimeric) embodiments, an antiscnse compound may contain both A- and B-form regions.
1001381 The antisense compounds in accordance with this invention can comprise an antiscnse 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 antiscnse strand or portion of the antiscnse compound. In other words, a single-stranded antiscnse compound of the invention comprises from 5 to about 80 nucleotides, and a double-stranded antiscnse 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44. 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length, or any range therewithin.
1001391 In one embodiment, the antiscnse compounds of the invention have antiscnse portions of 10 to 50 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies oligonucleotidcs having antiscnse portions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any range thcrcwithin. In some embodiments, the oligonuclcotidcs arc 15 nucleotides in length.
1001401 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 antiscnse compounds having antiscnse 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 therewithin.
1001411 In another preferred embodiment, the oligomeric 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 cytidinc at this position. This may be done at any of the positions of the antisense or dsRNA compounds. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of a target nucleic acid.

1001421 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 complcmcntarity, is from about 60% to about 70%. In some embodiments, homology, sequence identity or complcmcntarity, 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 complcmcntarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.
1001431 In another preferred embodiment, the antiscnse oligonuclcotidcs, such as for example, nucleic acid molecules set forth in SEQ ID NOS: 4 to 9 comprise one or more substitutions or modifications. In one embodiment, the nucleotides are substituted with locked nucleic acids (LNA).
1001441 In another preferred embodiment, the oligonucleotides target one or more regions of the nucleic acid molecules sense and/or antisensc of coding and/or non-coding sequences associated with TFE3 and/or IRS2 and the sequences set forth as SEQ ID NOS: 1, 2 and 3. The oligonucleotides are also targeted to overlapping regions of SEQ ID NOS: 1,2and3.
1001451 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, RNasc H is a cellular endonuclease 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 antisensc modulation of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotidcs when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides 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 preferred 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 RNAse 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 oligonucleotide/target pair, which is the temperature at which the oligonuclcotidc and target dissociate, dissociation is detected spectrophotomctrically. The higher the Till, the greater is the affinity of the oligonuclcotidc for the target.
1001461 Chimeric antiscnse compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonuclcotides 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 are not limited to, US patent nos.
5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,8 78; 5,403,711; 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.
1001471 In another preferred 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'-O-alkyl-O-alkyl or 2'-fluoro-modified nucleotide. In other preferred embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2' 0-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA.
Such modifications are routinely incorporated into oligonucleotidcs and these oligonucleotides have been shown to have a higher Tin (i.e., higher target binding affinity) than, 2'-deoxyoligonuclcotides against a given target. The effect of such increased affinity is to greatly enhance RNAi oligonucleotide inhibition of gene expression. RNAsc H is a cellular endonuclease that cleaves the RNA strand of RNA: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 another preferred embodiment, the chimeric oligonucleotide is also modified to enhance nuclease resistance. Cells contain a variety of cxo- and endo-nucleases 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 incubating oligonucleotidcs 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 phosphorothioate modification are presently more preferred. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance. Some desirable modifications can be found in De Mesmaeker e/ al. (1995) Acc. Chem. Res., 28:366-374.
1001481 Specific examples of some preferred oligonucleotidcs envisioned for this invention include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, 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--O--CH2, CH,--N(CH3)--O-CH2 (known as a methylene(methylimino) or MMI
backbone], CH2 --0--N (CH3) -CH2, CH2 -N (CH3)--N (CH3)--CH2 and O-N (CH3)--CH2 -CH2 backbones, wherein the native phosphodicster backbone is represented as O--P--0-CH,). The amide backbones disclosed by Dc Mesmaeker er al. (1995) Acc. Chem. Rev. 28:366-374 are also preferred. Also preferred are oligonucleotides having morpholino backbone structures (Summerton and Weller, U.S. Pat. No. 5,034,506). In other preferred embodiments, such as the peptide nucleic acid (PNA) backbone, the phosphodiestcr backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al. (1991) Science 254, 1497). 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 O(CH2)n CH3, O(CH2)n NH2 or O(CH2)n CH3 where n is from I
to about 10: C I to C 10 lower alkyl, alkoxyalkoxy, substituted lower alkyl. alkaryl or aralkyl;
Cl; Br; CN; CF3 ; OCF3; 0--, S-, or N-alkyl: 0--, S--, or N-alkenyl; SOCH3; S02 CH3; ON02: N02: N3: NH2;
hetcrocycloalkyl; hcterocycloalkaryl:
aminoalkylamino; polyalkylarnino: substituted silyl; an RNA cleaving group; a reporter group; an intcrcalator; a group for improving the pharmacokinctic properties of an oligonucleotide; or a group for improving the phannacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy 12'-O-CH2 CH2 OCH3. also known as 2'-O-(2-methoxyethyl)j (Martin et al., (1995) He/v. Chinn. Acta, 78, 486). Other preferred modifications include 2'-methoxy (2'-O--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. Oligonuclcotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
1001491 Oligonuclcotidcs 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-methyladeninc, 5-Mc pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosinc 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)adeninc, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other hetcrosubstitutcd alkyladcnincs, 2-thiouracil, 2-thiothyminc, 5- brornouracil, 5-hydroxymcthyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6-diarninopurine. (Kornbcrg, A., DNA Replication, W. H.
Freeman & Co., San Francisco, 1980, pp75-77; Gcbcychu, G., (1987) et aL Nucl.
Acids Res. 15:4513). A
"universal" base known in the art, e.g., inosine, may be included. 5-Me-C
substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2"C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions.
1001501 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 (Letsinger et a/., (1989) Proc. Nati. Acad. Sci. USA 86, 6553), cholic acid (Manoharan ei at.
(1994) Bioorgg. Mcc/. (hem. Let. 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al. (1992) Ann. MY

Acad. Sci. 660, 306; Manoharan et a!. (1993) Bioo . Med. (.:hem. Let. 3, 2765). a thiocholesterol (Oberhauser ei al., (1992) ,V,,cl. Acids Res. 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al. EMBO J. 1991, 10, 111; Kabanov el al. (1990) FEBS Lett. 259, 327;
Svinarchuk et al. (1993) Biochimie 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero- 3-H-phosphonate (Manoharan et al. (1995) "Tetrahedron Lett. 36, 3651; Shea et cit.
(1990) Nuc!. Acids Res. 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et a!. (1995) Nucleosides & Nucleotides, 14, 969), or adamantane acetic acid (Manoharan et al. (1995) Tetrahedron Lett. 36, 3651).
Oiigonucleotidcs comprising lipophilic moieties, and methods for preparing such oligonucleotides are known in the an, for example, U.S. Pat.
Nos. 5,138,045, 52.1 8,105 and 5,459,255.
1001511 It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide. The present invention also includes oligonuclcotides which are chimeric oligonucleotides as hercinbefore defined.
1001521 In another embodiment, the nucleic acid molecule of the present invention is conjugated with another moiety including but not limited to abasic nucleotides, polycthcr, polyamine, polyamides, 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.
1001531 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 phosphorothioates and alkylated derivatives. It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridinc or psoralen-modified amiditcs and/or CPG (available from Glen Research, Sterling VA) to synthesize fluorescently labeled, biotinylated or other modified oligonuclcotides such as cholesterol-modified oligonucleotides.
1001541. 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 broader. the routes of administration of oligonucleotides comprised of current chemistries such as MOE, ANA, FANA, PS etc (Uhlman, et a!. (2000) Current Opinions in Drug Discover- & Development Vol. 3 No 2). This can be achieved by substituting some of the monomers in the current oligonuclcotides 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.
1001551 Preferred modified oligonuclcotide backbones comprise, but not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylenc phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidatc and aminoalkylphosphoramidatcs, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotricsters, 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 are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.
1001561 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; 5,550,111; 5,563, 253:
5,571,799; 5,587,361: and 5,625,050, each of which is herein incorporated by reference.
1001571 Preferred modified oligonuclcotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intcmuclcoside linkages, mixed hetcroatom and alkyl or cycloalkyl internucleosidc linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones;
formacetyl and thiofomiacetyl backbones; methylene fomtacctyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methylcncimino and mcthylenchydrazino backbones; sulfonatc and sulfonamide backbones: amide backbones; and others having mixed N, 0, S and CH2 component parts.
1001581 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.
1001,591 In other preferred oligonuclcotide 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 oligonuclcotide 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 oligonuclcotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobascs arc 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 arc 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, ei a!. (1991) Science 254, 1497-1500.
1001601 In another preferred embodiment of the invention the oligonucleotidcs with phosphorothioate backbones and oligonucleosides with hetcroatom backbones, and in particular- CH2-NH-O-CH2-,-CH2-N (CH3)-O-CH2-known as a methylene (methylimino) or MMI backbone; CH2-O-N (CH3)-CH2-,-CH2N(CH3)-N(CH3) CH2-and-O-N(CH3)-CH2-CH2- wherein the native phosphodiester backbone is represented as-O-P-O-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 preferred are oligonucleotides having morpholino backbone structures of the above-referenced US
patent no. 5,034,506.
1001611 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-alkcnyl;
0-, S-or N-alkynyl; or 0 alkyl-O-alkyl, wherein the alkyl, alkcnyl and alkynyl may be substituted or unsubstitutcd C to CO alkyl or C2 to CO alkenyl and alkynyl. Particularly preferred are 0 (CH2)n OmCH3, O(CH2)n,OCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2nON(CH2)nCH3)2 where n and m can be from I 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, 0-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3, NH2, hcterocycloalkyl, hcterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the phannacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotidc, and other substituents having similar properties. A
preferred modification comprises 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2- methoxyethyl) or 2'-MOE) (Martin et a!.. (1995) Hely. ("hem. Acia, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification comprises 2'-dimcthylaminooxycthoxy, i.e., a O(CH2)20N(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'- dimcthylaminocthoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxycthyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N (CH2)2.
1001621 Other preferred modifications comprise 2'-mcthoxy (2'-0 CH3), 2'-aminopropoxy (2'-O
CH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonuclcotide, particularly the 3' position. of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotidcs and the 5' position of 5' terminal nucleotide.
Oligonucleotides may also have sugar mimctics 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;
5,118,800, 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514, 785;
5,519,134; 5,567,811; 5,576,427;

5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646, 265; 5,658,873;
5,670,633; and 5,700,920, each of which is herein incorporated by reference.
1001631 Oligonuclcotidcs may also comprise nucleobasc (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 pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nuclcotidcs comprise other synthetic and natural nuclcotidcs such as 5-mcthylcytosine (5-me-C), 5-hydroxymcthyl cytosine, xanthinc, hypoxanthinc, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosinc, 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-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-mcthylquanine and 7-mcthyladeninc, 8-azaguanine and 8-azaadcninc, 7-deazaguaninc and 7-dcazaadcninc and 3-deazaguanine and 3-deazaadcnine.
1001641 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, it., ed. John Wiley & Sons, 1990, those disclosed by Englisch et a!.,'Angewandle Chemic, International Edition', 1991, 30, page 613, and those disclosed by Sanghvi, Y.S., Chapter 15, 'Antisense Research and Applications', pages 289-302, Crooke, S.T. 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-substituted pyrimidines, 6-azapyrimidincs and N-2. N-6 and 0-6 substituted purities, comprising 2-aminopropyladcnine, 5- propynyluracil and 5-propynylcytosinc. 5-mcthylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 C (Sanghvi, Y.S., Crooke, S.T. and Leblcu. B., eds, 'Antisense Research and Applications', CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-Omethoxyethyl sugar modifications.
1001651 Representative United States patents that teach the preparation of the above noted modified nucleotides as well 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;
5,525,711; 5,552,54(1; 5,587,469: 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of which is herein incorporated by reference.
1001661 Another modification of the oligonucleotides of the invention involves chemically linking to the oligonuclcotidc one or more moieties or conjugates, which enhance the activity, cellular distribution, or cellular uptake of the oligonuclcotide.
1001671 Such moictics comprise but are not limited to, lipid moictics such.as a cholesterol moiety (Lctsingcr el al., (1989) Proc. Nail. Acacl. Sci. (USA, 86, 6553-6556), cholic acid (Manoharan el al., (1994) Bioorg. Mal. Chem.

Let., 4, 1053-1060), a thiocther, e.g., hexyl-S-tritylthiol (Manoharan et a!., (1992) Ann. N. Y. Acad. Sci., 660, 306-3(x); Manoharan et al., (1993) Bioorg. Med. Chem. Let., 3, 2765-2770), a thiocholestcrol (Obcrhauscr et at, (1992) Noel. Acids Rev., 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et a!., (1990) F=EBS Lett., 259, 327-330; Svinarchuk el al., (1993) Biochimie 75, 49-54), a phospholipid, e.g., di-hcxadccyl-rac-glycerol or tricthylammoniwn 1,2-di-O-hcxadccyl-rac-glyccro-3-H-phosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36, 3651-3654; Shea et al., (1990) Nucl. Acids Res., 18, 3777-3783), a polyaminc or a polyethylene glycol chain (Manoharan ei a!., (1995) Nucleosides & Nucleotides, 14, 969-973), or adamantanc acetic acid (Manoharan et a!., (1995) Tetrahedron Let;., 36, 3651-3654), a palmityl moiety (Mishra et al., (1995) Biochim. Biophvs. Acta, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-t oxycholestcrol moiety (Crooke et al., (1996) J. Pharmacol. k' p. "ITrer., 277, 923-937).
1001681 Representative United States patents that teach the preparation of such oligonucleotides conjugates comprise, but arc 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; 5,118,802;
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; 5,112,963; 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.
(00169( Drug discovery: 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 Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) and a disease state, phenotype, or condition. These methods include detecting or modulating TFE3 and/or IRS2 polynucleotides comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of TFE3 and/or IRS2 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 phenotype.
Assessing (Ip-regii!aiion or Inhibition o/'(,ene !. press!on:
(001701 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 polymerasc chain reaction (PCR) 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).
1001711 Expression of RNA from the exogenous nucleic acid can also be detected by measuring an enzymatic activity or a reporter protein activity. For example, antisensc 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 RNAi target in the 3' non-coding region. The effectiveness of individual antisense oligonueleotidcs 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), luciferase (Luc), nopaline synthase (NOS), octopinc synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, blcomycin, chlorarnphenicol, gcntamycin, hygrornycin, kanamycin, lincomycin, methotrexatc, phosphinothricin, puromycin, and tetracycline. Methods to determine modulation of a reporter gene are well known in the art, and include, but are not limited to, fluorometric methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), antibiotic resistance determination.
1721 TFE3 and/or IRS2 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 ELISA can be used to measure protein levels. TFE3 and/or IRS2 antibodies for ELISAs are available commercially, e.g., from Abnova (Walnut, CA), Abeam, (Cambridge, MA.) 1001731 In embodiments, TFE3 and/or IRS2 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 TFE3 and/or IRS2 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 antisensc 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 TFE3 and/or IRS2 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.

1001741 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 TFE3 and/or IRS2 mRNA or protein, in a sample treated with an antiscnsc oligonuclcotide 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 TFE3 and/or IRS2 mRNA or protein is increased or decreased by at least about I.25-fold, at least about I.3-fold, at least about I.4-fold, at least about 1.5-fold, at least about I.6-fold, at least about I.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 71ierapeuIic+' 1001751 The compounds of the present invention can be utilized for diagnostics, therapeutics, and prophylaxis, and as research reagents and components of kits. Furthermore, antiscnsc oligonuclcotides, 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.
1001761 For use in kits and diagnostics and in various biological systems, the compounds of the present invention, either 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.
1001771 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 Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) genes. These include, but are not limited to, humans, transgcnic animals, cells, cell cultures, tissues, xenografts, transplants and combinations thereof.
1001781 As one non limiting example, expression patterns within cells or tissues treated with one or more antiscnse compounds are 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.
1001791 Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, (2000) FY BS Lett., 480, 17-24, Cclis, ei a/., (2000) PT13S
Lea., 480, 2-16), SAGE (serial analysis of gene expression) (Madden, ei al., (2000) Drug Discov. Today, 5, 415- 425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, (1999) Methods / nzymol., 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, el al., (2000) Proc. Nail.
Acad. Sci. U.S.A., 97, 1976=81), protein arrays and protcomics (Cells, el al., (2000) !'IBS' Leii., 480, 2-16;
Jungblut, et at.. Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et at., J.
Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, el a!., (2000) Anal. Biochem. 286, 91-98; Larson, et al., (2000) (}viomeirv 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, (2000) Curr. Opin. Microbi(jl. 3, 316-21), comparative genomic hybridization (Carulli, ei al., (1998) J.
Cell Biochem. Suppl., 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson.
(1999) Eur. J. Cancer, 35, 1895-9(4) and mass spectrometry methods (To, Comb.
(2000) Chem. High 7hroughpui Screen, 3, 235-41).
1001801 The compounds of the invention are usefid for research and diagnostics, because these compounds hybridize to nucleic acids encoding Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2). For example, oligonucleotides that hybridize with such efficiency and under such conditions as disclosed herein as to be effective Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) 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 Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) and in the amplification of said nucleic acid molecules for detection or for use in further studies of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2). Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabeling of the oligonucleotide, or any other suitable detection means. Kits using such detection means for detecting the level of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) in a sample may also be prepared.
1001811 The specificity and sensitivity of antisense are also harnessed by those of skill in the art for therapeutic uses. Antiscnse 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.
1001821 For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) 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 Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) modulator. The Transcription factor E3 (TFE3) modulators of the present invention effectively modulate the activity of the Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) or modulate the expression of the TFE3 and/or IRS2. In one embodiment, the activity or expression of TFE3 and/or IRS2 in an animal is inhibited by about 10% as compared to a control. Preferably, the activity or expression of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) in an animal is inhibited by about 30%. More preferably, the activity or expression of TFE3 and/or IRS2 in an animal is inhibited by 50% or more. Thus, the oligorneric compounds modulate expression of TFE3 and/or IRS2 rRNA by at least 10%o, 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`Yn, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100% as compared to a control.
1001831 In one embodiment, the activity or expression of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) in an animal is increased by about 10% as compared to a control. Preferably, the activity or expression of TFE3 and/or IRS2 in an animal is increased by about 30%. More preferably, the activity or expression of TFE3 and/or IRS2 in an animal is increased by 50% or more. Thus, the oligomeric compounds modulate expression of Insulin TFE3 and/or IRS2 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 ]cast 98%, by at least 99%, or by 100% as compared to a control.
1001841 For example, the reduction of the expression of TFE3 and/or IRS2 may be measured in ser un, 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 TFE3 and/or IRS2 protein itself.
1001851 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.
Conjugales 1001861 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, polyethers, groups that enhance the phannacodynamic properties of oligomers, and groups that enhance the pharmacokinctic properties of oligomers.
Typicalconjugate groups include cholesterols, lipids, phospholipids, biotin, phcnazine, folatc, phcnanthridinc, anthraquinone, acridinc, fluoresceins, rhodamincs, coumarins, and dyes. Groups that enhance the phannacodynamic 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 pharmacokinctic 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 are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No.
6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thiocthcr, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undccyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or tricthylammonium 1,2-di-0-hexadecyl-rac-glyccro-3-Hphosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepinc, indomethicin, a barbiturate, a ccphalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
1001871 Representative United States patents that teach the 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; 5,118,802;
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; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022;
5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 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.
f nnnulations 1001881 The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as forexamplc, 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 formulations 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.
1001891 Although, the antiscnsc 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.

1001901 In an embodiment. invention practice involves administering at least.
one of the foregoing antisense oligonucleotidcs with a suitable nucleic acid delivery system. In one embodiment, that system includes a non-viral vector operably linked to the polynucleotidc. Examples of such nonviral vectors include the oligonucleotide alone (e.g. any one or more of SEQ ID NOS: 4 to 9) or in combination with a suitable protein, polysaccharide or lipid formulation.
1001911 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 hcmagglutinatin virus of Japan-liposomc (HVJ) complex. Preferably, the viral vector comprises a strong cukaryotic promoter operably linked to the polynucleotide e.g., a cytomegalovirus (CMV) promoter.
1001921 Additionally preferred vectors include viral vectors, fission proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia viruses and HIV-based viruses. One preferred HIV-based 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 arc preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector IGcller, A.I. et al., (1995) J.
Neurochem, 64: 487: Lim, F., ei a!., in DNA Cloning: Mammalian Systems, D.
Glover, Ed. (Oxford Univ. Press, Oxford England) (1995): Geller, A.I. et al., (1993) Proc Nail. Acad. Sci.:
U.S.A.:90 7603: Geller, Al., .el al., (1990) Proc Nail. Acad. Sci (.ISA: 87:11491, Adenovirus Vectors (LeGal LaSalle et al., Science, 259:988 (1993):
Davidson, et al., (1993) Na.'. Genet. 3: 219: Yang, et al., (1995).j Virol.
69: 2004) and Adeno-associated Virus Vectors (Kaplitt, M.G., et al., (1994) Nat. (.-;enet. 8:148).
1001931 The antiscnsc compounds of the invention encompass any pharmaceutically 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.
1001941 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 undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S.
Pat. No. 6,287,860, which is incorporated herein by reference.
1001951 The present invention also includes pharmaceutical compositions and formulations that include the antisense 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, intraartcrial, subcutaneous. intraperitoneal or intramuscular injection or infusion, or intracranial, e.g..
intrathecal or intravcntricular, administration.
1001961 For treating tissues in the central nervous system, administration can be made by, e.g., injection or infusion into the cerebrospinal fluid. Administration of antiscnse RNA into cerebrospinal fluid is described, e.g., in U.S. Pat. App. Pub. No. 2007/0117712, "Methods for slowing familial ALS
disease progression," incorporated herein by reference in its entirety.
(001971 When it is intended that the antiscnse oligonucleotidc 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 antiscnse oligonucleotide across the blood-brain barrier. Injection can be made, e.g., in the entorhinal 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, "Adcnovirus 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.
1001981 The subject antiscnse oligonucleotides can also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. For example, the antiscnse oligonuclcotide 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 transfcrrin receptor, and administered by intravenous injection. The antisense compound can be linked with a viral vector, for example, that makes the antiscnse compound more effective and/or increases the transport of the antiscnse 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 crythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiosc, D(+) maltose, D(+) raffinosc, L(+) rhamnose, D(+) mclibiosc, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fueose, L(-) fueose, D(-) lyxose, L(+) lyxose.
and L(-) lyxose, or amino acids including, but not limited to, glutwnine, lysine, arginine, asparagine, aspartic acid, cysteinc, glutamic acid, glycine, histidine. lcucinc, mcthionine, phenylalaninc, prolinc, scrinc, thrconine, tyrosine, valinc, and taurine. Methods and materials for enhancing blood brain barrier penetration are 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, "Parentcral delivery systems," all incorporated herein by reference in their entirety.
1001991 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 LIPOFECTIN (available from GTBCO-BRL, Bethesda, MD).
1002001 Oligonucleotides with at least one 2'-O-methoxycthyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdcrmal 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.
[002011 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 excipient(s). In general, the formulations are 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.
1002021 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 carboxymethylccllulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
1002031 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, cxcipients or other active or inactive ingredients.
1002041 Emulsions arc typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 pm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug that may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No.
6,297,860.
1002051 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 unilamcllar or multilamcllar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomcs that are believed to interact with negatively charged DNA molecules to form a stable complex.

Liposomcs that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it.
Both cationic and noncationic liposomes have been used to deliver DNA to cells.
1002061 Liposomcs 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 are those in which part of the vesicle-forming lipid portion of the liposorne 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 are further described in U.S. Pat. No. 6,287,860.
1002071 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.
1002081 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-chelating nonsurfactants. Penetration enhancers. and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.
1002091 One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.
1002101 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. diolcoyl-phosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholinc DMPC, distearolyphosphatidyl choline) negative (e.g.
dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.
dioleoyltetramethylaminopropyl DOTAP and diolcoyl-phosphatidyl ethanolamine DOTMA).
1002111 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 are further described in U.S. Pat. No.
6,287,860.
1002121 Compositions and formulations for oral administration include powders or gianules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
Preferred oral formulations are 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 are 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, polyoxycthylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexcd to form micro or nanoparticlcs. Oligonuclcotidc complexing agents and their uses are further described in U.S. Pat. No.
6,287,860, which is incorporated herein by reference.
1002131 Compositions and formulation's 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.
(002141 Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomcric compounds and one or more other chemotherapeutic agents that function by a non-antisensc mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esor ubicin, bleornycin, mafosfamide, ifosfamide, cytosine arabinoside, bischloroethyl-nitrosurea, busulfan, mitomycin C, actinonlycin D, mithramycin, prednisone, hydroxyprogcsterone, testosterone, tamoxifen. dacarbazine, procarbazinc, hexamethylmclaminc, pentamcthylmclaminc, mitoxantronc, amsacrinc, chlorambucil, methylcyelohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mereaptopurine, 6-thioguanine, cytarabinc, 5- azacytidine, hydroxyurea, dcoxycoformycin, 4-hydroxyperoxycyclo-phosphoramidc, 5-fluorouracil (5-FU), 5-fluorodcoxyuridinc (5-FUdR), methotrexate (MTX), colchicinc, taxol, vincristine, vinblastinc, ctoposidc (VP-16), trimetrexate, irinotecan, topotecan, gemcitabinc, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonuclcotide), sequentially (e.g., 5-FU and oligonuclcotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g.. 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisensc compounds and other non-antisensc drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.
1002151 In another related embodiment, compositions of the invention may contain one or more antisensc 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 TFE3 and/or IRS2, 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 Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) nucleic acid target.
Numerous examples of antisense 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,:
1002161 The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity 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 in vitro and in vivo animal models. In general, dosage is from 0.01 pg 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 Vg to 100 g per kg of body weight, once or more daily, to once every 20 years.
1002171 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 oligonuclcotides are described, e.g., in U.S.
Pat. No. 7.563,884, " Antisense modulation of PTP I B expression,"
incorporated herein by reference in its entirety.
1002181 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.
1002191 All documents mentioned herein are incorporated herein by reference.
All publications and patent documents cited in this application arc 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 are illustrated in the following examples.

EXAMPLES
1002201 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 l: Design of crntisense oligonucleotides specific for a nucleic acid molecule antisense to Transcription factor E3 (/71T 3) and/or Insulin Receptor Substrate 2 (I162) and/or sense strand (f T E3 and/or IRS?
polpnucleoiide 1002211 As indicated above the term "oligonucleotide specific for" or "oligonucleotidc 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.
1002221 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 GcnBank 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 oligonuclcotides that exhibit a high degree of complementarity to target nuclcic 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.
1002231 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 modulation of function and/or activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisensc 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 1002241 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 properties 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.

(00225) The binding strength between the target natural antisensc and a potential drug molecule (Molecule) can be estimated using any of the established methods of measuring the strength of intermolecular interactions, for example, a melting curve assay.
1002261 Melting curve assay determines the temperature at which a rapid transition from double-stranded to single-stranded conformation occurs for the natural antisense/Molecule complex. This temperature is widely accepted as a reliable measure of the interaction strength between the two molecules.
1002271 A melting curve assay can be performed using a eDNA copy of the actual natural antisense 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 are highly fluorescent when bound to dsDNA.
1002281 To perform the assay the eDNA 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 pre-formed dsDNA complexes, then slowly cooled to morn 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.ABI's StepOne Plus Real Time PCR
System or LightTypcr instrument, Roche Diagnostics, Lewes, UK).
1002291 Melting peaks are constructed by plotting the negative derivative of fluorescence with respect to temperature (-d(Fluorescencc)/dT) on the y-axis) against temperature (x-axis) using appropriate software (for example LightTypcr (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 T.
Example 2: Modulation of 7'1,`E3 and/or IRS2 polvnucleotidec Trewment of HepG2 cells with antisense oligonucleotides:
1002301 HepG2 cells from ATCC (cat# HB-8065) were grown in growth media (MEM/EBSS (Hyclonc cat #SH30024, or Mediatech cat # MT-10-010-CV) +10% FBS (Mediatech cat# MT35- 011-CV)+
penicillin/streptomycin (Mediatech cat# MT30-002-CI)) at 37 C and 5% CO, One day before the experiment the cells were rcplatcd at the density of 1.5 x 105/ml into 6 well plates and incubated at 37 C and 5% CO,. On the day of the experiment the media in the 6 well plates was changed to fresh growth media. All antisense oligonucleotidcs were diluted to the concentration of 20 p.M. Two l of this solution was incubated with 400 pi of Opti-MEM media (Gibco cat931985-070) and 4 l of Lipofectamine 2000 (Invitrogen cat#
11668019) at room temperature for 20 min and applied to each well of the 6 well plates with HcpG2 cells. A Similar mixture including 2 pl of water instead of the oligonucleotide solution was used for the mock-transfected 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 RNeasy Total RNA Isolation kit from Qiagcn (cat#
74181) following the manufacturers' instructions. 600 ng of RNA was added to the reverse transcription reaction performed using Verso eDNA kit from Thermo Scientific (cat#AB 14538) or High Capacity eDNA Reverse Transcription Kit (call 4368813) as described in the manufacturer's protocol. The eDNA 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:
Hs00275843_sl (IRS2) and Hs00232406_m I (TFE3) 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 I min) using StcpOne Plus Real Time PCR Machine (Applied Biosystems).
1002311 Fold change in gene expression after treatment with antisense oligonucleotides was calculated based on the difference in I 8S-normal ized dCt values between treated and mock-transfected samples.
Results:
1002321 Real Time PCR results show that levels of IRS2 mRNA in HepG2 cells are significantly increased 48h after treatment with siRNAs to TFE3 antisensc Hs.708291 (Fig 1).
100233$ Real Time PCR results show the fold change + standard deviation in TFE3 mRNA after treatment of HepG2 cells with siRNA oligonucleotides introduced using Lipofectaminc 2000, as compared to control (Fig 2).
100234$ Real time PCR results show that the levels of TFE3 mRNA in HepG2 cells is significantly increased 48h after treatment with one of the siRNAs designed to TFE3 antisense Hs.708291.
(Fig 3).
7reanneni of'518A2 cells with antisense oligonucleotides:
1002351518A2 cells obtained from Albert Einstein-Montcfiore Cancer Center, NY
were grown in growth media (MEM/EBSS (Hyclone cat #SH30024, or Mediatech cat # MT-10-010-CV) +10% FBS
(Mediatech cat# MT35-011-CV)+ penicillin/streptomycin (Mediatech cat# MT30-002-CI)) at 37 C and 5%
CO One day before the experiment the cells were replated at the density of 1.5 x 105/ml into 6 well plates and incubated at 37 C and 5%
CO,. On the day of the experiment the media in the 6 well plates was changed to fresh growth media. All antisensc oligonucleotides were diluted to the concentration of 20 1M. Two pl of this solution was incubated with 400 pl of Opti-MEM media (Gibco cat43I985-070) and 4 pl of Lipofectamine 2000 (Invitrogen cat# 11668019) at room temperature for 20 min and applied to each well of the 6 well plates with 518A2 cells. Similar mixture including 2 pi of water instead of the oligonuclcotide 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 antisensc oligonucleotides the media was removed and RNA was extracted from the cells using SV Total RNA Isolation System from Promega (cat # Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat# 74181) following the manufacturers' instructions. 600 ng of RNA was added to the reverse transcription reaction performed using Verso eDNA kit from Thermo Scientific (cat#AB1453B) or High Capacity eDNA 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 (cat94369510) and primers/probes designed by ABI (Applied Biosystcros Taqman Gene Expression Assay: Hs00275843_s l by Applied Biosystcms 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 l min) using StepOne Plus Real Time PCR Machine (Applied Biosystems).
1002361 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.
Results:
1002371 Real Time PCR results show that levels of IRS2 mRNA in 518A2 cells are significantly increased 48h after treatment with siRNAs to TFE3 antisense Hs.708291 (Fig 1).
1002381 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.
1002391 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.

Claims (37)

1. A method of modulating a function of and/or the expression of an Insulin Receptor Substrate 2 (IRS2) and/or Transcription factor E3 (TFE3)Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) 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 at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within nucleotides 1 to 497 of SEQ ID NO:3 thereby modulating a function of and/or the expression of the Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2(IRS2) polynucleotide in patient cells or tissues in vivo or in vitro.

2. A method of modulating a function of and/or the expression of an Transcription factor E3 (TFE3) mid/or Insulin Receptor Substrate 2(IRS2) polynucleotide in patient cells or tissues in vivo or in vitro comprising:
contacting said cells or tissues with at least one ant-sense 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 an Transcription factor E3 (TFE3) and/or insulin Receptor Substrate 2(IRS2) polynucleotide, thereby modulating a function of and/or the expression of the Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide in patient cells or tissues in vivo or in vitro.

3. A method of modulating a function of and/or the expression of an Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2(IRS2) 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 Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide; thereby modulating a function of and/or expression of the Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide in patient cells or tissues in vivo or in vitro.

4. A method of modulating a function of and/or the expression of an Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2(IRS2) polynucleotide in patient cells or tissues in vivo or in vitro.
comprising:
contacting said cells or tissues with at least one antisense oligonucleotide that targets a region of a natural antisense oligonucleotide of the Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide; thereby modulating a function of and/or the expression of the Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide in patient cells or tissues in vivo or in vitro.

5. The method of claim 4, wherein a function of and/or the expression of the Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) is increased in vivo or in vitro with respect to a control.

6. The method of claim 4, wherein the at least one antisense oligonucleotide targets a natural antisense sequence of an Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide.

7. The method of claim 4, wherein the at least one antisense oligonucleotide targets a nucleic acid sequence comprising coding and/or non-coding nucleic acid sequences of an Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide.

8. The method of claim 4, wherein the at least one antisense oligonucleotide targets overlapping and/or non-overlapping sequences of an Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide.

9. The method of claim 4, wherein the at least one antisense oligonucleotide comprises one or more modifications selected from: at least one modified sugar moiety, at least one modified internucleoside linkage, at least one modified nucleotide, and combinations thereof.

10. The -method of claim 9, wherein the one or more modifications comprise at least one modified sugar moiety selected from: a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and combinations thereof.

11. The method of claim 9, wherein the one or more modifications comprise at least one modified internucleoside linkage selected from: a phosphorothioate, 2'- Omethoxyethyl (MOE), 2'-fluoro, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.

12. The method of claim 9, 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.

13. The method of claim 1, wherein the at least one oligonucleotide comprises at least one oligonucleotide sequences set forth as SEQ ID NOS: 4 to 9.

14. A method of modulating a function of and/or the expression of an Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) 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 an Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2(IRS2) 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 Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide, and, modulating a function of and/or the expression of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) in mammalian cells or tissues in vivo or in vitro.

15. The method of claim 14, 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 Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide.

16. A method of modulating a function of and/or the expression of Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) 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 an Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide wherein said at least one antisense oligonucleotide has at least 50% sequence identity to at least one nucleic acid sequence set forth as SEQ ID NOS: 1, 2, 5; and, modulating the function and/or expression of the Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) in mammalian cells or tissues in vivo or in vitro.

17. 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 internucleotide 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 an Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) gene in vivo or in vitro as compared to a normal control.

18. The oligonucleotide of claim 17, wherein the at least one modification comprises an internucleotide linkage selected from the group consisting of: phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.

19. The oligonucleotide of claim 17, wherein said oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

20. The oligonucleotide of claim 17, wherein said oligonucleotide comprises a backbone of phosphorothioate internucleotide linkages.

21. The oligonucleotide of claim 17, 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.

22. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from:
phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and a combination thereof.

23. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from:
peptide nucleic acids, locked nucleic acids (LNA), analogues, derivatives, and a combination thereof.

24. The oligonucleotide of claim 17, wherein the oligonucleotide comprises at least one modified sugar moiety selected from: a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.

25. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified sugar moieties selected from: a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.

26. The oligonucleotide of claim 17, wherein the oligonucleotide is of at least about 5 to 30 nucleotides in length and hybridizes to an antisense and/or sense strand of an Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide wherein said oligonucleotide has at least about 20%
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 Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide.

27. The oligonucleotide of claim 17, 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 Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide.

28. The oligonucleotide of claim 17, wherein said oligonucleotide hybridizes to and modulates expression and/or function of at least one Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide in vivo or in vitro, as compared to a normal control.

29. The oligonucleotide of claim 17, wherein the oligonucleotide comprises the sequences set forth as SEQ ID
NOS: 4 to 9.

30. A composition comprising one or more oligonucleotides specific for one or more Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotides, said polynucleotides comprising antisense sequences, complementary sequences, alleles, homologs, isoforms, variants, derivatives, mutants, fragments, or combinations thereof.

31. The composition of claim 30, wherein the oligonucleotides have at least about 40% sequence identity as compared to any one of the nucleotide sequences set forth as SEQ ID NOS: 4 to 9.

32. The composition of claim 30, wherein the oligonucleotides comprise nucleotide sequences set forth as SEQ ID NOS: 4 to 9.

33. The composition of claim 32, wherein the oligonucleotides set forth as SEQ
ID NOS: 4 to 9 comprise one or more modifications or substitutions.

34. The composition of claim 33, wherein the one or more modifications are selected from; phosphorothioate, methylphosphonate, peptide nucleic acid, locked nucleic acid (LNA) molecules, and combinations thereof .

35. A method of preventing or treating a disease associated with at least one Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide and/or at least one encoded product thereof, comprising:
administering to a patient a therapeutically effective dose of at least one antisense oligonucleotide that binds to a natural antisense sequence of said at least one Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide and modulates expression of said at least one Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2(IRS2) polynucleotide;
thereby preventing or treating the disease associated with the at least one Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2(IRS2) polynucleotide and/or at least one encoded product thereof.

36. The method of claim 35, wherein a disease associated with the at least one Transcription factor E3 (TFE3) and/or Insulin Receptor Substrate 2 (IRS2) polynucleotide is selected from, type 1 and type 2 diabetes, an insulin resistant non diabetic state (e.g., obesity, impaired glucose tolerance (IGT) and Metabolic Syndrome), Polycystic Ovary Syndrome, atherosclerosis, cancer, a disease associated with apoptosis, aging and senescence, and a neurodegenerative disease or disorder (e.g Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis etc.

37. A method of identifying and selecting at least one oligonucleotide for in vivo administration comprising selecting a target polynucleotide associated with a disease state, identifying at least one oligonucleotide comprising at least five consecutive nucleotides which are complementary to the selected target polynucleotide or to a polynucleotide that is antisense 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.