AU2007221812B2 - Antisense modulation of PTP1B expression - Google Patents

Description

Australian Patents Act 1990 - Regulation 3.2 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Antisense modulation of PTP I B expression The following statement is a full description of this invention, including the best method of performing it known to me: P/00/011 5951 P\WPDOCS\CRN\RMH\Spc\20287356div spe dck -3/10/2007 ANTISENSE MODULATION OF PTP1B EXPRESSION This application is a divisional application of Australian patent application No. 2002309819, itself a 5 divisional application of both Australian patent application Nos. 2001226246 and Australian patent application Nos. 2001278077. BACKGROUND OF THE INVENTION 10 The process of phosphorylation, defined as the attachment of a phosphate moiety to a biological molecule through the action of enzymes called kinases, represents one course by which intracellular signals are propagated resulting finally in a cellular response. Within the 15 cell, proteins can be phosphorylated on serine, threonine or tyrosine residues and the extent of phosphorylation is regulated by the opposing action of phosphatases, which remove the phosphate moieties*. While the majority of protein phosphorylation within the cell is on serine and 20 threonine residues, tyrosine phosphorylation is modulated to the greatest extent during oncogenic transformation and growth factor stimulation.(Zhang, Crit. Rev. Biochem. Mol. Biol., 1998, 33, 1-52). Because phosphorylation is such a ubiquitous process 25 within cells and because cellular phenotypes are largely influenced by the activity of these pathways, it is currently believed that a number of disease states and/or disorders are a result of either aberrant activation of, P \WDOCS\CRN\RMH\Spc\20287356 div spccdc - 3/10/2007 or functional mutations in, kinases and phosphatases. Consequently, considerable at-tention has been devoted recently to the characterization of tyrosine kinases and tyrosine phosphatases. 5 PTPlB (also known as protein phosphatase 1B and PTPN1) is an endoplasmic reticulum (ER)-associated enzyme originally isolated as the major protein tyrosine phosphatase of the human placenta (Tonks et al., J. Biol. Chem., 1988, 263, 6731-6737; Tonks et al., J. Biol. 10 Chem., 1988, 263, 6722-6730).

]A

P:\WPDOCS\CRN\RMfkSpc\20287356 div spcc doc - 3/10/2007 An essential regulatory role in signaling mediated by the insulin receptor has been established for PTP1B. PTP1B interacts with and dephosphorylates the activated insulin receptor both in vitro and in intact cells 5 resulting in the downregulation of the signaling pathway (Goldstein et al., Mol. Cell. Biochem., 1998, 182, 91-99; Seely et al., Diabetes, 1996, 45, 1379-1385). In addition, PTP1B modulates the mitogenic actions of insulin (Goldstein et al., Mol. Cell. Biochem., 1998, 10 182, 91-99). In rat adipose.cells overexpressing PTP1B, the translocation of the GLUT4 glucose transporter was inhibited, implicating PTP1B as a negative regulator of glucose transport as well (Chen et al., J. Biol. Chem., 1997, 272, 8026-8031). 15 Mouse knockout models lacking the PTP1B gene also point toward the negative regulation of insulin signaling by PTP1B. Mice harboring a disrupted PTP1B gene showed increased insulin sensitivity, increased phosphorylation of the insulin receptor and when placed on a high-fat 20 diet, PTP1B -/- mice were resistant to weight gain and remained insulin sensitive (Eichebly et al., Science, 1999, 283, 1544-1548). These studies clearly establish PTP1B as a therapeutic target in the treatment of diabetes and obesity. 25 PTP1B, which is differentially regulated during the cell cycle (Schievella et al., Cell. Growth Differ., 1993, 4, 239-246), is expressed in insulin sensitive tissues as two different isoforms that arise from alternate splicing of the pre-mRNA (Shifrin and Neel, J. 30 Biol. Chem., 1993, 268, 25376-25384). It was recently demonstrated that the ratio of the alternatively spliced products is affected by growth factors such as insulin 2 P \WPDOCS'CRN\RMfSpc\20287356 div sp doc - 3/10/2007 and differs in various tissues examined (Sell and Reese, Mol. Genet. Metab., 1999, 66, 189-192). In these studies it was also found that the levels of the variants correlated with the plasma insulin concentration and 5 percentage body fat and may therefore be used as a biomarker for patients with chronic hyperinsulinemia or type 2 diabetes. Liu and Chernoff have shown that PTP1B binds to and serves as a substrate for the epidermal growth factor 10 receptor (EGFR) (Liu and Chernoff, Biochem. J., 1997, 327, 139-145). Furthermore, in A431 human epidermoid carcinoma cells, PT1B was found to be inactivated by the presence of H 2 0 2 generated by the addition of EGF. These studies indicate that PTP1B can be negatively regulated 15 by the oxidation state of the cell, which is often deregulated during tumorigenesis (Lee et al., J. Biol. Chem., 1998, 273, 15366-15372-). Overexpression of PTP1B has been demonstrated in malignant ovarian cancers and this correlation was 20 accompanied by a concomitant increase in the expression of the associated growth factor receptor (Wiener et al., Am. J. Obstet. Gynecol., 1994, 170, 1177-1183). PTP1B has been shown to suppress transformation in NIH3T3 cells induced by the reu oncogene (Brown-Shimer et 25 al., Cancer Res., 1992, 52, 478-482), as well as in rat 3Y1 fibroblasts induced by v-srk, v-src, and v-ras (Liu et al., Mol. Cell. Biol., 1998, 18, 250-259) and rat-1 fibroblasts induced by bcr-abl (LaMontagne et al., Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 14094-14099). It 30 has also been shown that PTP1B promotes differentiation of K562 cells, a chronic myelogenous leukemia cell line, in a similar manner as does an inhibitor of the bcr-abl 3 P\WPDOCSICRN\RMH\Spec\20287356div spec doc - 3/1/2007 oncoprotein. These studies describe the possible role of PTP1B in controlling the pathogenesis of chronic myeloid leukemia (LaMontagne et al., Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 14094-14099). 5 PTP1B negatively regulates integrin signaling by interacting with one or more adhesion-dependent signaling components and repressing int'egrin-mediated MAP kinase activation (Liu et al., Curr. Biol., 1998, 8, 173-176). Other studies designed to study integrin signaling, using 10 a catalytically inactive form of PTP1B, have shown that PTP1B regulates cadherin-mediated cell adhesion (Balsamo et al., J. Cell. Biol., 1998, 143, 523-532) as well as cell spreading, focal adhesion and stress fiber formation and tyrosine phosphorylation (Arregui et al., J. Cell. 15 Biol., 1998, 143, 861-873). Currently, therapeutic agents designed to inhibit the synthesis or action of PTP1B include small molecules (Ham et al., Bioorg. Med. Che'm. Lett., 1999, 9, 185-186; Skorey et al., J. Biol. Chem.., 1997, 272, 22472-22480; 20 Taing et al., Biochemistry, 1999, 38, 3793-3803; Taylor et al., Bioorg. Med. Chem., 1998, 6, 1457-1468; Wang et al., Bioorg. Med. Chem. Lett., 1998, 8, 345-350; Wang et al., Biochem. Pharmacol., 199.7, 54, 703-711; Yao et al., Bioorg. Med. Chem., 1998, 6, 1799-1810) and peptides 25 (Chen et al., Biochemistry, 1999, 38, 384-389; Desmarais et al., Arch. Biochem. Biophys., 1998, 354, 225-231; Roller et al., Bioorg. Med. Cbem. Lett., 1998, 8, 2149 2150). In addition, disclosed in the PCT publication WO 97/32595 are phosphopeptides and antibodies that inhibit 30 the association of PTP1B with the activated insulin receptor for the treatment of disorders associated with 4 C.\NRPobl\DCOCGRS\2778599_I DOC-3/1812010 -5 insulin resistance. Antisense nucleotides against PTP1B are also generally disclosed in that document. There remains a long felt need for additional agents capable of effectively inhibiting PTP1B function and 5 antisense technology is emerging as an effective means for reducing the expression of specific gene products. This technology may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of PTP1B expression. 10 SUMMARY OF THE INVENTION According to a first aspect of the present invention there is provided an antisense oligonucleotide up to 50 nucleobases in length targeted to a nucleic acid molecule 15 encoding PTP1B, wherein said oligonucleotide specifically hybridizes with exon 10 of PTP1B thereby inhibiting the expression of PTP1B, and wherein said oligonucleotide comprises a sequence set forth in SEQ ID NO: 315 or 316. According to a second aspect of the present invention 20 there is provided a composition comprising an antisense oligonucleotide of the first aspect and a pharmaceutically acceptable carrier or diluent. According to a third aspect of the present invention there is provided a method of inhibiting the expression of 25 PTP1B in cells or tissues comprising contacting said cells or tissues with an antisense oligonucleotide of the first aspect or a composition of the second aspect such that expression of PTP1B is inhibited.According to a fourth aspect of the present invention there is provided a method 30 of treating an animal having or C:\NRPoblDCCRS\2778599_ DOC-3/18/2010 - 5a suspected of having a disease or condition associated with PTP1B comprising administering to said animal a therapeutically or prophylactically effective amount of an antisense oligonucleotide of the first aspect or a 5 composition of the second aspect such that expression of PTP1B is inhibited. According to a fifth aspect of the present invention there is provided a method of decreasing blood glucose levels in an animal comprising administering to said animal 10 an antisense oligonucleotide of the first aspect or a composition of the second aspect. According to sixth aspect of the present invention there is provided a method of preventing or delaying the onset of a disease or condition associated with PTP1B in an 15 animal comprising administering to said animal a therapeutically or prophylactically effective amount of an antisense oligonucleotide of the first aspect or a composition of the second aspect. According to a seventh aspect of the present invention 20 there is provided a method of preventing or delaying the onset of an increase in blood glucose levels in an animal comprising administering to said animal an antisense oligonucleotide of the first aspect or a composition of the second aspect. 25 According to an eighth aspect of the present invention there is provided use of an antisense oligonucleotide of the first aspect for the manufacture of a medicament for inhibiting the expression of PTP1B in cells or tissues. According to a ninth aspect of the present invention 30 there is provided use of an antisense oligonucleotide of the first apsect for the manufacture of a medicament for C:\NRPorbl\DCC1GRS\2778599 1.DOC-3/1s/2010 - 5b treating an animal having or suspected of having a disease or condition associated with PTP1B, or preventing or delaying the onset of such a disease or condition. According to a tenth aspect of the present invention 5 there is provided use of an antisense oligonucleotide of the first aspect for the manufacture of a medicament for decreasing blood glucose levels or preventing or delaying the onset of an increase in blood glucose levels. The present invention provides compositions and methods 10 for modulating the expression of PTP1B. In particular, the present invention provides compositions and methods for modulating the alternatively spliced form of PTP1B. This invention relates to antisense compounds, particularly antisense oligonucleotides, specifically hybridizable with 15 and targeted to a nucleic acid encoding PTP1B. Such oligonucleotides have been shown to modulate the expression of PTPlB. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. 20 Further provided are methods of modulating the expression of PTP1B in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, 25 particularly a human, suspected of having or being prone to a disease or condition associated with expression of PTP1B by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.

P.\WDOC\CRN\RMfSpc\2028735b div spc doc - 3/10/2007 Other aspects and advantages of this invention are encompassed in the following detailed description of the invention. 5 DETAILED DESCRIPTION OF THE INVENTION The present invention employs oligomeric antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding PTP1B, ultimately modulating the amount of PTP1 10 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding PTP1B. As used herein, the terms "target nucleic acid" and "nucleic acid encoding PTPlB".encompass DNA encoding 15 PTP1B, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. 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 20 target nucleic acid by compounds which specifically hybridize to it is generally referred to as "antisense". The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, 25 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 30 interference with target nucleic acid function is modulation of the expression of PTP1B. In the context of the present invention, "modulation" means either an increase (stimulation) or a 6' P:\WPDOCSCRN\RMI'Spcc0287356divsp doc- 3/1(1'2007 decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target. 5 It is preferred to target specific nucleic acids for antisense. "Targeting" an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence 10 whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the 15 target is a nucleic acid molecule encoding PTP1B. The targeting process also includes determination of a site or sites within this gene for. the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. 20 Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation 25 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 have a translation initiation codon 30 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 7 P:\WPDOCSCRN\RMH\Spec\20287356 div spc doc -3/10/2007 though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more 5 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 10 codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding PTP1B, regardless of-the sequence(s) of such codons. It is also known in the art that a translation 15 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 are 5'-TAA, 5'-TAG and 5'-TGA, respectively). The terms "start codon region" and "translation initiation codon region" refer to a 20 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 25 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. The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between 30 the translation initiation codon and the translation termination codon, is also a -region which may be targeted effectively. Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to P \WPDOCSCRN\RM I Spoc\20287356 div spec do -3/10/2007 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 5 on the gene, and the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding 10 nucleotides on the gene. The 5' cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 15 nucleotides adjacent to the cap. The 5' cap region may also be a preferred target region. Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," which are-excised from a transcript 20 before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations 25 where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. It has also been found that introns 30 can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA. 9 P;\WPDOCS\CRN\RMHfSpec\20287356 div specdoc - 3/10/2007 Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired 5 effect. In the context of this invention, "hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For 10 example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. "Complementary," as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an is oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and -the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are 20 complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of 25 complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is' understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be 30 specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of P.WPDOCS\CRN\RMH Spe\202730 div sp mdo - 3/1012007 utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., 5 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. Antisense compounds are commonly used as research 10 reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for 15 example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use. The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic 20 uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is 25 thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especial-ly humans. In the context of this invention, the term 30 "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and 11 P WPDOCS\CRN\RMI Spe\20287356 di, spe do -3/10'2007 covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms 5 because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. While antisense oligonucleotides are a preferred 10 form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from 15 about 8 to about 50 nucleobases (i.e. from about 8 to about 50 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 30 nucleobases. As is known in the art, a nucleoside is 20 a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to 25 the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar.' In forming oligonucleo tides, the phosphate groups covalently link adjacent 30 nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are 12 P:\WPDOCS\CRN\RMH\Spcc2O287356 div spcc doc - 3/10/2007 generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA 5 and DNA is a 3' to 5' phosphodiester linkage. Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleo side linkages. As defined in this specification, 10 oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modifi.ed oligonucleotides that do 15 not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, 20 aminoalkylphosphotriesters, Tethyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphos phoramidates, thionophosphoramidates, thionoalkylphos 25 phonates, thionoalkylphosphotriesters, and boranophos phates 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 30 free acid forms are also included. Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S.: 3,687,808; P:\WPDOCS\CRN\RMHSpec\20287356 div spcdoc -3/10/2007 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 5,563,253; 5,571,799; 5,587,361; and [COMMENT][a]Page: 1 The patent No. 5,697,248 was found to be wrong. On 9 3-98 deleted it and changed the wording from "5,625,050; and 5,697,248" to " and 5,625;050" 5,625,050, certain of which are commonly owned with this application, and each 10 of which is herein incorporated by reference. Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or 15 cycloalkyl internucleoside li-nkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone 20 backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having 25 mixed N, 0, S and CH 2 component parts. Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited' to, U.S.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 30 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,&46; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 14.

PNWPDOCS\CR\R M fSpm20287356 div specdoc .3!10'2007 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference. In other preferred oligonucleotide mimetics, both 5 the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that 10 has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. 15 The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S.: 5,539,082; 5,714,331; and 20 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500. Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and 25 oligonucleosides with heteroatom backbones, and in particular -CH 2

-NH-O-CH

2 -, -CH 2

-N(CH

3

)-O-CH

2 - [known as a methylene (methylimino) or MMI backbone], -CH 2 -0-N(CH 3 ) CH 2 -, -CH 2

-N(CH

3

)-N(CH

3

)-CH

2 - and -O-N(CH 3

)-CH

2

-CH

2 [wherein the native phosphodiester backbone is 30 represented as -O-P-0-CH 2 -] of the above referenced U.S. patent 5,489,677, and the amide backbones of the above referenced U.S. patent 5,602,240. Also preferred are 15 P :WPDOCS\CRN\RMH\Spc\2O0287356 di sp doc - 3/10/2007 oligonucleotides having morph.olino backbone structures of the above-referenced U.S. patent 5,034,506. Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligo 5 nucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N alkenyl; 0-, S- or N-alkynyl;' or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and 10 alkynyl. Particularly preferred are O[(CH 2 )nO]mCH 3 , O (CH 2 ) nOCH 3 , O (CH 2 )nNH 2 , O (CH 2 ) nCH 3 , O (CH 2 ) nONH 2 , and

O(CH

2 )nON[(CH 2 )nCH 3

)]

2 , where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position:- C 1 to C 10 lower alkyl, 15 substituted lower alkyl, alkaryl, aralkyl, 0-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 ,

SO

2

CH

3 , ON0 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocyclo alkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an 20 intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2' 25 methoxyethoxy (2'-O-CH 2

CH

2 0CH 3 , also known as 2'-O-(2 methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2'-dimethylamino oxyethoxy, i.e., a O(CH 2

)

2 0N(CH 3

)

2 group, also known as 2' 30 DMAOE, as described in examples hereinbelow, and 2' dimethylaminoethoxyethoxy (also known in the art as 2'-0 dimethylaminoethoxyethyl or 2' -DMAEOE), i.e., 2 '-O-CH 2 -0

CH

2

-N(CH

2

)

2 , also described in examples hereinbelow. 16 P \WPDOCS\CRN\RMHSpcc\20287356div spc doc -3/10/2007 Other preferred modifications include 2'-methoxy (2 ' -O-CH 3 ) , 2 ' -aminopropoxy (2.' -OCH 2

CH

2

CH

2

NH

2 ) and 2' fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 5 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States 10 patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S.: 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; 15 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety. Oligonucleotides may also include nucleobase (often 20 referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). -Modified nucleobases 25 include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2 30 thiouracil, 2-thiothymine and-2-thiocytosine, 5 halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8 17 P \WPDOCS\CRN\RMH\Spc\2f)287356 di s doc 3/10/2O7 thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoro methyl and other 5-substituted uracils and cytosines, 7 methylguanine and 7-methyladenine, 8-azaguanine and 8 5 azaadenine, 7-deazaguanine and 7-deazaadenine and 3 deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, 10 Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B. 15 , ed., CRC Press, 1993. Cert-ain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 20 aminopropyladenine, 5-propynyluracil and 5-propynyl cytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca 25 Raton, 1993, pp. 276-278) and'are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Representative United States patents that teach the preparation of certain of the above noted modified 30 nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. 3,687,808, as well as U.S.: 4',845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 18 P-\WPDOCSkCRNJRM\fSpcc\20287356 di, s doc - 3/10/2007 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, certain of which are commonly owned with the instant application, and each'of which is herein 5 incorporated by reference, and United States patent 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference. Another modification of the oligonucleotides of the invention involves chemically linking to the oligo 10 nucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 15 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994; 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a 20 thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov.et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49 25 54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-0-hexadecyl-rac-glycero-3-H phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain 30 (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl 19 P\WPDOCS'CRNNRMrSpec\20287356 div s doc - 3/10/2007 moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937. 5 Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S.: 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; 10 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; 4,824,9'41; 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; 15 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, certain of which are commonly 20 owned with the instant application, and each of which is herein incorporated by reference. It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be 25 incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. "Chimeric" antisense compounds or "chimeras," in the context of this invention, are 30 antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These 20 P.\WPDOCS\CRN\RMMrSpe\20287356 di s doc -3/10/2007 oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased 5 binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an 10 RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when 15 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 20 techniques known in the art. Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as 25 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 include, but are not limited to, U.S.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 30 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety. 21 P:WPDOCS\CRN\RMHSpe 273 d sp doc - 3/10/2007 The antisense compounds 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 5 several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as 10 the phosphorothioates and alkylated derivatives. The antisense compounds of the invention are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of 15 antisense molecules. The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or 20 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.: 5,108,921; 5,354,844; 5,416,016; 25 5,459,127; 5,521,291; 5,543,158; 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; 30 and 5,595,756, each of which is herein incorporated by reference. The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts P,\WPDOCS\CRN\RM Spc\2027356 di spe doc - 3/10/2007 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. Accordingly, for 5 example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. The term "prodrug" indicates a therapeutic agent 10 that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are 15 prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., 'published December 9, 1993 or in WO 94/26764 to Imbach et al. The term "pharmaceutically acceptable salts" refers 20 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. 25 Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N'-dibenzyl 30 ethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., "Pharmaceutical Salts," J. of.Pharma Sci., 1977, 66, 1- P.\WPDOCS\CRN\RMHf\Spc\22873S6div sp cdo 3/10/2007 19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be 5 regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are 10 equivalent to their respective free acid for purposes of the present invention. As used herein, a "pharmaceutical addition salt" includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or 15 inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and 20 organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids,- for example acetic acid, 25 propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, 30 salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of P \WPDOCSCRNRMlfHSpc\20287356 di spc doc - 310'2007 proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 5 4-methylbenzenesulfoc acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phospho glycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. 10 Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. 15 Carbonates or hydrogen carbonates are also possible. For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such 20 as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic 25 acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, 30 naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

P \WPDOCSCRN\RMII\Sp-\20287356 div padc - 3/I0/2007 The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of 5 having a disease or disorder which can be treated by modulating the expression of PTP1B is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an 10 effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for 15 example. The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding PTP1B, enabling sandwich and other assays to easily be constructed to 20 exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding PTP1B can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or 25 any other suitable detection means. Kits using such detection means for detecting the level of PTP1B in a sample may also be prepared. The present invention also includes pharmaceutical compositions and formulations which include the antisense 30 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 P\WPDOCSICRN\RMfSpc\20287356 div s doc - 3/10!2007 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 5 nebulizer; intratracheal, int'ranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or 10 intraventricular, administration. Oligonucleotides with at least one 2'-0-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, 15 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. 20 Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be 25 desirable. Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, 30 but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. 27 P:\WPDOCS\CRN\RMH\Spcc\20287356 d spe dc -3/10/2007 Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of 5 components that include, but are not limited to, preformed liquids, self-emulsifying solids and self emulsifying semisolids. The pharmaceutical formulations of the present invention, which may conveniently be presented in unit 10 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 pharma ceutical carrier(s) or excipient(s). In general the 15 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, shapi-ng the product. The compositions of the present invention may be 20 formulated into any of many possible dosage forms such as, but not limited to, tablets, 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. 25 Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. In one embodiment of the present invention the 30 pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar 28 P:\WPDOCS\CRN\RMHI\Spc\20287356 di spedoc - 3/10'2007 in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts 5 and may be applied to the formulation of the compositions of the present invention. Emulsions: The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one 10 liquid dispersed in another in the form of droplets usually exceeding 0.1 m in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, 15 Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical 20 Sciences, Mack Publishing Co., Easton, PA, 1985, p. 301). Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other. In general,.emulsions may be either water-in-oil (w/o) or of the oil-in-water (o/w) variety. 25 When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as'minute droplets into a bulk 30 aqueous phase the resulting composition is called an oil in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in 29 P-\WPDOCSCRN\RMH\Spcc\2027356div spa dc - 3/10/2007 either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical 5 emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple 10 binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o 15 emulsion. Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in 20 this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of 25 emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, 30 Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Synthetic surfactants,.also known as surface active agents, have found wide applicability in the formulation 30 P.\WPDOCS\CRN\RMH\Spec'20287356 div sp d - 3/10'2007 of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical 5 Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant 10 has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, 15 anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, .Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285). Naturally occurring emulsifiers used in emulsion 20 formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic 25 petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, 30 hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate. 31.

P.\WPDOCS\CRN\RMH\Spec\20287356 div scdoc - 3/10/2007 A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, 5 humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker *(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker 10 (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, 15 carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethyl cellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water 20 to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed phase droplets and by increasing the viscosity of the external phase. Since emulsions often contain a number of 25 ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used-preservatives included in emulsion formulations include methyl paraben, propyl 30 paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.

P.\WPDOCS\CRN\RM \Spe\20287356 div pc doc - 3/10/2007 Antioxidants used may be free-radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant 5 synergists such as citric acid, tartaric acid, and lecithin. The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the 10 literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume.1, p. 199). Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from 15 an absorption and bioavailability standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker.(Eds.), 1988, Marcel Dekker, 20 Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions. In one embodiment of the present invention, the 25 compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, 30 Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and P\WPDO CS\CRN\RM\ Spec20287356 d doc - 3/10/2007 then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically 5 clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Micro 10 emulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in water (o/w) type is dependent on the properties of the 15 oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 1985, p. 271). 20 The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker 25 (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the 30 advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously. 34' P.WPDOCSkCRN\M H\iSpc\20287356 div sedoc -3/10/2007 Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers,- polyglycerol fatty acid 5 esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (M0310), hexaglycerol monooleate (P0310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (M0750), decaglycerol sequioleate (S0750), decaglycerol decaoleate 10 (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered 15 film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited 20 to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium 25 chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil. 30 Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral 35 P-\WPDOCS\CRN\RMH\Spec\20287356 div spec doc - 3!10'2007 bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford 5 advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, 10 improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. 15 This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been-effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected 20 that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligon~icleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic 25 acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration. Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and 30 penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the micro- P \WPDOCS\CRN\RM f\Spe\20287356div spe doc - 3110!2007 emulsions of the present invention may be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non chelating non-surfactants (Lee et al., Critical Reviews 5 in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above. Liposomes: There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include 10 monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of *drug delivery. As used in the present invention, the term "liposome" means a 15 vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains 20 the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo. 25 In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable 30 and able to pass through such fine pores. Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of 37 P WPDOCS\CRNRMH\Spa\20287356 div spc doc -3/10/2007 water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, 5 Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the.liposomes. Liposomes are useful for the transfer and delivery 10 of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with-the cellular membranes. As the merging of the liposome and cell progresses, the 15 liposomal contents are emptied into the cell where the active agent may act. Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical 20 administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability 25 to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin. Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including 30 analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.

P \WPDOCSCRN\RMH\Spec\2027356 div spec do- 3/I 02007 Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome 5 complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985). 10 Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous 15 interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274). 20 One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidyl choline. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome 25 compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanol amine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for 30 example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol. 39 P:\WPDOCS\CRN\RMH\Spe 287356 div spc cdoc -3/10/2007 Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while 5 delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal 10 formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265). Non-ionic liposomal systems have also been examined 15 to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising NovasomeJ I (glyceryl dilaurate/ cholesterol/polyoxyethylene-10-stearyl ether) and 20 NovasomeJ II (glyceryl distearate/cholesterol/ polyoxy ethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A 25 into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466). Liposomes also include ''sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, 30 when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming 40 P:\WPDOCS\CRN\RMI\Spe\2O287356 di sp doc -3/10/2007 lipid portion of the liposomes (A) comprises one or more glycolipids, such as monosialoganglioside Gmj, or (2) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing 5 to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a 10 reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765). Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 15 1987, 507,64) reported the ability of monosialoganglio side GmI, galactocerebroside sulfate and phosphatidyl inositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988', 85, 6949). U.S. Patent 20 No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside Gm or a galactocerebroside sulfate ester. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 25 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.). Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et 30 al. (Bull. Chem. Soc. Jpn., 1-980, 53, 2778) described liposomes comprising a nonionic detergent, 2C 12 15G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 41 P:\WPDOCS\CRN\RMISpeO 287356 div spc dm - 3/10/2007 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of 5 polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Patent Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidyl ethanolamine (PE) derivatized with PEG or PEG stearate 10 have significant increases in blood circulation half lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine 15 (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use 20 thereof, are described by Woodle et al. (U.S. Patent Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Patent No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Patent 25 No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Patent Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe PEG-containing 30 liposomes that can be further-derivatized with functional moieties on their surfaces. A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et 42 P \WPDOCS\CRN\RMH\Spa%20287356 div spe - 3/10/2007 al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Patent No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes 5 may include an antisense RNA.- U.S. Patent No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising antisense oligonucleotides targeted to the raf gene. 10 Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to 15 penetrate through pores which-are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self repairing, frequently reach their targets without 20 fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated 25 delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin. Surfactants find wide application in formulations such as emulsions (including microemulsions) and 30 liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and'synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of P.\WPDOCSiCRN\RMi\Spec\227356 di specdoc -3/10/2007 the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New 5 York, NY, 1988, p. 285). If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH 10 values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic.esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and 15 ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant 20 class. If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl 25 amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates,'and phosphates. The most important members of the anionic surfactant class are the 30 alkyl sulfates and the soaps. If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic 44 P:\WPDOCS\CRN\RMH\Spec2O287356 d spe doc - 3/10/2007 surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class. If the surfactant molecule has the ability to carry 5 either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N alkylbetaines and phosphatides. The use of surfactants in drug products, 10 formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285). Penetration Enhancers:- In one embodiment, the present invention employs various penetration enhancers 15 to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has 20 been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of 25 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 non-surfactants (Lee et al., Critical 30 Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

PAWPDOCS\CRN\RMH\Spcc\20287356 div s doc -3/102007 Surfactants: In connection with the present invention, surfactants (or "surface-active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or 5 the interracial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, 10 polyoxyethylene-9-lauryl ether and polyoxyethylene-20 cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252). 15 Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, 20 tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1 monocaprate, 1-dodecylazacycloheptan-2-one, acylcarni tines, acylcholines, Ci 10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and diglycer 25 ides thereof (i.e., oleate, lAurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. 30 Pharm. Pharmacol., 1992, 44, 651-654). Bile salts: The physiological role of bile includes the facilitation of.dispersion and absorption of 46 P.\WPI)OCS\CRN\RMH\Spec\20287356 div sp dc -3/10/2007 lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, 5 and their synthetic derivatives, act as penetration enhancers. Thus the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its 10 pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), 15 taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether 20 (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, pages 782 783; Muranishi, Critical Reviews in Therapeutic Drug 25 Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263,-25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583). Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as 30 compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is 47 P \WPDOCS\CRN\RMH\Spe\20287356 div spcdc 3,1W/2007 enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent 5 metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J., Chromatogr., 1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium 10 salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones' (enamines) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic 15 Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51). Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate 20 insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews'in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration 25 enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti inflammatory agents such as diclofenac sodium, 30 indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626). 48 P \WPDOCSICRN\RMH\Spec2028 7 356 d spcc doc - 3110'2007 Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as 5 lipofectin (Junichi et al, U.S. Patent No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides. 10 Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone. 15 Carriers: Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological 20 activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The 25 coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to 30 competition between the carri-er compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with 49 P:\WPDOCS\CRN\RMif\Spcc\20287356 div s dc - 3/1102007 polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., Antisense Res..Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 5 6, 177-183). Excipients: In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering 10 one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given 15 pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, 20 pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, 25 polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.). Pharmaceutically acceptable organic or inorganic 30 excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the- compositions of the present invention. Suitable pharmaceutically acceptable carriers 50 P.\WPDOCSiCRN\AR.M \Spc\20287356 di spc doc -310/2007 include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellul-ose, polyvinylpyrrolidone 5 and the like. Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid 10 or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can 15 be used. Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, 20 hydroxymethylcellulose, polyvinylpyrrolidone and the like. Other Components: The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical 25 compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain 30 additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and 51 P \WPDOCS\CRN\RMI\Spec\20287356 div spcc doc - 3/10/2007 stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if 5 desired, mixed with auxiliary.agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic 10 acid(s) of the formulation. Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. 15 Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such.chemotherapeutic agents 20 include, but are not limited to, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine 25 (5-FUdR), methotrexate (MTX),' colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 1206-1228). Anti 30 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 52 P.WPDOCS\CRN\R *MH 0287356 s d s doc. 3/102007 also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense 5 chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially. In another related embodiment, compositions of the invention may contain one or more antisense compounds, 10 particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together 15 or sequentially. The formulation of therapeutic compositions and their subsequent administration 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 20 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 25 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 EC 50 s found to be 30 effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug 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. 53 P \WPDOCSiCRN\ARMH\Spcc\2287356 di s doc -3/1W2007 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 5 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 pg to 100 g per kg of body weight, once or more daily, to once every -20 years. 10 While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. 15 EXAMPLES EXAMPLE 1: NUCLEOSIDE PHOSPHORAMIDITES FOR OLIGONUCLEOTIDE SYNTHESIS DEOXY AND 2'-ALKOXY AMIDITES 2'-Deoxy and 2'-methoxy'beta-cyanoethyldiisopropyl 20 phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham, MA or Glen Research, Inc., Sterling, VA). Other 2'-0-alkoxy substituted nucleoside amidites are prepared as described in U.S. Patent 5,506,351, herein incorporated by reference. For 25 oligonucleotides synthesized using 2'-alkoxy amidites, the standard cycle for unmodi-fied oligonucleotides was utilized, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds. Oligonucleotides containing 5-methyl-2' 30 deoxycytidine (5-Me-C) nucleotides were synthesized according to published methods (Sanghvi, et al, Nucleic Acids Research, 1993, 21, 3197-3203) using commercially 54 P:\WPDOCS\CRN\RMH\Sp:\20287356 di sp do -3/10,2007 available phosphoramidites (Glen Research, Sterling, VA, or ChemGenes, Needham, MA). 2'-FLUORO AMIDITES (a) 2'-FLUORODEOXYADENOSINE AMIDITES 5 2'-fluoro oligonucleotides were synthesized as described previously (Kawasaki, et al, J. Med. Chem., 1993, 36, 831-841) and United States patent 5,670,633, herein incorporated by reference. Briefly, the protected nucleoside N6-benzoyl-2'-deoxy-2'-fluoroadenosine was 10 synthesized utilizing commercially available 9-beta-D arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2'-alpha fluoro atom is introduced by a SN2-displacement of a 2' beta-trityl group. Thus N6-benzoyl-9-beta-D 15 arabinofuranosyladenine was selectively protected in moderate yield as the 3',5'-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies and standard methods were used to obtain the 5' 20 dimethoxytrityl-(DMT) and 5'-DMT-3'-phosphoramidite intermediates. (b) 2'-FLUORODEOXYGUA14OSINE The synthesis of 2'-deoxy-2'-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) 25 protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate diisobutyrylarabinofuranosylguanosine. Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected 30 arabinofuranosylguanine. Selective 0-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups.

P.\WPDOCS\CRN\RMIf\Spe\20287356 div s doc -3/1 C/2007 Standard methodologies were used to obtain the 5'-DMT and 5'-DMT-3'-phosphoramidites. (c) 2'-FLUOROURIDINE Synthesis of 2'-deoxy-2''-fluorouridine was 5 accomplished by the modificat.ion of a literature procedure in which 2,2'-anhydro-l-beta-D arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5'-DMT and 5'-DMT-3'phosphoramidites. 10 (d) 2'-FLUORODEOXYCYTIDINE 2'-deoxy-2'-fluorocytidine was synthesized via amination of 2'-deoxy-2'-fluorouridine, followed by selective protection to give N4-benzoyl-2'-deoxy-2' fluorocytidine. Standard procedures were used to obtain 15 the 5'-DMT and 5'-DMT-3'phosphoramidites. 2'-0-(2-METHOXYETHYL) MODIFIED AMIDITES 2'-0-Methoxyethyl-substituted nucleoside amidites are prepared as follows, or alternatively, as per the methods of Martin, P., Helvetica Chimica Acta, 1995, 78, 20 486-504. (a) 2,2'-ANHYDRO[1-(BETA-D-ARABINOFURANOSYL)-5 METHYLURIDINE] 5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 25 M), diphenylcarbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened 30 solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum P \WPDOCS\CRN\RMIf\Spc\20287356 div sp dc - 3/1/O2007 amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60 C at 1 mm Hg for 24 h) to give a solid that was crushed to 5 a light tan powder (57 g, 85% crude yield). The NMR spectrum was consistent with the structure, contaminated with phenol as its sodium salt (ca. 5%). The material was used as is for further reactions (or it can be purified further by column chromatography using a 10 gradient of methanol in ethyl acetate (10-25%) to give a white solid, mp 222-4 C) (b) 2'-0-METHOXYETHYL-5-METHYLURIDINE 2,2'-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2 15 methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160 C. After heating for 48 hours at 155-16 C, the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was 20 suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH 3 CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH 2 Cl 2 /acetone/MeOH.(20:5:3) containing 0.5% 25 Et 3 NH. The residue was dissolved in CH 2 Cl 2 (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product. Additional material was obtained by reworking impure fractions. 30 (c) 2'-O-METHOXYETHYL-5'-0-DIMETHOXYTRITYL-5 METHYLURIDINE 2'-0-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried ';7 P\WPDOCSCR\RMH\Spm\20287356di spcc doc - 3/10/2007 residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 5 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH 3 CN (200 mL). The residue was 10 dissolved in CHCl 3 (1.5 L) and extracted with 2x500 mL of saturated NaHCO 3 and 2x500 mL of saturated NaCl. The organic phase was dried over Na 2

SO

4 , filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and 15 eluted with EtOAc/hexane/acet'one (5:5:1) containing 0.5% Et 3 NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%). 20 (d) 3'-O-ACETYL-2'-O-METHOXYETHYL-5'-0 DIMETHOXYTRITYL-5-METHYLURIDINE 2'-0-Methoxyethyl-5'-0-dimethoxytrityl-5 methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of 25 pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room-temperature for 24 hours. The reaction was monitored by TLC by first quenching the TLC sample with the addition of MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL) was added 30 and the mixture evaporated at 35 C. The residue was dissolved in CHCl 3 (800 mL) and extracted with 2x200 mL of saturated sodium bicarbonate and 2x200 mL of saturated NaCl. The water layers were back extracted with 200 mL 58 P \WPDOCS\CRN\RMH\Spe\20287356 div s doc - 3O'2007 of CHCl 3 . The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/hexane(4:1). 5 Pure product fractions were evaporated to yield 96 g (84%). An additional 1.5 g was recovered from later fractions. (e) 3'-0-ACETYL-2'-0-METHOXYETHYL-5'-0 DIMETHOXYTRITYL-5-METHYL-4-TRIAZOLEURIDINE 10 A first solution was prepared by dissolving 3'-0 acetyl-2'-0-methoxyethyl-5'-0-dimethoxytrityl-5 methyluridine (96 g, 0.144 M) in CH 3 CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH 3 CN (1 L), cooled 15 to -5 C and stirred for 0.5 h using an overhead stirrer. POCl 3 was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10 C, and the resulting mixture stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to 20 the latter solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The 25 filtrate was washed with 1x300 mL of NaHCO 3 and 2x300 mL of saturated NaCl, dried over'sodium sulfate and evaporated. The residue was.triturated with EtOAc to give the title compound. (f) 2'-0-METHOXYETHYL-5'-0-DIMETHOXYTRITYL-5 30 METHYLCYTIDINE A solution of 3'-0-acetyl-2'-0-methoxyethyl-5'-0 dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH 4 0H (30 mL) was stirred at 59.

P \WPDOCS\CRMRMK\Spec20287356 d.v pc -. 3/10/2007 room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2x200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. 5 MeOH (400 mL) saturated with NH 3 gas was added and the vessel heated to 100 C for 2 hours (TLC showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The 10 organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound. (g) N4-BENZOYL-2'-0-METHOXYETHYL-5'-0 DIMETHOXYTRITYL-5-METHYLCYTIDINE 2'-0-Methoxyethyl-5'-0-dimethoxytrityl-5-methyl 15 cytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, TLC showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 20 mL). The residue was dissolved in CHC1 3 (700 mL) and extracted with saturated NaHCO 3 (2x300 mL) and saturated NaCl (2x300 mL), dried over MgSO 4 and evaporated to give a residue (96 g). The residue.was chromatographed on a 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% 25 Et 3 NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound. (h) N4-BENZOYL-2'-0-METHOXYETHYL-5'-0 DIMETHOXYTRITYL-5-METHYLCYTIDINE-3'-AMIDITE N4-Benzoyl-2'-O-methoxyethyl-5'-0-dimethoxytrityl 30 5-methylcytidine (74 g, 0.10 M) was dissolved in CH 2 Cl 2 (1 L). Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy tetra(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The 60 P \WPDOCS\CRN\RMH\Spcc\202S7356 div p d -3/10/2007 resulting mixture was stirred for 20 hours at room temperature (TLC showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO 3 (1x300 mL) and saturated NaCl (3x300 mL). The aqueous 5 washes were back-extracted with CH 2 Cl 2 (300 mL), and the extracts were combined, dried-over MgSO 4 and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g 10 (87%) of the title compound. 2'-O-(AMINOOXYETHYL) NUCLEOSIDE AMIDITES AND 2'-0 (DIMETHYLAMINOOXYETHYL) NUCLEOSIDE AMIDITES (a) 2'-(DIMETHYLAMINOOXYETHOXY) NUCLEOSIDE AMIDITES 15 2'-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the art as 2'-0-(dimethylaminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs[COMMENT] [1]Page: 1 T = cpds 102-108 from ISIS-2824.. Adenosine, cytidine and 20 guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine. 25 (b) 5'-O-TERT-BUTYLDIPHENYLSILYL-0 2 -2 ' -ANHYDRO-5 METHYLURIDINE 0 2 -2'-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylamino pyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved 30 in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was 61 P \WPDOCS\CRN\RMH\Spec20287356 div sp doc -3,10,2007 stirred for 16 h at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between dichloromethane 5 (1 L) and saturated sodium bicarbonate (2x1 L) and brine (1 L). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) and the solution was cooled to 10 10 0 C. The resulting crystalline product was collected by filtration, washed with ethyl ether (3x200 mL) and dried (40 0 C, 1mm Hg, 24 h) to 149g (74.8%) of white solid. TLC and NMR were consistent with pure product. (c) 5'-O-TERT-BUTYLDIPHENYLSILYL-2'-0-(2 15 HYDROXYETHYL)-5-METHYLURIDINE In a 2 L stainless steel, unstirred pressure reactor was added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and with manual stirring, ethylene glycol (350 mL, excess) was added cautiously at 20 first until the evolution of -hydrogen gas subsided. 5'-0 tert-Butyldiphenylsilyl-0 2 -2'-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature 25 of 160 0 C was reached and then maintained for 16 h (pressure < 100 psig). The reaction vessel was cooled to ambient and opened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T side product, ethyl acetate) indicated about 70% conversion to the product. In order to avoid 30 additional side product formation, the reaction was stopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100 0 C) with the more extreme conditions used to remove the ethylene glycol.

P-\WPDOCS\CRMRM1\Spc\20287356div spe doc -3/102007 [Alternatively, once the low boiling solvent is gone, the remaining solution can be partitioned between ethyl acetate and water. The product will be in the organic phase.] The residue was purified by column 5 chromatography (2 kg silica gel, ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, stripped and dried to product as a white crisp foam (84 g, 50%), contaminated starting material (17.4 g) and pure reusable starting material 20 g. The yield 10 based on starting material less pure recovered starting material was 58%. TLC and NMR were consistent with 99% pure product. (d) 2'-0-([2-PHTHALIMIDOXY)ETHYL]-5'-T BUTYLDIPHENYLSILYL-5-METHYLURIDINE 15 5'-0-tert-Butyldiphenylsilyl-2'-0-(2-hydroxyethyl) 5-methyluridine (20 g, 36.98 'mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried over P 2 0 5 under high vacuum for two days at 40 C. 20 The reaction mixture was flushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture. The rate of addition is maintained such that resulting deep 25 red coloration is just discharged before adding the next drop. After the addition was'complete, the reaction was stirred for 4 hrs. By that t.ime TLC showed the completion of the reaction (ethylacetate:hexane, 60:40). The solvent was evaporated in vacuum. Residue obtained 30 was placed on a flash column and eluted with ethyl acetate:hexane (60:40), to get 2'-0-([2 phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5 methyluridine as white foam ('21.819 g, 86%).

P.WPDOCS\CR.N\RMHIfSpc\20287356 di, sp do 3/10/2007 (e) 5'-0-TERT-BUTYLDIPHENYLSILYL-2'-0-[(2 FORMADOXIMINOOXY)ETHYL]-5-METHYLURIDINE 2'-0-([2-phthalimidoxy)ethyl]-5'-t butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was 5 dissolved in dry CH 2 C1 2 (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added drop'wise at -10 C to 0 C. After 1 h the mixture was filtered,. the filtrate was washed with ice cold CH 2 Cl 2 and the combined organic phase was washed with water, brine and dried over anhydrous Na 2

SO

4 . 10 The solution was concentrated.to get 2'-0-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was strirred for 1 h. Solvent was removed under vacuum; residue chromatographed 15 to get 5'-0-tert-butyldiphenylsilyl-2'-0-[(2 formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%). (f) 5' -0-TERT-BUTYLDIPHENYLSILYL-2' -0- [N,N DIMETHYLAMINOOXYETHYL]-5-METHYLURIDINE 20 5'-0-tert-butyldiphenylsilyl-2'-0-[(2 formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added to this 25 solution at 10 C under inert atmosphere. The reaction mixture was stirred for 10 minutes at 10 C. After that the reaction vessel was removed from the ice bath and stirred at room temperature for 2 h, the reaction monitored by TLC (5% MeOH in CH 2 C1 2 ). Aqueous NaHCO 3 30 solution (5%, 10 mL) was added and extracted with ethyl acetate (2x20 mL). Ethyl acetate phase was dried over anhydrous Na 2

SO

4 , evaporated to dryness. Residue was dissolved in a solution of 1M PPTS in MeOH (30.6 mL). 04 P .WPDOCS\CRN\RM1rspcc\20287356 div spc dc - 3/10/2007 Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and the reaction mixture was stirred at room temperature for 10 minutes. Reaction mixture cooled to 10 C in an ice bath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was 5 added and reaction mixture stirred at 10 C for 10 minutes. After 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature for 2 hrs. To the reaction mixture 5% NaHCO 3 (25 mL) solution was added and extracted with ethyl acetate (2x25 mL). Ethyl 10 acetate layer was dried over anhydrous Na 2

SO

4 and evaporated to dryness . The residue obtained was purified by flash column chromatography and eluted with 5% MeOH in CH 2 Cl 2 to get 5'-0-tert-butyldiphenylsilyl-2' 0-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white 15 foam (14.6 g, 80%). (g) 2'-0-(DIMETHYLAMINOOXYETHYL)-5-METHYLURIDINE Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). This mixture of triethyl 20 amine-2HF was then added to 5'-O-tert-butyldiphenylsilyl 2'-0-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reaction was monitored by TLC. (5% MeOH in CH 2 Cl 2 ) Solvent was removed under vacuum and the residue placed 25 on a flash column and eluted with 10% MeOH in CH 2 Cl 2 to get 2'-0-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%). (h) 5'-0-DMT-2'-0-(DIMETHYLAMINOOXYETHYL)-5 METHYLURIDINE 30 2'-0-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmcl) was dried over P 2 0 5 under high vacuum overnight at 40 C. It was then co-evaporated with anhydrous pyridine (20 mL). The residue obtained 65 P \WPDOCS\CRN\RMH\Spe\20287356 div spec dc - 3/10/2007 was dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4'-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the mixture and the reaction 5 mixture was stirred at room temperature until all of the starting material disappeared. Pyridine was removed under vacuum and the residue chromatographed and eluted with 10% MeOH in CH 2 Cl 2 (containing a few drops of pyridine) to get 5'-0 10 DMT-2'-0-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%). (i) 5'-0-DMT-2'-0-(2-N,N DIMETHYLAMINOOXYETHYL)-5- METHYLURIDINE-3'-[(2 CYANOETHYL)-N,N-DIISO- - PROPYLPHOSPHORAMIDITE] 5'-0-DMT-2'-0-(dimethylaminooxyethyl)-5 15 methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and dried over

P

2 0 5 under high vacuum overnight at 40 C. Then the reaction mixture was dissolved in anhydrous acetonitrile 20 (8.4 mL) and 2-cyanoethyl-N,.N,N ,N -tetraisopropyl phosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 hrs under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:ethyl acetate 1:1). The 25 solvent was evaporated, then the residue was dissolved in ethyl acetate (70 mL) and washed with 5% aqueous NaHCO 3 (40 mL). Ethyl acetate layer was dried over anhydrous Na 2

SO

4 and concentrated. Residue obtained was chromatographed (ethyl acetate as eluent) to get 5'-0 30 DMT-2'-0-(2-N,N-dimethylaminooxyethyl)-5-methyluridine 3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%). 2'- (AMINOOXYETHOXY) NUCLEOSIDE AMIDITES 66 P \WPDOCS\CR N\RMH\Spcc\20287356 div sp doc -3/10/2DO7 2'-(Aminooxyethoxy) nucleoside amidites [also known in the art as 2'-O-(aminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs[COMMENT] [1]Page: 1. 5 G= cpds 26-31 from ISIS 2824.' Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly. N2-ISOBUTYRYL-6-0-DIPHENYLCARBAMOYL-2'-0-(2 ETHYLACETYL)-5'-0-(4,4'-DIMETHOXYTRITYL)GUANOSINE 3'-[(2-CYANOETHYL)-N,N-DIISOPROPYLPHOSPHORAMIDITE] 10 The 2'-0-aminooxyethyl guanosine analog may be obtained by selective 2'-0-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2' O-(2-ethylacetyl) diaminopurine riboside along with a 15 minor amount of the 3'-0-isomer. 2'-0-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2'-0-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 Al 940203.) Standard protection 20 procedures should afford 2'-0-(2-ethylacetyl)-5'-0-(4,4' dimethoxytrityl)guanosine and.2-N-isobutyryl-6-0 diphenylcarbamoyl-2'-0-(2-ethylacetyl)-5'-0-(4,4' dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-0-diphenylcarbamoyl-2'-0-(2 25 ethylacetyl)-5'-0-(4,4'-dimethoxytrityl)guanosine. As before the hydroxyl group may-be displaced by N hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may phosphitylated as usual to yield 2-N-isobutyryl-6-0-diphenylcarbamoyl-2'-0-(2 30 ethylacetyl)-5'-0-(4,4'-dimethoxytrityl)guanosine-3'-[(2 cyanoethyl)-N,N-diisopropylphosphoramiditel. 2'-DIMETHYLAMINOETHOXYETHOXY (2'-DMAEOE) NUCLEOSIDE AMIDITES r7 P.\WPDOCS\CRN\Rhf Spec\227356 div specdoc -3/1O2007 2'-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2'-0-dimethylaminoethoxyethyl, i.e., 2'-0-CH 2 -0-CH 2

-N(CH

2

)

2 , or 2'-DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside 5 amidites are prepared similarly. 2'-0-[2 (2-N,N-DIMETHYLAMINOETHOXY)ETHYL]-5-METHYL URIDINE 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowly added to a solution of borane in 10 tetrahydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the solid dissolves. O2-,2'-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oil bath and heated to 155 C for 26 15 hours. The bomb is cooled to room temperature and opened. The crude solution is concentrated and the residue partitioned between water (200 mL) and hexanes (200 mL). The excess phenol is extracted into the hexane layer. The aqueous layer is extracted with ethyl acetate 20 (3x200 mL) and the combined organic layers are washed once with water, dried over anhydrous sodium sulfate and concentrated. The residue is columned on silica gel using methanol/methylene chloride 1:20 (which has 2% triethylamine) as the eluent. As the column fractions 25 are concentrated a colorless solid forms which is collected to give the title compound as a white solid. 5'-O-DIMETHOXYTRITYL-2' -0-[2(2-N,N-DIMETHYL AMINOETHOXY)ETHYL)]-5-METHYL URIDINE To 0.5 g (1.3 mmol) of 2'-0-[2(2-N,N-dimethylamino 30 ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reaction mixture is poured into water (200 mL) and 69 P:\WPDOCSCRN\R MIf'Spcc\20287356 div spe doc - 3/10:20W7 extracted with CH 2 C1 2 (2x200 mL). The combined CH 2 Cl 2 layers are washed with saturated NaHCO 3 solution, followed by saturated NaCl solution and dried over anhydrous sodium sulfate. Evaporation of the solvent followed by 5 silica gel chromatography using MeOH:CH 2 Cl 2 :Et 3 N (20:1, v/v, with 1% triethylamine) gives the title compound. 5'-0-DIMETHOXYTRITYL-2'-0-[2(2-N,N DIMETHYLAMINOETHOXY)ETHYL)]-5-METHYL URIDINE-3 1 -0 (CYANOETHYL-N,N-DIISOPROPYL)PHOSPHORAMIDITE 10 Diisopropylaminotetrazolide (0.6 g) and 2 cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are added to a solution af 5'-0-dimethoxytrityl-2' 0-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH 2 Cl 2 (20 mL) under an 15 atmosphere of argon. The reaction mixture is stirred overnight and the solvent evaporated. The resulting residue is purified by silica gel flash column chromatography with ethyl acetate as the eluent to give the title compound. 20 EXAMPLE 2: OLIGONUCLEOTIDE SYNTHESIS Unsubstituted and substituted phosphodiester (P=0) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using 25 standard phosphoramidite chemistry with oxidation by iodine. Phosphorothioates (P=S) are synthesized as for the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H 30 1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation wait step was increased to 68 sec and was followed by the capping step. After cleavage from the 69 P \WPDOCSkCRN\RMH\SpU\2O27356 dv s doc -3/10/2007 CPG column and deblocking in concentrated ammonium hydroxide at 55 C (18 h) , the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution. Phosphinate 5 oligonucleotides are prepared as described in U.S. Patent 5,508,270, herein incorporated by reference. Alkyl phosphonate oligonucleotides are prepared as described in U.S. Patent 4,469,863, herein incorporated by reference. 3'-Deoxy-3'-met-hylene phosphonate 10 oligonucleotides are prepared as described in U.S. Patents 5,610,289 or 5,625,050, herein incorporated by reference. Phosphoramidite oligonucleotides are prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878, herein incorporated by reference. Alkylphos 15 phonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference. 3' Deoxy-3'-amino phosphoramidate oligonucleotides are 20 prepared as described in U.S.. Patent 5,476,925, herein incorporated by reference. Phosphotriester oligonucleo tides are prepared as described in U.S. Patent 5,023,243, herein incorporated by reference. Borano phosphate oligonucleotides are prepared as described in U.S. 25 Patents 5,130,302 and 5,177,198, both herein incorporated by reference. EXAMPLE 3: OLIGONUCLEOSIDE SYNTHESIS Methylenemethylimino linked oligonucleosides, also 30 identified as MMI linked oligonucleosides, methylenedi methylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 70 P:\WPDOCS\CRN\RMH\Spc\20287356 div d - 3/10/2007 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P=O 5 or P=S linkages are prepared as described in U.S. Patents 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference. Formacetal and thioformacetal linked oligonucleo 10 sides are prepared as described in U.S. Patents 5,264,562 and 5,264,564, herein incorporated by reference. Ethylene oxide linked oligonucleosides are prepared as described in U.S. Patent 5,223,618, herein incorporated by reference. 15 EXAMPLE 4: PNA SYNTHESIS Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and 20 Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Patents 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference. 25 EXAMPLE 5: SYNTHESIS OF CHIMERIC OLIGONUCLEOTIDES Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligon'ucleosides of the invention can be of several different types. These include a first type wherein the "gap" segment of linked nucleosides is 30 positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open end" type wherein the "gap" segment is located at either the 3' or the 5' terminus of the oligomeric compound. Oligonucleotides of 71 P \WPDOCS\CRN\R MfHSpc20287356d s doc - 3/10'2007 the first type are also known in the art as "gapmers" or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as "hemimers" or "wingmers". 5 [2'-O-ME]--[2'-DEOXY] -- [2'-0-ME] CHIMERIC PHOSPHOROTHIOATE OLIGONUCLEOTIDES Chimeric oligonucleotides having 2'-0-alkyl phosphorothioate and 2'-deoxy phosphorothioate oligo nucleotide segments are synthesized using an Applied 10 Biosystems automated DNA synthesizer Model 380B, as above. Oligonucleotides are synthesized using the automated synthesizer and 2'--deoxy-5'-dimethoxytrityl-3' 0-phosphoramidite for the DNA portion and 5'-dimethoxy trityl-2'-0-methyl-3'-0-phosphoramidite for 5' and 3' 15 wings. The standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for RNA and twice for 2'-0-methyl. The fully protected oligonucleotide is cleaved from the support and the phosphate group is 20 deprotected in 3:1 ammonia/ethanol at room temperature overnight then lyophilized to dryness. Treatment in methanolic ammonia for 24 hrs'at room temperature is then done to deprotect all bases a.nd sample was again lyophilized to dryness. The pellet is resuspended in 1M 25 TBAF in THF for 24 hrs at room temperature to deprotect the 2' positions. The reaction is then quenched with 1M TEAA and the sample is then reduced to 1/2 volume by rotovac before being desalted on a G25 size exclusion column. The oligo recovered is then analyzed 30 spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry. 72 P:\WPDOCSiCRN\RMH\Spa\20287356 div spc doc - 3/10/2007 [2'-O-(2-METHOXYETHYL)]--[2'-DEOXY]--[2'-0 (METHOXYETHYL)] CHIMERIC PHOSPHOROTHIOATE OLIGONUCLEOTIDES [2'-0-(2-methoxyethyl)]--[2'-deoxy]--[-2'-0 5 (methoxyethyl)] chimeric phosphorothioate oligonucleo tides were prepared as per the procedure above for the 2'-0-methyl chimeric oligonucleotide, with the substitution of 2'-0-(methoxyethyl) amidites for the 2' O-methyl amidites. 10 [2'-0-(2-METHOXYETHYL)PHOSPHODIESTER]--[2'-DEOXY PHOSPHOROTHIOATE]--[2'-0-(2-METHOXYETHYL) PHOSPHODIESTER] CHIMERIC OLIGONUCLEOTIDES [2'-0-(2-methoxyethyl phosphodiester]--[2'-deoxy phosphorothioate]--[2'-0-(methoxyethyl) phosphodiester] 15 chimeric oligonucleotides are prepared as per the above procedure for the 2'-0-methyl chimeric oligonucleotide with the substitution of 2'-0-(methoxyethyl) amidites for the 2'-0-methyl amidites, oxidization with iodine to generate the phosphodiester 'internucleotide linkages 20 within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap. 25 Other chimeric oligonucleotides, chimeric oligo nucleosides and mixed chimeric oligonucleotides/oligo nucleosides are synthesized according to United States patent 5,623,065, herein incorporated by reference. 30 EXAMPLE 6: OLIGONUCLEOTIDE ISOLATION After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55 C for 18 hours, the 73 P\WPDOCS\CRN\RMHI\Sp,\20287356 div s doc -3/10/2007 oligonucleotides or oligonucleosides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and 5 judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by 31 P nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as 10 described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material. 15 EXAMPLE 7: OLIGONUCLEOTIDE SYNTHESIS - 96 WELL PLATE FORMAT Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences 20 simultaneously in a standard '96 well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide 25 (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyldiisopropyl phosphor amidites were purchased from commercial vendors (e.g. PE Applied Biosystems, Foster City, CA, or Pharmacia, Piscataway, NJ). Non-standard nucleosides are 30 synthesized as per known literature or patented methods. They are utilized as base protected beta cyanoethyldiisopropyl phosphoramidites. 74 P \WPDOCS\CRN\RMf\Spc\0287356 div spc doc -3/ 0/2OU7 Oligonucleotides were cleaved from support and deprotected with concentrated NH 4 0H at elevated temperature (55-60 C) for 12-16 hours and the released product then dried in vacuo.. The dried product was then 5 re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors. EXAMPLE 8: OLIGONUCLEOTIDE ANALYSIS - 96 WELL PLATE 10 FORMAT The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary 15 electrophoresis (CE) in either the 96 well format (Beckman P/ACEJ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACEJ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing 20 electrospray-mass spectroscopy. All assay test plates were diluted from the master-plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length. 25 EXAMPLE 9: CELL CULTURE AND OLIGONUCLEOTIDE TREATMENT The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at 30 measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following 5 cell types are provided for illustrative purposes, but other cell types can be routinely used, 75 P WPDOCS\CRN\RM apcc\20287356 div spa do - 3/10/2007 provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, Ribonuclease protection assays, or RT-PCR. 5 T-24 CELLS: The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, VA). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Gibco/Life Technologies, Gaithersburg, MD) 10 supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, MD), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, MD). Cells were routinely passaged by trypsinization and dilution when they reached 15 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis. . For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture 20 plates and treated similarly, using appropriate volumes of medium and oligonucleotide. A549 CELLS: The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, VA). A549 cells were 25 routinely cultured in DMEM basal media (Gibco/Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, MD), penicillin 100 units per-mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, 30 MD). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. NHDF CELLS: Human neonatal dermal fibroblast (NHDF) were obtained from the'Clonetics Corporation 76 P:\WPDOCSCRNRM1H\Spe\20287356 div sp doc - 3/10/2007 (Walkersville MD). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, MD) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as 5 recommended by the supplier. HEK CELLS: Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, MD). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, 10 Walkersville MD) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier. PC-12 CELLS: The rat neuronal cell line PC-12 was obtained from the American Type Culure Collection 15 (Manassas, VA). PC-12 cells were routinely cultured in DMEM, high glucose (Gibco/Life Technologies, Gaithersburg, MD) supplemented with 10% horse serum + 5% fetal calf serum (Gibco/Life.Technologies, Gaithersburg, MD). Cells were routinely passaged by trypsinization and 20 dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 20000 cells/well for use in RT-PCR analysis. For Northern blotting or other analysis, cells may be seeded onto 100 mm or othe-r standard tissue culture 25 plates and treated similarly, using appropriate volumes of medium and oligonucleotide. TREATMENT WITH ANTISENSE COMPOUNDS: When cells reached 80% confluency, they were treated with oligonucleotide. For cells grown in 96-well 30 plates, wells were washed once with 200 L OPTI-MEMJ-1 reduced-serum medium (Gibco BRL) and then treated with 130 L of OPTI-MEMJ-1 containing 3.75 g/mL LIPOFECTINJ (Gibco BRL) and the desired concentration of 77 P-WPDOCS\CRN\RM \ Spcc\20287356divsp dm - 3/10/2007 oligonucleotide. After 4-7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment. The concentration of oligonucleotide used varies 5 from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of.concentrations. For human cells the positive control oligonucleotide is ISIS 13920, 10 TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1, a 2'-0-methoxyethyl gapmer (2'-0-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to human H ras. For mouse or rat cells the positive control oligonucleotide is ISIS 15770., ATGCATTCTGCCCCCAAGGA, SEQ 15 ID NO: 2, a 2'-0-methoxyethyl gapmer (2'-0-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for 20 ISIS 15770) mRNA is then util-ized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras 25 or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. 30 EXAMPLE 10: ANALYSIS OF OLIGONUCLEOTIDE INHIBITION OF PTP1B EXPRESSION 78 P:\WDOCS\CRNRMHSpc\2O287356 div spcc doc - 3/10/2007 Antisense modulation of PTP1B expression can be assayed in a variety of ways known in the art. For example, PTP1B mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain 5 reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are taught in, for example, Ausubel, F.M. et al., Current. Protocols in Molecular 10 Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, 15 Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISMJ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, CA and used according to manufacturer's instructions. Prior to 20 quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be "multiplexed" with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently 25 in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only ("single-plexing"), or both (multiplexing). Following PCR amplification, 30 standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals 79 P .WPDOCSCRN\RMH\Spcc\20287356 div spec doc -3/ 0/2007 generated from the multiplexed samples fall within 10% of their corresponding values generated from the single plexed samples, the primer-probe set specific for that target is deemed as multiplexable. Other methods of PCR 5 are also known in the art. Protein levels of PTP1B can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell 10 sorting (FACS). Antibodies directed to PTP1B can be identified and obtained from.a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional antibody generation methods. Methods for preparation of 15 polyclonal antisera are taught in, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F.M. et al., Current Protocols in 20 Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997. Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 25 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1 10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked 30 immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F.M. et al., Rn P \WPDOCSCRN\RMfrSpcm\20287356 di sp do - 3/10/2007 Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991. EXAMPLE 11: POLY(A)+ MRNA ISOLATION 5 Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, 10 Inc., 1993. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 L cold PBS. 60 L lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each 15 well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 L of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine CA). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 L of wash 20 buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70 C was added to each well, the plate 25 was incubated on a 90 C hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate. Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions. 30 EXAMPLE 12: TOTAL RNA ISOLATION Total mRNA was isolated using an RNEASY 96J kit and buffers purchased from Qiagen Inc. (Valencia, CA) 81 P \WPDOCS\CRN\RMIf\Spec\20287356 div spc doc3/1012007 following the manufacturer's recommended procedures. Briefly, for cells grown on 9.6-well plates, growth medium was removed from the cells and each well was washed with 200 L cold PBS. 100 L Buffer RLT was added to each well 5 and the plate vigorously agitated for 20 seconds. 100 L of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96J well plate attached to a QIAVACJ manifold fitted with a waste 10 collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96J plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY 96J plate and the vacuum 15 applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAVACJ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVACJ manifold 20 fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 L water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 L water. 25 The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia, CA). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution 30 steps are carried out. EXAMPLE 13: REAL-TIME QUANTITATIVE PCR ANALYSIS OF PTP1B MRNA LEVELS 82 P \WPDOCSCRN\RMIH\Spcv\20287356 div spc doc - 10:3'2007 Quantitation of PTP1B mRNA levels was determined by real-time quantitative PCR using the ABI PRISMJ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to manufacturer's instructions. This 5 is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, 10 products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., 15 JOE, FAM, or VIC, obtained from either Operon Technologies Inc., Alameda, CA or PE-Applied Biosystems, Foster City, CA) is attached to the 5' end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, CA or PE-Applied 20 Biosystems, Foster City, CA) is attached to the 3' end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3' quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can 25 be cleaved by the 5'-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a 30 sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes,. and the fluorescence 83 P\WPDOCS\CRN\RM rpc20287356 dvspe doc - 10/3/2007 intensity is monitored at regular intervals by laser optics built into the ABI PRISMJ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated 5 control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples. PCR reagents were obtained from PE-Applied Biosystems, Foster City, CA. RT-PCR reactions were 10 carried out by adding 25 L PCR cocktail (1x TAQMANJ buffer A, 5.5 mM MgCl 2 , 300 M each of dATP, dCTP and dGTP, 600 M of dUTP, 100 nM -each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLDJ, and 12.5 Units MuLV reverse 15 transcriptase) to 96 well plates containing 25 L poly(A) mRNA solution. The RT reaction was carried out by incubation for 30 minutes at 48 C. Following a 10 minute incubation at 95 C to activate the AMPLITAQ GOLDJ, 40 cycles of a two-step PCR protocol were carried out: 95 C 20 for 15 seconds (denaturation) followed by 60 C for 1.5 minutes (annealing/extension). Probes and primers to human PTP1B were designed to hybridize to a human PTP1B sequence, using published sequence information (GenBank accession number M31724, 25 incorporated herein as SEQ ID NO:3). For human PTP1B the PCR primers were: forward primer: GGAGTTCGAGCAGATCGACAA (SEQ ID NO: 4) reverse primer: GGCCACTCTACATGGGAAGTC (SEQ ID NO: 5) and the PCR probe was: FAM-AGCTGGGCGGCCATTTACCAGGAT-TAMRA 30 (SEQ ID NO: 6) where FAM (PE-Applied Biosystems, Foster City, CA) is the fluorescent reporter dye) and TAMRA (PE Applied Biosystems, Foster City, CA) is the quencher dye. For human GAPDH the PCR primers were: 84- P \WPDOCS\CRN\RMHLSpcc\20287356 div spec doc - 103/2007 forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC- TAMRA 3' (SEQ ID NO: 9) where JOE (PE-Applied Biosystems, Foster 5 City, CA) is the fluorescent reporter dye) and TAMRA (PE Applied Biosystems, Foster City, CA) is the quencher dye. Probes and primers to 2at PTP1B were designed to hybridize to a rat PTP1B sequence, using published sequence information (GenBank accession number M33962, 10 incorporated herein as SEQ ID NO:10). For rat PTP1B the PCR primers were: forward primer: CGAGGGTGCAAAGTTCATCAT (SEQ ID NO:11) reverse primer: CCAGGTCTTCATGGGAAAGCT (SEQ ID NO: 12) and the PCR probe was: FAM-CGACTCGTCAGTGCAGGATCAGTGGA-TAMRA 15 (SEQ ID NO: 13) where FAM (PE-Applied Biosystems, Foster City, CA) is the fluorescent reporter dye) and TAMRA (PE Applied Biosystems, Foster City, CA) is the quencher dye. For rat GAPDH the PCR primers-were: forward primer: TGTTCTAGAGACAGCCGCATCTT (SEQ ID NO: 14) 20 reverse primer: CACCGACCTTCACCATCTTGT (SEQ ID NO: 15) and the PCR probe was: 5' JOE-TTGTGCAGTGCCAGCCTCGTCTCA- TAMRA 3' (SEQ ID NO: 16) where JOE (PE-Applied Biosystems, Foster City, CA) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems,. Foster City, CA) is the 25 quencher dye. EXAMPLE 14: NORTHERN BLOT ANALYSIS OF PTP1B MRNA LEVELS Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 30 mL RNAZOLJ (TEL-TEST "B" Inc.', Friendswood, TX). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels P-\WPDOCS\CRN\RMH\Spe\20287356 div spec dc - 10/3/2007 containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, OH). RNA was transferred from the gel to HYBONDJ-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ) by overnight capillary transfer 5 using a Northern/Southern Transfer buffer system (TEL TEST "B" Inc., Friendswood, TX). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKERJ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, CA) and then robed using 10 QUICKHYBJ hybridization solution (Stratagene, La Jolla, CA) using manufacturer's recommendations for stringent conditions. To detect human PTPlB, a human PTP1B specific probe was prepared by PCR using the forward primer 15 GGAGTTCGAGCAGATCGACAA (SEQ ID NO: 4) and the reverse primer GGCCACTCTACATGGGAAGTC (SEQ ID NO: 5). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA 20 (Clontech, Palo Alto, CA). To detect rat PTP1B, a rat PTP1B specific probe was prepared by PCR using the forward primer CGAGGGTGCAAAGTTCATCAT (SEQ ID NO:ll) and the reverse primer CCAGGTCTTCATGGGAAAGCT (SEQ ID NO: 12). To 25 normalize for variations in loading and transfer efficiency membranes were stripped and probed for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, CA). Hybridized membranes were visualized and 30 quantitated using a PHOSPHORIMAGERJ and IMAGEQUANTJ Software V3.3 (Molecular Dynamics, Sunnyvale, CA). Data was normalized to GAPDH levels in untreated controls.

P \WPDOCS\CRN\RMH\Spc20287356 dv sp doc - 10/3/2007 EXAMPLE 15: ANTISENSE INHIBITION OF HUMAN PTP1B EXPRESSION BY CHIMERIC PHOSPHOROTHIOATE OLIGONUCLEOTIDES HAVING 2'-MOE WINGS AND A DEOXY GAP In accordance with the -present invention, a series 5 of oligonucleotides were designed to target different regions of the human PTP1B RNA, using published sequences (GenBank accession number M31724, incorporated herein as SEQ ID NO: 3). The oligonucleotides are shown in Table 1. ATarget siteA indicates the first (5'-most) 10 nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 1 are chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap" region consisting of ten 2'-deoxynucleotides, which is flanked 15 on both sides (5' and 3' directions) by five-nucleotide "wings". The wings are composed of 2'-methoxyethyl (2' MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. All cytidine residues are 5 20 methylcytidines. The compounds were analyzed for their effect on human PTP1B mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, "N.D." indicates "no data". 25 TABLE 1: INHIBITION OF HUMAN.PTP1B MRNA LEVELS BY CHIMERIC PHOSPHOROTHIOATE OLIGONUCLEOTIDES HAVING 2'-MOE WINGS AND A DEOXY GAP ISIS REGION TARGET TARGET SEQUENCE % SEQ # SEQ ID SITE INHI ID NO BIT. NO 107769 5' UTR 3 1 cttagccccgaggcccgccc 0 17 107770 5' UTR 3 41 ctcggcccactgcgccgtct 58 18 107771 Start 3 74 catgacgggccagggcggct 60 19 Codon 107772 Coding 3 113 'cccggacttgtcgatctgct 95 20 107773 Coding 3 154 ctggcttcatgtcggatatc 88 21 27 P \WPDOCSCR RMIf\Spc\20287356 div spe doc - 10/32007 107774 Coding 3 178 ttggccactctacatgggaa 77 22 107775 Coding 3 223 ggactgacgtctctgtacct 75 23 107776 Coding 3 252 gatgtagtttaatccgacta 82 24 107777 Coding 3 280 ctagcgttgatatagtcatt 29 25 107778 Coding 3 324 gggtaagaatgtaactcctt 86 26 107779 Coding 3 352 tgaccgcatgtgttaggcaa 75 27 107780 Coding 3 381 .ttttctgctcccacaccatc 30 28 107781 Coding 3 408 ctctgttgagcatgacgaca 78 29 107782 Coding 3 436 gcgcattttaacgaaccttt 83 30 107783 Coding 3 490 aaatttgtgtcttcaaagat 0 31 107784 Coding 3 519 tgatatcttcagagatcaat 57 32 107785 Coding 3 547 tctagctgtcgcactgtata 74 33 107786 Coding 3 575 agtttcttgggttgtaaggt 33 34 107787 Coding 3 604 gtggtatagtggaaatgtaa 51 35 107788 Coding 3 632 tgattcagggactccaaagt 55 36 107789 Coding 3 661 'ttgaaaagaaagttcaagaa 17 37 107790 Coding 3 688 gggctgagtgaccctgactc 61 38 107791 Coding 3 716 gcagtgcaccacaacgggcc 81 39 107792 Coding 3 744 aggttccagacctgccgatg 81 40 107793 Coding 3 772 agcaggaggcaggtatcagc 2 41 107794 Coding 3 799 gaagaagggtctttcctctt 53 42 107795 Coding 3 826 tctaacagcactttcttgat 18 43 107796 Coding 3 853 atcaaccccatccgaaactt 0 44 107797 Coding 3 880 gagaagcgcagctggtcggc 82 45 107798 Coding 3 908 tttggcaccttcgatcacag 62 46 107799 Coding 3 952 agctccttccactgatcctg 70 47 107800 Coding 3 1024 tccaggattcgtttgggtgg 72 48 107801 Coding 3 1052 gaactccctgcatttcccat 68 49 107802 Coding 3 1079 ttccttcacccactggtgat 40 50 107803 Coding 3 1148 gtagggtgcggcatttaagg 0 51 107804 Coding 3 1176 cagtgtcttgactcatgctt 75 52 107805 Coding 3 1222 gcctgggcacctcgaagact 67 53 107806 Coding 3 1268 ctcgtccttctcgggcagtg 37 54 107807 Coding 3 1295 gggcttccagtaactcagtg 73 55 107808 Coding 3 1323 ccgtagccacgcacatgttg 80 56 107809 Coding 3 1351 tagcagaggtaagcgccggc 72 57 107810 Stop 3 1379 ctatgtgttgctgttgaaca 85 58 Codon 107811 3' UTR 3 1404 Qgaggtggagtggaggaggg 51 59 107812 3' UTR 3 1433 ggctctgcgggcagaggcgg 81 60 107813 3' UTR 3 1460 'ccgcggcatgcctgctagtc 84 61 107814 3' UTR 3 1489 tctctacgcggtccggcggc 84 62 107815 3' UTR 3 1533 -aagatgggttttagtgcaga 65 63 107816 3' UTR 3 1634 gtactctctttcactctcct 69 64 107817 3' UTR 3 1662 ggccccttccctctgcgccg 59 65 107818 3' UTR 3 1707 ctccaggagggagccctggg 57 66 107819 3' UTR 3 1735 gggctgttggcgtgcgccgc 54 67 107820 3' UTR 3 1783 tttaaataaatatggagtgg 0 68 107821 3' UTR 3 1831 gttcaagaaaatgctagtgc 69 69 P.\WPDOCS CRN\RM\{Spc\20287356 div spc doc - 10/3/2007 107822 3' UTR 3 1884 .ttgataaagcccttgatgca 74 70 107823 3' UTR 3 1936 atggcaaagccttccattcc 26 71 107824 3' UTR 3 1973 gtcctccttcccagtactgg 60 72 107825 3' UTR 3 2011 ttacccacaatatcactaaa 39 73 107826 3' UTR 3 2045 attatatattatagcattgt 24 74 107827 3' UTR 3 2080 tcacatcatgtttcttatta 48 75 107828 3' UTR 3 2115 ataacagggaggagaataag 0 76 107829 3' UTR 3 2170 ttacatgcattctaatacac 21 77 107830 3' UTR 3 2223 gatcaaagtttctcatttca 81 78 107831 3' UTR 3 2274 ggtcatgcacaggcaggttg 82 79 107832 3' UTR 3 2309 caacaggcttaggaaccaca 65 80 107833 3' UTR 3 2344 aactgcaccctattgctgag 61 81 107834 3' UTR 3 2380 gtcatgccaggaattagcaa 0 82 107835 3' UTR 3 2413 acaggctgggcctcaccagg 58 83 107836 3' UTR 3 2443 tgagttacagcaagaccctg 44 84 107837 3' UTR 3 2473 gaatatggcttcccataccc 0 85 107838 3' UTR 3 2502 ccctaaatcatgtccagagc 87 86 107839 3' UTR 3 2558 gacttggaatggcggaggct 74 87 107840 3' UTR 3 2587 'caaatcacggtctgctcaag 31 88 107841 3' UTR 3 2618 gaagtgtggtttccagcagg 56 89 107842 3' UTR 3 2648 cctaaaggaccgtcacccag 42 90 107843 3' UTR 3 2678 gtgaaccgggacagagacgg 25 91 107844 3' UTR 3 2724 gccccacagggtttgagggt 53 92 107845 3' UTR 3 2755 cctttgcaggaagagtcgtg 75 93 107846 3' UTR 3 2785 aaagccacttaatgtggagg 79 94 107847 3' UTR 3 2844 .gtgaaaatgctggcaagaga 86 95 107848 3' UTR 3 2970 tcagaatgcttacagcctgg 61 96 As shown in Table 1, SEQ ID NOs 18, 19, 20, 21, 22, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 40, 42, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 5 60, 61, 62, 63, 64, 65, 66, 67, 69, 70, 72, 73, 75, 78, 79, 80, 81, 83, 84, 86, 87, 89, 90, 92, 93, 94, 95, and 96 demonstrated at least 35% inhibition of human PTP1B expression in this assay and are therefore preferred. 10 EXAMPLE 16: ANTISENSE INHIBITION OF RAT PTP1B EXPRESSION BY CHIMERIC PHOSPHOROTHIOATE OLIGONUCLEOTIDES HAVING 2' MOE WINGS AND A DEOXY GAP. In accordance with the present invention, a second series of oligonucleotides were designed to target 15 different regions of the rat PTP1B RNA, using published 89 P\WPDOCSCRNRMH\Spc\0287356 div spe doc - 10/3/2007 sequences (GenBank accession number M33962, incorporated herein as SEQ ID NO: 10). The oligonucleotides are shown in Table 2. ATarget siteA indicates the first (5'-most) nucleotide number on the particular target sequence to 5 which the oligonucleotide binds. All compounds in Table 2 are chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap" region consisting of ten 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) by five-nucleotide 10 "wings". The wings are composed of 2'-methoxyethyl (2' MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleo tide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on rat PTP1B 15 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, "N.D." indicates "no data". 20 TABLE 2: INHIBITION OF RAT PTP1B MRNA LEVELS BY CHIMERIC PHOSPHOROTHIOATE OLIGONUCLEOTIDES HAVING 2'-MOE WINGS AND A DEOXY GAP ISIS REGION TARGET TARGET SEQUENCE %IN- SEQ # SEQ ID SITE HIB ID NO NO 111549 5' UTR 10 1 caacctccccagcagcggct 32 97 111550 5' UTR 10 33 tcgaggcccgtcgcccgcca 27 98 111551 5' UTR 10 73 cctcggccgtccgccgcgct 34 99 111552 Coding 10 132 tcgatctgctcgaattcctt 49 100 113669 Coding 10 164 cctggtaaatagccgcccag 36 101 113670 Coding 10 174 tgtcgaatatcctggtaaat 63 102 113671 Coding 10 184 actggcttcatgtcgaatat 58 103 113672 Coding 10 189 aagtcactggcttcatgtcg 40 104 111553 Coding 10 190 gaagtcactggcttcatgtc 27 105 113673 Coding 10 191 ggaagtcactggcttcatgt 54 106 113674 Coding 10 192 gggaagtcactggcttcatg 41 107 113675 Coding 10 193 tgggaagtcactggcttcat 56 108 113676 Coding 10 194 atgggaagtcactggcttca 31 109 P:\WPDOCSCRN\RMH\Spec\20287356 div specdoc - 10/3/2007 113677 Coding 10 195 catgggaagtcactggcttc 59 110 113678 Coding 10 225 tttttgttcttaggaagttt 24 111 111554 Coding 10 228 cggtttttgttcttaggaag 45 112 111555 Coding 10 269 tccgactgtggtcaaaaggg 39 113 113679 Coding 10 273 ttaatccgactgtggtcaaa 45 114 113680 Coding 10 298 atagtcattatcttcctgat 49 115 111556 Coding 10 303 ttgatatagtcattatcttc 29 116 113681 Coding 10 330 gcttcctccatttttatcaa 67 117 111557 Coding 10 359 ggccctgggtgaggatatag 20 118 113682 Coding 10 399 cacaccatctcccagaagtg 29 119 111558 Coding 10 405 tgctcccacaccatctccca 48 120 113683 Coding 10 406 ctgctcccacaccatctccc 51 121 113684 Coding 10 407 tctgctcccacaccatctcc 37 122 113685 Coding 10 408 ttctgctcccacaccatctc 54 123 113686 Coding 10 417 cccctgctcttctgctccca 60 124 111559 Coding 10 438 atgcggttgagcatgaccac 15 125 113687 Coding 10 459 tttaacgagcctttctccat 33 126 113688 Coding 10 492 ttttcttctttctgtggcca 54 127 113689 Coding 10 502 gaccatctctttttcttctt 58 128 111560 Coding 10 540 tcagagatcagtgtcagctt 21 129 113690 Coding 10 550 cttgacatcttcagagatca 64 130 113691 Coding 10 558 taatatgacttgacatcttc 46 131 111561 Coding 10 579 aactccaactgccgtactgt 14 132 111562 Coding 10 611 tctctcgagcctcctgggta 38 133 113692 Coding 10 648 ccaaagtcaggccaggtggt 63 134 111563 Coding 10 654 gggactccaaagtcaggcca 31 135 113693 Coding 10 655 agggactccaaagtcaggcc 50 136 113694 Coding 10 656 cagggactccaaagtcaggc 45 137 113695 Coding 10 657 tcagggactccaaagtcagg 49 138 113696 Coding 10 663 ggtgactcagggactccaaa 34 139 111564 Coding 10 705 cctgactctcggactttgaa 53 140 113697 Coding 10 715 gctgagtgagcctgactctc 57 141 113698 Coding 10 726 ccgtgctctgggctgagtga 48 142 111565 Coding 10 774 aaggtccctgacctgccaat 28 143 111566 Coding 10 819 tctttcctcttgtccatcag 34 144 113699 Coding 10 820 gtctttcctcttgtccatca 41 145 113700 Coding 10 821 ggtctttcctcttgtccatc 66 146 113701 Coding 10 822 gggtctttcctcttgtccat 71 147 113702 Coding 10 852 aacagcactttcttgatgtc 39 148 111567 Coding 10 869 ggaacctgcgcatctccaac 0 149 111568 Coding 10 897 tggtcggccgtctggatgag 29 150 113703 Coding 10 909 gagaagcgcagttggtcggc 48 151 113704 Coding 10 915 aggtaggagaagcgcagttg 31 152 113705 Coding 10 918 gccaggtaggagaagcgcag 41 153 111569 Coding 10 919 agccaggtaggagaagcgca 56 154 113706 Coding 10 920 cagccaggtaggagaagcgc 58 155 113707 Coding 10 921 acagccaggtaggagaagcg 43 156 113708 Coding 10 922 cacagccaggtaggagaagc 49 157 113709 Coding 10 923 tcacagccaggtaggagaag 47 158 111570 Coding 10 924 atcacagccaggtaggagaa 51 159 91.

P \WDOCS\CRN\RMHi\Spcc\20287356div spc do - W03/2007 113710 Coding 10 925 gatcacagccaggtaggaga 51 160 113711 Coding 10 926 cgatcacagccaggtaggag 63 161 113712 Coding 10 927 tcgatcacagccaggtagga 71 162 113713 Coding 10 932 caccctcgatcacagccagg 75 163 113714 Coding 10 978 tccttccactgatcctgcac 97 164 111571 Coding 10 979 ctccttccactgatcctgca 89 165 113715 Coding 10 980 gctccttccactgatcctgc 99 166 107799 Coding 10 981 agctccttccactgatcctg 99 167 113716 Coding 10 982 aagctccttccactgatcct 97 168 113717 Coding 10 983 aaagctccttccactgatcc 95 169 113718 Coding 10 984 gaaagctccttccactgatc 95 170 113719 Coding 10 985 ggaaagctccttccactgat 95 171 111572 Coding 10 986 gggaaagctccttccactga 89 172 113720 Coding 10 987 tgggaaagctccttccactg 97 173 113721 Coding 10 1036 tggccggggaggtgggggca 20 174 111573 Coding 10 1040 tgggtggccggggaggtggg 20 175 113722 Coding 10 1046 tgcgtttgggtggccgggga 18 176 111574 Coding 10 1073 tgcacttgccattgtgaggc 38 177 113723 Coding 10 1206 acttcagtgtcttgactcat 67 178 113724 Coding 10 1207 aacttcagtgtcttgactca 60 179 111575 Coding 10 1208 taacttcagtgtcttgactc 50 180 113725 Coding 10 1209 ctaacttcagtgtcttgact 53 181 111576 Coding 10 1255 gacagatgcctgagcacttt 32 182 106409 Coding 10 1333 gaccaggaagggcttccagt 32 183 113726 Coding 10 1334 tgaccaggaagggcttccag 39 184 111577 Coding 10 1335 ttgaccaggaagggcttcca 32 185 113727 Coding 10 1336 gttgaccaggaagggcttcc 41 186 113728 Coding 10 1342 gcacacgttgaccaggaagg 59 187 111578 Coding 10 1375 gaggtacgcgccagtcgcca 45 188 111579 Coding 10 1387 tacccggtaacagaggtacg 32 189 111580 Coding 10 1397 agtgaaaacatacccggtaa 30 190 111581 3' UTR 10 1456 caaatcctaacctgggcagt 31 191 111582 3' UTR 10 1519 ttccagttccaccacaggct 24 192 111583 3' UTR 10 1552 ccagtgcacagatgcccctc 47 193 111584 3' UTR 10 1609 acaggttaaggccctgagat 29 194 111585 3' UTR 10 1783 gcctagcatcttttgttttc 43 195 111586 3' UTR 10 1890 aagccagcaggaactttaca 36 196 111587 3' UTR 10 2002 gggacacctgagggaagcag 16 197 111588 3' UTR 10 2048 ggtcatctgcaagatggcgg 40 198 111589 3' UTR 10 2118 gccaacctctgatgaccctg 25 199 111590 3' UTR 10 2143 tggaagccccagctctaagc 25 200 111591 3' UTR 10 2165 tagtaatgactttccaatca 44 201 111592 3' UTR 10 2208 tgagtcttgctttacacctc 41 202 111593 3' UTR 10 2252 cctgcgcgcggagtgacttc 22 203 111594 3' UTR 10 2299 aggacgtcactgcagcagga 43 204 111595 3' UTR 10 2346 tcaggacaagtcttggcagt 32 205 111596 3' UTR 10 2405 gaggctgcacagtaagcgct 34 206 111597 3' UTR 10 2422 tcagccaaccagcatcagag 20 207 111598 3' UTR 10 2449 acccacagtgtccacctccc 30 208 111599 3' UTR 10 2502 agtgcgggctgtgctgctgg 30 209

Q?

P:WPDOCS\CRNRMH\Spec\20287356 div spe mdoc - 10/3/2007 111600 3' UTR 10 2553 6agctcgctctggcggcctc 8 210 111601 3' UTR 10 2608 aggaagggagctgcacgtcc 32 211 111602 3' UTR 10 2664 ccctcacgattgctcgtggg 24 212 111603 3' UTR 10 2756 cagtggagcggctcctctgg 18 213 111604 3' UTR 10 2830 caggctgacaccttacacgg 30 214 111605 3' UTR 10 2883 gtcctacctcaaccctagga 37 215 111606 3' UTR 10 2917 ctgccccagcaccagccaca 12 216 111607 3' UTR 10 2946 attgcttctaagaccctcag 33 217 111608 3' UTR 10 2978 ttacatgtcaccactgttgt 28 218 111609 3' UTR 10 3007 tacacatgtcatcagtagcc 37 219 111610 3' UTR 10 3080 ttttctaactcacagggaaa 30 220 111611 3' UTR 10 3153 gtgcccgccagtgagcaggc 23 221 111612 3' UTR 10 3206 cggcctcggcactggacagc 27 222 111613 3' UTR 10 3277 gtggaatgtctgagatccag 31 223 111614 3' UTR 10 3322 agggcgggcctgcttgccca 23 224 111615 3' UTR 10 3384 cggtcctggcctgctccaga 31 225 111616 3' UTR 10 3428 tacactgttcccaggagggt 42 226 111617 3' UTR 10 3471 tggtgccagcagcgctagca 10 227 111618 3' UTR 10 3516 cagtctcttcagcctcaaga 43 228 113729 3' UTR 10 3537 aagagtcatgagcaccatca 56 229 111619 3' UTR 10 3560 tgaaggtcaagttcccctca 40 230 111620 3' UTR 10 3622 ctggcaagaggcagactgga 30 231 111621 3' UTR 10 3666 ggctctgtgctggcttctct 52 232 111622 3' UTR 10 3711 gccatctcctcagcctgtgc 39 233 111623 3' UTR 10 3787 agcgcctgctctgaggcccc 16 234 111624 3' UTR 10 3854 tgctgagtaagtattgactt 35 235 111625 3' UTR 10 3927 6tatggccatttagagagag 36 236 113730 3' UTR 10 3936 tggtttattctatggccatt 59 237 111626 3' UTR 10 3994 cgctcctgcaaaggtgctat 11 238 111627 3' UTR 10 4053 gttggaaacggtgcagtcgg 39 239 111628 3' UTR 10 4095 atttattgttgcaactaatg 33 240 As shown in Table 2, SEQ ID NOs 97, 99, 100, 101, 102, 103, 104, 106, 107, 108,. 109, 110, 112, 113, 114, 115, 117, 120, 121, 122, 123, 124, 126, 127, 128, 130, 5 131, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 144, 145, 146, 147, 148, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, .177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 191, 193, 195, 10 196, 198, 201, 202, 204, 205,. 206, 211, 215, 217, 219, 223, 225, 226, 228, 229, 230, 232, 233, 235, 236, 237, 239 and 240 demonstrated at least 30% inhibition of rat P.\WPDOCSCRN\RMifSpec\2O287356 div sp doc - 10/3/207 PTP1B expression in this experiment and are therefore preferred. EXAMPLE 17: WESTERN BLOT ANALYSIS OF PTP1B PROTEIN 5 LEVELS Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), 10 boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to PTP1B is.used, with a radiolabelled or fluorescently labeled secondary antibody directed 15 against the primary antibody species. Bands are visualized using a PHOSPHORIMAGERJ (Molecular Dynamics, Sunnyvale CA). EXAMPLE 18: EFFECTS OF ANTISENSE INHIBITION OF PTP1B 20 (ISIS 113715) ON BLOOD GLUCOSE LEVELS db/db mice are used as a model of Type 2 diabetes. These mice are hyperglycemic, obese, hyperlipidemic, and insulin resistant. The db/db phenotype is due to a mutation in the leptin receptor on a C57BLKS background. 25 However, a mutation in the leptin gene on a different mouse background can produce obesity without diabetes (ob/ob mice). Leptin is a hormone produced by fat that regulates appetite and animals or humans with leptin deficiencies become obese. Heterozygous db/wt mice 30 (known as lean littermates) do not display the hyperglycemia/hyperlipidemia or obesity phenotype and are used as controls. 94 P.\WPDOCS\CRN\RMH\Sp:\202&7356 div sp dc - 10/2007 In accordance with the present invention, ISIS 113715 (GCTCCTTCCACTGATCCTGC, SEQ ID No: 166) was investigated in experiments designed to address the role of PTPlB in glucose metabolism and homeostasis. ISIS 5 113715 is completely complementary to sequences in the coding region of the human, rat, and mouse PTP1B nucleotide sequences incorporated herein as SEQ ID No: 3 (starting at nucleotide 951 of human PTPlB; Genbank Accession No. M31724), SEQ ID No: 10 (starting at 10 nucleotide 980 of rat PTPlB; Genbank Accession No. M33962) and SEQ ID No: 241 (starting at nucleotide 1570 of mouse PTP1B; Genbank Accession No. U24700). The control used is ISIS 29848 (NNN NNNNNNN, SEQ ID No: 242) where N is a mixture of A, G, T and C. 15 Male db/db mice and lean (heterozygous, i.e., db/wt) littermates (age 9 weeks at time 0) were divided into matched groups (n=6) with the same average blood glucose levels and treated by intraperitoneal injection once a week with saline, ISIS 29848 (the control 20 oligonucleotide) or ISIS 113715. db/db mice were treated at a dose of 10, 25 or 50 mg/kg of ISIS 113715 or 50 mg/kg of ISIS 29848 while lean littermates were treated at a dose of 50 or 100 mg/kg of ISIS 113715 or 100 mg/kg of ISIS 29848. Treatment was continued for 4 weeks with 25 blood glucose levels being measured on day 0, 7, 14, 21 and 28. By day 28 in db/db mice, blood glucose levels were reduced at all doses from a starting level of 300 mg/dL to 225 mg/dL for the 10 mg/kg dose, 175 mg/dL for the 25 30 mg/kg dose and 125 mg/dL for the 50 mg/kg dose. These final levels are within normal range for wild-type mice (170 mg/dL). The mismatch control and saline treated 95.

P \WPDOCS\CRN\RMH\Spc\20287356 div spe doc -I 03/2007 levels levels were 320 mg/dL and 370 mg/dL at day 28, respectively. In lean littermates, blood glucose levels remained constant throughout the study for all treatment groups 5 (average 120 mg/dL). These results indicate that treatment with ISIS 113715 reduces blood glucose in db/db mice and that there is no hypoglycemia induced in the db/db or the lean littermate mice as a result of the oligonucleotide treatment. 10 In a similar experiment, ob/ob mice and their lean littermates (heterozygous, i.e., ob/wt) were dosed twice a week at 50 mg/kg with ISIS 113715, ISIS 29848 or saline control and blood glucose levels were measured at the end of day 7, 14 and 21. Treatment of ob/ob mice with ISIS 15 113715 resulted in the largest decrease in blood glucose over time going from 225 mg/d.L at day 7 to 95 mg/dL at day 21. Ob/ob mice displayed an increase in plasma glucose over time from 300 mg/dL to 325 mg/dL while treatment with the control oligonucleotide reduced plasma 20 glucose from an average of 280 mg/dL to 130 mg/dL. In the lean littermates plasma glucose levels remained unchanged in all treatment groups (average level 100 mg/dL). 25 EXAMPLE 19: EFFECTS OF ANTISENSE INHIBITION OF PTP1B (ISIS 113715) ON MRNA EXPRESSION IN LIVER Male db/db mice and lean-littermates (age 9 weeks at time 0) were divided into matched groups (n=6) with the same average blood glucose levels and treated by 30 intraperitoneal injection once a week with saline, ISIS 29848 (the control oligonucleotide) or ISIS 113715. db/db mice were treated at a dose of 10, 25 or 50 mg/kg of ISIS 113715 or 50 mg/kg of. ISIS 29848 while lean 96 P \WPDOCS\CRN\RMifSpcc\20287356 di Sp doc - 10/3/207 littermates were treated at a dose of 50 or 100 mg/kg of ISIS 113715 or 100 mg/kg of ISIS 29848. Treatment was continued for 4 weeks after which the mice were sacrificed and tissues collected for mRNA analysis. RNA 5 values were normalized and are expressed as a percentage of saline treated control. ISIS 113715 successfully reduced PTP1B mRNA levels in the livers of db/db mice at all doses examined (60% reduction of PTP1B mRNA), whereas the control 10 oligonucleotide treated animals showed no reduction in PTP1B mRNA, remaining at the level of the saline treated control. Treatment of lean littermates with ISIS 113715 also reduced mRNA levels to 45% of control at the 50 mg/kg dose and 25% of control at the 100 mg/kg dose. The 15 control oligonucleotide (ISIS 29848) failed to show any reduction in mRNA levels. EXAMPLE 20: EFFECTS OF ANTISENSE INHIBITION OF PTP1B (ISIS 113715) ON BODY WEIGHT 20 Male db/db mice and lean littermates (age 9 weeks at time 0) were divided into matched groups (n=6) with the same average blood glucose levels and treated by intraperitoneal injection once a week with saline, ISIS 29848 (the control oligonucleotide) or ISIS 113715. 25 db/db mice were treated at a dose of 10, 25 or 50 mg/kg of ISIS 113715 or 50 mg/kg of ISIS 29848 while lean littermates were treated at a dose of 50 or 100 mg/kg of ISIS 113715 or 100 mg/kg of ISIS 29848. Treatment was continued for 4 weeks. At day 28 mice were sacrificed 30 and final body weights were measured. Treatment of ob/ob mice with ISIS 113715 resulted in an increase in body weight which was constant over the dose range with animals gaining an average of 11.0 grams 97 P.\WPDOCS\CRN\RMI\Sp \20217356 di sp doc - 10/3/2007 while saline treated controls-gained 5.5 grams. Animals treated with the control oligonucleotide gained an average of 7.8 grams of body weight. Lean littermate animals treated with 50 or 100 mg/kg 5 of ISIS 113715 gained 3.8 grams of body weight compared to a gain of 3.0 grams for the saline controls. In a similar experiment, ob/ob mice and their lean littermates were dosed twice -a week at 50 mg/kg with ISIS 113715, ISIS 29848 or saline control and body weights 10 were measured at the end of day 7, 14 and 21. Treatment of the ob/ob mice with ISIS 113715, ISIS 29848 or saline control all resulted in a similar increase in body weight across the 21-day timecourse. At the end of day 7 all ob/ob treatment groups had an 15 average weight of 42 grams. By day 21, animals treated with ISIS 113715 had an average body weight of 48 grams, while those in the ISIS 29848 (control oligonucleotide) and saline control group each-had an average body weight of 52 grams. All of the lean littermates had an average 20 body weight of 25 grams at the beginning of the timecourse and all lean littermate treatment groups showed an increase in body weight, to 28 grams, by day 21. 25 EXAMPLE 21: EFFECTS OF ANTISENSE INHIBITION OF PTP1B (ISIS 113715) ON PLASMA INSULIN LEVELS Male db/db mice (age 9 weeks at time 0) were divided into matched groups (n=6) with the same average blood glucose levels and treated by intraperitoneal injection 30 twice a week with saline, ISIS 29848 (the control oligonucleotide) or ISIS 113715 at a dose of 50 mg/kg. Treatment was continued for 3 weeks with plasma insulin levels being measured on day 7, 14, and 21. 99 P \WPDOCS\CRRMH\pcc\20287356 div spc doc - 10/3/2007 Mice treated with ISIS 113715 showed a decrease in plasma insulin levels from 15.ng/mL at day 7 to 7.5 ng/mL on day 21. Saline treated animals has plasma insulin levels of 37 ng/mL at day 7 which dropped to 25 ng/mL on 5 day 14 but rose again to 33 ng/mL by day 21. Mice treated with the control oligonucleotide also showed a decrease in plasma insulin levels across the timecourse of the study from 25 ng/mL at'day 7 to 10 ng/mL on day 21. However, ISIS 113715 was. the most effective at 10 reducing plasma insulin over time. EXAMPLE 22: ANTISENSE INHIBITION OF HUMAN PTP1B EXPRESSION BY ADDITIONAL CHIMERIC PHOSPHOROTHIOATE OLIGONUCLEOTIDES HAVING 2'-MOE WINGS AND A DEOXY GAP 15 In accordance with the present invention, an additional series of oligonucleotides were designed to target different genomic regions of the human PTP1B RNA, using published sequences (GenBank accession number M31724, incorporated herein as SEQ ID NO: 3), and 20 concatenated genomic sequence -derived from nucleotide residues 1-31000 of Genbank accession number AL034429 followed by nucleotide residues 1-45000 of Genbank accession number AL133230, incorporated herein as SEQ ID NO: 243). The oligonucleotides are shown in Table 3. 25 "Target site" indicates the first (5'-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 3 are chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap" region consisting of 30 ten 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) by five-nucleotide "wings". The wings are composed of 2'-methoxyethyl (2'- MOE) nucleotides. The internucleoside (backbone) linkages are 99 P \WPDOCS\CRN\RMH\Spcc\20287356 div spcc dc - 10/3/2007 phosphorothioate (P=S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human PTP1B mRNA levels by quantitative real-time PCR as described in 5 other examples herein. Data.are averages from two experiments. If present, "N.D." indicates "no data". TABLE 3: INHIBITION OF HUMAN PTP1B MRNA LEVELS BY CHIMERIC PHOSPHOROTHIOATE OLIGONUCLEOTIDES HAVING 2 ' -MOE WINGS AND A DEOXY GAP 10 Isis # REGION TARGET TARGET SEQUENCE % SEQ ID 142020 5' UTR 3 6 GCGCTCTTAGCCCCGAGGCC 61 244 142021 5' UTR 3 65 CCAGGGCGGCTGCTGCGCCT 56 245 142022 Start 3 80 CATCTCCATGACGGGCCAGG 4 246 Codon 142023 Start 3 85 TTTTCCATCTCCATGACGGG 67 247 Codon 142024 Start 3 90 ACTCCTTTTCCATCTCCATG 71 248 Codon 142025 Exon 1 3 106 TTGTCGATCTGCTCGAACTC 61 249 142026 Exon 1 3 109 GACTTGTCGATCTGCTCGAA 66 250 142027 Exon 1 3 116 GCTCCCGGACTTGTCGATCT 95 251 142028 Exon 1 3 119 CCAGCTCCCGGACTTGTCGA 92 252 142029 Exon:Exo 3 945 TCCACTGATCCTGCACGGAA 44 253 n Junction 142030 Exon:Exo 3 948 CCTTCCACTGATCCTGCACG 55 254 n 142031 3' UTR 3 1453 ATGCCTGCTAGTCGGGCGTG 67 255 142032 3' UTR 3 1670 CGGGTGTAGGCCCCTTCCCT 74 256 142033 3' UTR 3 1772 ATGGAGTGGAGAGTTGCTCC 63 257 142034 3' UTR 3 1893 TTGTACTTTTTGATAAAGCC 61 258 142035 3' UTR 3 1962 CAGTACTGGTCTGACGCAGC 68 259 142036 3' UTR 3 2018 TCTCACGTTACCCACAATAT 74 260 142037 3' UTR 3 2070 TTTCTTATTAAATACCCACG 61 261 142038 3' UTR 3 2088 AAGTAATCTCACATCATGTT 79 262 142039 3' UTR 3 2314 TTCAGCAACAGGCTTAGGAA 51 263 142040 3' UTR 3 2323 GACAATGACTTCAGCAACAG 43 264 142041 3' UTR 3 2359 TGCCTATTCCTGGAAAACTG 43 265 142042 3' UTR 3 2395 GGAAGTCACTAGAGTGTCAT 14 266 142043 3' UTR 3 2418 CCAGGACAGGCTGGGCCTCA 67 267 142044 3' UTR 3 2426 CTGCTGTACCAGGACAGGCT 73 268 142045 3' UTR 3 2452 TGGAATGTCTGAGTTACAGC 74 269 142046 3' UTR 3 2566 AGAGTGTTGACTTGGAATGG 43 270 100 P \WPDOCSXCRN\RMH\Spc\20287356 div spe doc 10/3/2007 142047 3' UTR 3 2574 GCTCAAGAAGAGTGTTGACT 76 271 142048 3' UTR 3 2598 TGCCTCTCTTCCAAATCACG 43 272 142049 3' UTR 3 2800 TGTTTTTCATGTTAAAAAGC 44 273 142050 3' UTR 3 2895 TCCCACCACAGAATTTCTCT 21 274 142051 3' UTR 3 2921 GCTCTGCAGGGTGACACCTC 74 275 142052 3' UTR 3 3066 AGGAGGTTAAACCAGTACGT 78 276 142053 3' UTR 3 3094 GGTGGAGAGCCAGCTGCTCT 59 277 142054 3' UTR 3 3153 TATTGGCTTAAGGCATATAG 72 278 142055 3' UTR 3 3168 GACCTGATGAGTAAATATTG 58 279 142084 5' UTR 243 859 TTCTTCATGTCAACCGGCAG 11 280 142085 5' UTR 243 919 GCCCCGAGGCCCGCTGCAAT 83 281 142056 Intron 1 243 4206 TAGTGAACTATTGTTACAAC 70 282 142057 Intron 1 243 27032 TGCTAAGCCACTTCTAATCA 72 283 142058 Intron 1 243 27203 CAGGATTCTAAGTTATTAAA 32 284 142059 Intron 1 243 33720 TGGGCAGGATGGCTCTGGTA 21 285 142060 Intron 1 243 48065 TACAATACTATCTGTGACTA 34 286 142061 Exon: 243 51931 GATACTTACAGGGACTGACG 39 287 Intron 142066 Intron 2 243 52005 AACCCTGAGGCGAAAGGAGT 64 288 142062 Intron 2 243 54384 CCCCAGGTCACTAAAATTAA 48 289 142063 Intron 2 243 55362 AAAGCAAAGGTGAGTTGGTG 56 290 142064 Intron 3 243 56093 GCTCAATTATTAAACCACTT 64 291 142065 Intron 3 243 56717 AGTCCTCAAGAAGTCACTTT 70 292 142066 Intron 4 243 61780 GAAAGCAGGGACTGCTGGCA 39 293 142067 Intron 4 243 64554 AAAACTGGGAGAGACAGCAG 71 294 142068 Intron 4 243 64869 ACATGGAAGCCATGGTCAGC 24 295 142069 Intron 5 243 67516 ATTGCTAGACTCACACTAGG 68 296 142070 Intron 5 243 68052 GGCTGTGATCAAAAGGCAGC 51 297 142087 Intron 5 243 68481 CACTGGCTCTGGGCAACTTT 70 298 142088 Intron 5 243 68563 GCTGGGCAGCCACCCATAAA 71 299 142071 Intron 5 243 68648 AGTCCCCTCACCTCTTTTCT 59 300 142072 Exon: 243 69107 CCTCCTTACCAGCAAGAGGC 26 301 Intron 142089 Intron 6 243 69198 TGTATTTTGGAAGAGGAGCG 53 302 142090 Intron 6 243 69220 ACAGACTAACACAGTGAGTC 53 303 142073 Intron 6 243 69264 ACAAATTACCGAGTCTCAGG 47 304 142074 Intron 6 243 69472 TCATGAAAGGCTTGGTGCCC 41 305 142075 Intron 7 243 70042 TTGGAAGATGAAATCTTTTG 30 306 142076 Intron 7 243 70052 AGCCATGTACTTGGAAGATG 69 307 142077 Intron 8 243 70661 CGAGCCCCTCATTCCAACAA 42 308 142078 Intron 8 243 71005 CACCTCAGCGGACACCTCTA 6 309 142079 Exon: 243 71938 GAAACATACCCTGTAGCAGA 52 310 Intron 142091 Intron 9 243 72131 CAGAGGGCTCCTTAAAACCC 61 311 142092 Intron 9 243 72430 ATTCGTAAAAGTTTGGGATT 34 312 142080 Intron 9 243 72453 CCCTCTTCTCCAAGGGAGTT 73 313 142081 Intron 9 243 73158 GGAATGAAACCAAACAGTTC 42 314 142082 Exon 10 243 75012 AAATGGTTTATTCCATGGCC 66 315 101 P:\WPDOCSiCRN\RM f\Spc\0287356 dv se doc - 10/312007 142083 Exon 10 243 75215 AAAAATTTTATTGTTGCAGC 48 316 142093 3' UTR 243 75095 CCGGTCATGCAGCCACGTAT 85 317 142094 3' UTR 243 75165 GTTGGAAAACTGTACAGTCT 77 318 142095 3' UTR 243 75211 ATTTTATTGTTGCAGCTAAA 46 319 As shown in Table 3, SEQ ID NOs: 244, 245, 247, 248, 249, 250, 251, 252, 254, 255,.256, 257, 258, 259, 260, 261, 262, 263, 267, 268, 269, 271, 275, 276, 277, 278, 5 279, 281, 282, 283, 288, 290, 291, 292, 294, 296, 297, 298, 299, 300, 302, 303, 307,- 310, 311, 313, 315, 317, and 318, demonstrated at least 50% inhibition of human PTP1B expression in this assay and are therefore preferred. 10 EXAMPLE 23: ANTISENSE INHIBITION OF HUMAN PTP1B EXPRESSION BY ADDITIONAL CHIMERIC PHOSPHOROTHIOATE OLIGONUCLEOTIDES HAVING 2'-MOE WINGS AND A DEOXY GAP In accordance with the present invention, an 15 additional series of oligonucleotides were designed to target either the 3'UTR or the 5'UTR of the human PTP1B RNA, using published sequences (GenBank accession number M31724, incorporated herein as SEQ ID NO: 3) and concatenated genomic sequence derived from nucleotide 20 residues 1-31000 of Genbank accession number AL034429 followed by nucleotide residues 1-45000 of Genbank accession number AL133230, incorporated herein as SEQ ID NO: 243. The oligonucleotides are shown in Table4. "Target site" indicates the first (5'-most) nucleotide 25 number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 3 are chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap" region consisting of ten 2'-deoxynucleotides, which is flanked on both sides 30 (5' and 3' directions) by five-nucleotide "wings". The P-\WPDOCSCRN\RMH\Sp\20287356 div spe doc. 10/3/2007 wings are composed of 2'-methoxyethyl (2'-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The 5 compounds were analyzed for their effect on human PTP1B mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, "N.D." indicates "no data". 10 TABLE 4: INHIBITION OF HUMAN PTP1B MRNA LEVELS BY CHIMERIC PHOSPHOROTHIOATE OLIGONUCLEOTIDES HAVING 2'-MOE WINGS AND A DEOXY GAP Isis # REGION TARGET TARGET SEQUENCE SEQ ID 146879 5' UTR 3 50 CGCCTCCTTCTCGGCCCACT 29 320 146880 5' UTR 3 62 GGGCGGCTGCTGCGCCTCCT 34 321 146881 3' UTR 3 1601 GTGGATTTGGTACTCAAAGT 72 322 146882 3' UTR 3 1610 AAATGGCTTGTGGATTTGGT 72 323 146883 3' UTR 3 1637 ATGGTACTCTCTTTCACTCT 61 324 146884 3' UTR 3 1643 GCCAGCATGGTACTCTCTTT 63 325 146885 3' UTR 3 1764 GAGAGTTGCTCCCTGCAGAT 62 326 146886 3' UTR 3 1770 GGAGTGGAGAGTTGCTCCCT 57 327 146887 3' UTR 3 1874 CCTTGATGCAAGGCTGACAT 65 328 146888 3' UTR 3 1879 AAAGCCCTTGATGCAAGGCT 59 329 146889 3' UTR 3 1915 AGTACTACCTGAGGATTTAT 46 330 146890 3' UTR 3 1925 TTCCATTCCCAGTACTACCT 41 331 146891 3' UTR 3 1938 CCATGGCAAAGCCTTCCATT 65 332 146892 3' UTR 3 1943 CAGGCCCATGGCAAAGCCTT 52 333 146893 3' UTR 3 1988 CAACTGCTTACAACCGTCCT 60 334 146894 3' UTR 3 2055 CCACGTGTTCATTATATATT 42 335 146895 3' UTR 3 2063 TTAAATACCCACGTGTTCAT 27 336 146896 3' UTR 3 2099 TAAGCGGGACAAAGTAATCT 47 337 146897 3' UTR 3 2118 CAGATAACAGGGAGGAGAAT 31 338 146898 3' UTR 3 2133 GAGAACTAGATCTAGCAGAT 0 339 146899 3' UTR 3 2140 AGTGATTGAGAACTAGATCT 62 340 146900 3' UTR 3 2184 GACACAAGAAGACCTTACAT 49 341 146901 3' UTR 3 2212 CTCATTTCAAGCACATATTT 60 342 146902 3' UTR 3 2263 GGCAGGTTGGACTTGGACAT 49 343 146903 3' UTR 3 2296 AACCACAGCCATGTAATGAT 43 344 146904 3' UTR 3 2332 TTGCTGAGCGACAATGACTT 42 345 146905 3' UTR 3 2350 CTGGAAAACTGCACCCTATT 31 346 146906 3' UTR 3 2409 GCTGGGCCTCACCAGGAAGT 77 347 146907 3' UTR 3 2439 TTACAGCAAGACCCTGCTGT 28 348 146908 3' UTR 3 2457 ACCCTTGGAATGTCTGAGTT 65 349 146909 3' UTR 3 2464 TTCCCATACCCTTGGAATGT 62 350 146910 3' UTR 3 2471 ATATGGCTTCCCATACCCTT 47 351 146911 3' UTR 3 2477 GTGTGAATATGGCTTCCCAT 54 352 146912 3' UTR 3 2509 CCTGCTTCCCTAAATCATGT 65 353 103 P \WPDOCSICRN\RM \Spc\20287356 div sp d . 10/3/2007 146913 3' UTR 3 2514 GTGTCCCTGCTTCCCTAAAT 55 354 146914 3' UTR 3 2546 CGGAGGCTGATCCCAAAGGT 55 355 146915 3' UTR 3 2602 CAGGTGCCTCTCTTCCAAAT 60 356 146916 3' UTR 3 2613 GTGGTTTCCAGCAGGTGCCT 63 357 146917 3' UTR 3 2628 GCTGTTTCAAGAAGTGTGGT 43 358 146918 3' UTR 3 2642 GGACCGTCACCCAGGCTGTT 32 359 146919 3' UTR 3 2655 CAGGCTGCCTAAAGGACCGT 60 360 146920 3' UTR 3 2732 ACCATCAGGCCCCACAGGGT 58 361 146921 3' UTR 3 2759 GTTCCCTTTGCAGGAAGAGT 69 362 146922 3' UTR 3 2772 GTGGAGGTCTTCAGTTCCCT 64 363 146923 3' UTR 3 2781 CCACTTAATGTGGAGGTCTT 54 364 146924 3' UTR 3 2814 AGCTACAGCTGCCGTGTTTT 51 365 146925 3' UTR 3 2862 CCACGAGAAAGGCAAAATGT 50 366 146926 3' UTR 3 2885 GAATTTCTCTGTACTGGCTT 23 367 146927 3' UTR 3 2890 CCACAGAATTTCTCTGTACT 61 368 146928 3' UTR 3 2901 GAATGTTCCCACCACAGAAT 61 369 146929 3' UTR 3 2956 GCCTGGCACCTAAGCCTTAT 0 370 146930 3' UTR 3 2965 ATGCTTACAGCCTGGCACCT 55 371 146931 3' UTR 3 3008 CTACATACATATACAGGACT 65 372 146932 3' UTR 3 3042 TTTGAAATGCTACTATATAT 44 373 146933 3' UTR 3 3070 GGATAGGAGGTTAAACCAGT 67 374 146934 3' UTR 3 3086 GCCAGCTGCTCTCCAAGGAT 42 375 146935 3' UTR 3 3121 CTACCTCTCTAACATAATGT 39 376 146936 3' UTR 3 3126 GCTCGCTACCTCTCTAACAT 68 377 146937 3' UTR 3 3143 AGGCATATAGCAGAGCAGCT 61 378 146938 5' UTR 243 851 GTCAACCGGCAGCCGGAACT 14 379 146942 5' UTR 243 891 CCTGCAGCTACCGCCGCCCT 69 380 146943 5' UTR 243 908 CGCTGCAATCCCCGACCCCT 87 381 146944 3' UTR 243 75050 ACCAAAACACCTTGCTTTTT 27 382 146945 3' UTR 243 75057 GTATTATACCAAAACACCTT 39 383 146946 3' UTR 243 75072 CACACACCTGAAAAGGTATT 42 384 146947 3' UTR 243 75097 ACCCGGTCATGCAGCCACGT 49 385 146948 3' UTR 243 75136 GTGAGGTCACAGAAGACCCT 49 386 146949 3' UTR 243 75154 GTACAGTCTGACAGTTCTGT 40 387 146950 3' UTR 243 75172 ATGGCAAGTTGGAAAACTGT 65 388 146951 3' UTR 243 75192 AATGCAAACCCATCATGAAT 43 389 As shown in Table 4, SEQ ID NOs, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 5 340, 341, 342, 343, 344, 345, 347, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358,.360, 361, 362, 363, 364, 365, 366, 368, 369, 371, 372, 373, 374, 375, 377, 378, 380, 381, 384, 385, 386, 387, 388, and 389 demonstrated at least 40% inhibition of human PTP1B expression in this 10 assay and are therefore preferred. 104 P.\WPDOCS\CRN\RMIH\Spec\20287356 div spo dc - 10/3/2007 EXAMPLE 24: ANTISENSE INHIBITION OF PTP1B EXPRESSION (ISIS 113715) IN LIVER, MUSCLE AND ADIPOSE TISSUE OF THE CYNOMOLGUS MONKEY In a further embodiment, male cynomolgus monkeys 5 were treated with ISIS 113715 (SEQ ID NO: 166) and levels of PTP1B mRNA and protein were measured in muscle, adipose and liver tissue. Serum samples were also measured for insulin levels. Male cynomolgus monkeys were divided into two 10 treatment groups, control animals (n=4; saline treatment only) and treated animals (n=8; treated with ISIS 113715). All animals had two pre-dosing glucose tolerance tests (GTTs) performed to establish insulin and glucose baseline values. Animals in the treatment group 15 were dosed subcutaneously on days 1, 8, and 15 with 3mg/kg, 6 mg/kg and 12 mg/kg of ISIS 113715, respectively. Animals in the. control group were untreated. All animals had GTTs performed on days 5, 13 and 19, four days post-dosing. Ten days after the last 20 dose of 12 mg/kg, all animals in the treatment group (ISIS 113715) received a one-time dose of 6 mg/kg of ISIS 113715. Three days later, all animals were sacrificed and tissues were taken for analysis of PTP1B mRNA and protein levels. Levels of mRNA and protein were 25 normalized to those of the saline treated animals. Of the tissue examined, PTP1B mRNA levels were reduced to the greatest extent in the fat and liver, being reduced by 41% and 40%, respectively. .mRNA levels in muscle were reduced by 10%. Protein levels were reduced by 60% in 30 the liver and by 45% in the muscle but were shown to increase by 10% in the fat. 105 P\WPDOCSCRNIRMH\Spec\20287356div spccdoc - 10/3/2007 Levels of the liver enzymes ALT and AST were measured weekly and showed no. change, indicating no ongoing toxic effects of the oligonucleotide treatment. The results of this study demonstrate a significant 5 reduction in liver PTP1B mRNA and protein upon treatment with ISIS 113715. Furthermore, there was no change seen in the fasting insulin levels either between groups or between pre-treatment and post-treatment of the same group. There was, however, a significant lowering of 10 insulin levels with no decrease in fasting glucose levels in all groups suggesting that insulin efficiency (sensitivity) was increased upon treatment with ISIS 113715. 15 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group.of integers or steps but 20 not the exclusion of any other integer or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any 25 matter which is known, is not', and should not be taken as an acknowledgment or admission or any form of suggestion that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to 30 which this specification relates. 106

Claims (36)

1. An antisense oligonucleotide up to 50 nucleobases in length targeted to a nucleic acid molecule encoding PTP1B, wherein said oligonucleotide specifically hybridizes with exon 10 of PTP1B thereby inhibiting the expression of PTP1B, and wherein said oligonucleotide comprises a sequence set forth in SEQ ID NO: 315 or 316.

2. The oligonucleotide according to claim 1, comprising at least one modified internucleoside linkage, at least one modified sugar moiety, and/or at least one modified nucleobase.

3. The oligonucleotide according to claim 2, wherein the modified internucleoside linkage is a phosphorothioate linkage.

4. The oligonucleotide of claim 2, wherein the modified sugar moiety is a 2'-0-methoxyethyl sugar moiety.

5. The oligonucleotide according to claim 2, wherein the modified nucleobase is a 5-methylcytosine.

6. The oligonucleotide according to any one of claims 1 to 5, wherein the oligonucleotide is a chimeric oligonucleotide.

7. A composition comprising an antisense oligonucleotide of any one of claims 1 to 6 and a pharmaceutically acceptable carrier or diluent.

8. The composition according to claim 7, further comprising a colloidal dispersion system. C:\NRPorbl\DCCGRS\2778599 1. DOC-.18/2010 - 108

9. An oligonucleotide according to any one of claims 1 to 6, which specifically hybridizes with at least an 8-nucleobase portion of an active site in exon 10 of PTP1B.

10. A method of inhibiting the expression of PTP1B in cells or tissues comprising contacting said cells or tissues with an antisense oligonucleotide of any one of claims 1 to 6 or 9 or a composition of claim 7 or 8 such that expression of PTP1B is inhibited.

11. The method according to claim 10, wherein the cells or tissues are human or rodent cells or tissues.

12. The method according to claim 11, wherein the rodent cells or tissues are mouse or rat cells or tissues.

13. The method according to any one of claims 10 to 12, wherein the cells or tissues are liver, kidney or adipose cells or tissues.

14. A method of treating an animal having or suspected of having a disease or condition associated with PTP1B comprising administering to said animal a therapeutically or prophylactically effective amount of an antisense oligonucleotide of any one of claims 1 to 6 or 9 or a composition of claim 7 or 8 such that expression of PTP1B is inhibited.

15. The method according to claim 14, wherein the animal is a human.

16. The method according to claim 14 or 15, wherein the disease or condition is a metabolic disease or condition. C\NRPrtbDCCIRS\2778599 OC-3/IB/2010 - 109

17. The method according to claim 14 or 15, wherein the disease or condition is selected from diabetes, obesity and a hyperproliferative condition.

18. The method according to claim 17, wherein the diabetes is Type 2 diabetes.

19. The method according to claim 17, wherein the hyperproliferative condition is cancer.

20. A method of decreasing blood glucose levels in an animal comprising administering to said animal an antisense oligonucleotide of any one of claims 1 to 6 or 9 or a composition of claim 7 or 8.

21. The method according to claim 20, wherein the animal is a human or a rodent.

22. The method according to claim 20 or 21, wherein the blood glucose levels are plasma glucose levels or serum glucose levels.

23. The method according to any one of claims 20 to 22, wherein the animal is a diabetic animal.

24. A method of preventing or delaying the onset of a disease or condition associated with PTP1B in an animal comprising administering to said animal a therapeutically or prophylactically effective amount of an antisense oligonucleotide of any one of claims 1 to 6 or 9 or a composition of claim 7 or 8.

25. The method according to claim 24, wherein the animal is a human.

26. The method according to claim 24 or 25, wherein the disease or condition is a metabolic disease or C:\NRPotbilDCCIGRS\2778599 I.DOC-3/18 2030 - 110 condition.

27. The method according to claim 24 or 25, wherein the disease or condition is selected from diabetes, obesity and a hyperproliferative condition.

28. The method according to claim 27, wherein the diabetes is Type 2 diabetes.

29. The method according to claim 27, wherein the hyperproliferative condition is cancer.

30. A method of preventing or delaying the onset of an increase in blood glucose levels in an animal comprising administering to said animal an antisense oligonucleotide of any one of claims 1 to 6 or 9 or a composition of claim 7 or 8.

31. The method according to claim 30, wherein the animal is a human or a rodent.

32. The method according to claim 30 or 31, wherein the blood glucose levels are plasma glucose levels or serum glucose levels.

33. The method according to any one of claims 30 to 32, wherein the animal is a diabetic animal.

34. Use of an antisense oligonucleotide of any one of claims 1 to 6 or 9 for the manufacture of a medicament for inhibiting the expression of PTP1B in cells or tissues.

35. Use of an antisense oligonucleotide of any one of claims 1 to 6 or 9 for the manufacture of a medicament for treating an animal having or suspected of having a disease or condition associated with PTP1B, or preventing or delaying the onset of such a disease or condition. CNRPortbl\DCC\(RS\2778599_1 DOC-3/18/2010 - 111

36. Use of an antisense oligonucleotide of any one of claims 1 to 6 or 9 for the manufacture of a medicament for decreasing blood glucose levels or preventing or delaying the onset of an increase in blood glucose levels. 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007 2007221812 03 Oct 2007