Modulation Of Eif4e-bp2

Abstract

Compounds, compositions and methods are provided for modulating the expression of eIF4E-BP2. The compositions comprise oligonucleotides, targeted to nucleic acid encoding eIF4E-BP2. Methods of using these compounds for modulation of eIF4E-BP2 expression and for diagnosis and treatment of diseases and conditions associated with expression of eIF4E-BP2 are provided.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application of U.S. patent application Ser. No. 12/274,030, filed Nov. 19, 2008, which is a continuation of application of U.S. patent application Ser. No. 11/042,899 filed Jan. 24, 2005, now U.S. Pat. No. 7,468,431 issued Dec. 23, 2008 which claims priority to U.S. Application Ser. No. 60/538,752, filed on Jan. 22, 2004, each of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled RTS0731USC2SEQ.txt, created on Oct. 6, 2011 which is 100 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention provides compositions and methods for modulating the expression of eIF4E-BP2. In particular, this invention relates to antisense compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding eIF4E-BP2. Such compounds are shown herein to modulate the expression of eIF4E-BP2.

BACKGROUND OF THE INVENTION

[0004] Eukaryotic gene expression must be regulated such that cells can rapidly respond to a wide range of different conditions. The process of mRNA translation is one step at which gene expression is highly regulated. In response to hormones, growth factors, cytokines and nutrients, animal cells generally activate translation in preparation for the proliferative response. The rate of protein synthesis typically decreases under stressful conditions, such as oxidative or osmotic stress, DNA damage or nutrient withdrawal. Activation or suppression of mRNA translation occurs within minutes and control over this process is thought to be exerted at the initiation phase of protein synthesis (Rosenwald et al., Oncogene, 1999, 18, 2507-2517; Strudwick and Borden, Differentiation, 2002, 70, 10-22).

[0005] Translation initiation necessitates the coordinated activities of several eukaryotic initiation factors (eIFs), proteins which are classically defined by their cytoplasmic location and ability to regulate the initiation phase of protein synthesis. One of these factors, eukaryotic initiation factor 4E (eIF4E), is present in limiting amounts relative to other initiation factors and is one component of the eIF4F initiation complex, which is also comprised of the scaffold protein eIF4G and the RNA helicase eIF4A. In the cytoplasm, eIF4E catalyzes the rate-limiting step of cap-dependent protein synthesis by specifically binding to the 5' terminal 7-methyl GpppX cap structure present on nearly all mature cellular mRNAs, which serves to deliver the mRNAs to the eIF4F complex. Once bound, the eIF4F complex scans from the 5' to the 3' end of the cap, permitting the RNA helicase activity of eIF4A to resolve any secondary structure present in the 5' untranslated region (UTR), thus revealing the translation initiation codon and facilitating ribosome loading onto the mRNA (Graff and Zimmer, Clin. Exp. Metastasis, 2003, 20, 265-273; Strudwick and Borden, Differentiation, 2002, 70, 10-22).

[0006] eIF4E availablity for incorporation into the eIF4E complex is regulated through phosphorylation as well as through the binding of inhibitory proteins. eIF4E is a phosphoprotein that is phosphorylated on serine 209 by the mitogen-activated protein kinase-interacting kinase Mnk1, as well as by protein kinase C (Flynn and Proud, J. Biol. Chem., 1995, 270, 21684-21688; Wang et al., J. Biol. Chem., 1998, 273, 9373-9377; Waskiewicz et al., Embo J., 1997, 16, 1909-1920). The inhibitory eIF4E-binding proteins 1 and 2 (eIF4E-BP1 and eIF4E-BP2) act as effective inhibitors of cap-dependent translation by competing with eIF4G for binding to the dorsal surface of eIF4E (Pause et al., Nature, 1994, 371, 762-767; Ptushkina et al., Embo J., 1999, 18, 4068-4075). When complexed with bp1, eIF4E is not a substrate for phosphorylation by protein kinase C or Mnk1, indicating that dissociation of bp1 from eIF4E is a prerequisite for eIF4E phosphorylation (Wang et al., J. Biol. Chem., 1998, 273, 9373-9377; Whalen et al., J Biol Chem, 1996, 271, 11831-11837). Phosphorylation of eIF4E increases its affinity for mRNA caps, thus elevating translation rates (Waskiewicz et al., Mol. Cell. Biol., 1999, 19, 1871-1880).

[0007] eIF4E-BP2 (also known as PHAS-II; 4EBP2; 4E-binding protein 2; EIF4EBP2) was cloned through use of the eIF4E protein in probing a cDNA expression library (Hu et al., Proc Natl Acad Sci USA, 1994, 91, 3730-3734; Pause et al., Nature, 1994, 371, 762-767). eIF4E-BP2 is ubiquitously expressed in human tissues, including heart, brain, placenta, lung, liver, kidney and spleen, as well as adipose tissue and skeletal muscle, the major insulin-responsive tissues (Hu et al., Proc Natl Acad Sci USA, 1994, 91, 3730-3734; Tsukiyama-Kohara et al., Genomics, 1996, 38, 353-363). The human gene maps to chromosome 10q21-q22 (Tsukiyama-Kohara et al., Genomics, 1996, 38, 353-363). The mouse bp1 gene consists of three exons, spans approximately 20 kb and maps to mouse chromosome 10 (Tsukiyama-Kohara et al., Genomics, 1996, 38, 353-363). The expression of eIF4E-BP2 does not appear to be altered in mice bearing a systemic disruption of bp1 (Blackshear et al., J Biol Chem, 1997, 272, 31510-31514).

[0008] Rather than preventing the binding of eIF4E to mRNA caps, eIF4E-BP2 prohibits the binding of eIF4E to eIF4G, thereby preventing formation of a complex that is necessary for efficient binding and proper positioning of the 40S ribosomal subunit on the target mRNA. When eIF4E-BP2 is bound to eIF4E, eIF4E does not serve as a substrate for phosphorylation by protein kinase C, suggesting that dissociation of eIF4E-BP2 from eIF4E is a prerequisite for phosphorylation of eIF4E (Whalen et al., J Biol Chem, 1996, 271, 11831-11837). The region to which eIF4E binds is a common motif shared by eIF4G and eIF4E-BP2, and point mutations in this region of eIF4E-BP2 abolish binding to eIF4E (Mader et al., Mol Cell Biol, 1995, 15, 4990-4997). Two conserved motifs are present in the eIF4E-BP2: the RAIP motif, which is found in the NH2-terminal region of EIF4E-BP2 and the TOS motif, which is formed by the last five amino acids of eIF4E-BP2 (Schalm and Blenis, Curr Biol, 2002, 12, 632-639; Tee and Proud, Mol Cell Biol, 2002, 22, 1674-1683).

[0009] Like eIF4E-BP1, insulin stimulates the phosphorylation of eIF4E-BP2 in cultured cells, which promotes the release of eIF4E-BP2 from eIF4E and allows for cap-dependent translation to proceed (Ferguson et al., J Biol Chem, 2003, 278, 47459-47465). Mitogen-activated protein kinase, the major insulin-stimulated kinase in rat adipocytes, can phosphorylate recombinant eIF4E-BP2 in vitro. However, treatment of 3T3-L1 rat adipocytes with rapamycin attenuates the effects of insulin on the phosphorylation of eIF4E-BP2, indicating that elements of the mTOR signaling pathway mediate the actions of insulin on eIF4E-BP2 (Lin and Lawrence, J Biol Chem, 1996, 271, 30199-30204). Additionally, serine-65 of eIF4E-BP2 represents an ideal consensus site for phosphorylation by cyclicAMP-dependent protein kinase. In rat 3T3-L1 adipocytes, where insulin or epidermal growth factor markedly increased the phosphorylation of eIF4E-BP2, compounds that increase cyclic AMP decrease the amount of radiolabeled phosphate incorporated into eIF4E-BP2, and attenuate the effects of insulin on increasing the phosphorylation of eIF4E-BP2. Incubation of eIF4E-BP2 with the catalytic subunit of cyclic AMP-dependent protein kinase results in the rapid phosphorylation of eIF4E-BP2. Together, these data suggest that increasing cyclic AMP may selectively increase eIF4E-BP2 phosphorylation (Lin and Lawrence, J Biol Chem, 1996, 271, 30199-30204).

[0010] Induction of cellular differentiation and reduction of cellular proliferation are concomitant with a reduction in translation rates, as is observed in conjuction with differential regulation of eIF4E-BPs during human myeloid cell differentiation. When induced to differentiate into monocytes/macrophages, cells from the HL-60 promyelocytic leukemia cell or U-937 monoblastic cell lines exhibit a decrease in the phosphorylation of bp1. In contrast, when HL-60 cells are stimulated to differentiate into granulocytic cells, the amount of bp1 is decreased, whereas phosphorylation of bp1 is not affected. Conversely, eIF4E-BP2 levels are markedly increased. These findings suggest that translation machinery is differentially regulated during human myeloid cell differentiation (Grolleau et al., J Immunol, 1999, 162, 3491-3497).

[0011] The disregulation of signaling networks that promote cell proliferation is often observed in association with cancer (Lawrence and Abraham, Trends Biochem Sci, 1997, 22, 345-349). Expression of excess eIF4E-BP2 in cells transformed by eIF4E or v-src results in significant reversion of the transformed phenotype, demonstrating that eIF4E-BP2 can function as an inhibitor of cell growth (Rousseau et al., Oncogene, 1996, 13, 2415-2420).

[0012] The U.S. Pat. No. 6,410,715 describes a purified human nucleic acid sequence encoding a cellular component that binds to eIF4E comprising a coding sequence for the protein eIF4E-BP2, and discloses a method for screening a non-hormone agent potentially useful to treat a hormone disorder (Sonenberg et al., 2000).

[0013] Currently, there are no known therapeutic agents that target eIF4E-BP2. Consequently, there remains a long felt need for agents capable of effectively inhibiting eIF4E-BP2. Antisense technology is an effective means of reducing the expression of specific gene products and therefore is uniquely useful in a number of therapeutic, diagnostic and research applications for the modulation of eIF4E-BP2 expression.

[0014] The present invention provides compositions and methods for inhibiting eIF4E-BP2 expression.

SUMMARY OF THE INVENTION

[0015] The present invention is directed to antisense compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding eIF4E-BP2, and which modulate the expression of eIF4E-BP2. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of eIF4E-BP2 and methods of modulating the expression of eIF4E-BP2 in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of eIF4E-BP2, thereby in some instances delaying onset of said disease or condition, are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

A. Overview of the Invention

[0016] The present invention employs antisense compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding eIF4E-BP2. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding eIF4E-BP2. As used herein, the terms "target nucleic acid" and "nucleic acid molecule encoding eIF4E-BP2" have been used for convenience to encompass DNA encoding eIF4E-BP2, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of this invention with its target nucleic acid is generally referred to as "antisense". Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as "antisense inhibition." Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.

[0017] The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of eIF4E-BP2. In the context of the present invention, "modulation" and "modulation of expression" mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.

[0018] In the context of this invention, "hybridization" means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

[0019] An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

[0020] In the present invention the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, "stringent conditions" under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.

[0021] "Complementary," as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.

[0022] 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 specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70%, or at least 75%, or at least 80%, or at least 85% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise at least 90% sequence complementarity and even more preferably comprise at least 95% or at least 99% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0023] Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In some preferred embodiments, homology, sequence identity or complementarity, between the oligomeric and target is between about 50% to about 60%. In some embodiments, homology, sequence identity or complementarity, is between about 60% to about 70%. In preferred embodiments, homology, sequence identity or complementarity, is between about 70% and about 80%. In more preferred embodiments, homology, sequence identity or complementarity, is between about 80% and about 90%. In some preferred embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.

B. Compounds of the Invention

[0024] According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, siRNAs, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid.

[0025] One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.

[0026] While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.

[0027] The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).

[0028] The antisense compounds of the present invention also include modified compounds in which a different base is present at one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenosine, modified compounds may be produced which contain thymidine, guanosine or cytidine at this position. This may be done at any of the positions of the antisense compound. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of eIF4E-BP2 mRNA.

[0029] In the context of this invention, the term "oligomeric compound" refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and 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 because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

[0030] While oligonucleotides are a preferred form of the antisense compounds of this invention, the present invention comprehends other families of antisense compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.

[0031] The antisense compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

[0032] In one preferred embodiment, the antisense compounds of the invention are 13 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases in length.

[0033] In another preferred embodiment, the antisense compounds of the invention are 15 to 30 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

[0034] Particularly preferred compounds are oligonucleotides from about 12 to about 50 nucleobases, even more preferably those comprising from about 15 to about 30 nucleobases.

[0035] Antisense compounds 13-80 nucleobases in length comprising a stretch of at least thirteen (13) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0036] While oligonucleotides are one 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 compounds in accordance with this invention can comprise from about 8 to about 80 nucleobases. In another embodiment, the oligonucleotide is about 10 to 50 nucleotides in length. In yet another embodiment, the oligonucleotide is 12 to 30 nucleotides in length. In yet another embodiment, the oligonucleotide is 12 to 24 nucleotides in length. In yet another embodiment, the oligonucleotide is 19 to 23 nucleotides in length. Some embodiments comprise at least an 8-nucleobase portion of a sequence of an oligomeric compound which inhibits expression of eIF4E-BP1. dsRNA or siRNA molecules directed to eIF4E-BP1, and their use in inhibiting eIF4E-BP1 mRNA expression, are also embodiments within the scope of the present invention.

[0037] The oligonucleotides of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the oligonucleotide. For example, if the first nucleotide is an adenosine, variants may be produced which contain thymidine (or uridine if RNA), guanosine or cytidine at this position. This may be done at any of the positions of the oligonucleotide. Thus, a 20-mer may comprise 60 variations (20 positions.times.3 alternates at each position) in which the original nucleotide is substituted with any of the three alternate nucleotides. These oligonucleotides are then tested using the methods described herein to determine their ability to inhibit expression of eIF4E-BP1 mRNA.

[0038] Exemplary preferred antisense compounds include oligonucleotide sequences that comprise at least the 13 consecutive nucleobases from the 5'-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately upstream of the 5'-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 13 to about 80 nucleobases). Similarly preferred antisense compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3'-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3'-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). It is also understood that preferred antisense compounds may be represented by oligonucleotide sequences that comprise at least 13 consecutive nucleobases from an internal portion of the sequence of an illustrative preferred antisense compound, and may extend in either or both directions until the oligonucleotide contains about 13 to about 80 nucleobases.

[0039] One having skill in the art armed with the preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.

C. Targets of the Invention

[0040] "Targeting" an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target nucleic acid encodes eIF4E-BP2.

[0041] The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. Within the context of the present invention, the term "region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. "Segments" are defined as smaller or sub-portions of regions within a target nucleic acid. "Sites," as used in the present invention, are defined as positions within a target nucleic acid.

[0042] Since, as is known in the art, the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon". A minority of genes have a translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, "start codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding eIF4E-BP2, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively).

[0043] The terms "start codon region" and "translation initiation codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon. Consequently, the "start codon region" (or "translation initiation codon region") and the "stop codon region" (or "translation termination codon region") are all regions which may be targeted effectively with the antisense compounds of the present invention.

[0044] The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a preferred region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

[0045] Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), 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 nucleotides on the gene). The 5' cap site 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 nucleotides adjacent to the cap site. It is also preferred to target the 5' cap region.

[0046] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts". It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.

[0047] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as "variants". More specifically, "pre-mRNA variants" are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.

[0048] Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA variants". Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as "alternative splice variants". If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0049] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as "alternative stop variants" of that pre-mRNA or mRNA. One specific type of alternative stop variant is the "polyA variant" in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also preferred target nucleic acids.

[0050] The locations on the target nucleic acid to which the preferred antisense compounds hybridize are hereinbelow referred to as "preferred target segments." As used herein the term "preferred target segment" is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.

[0051] While the specific sequences of certain preferred target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target segments may be identified by one having ordinary skill.

[0052] Target segments 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.

[0053] Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5'-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5'-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3'-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3'-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). It is also understood that preferred antisense target segments may be represented by DNA or RNA sequences that comprise at least 8 consecutive nucleobases from an internal portion of the sequence of an illustrative preferred target segment, and may extend in either or both directions until the oligonucleotide contains about 8 to about 80 nucleobases. One having skill in the art armed with the preferred target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.

[0054] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0055] The oligomeric antisense compounds may also be targeted to regions of the target nucleobase sequence (e.g., such as those disclosed in Example 13) comprising nucleobases 1-80, 81-160, 161-240, 241-320, 321-400, 401-480, 481-560, 561-640, 641-720, 721-800, 801-880, 881-960, 961-1040, 1041-1120, 1121-1200, 1201-1280, 1281-1360, 1361-1440, 1441-1520, 1521-1600, 1601-1680, 1681-1760, 1761-1840, 1841-1920, 1921-2000, 2001-2080, 2081-2160, 2161-2240, 2241-2320, 2321-2400, 2401-2480, 2481-2560, 2561-2640, 2641-2720, 2721-2782, or any combination thereof.

[0056] In one embodiment of the invention, the antisense compounds are targeted to a nucleic acid molecule encoding human eIF4E-BP2, for example nucleotides 146-165 in the 5' UTR, nucleotides 372-391, 420-520 or 544-593 in the coding region, nucleotides 589-608 in the stop codon region, nucleotides 623-766, 803-940, 1105-1599, 1868-1887, 1900-1919, 1962-1981, 2218-2242, 2377-2401, 2449-2490, 2536-2555 or 2578-2597 in the 3' UTR, all of SEQ ID NO: 4; nucleotides 8892-8911 and 11559-11937 in intron 1, and nucleotides 17941-17960 in the intron 1:exon 2 junction, all of SEQ ID NO: 25; nucleotides 2088-2107 in the 3' UTR of SEQ ID NO: 26; and nucleotides 697-716 in the 3'UTR of SEQ ID NO: 27, wherein said compound inhibits the expression of human eIF4E-BP2 mRNA.

[0057] In another embodiment of the invention, the antisense compounds are targeted to a nucleic acid molecule encoding mouse eIF4E-BP2, for example nucleotides 9-105 in the 5'UTR; nucleotides 132-480 in the coding region; nucleotides 473-492 in the stop codon region; and nucleotides 500-1175, 1222-1638, 1662-1780 in the 3' UTR, all of SEQ ID NO: 11; nucleotides 365-384 in the 3' UTR of SEQ ID NO: 107; and nucleotides 36-55 in the 5' UTR of SEQ ID NO: 108; wherein said compound inhibits the expression of mouse eIF4E-BP2 mRNA.

[0058] In a further embodiment of the invention, antisense compounds are targeted to a nucleic acid molecule encoding rat eIF4E-BP2, for example nucleotides 7-26 in the 5'UTR, nucleotides 7-151, 164-247, 270-313, or 303-388 in the coding region; nucleotides 390-409 in the stop codon region and nucleotides 402-490 in the 3' UTR, all of SEQ ID NO: 18;

wherein said compound inhibits the expression of rat eIF4E-BP2 mRNA.

D. Screening and Target Validation

[0059] In a further embodiment, the "preferred target segments" identified herein may be employed in a screen for additional compounds that modulate the expression of eIF4E-BP2. "Modulators" are those compounds that decrease or increase the expression of a nucleic acid molecule encoding eIF4E-BP2 and which comprise at least an 8-nucleobase portion which is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding eIF4E-BP2 with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding eIF4E-BP2. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding eIF4E-BP2, the modulator may then be employed in further investigative studies of the function of eIF4E-BP2, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

[0060] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.

[0061] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., Science, 2002, 295, 694-697).

[0062] The antisense compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between eIF4E-BP2 and a disease state, phenotype, or condition. These methods include detecting or modulating eIF4E-BP2 comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of eIF4E-BP2 and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

[0063] The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.

[0064] For use in kits and diagnostics, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0065] As one nonlimiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0066] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0067] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding eIF4E-BP2. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective eIF4E-BP2 inhibitors will also be effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding eIF4E-BP2 and in the amplification of said nucleic acid molecules for detection or for use in further studies of eIF4E-BP2. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding eIF4E-BP2 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 any other suitable detection means. Kits using such detection means for detecting the level of eIF4E-BP2 in a sample may also be prepared.

[0068] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

[0069] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of eIF4E-BP2 is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of a eIF4E-BP2 inhibitor. The eIF4E-BP2 inhibitors of the present invention effectively inhibit the activity of the eIF4E-BP2 protein or inhibit the expression of the eIF4E-BP2 protein. In one embodiment, the activity or expression of eIF4E-BP2 in an animal is inhibited by about 10%. Preferably, the activity or expression of eIF4E-BP2 in an animal is inhibited by about 30%. More preferably, the activity or expression of eIF4E-BP2 in an animal is inhibited by 50% or more. Thus, the oligomeric antisense compounds modulate expression of eIF4E-BP2 mRNA by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%.

[0070] For example, the reduction of the expression of eIF4E-BP2 may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding eIF4E-BP2 protein and/or the eIF4E-BP2 protein itself.

[0071] The antisense compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.

[0072] The compounds of the present inventions are inhibitors of eIF4E-BP2 expression. Thus, the compounds of the present invention are believed to be useful for treating metabolic diseases and conditions, particularly diabetes, obesity, hyperlipidemia or metabolic syndrome X. The compounds of the invention are also believed to be useful for preventing or delaying the onset of metabolic diseases and conditions, particularly diabetes, obesity, hyperlipidemia or metabolic syndrome X. Metabolic syndrome, metabolic syndrome X or simply Syndrome X refers to a cluster of risk factors that include obesity, dyslipidemia, particularly high blood triglycerides, glucose intolerance, high blood sugar and high blood pressure. Scott, C. L., Am J. Cardiol. 2003 Jul. 3; 92(1A):35i-42i. The compounds of the invention have surprisingly been found to be effective for lowering blood glucose, including plasma glucose, and for lowering blood lipids, including serum lipids, particularly serum cholesterol and serum triglycerides. The compounds of the invention are therefore particularly useful for the treatment, prevention and delay of onset of type 2 diabetes, high blood glucose and hyperlipidemia.

F. Modifications

[0073] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base sometimes referred to as a "nucleobase" or simply a "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 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 oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

Modified Internucleoside Linkages (Backbones)

[0074] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, 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, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0075] Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriaminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0076] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0077] 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 cycloalkyl internucleoside linkages, 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 backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts.

[0078] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

Modified Sugar and Internucleoside Linkages-Mimetics

[0079] In other preferred antisense compounds, e.g., oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate target nucleic acid. One such compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The 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. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0080] Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH.sub.2--NH--O--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--O--CH.sub.2-[known as a methylene (methylimino) or MMI backbone], --CH.sub.2--O--N(CH.sub.3)--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and --O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified Sugars

[0081] Modified antisense compounds may also contain one or more substituted sugar moieties. Preferred are antisense compounds, preferably antisense oligonucleotides, comprising one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3].sub.2, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C.sub.1 to C.sub.10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, poly-alkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy(2'-O--CH.sub.2CH.sub.2OCH.sub.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'-dimethylaminooxyethoxy, i.e., a O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.3).sub.2, also described in examples hereinbelow.

[0082] Other preferred modifications include 2'-methoxy(2'-O--CH.sub.3), 2'-aminopropoxy(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl (2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl (2'-O--CH.sub.2--CH.dbd.CH.sub.2) and 2'-fluoro (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. A preferred 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Antisense compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 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.

[0083] A further preferred modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (--CH.sub.2--).sub.n, group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

Natural and Modified Nucleobases

[0084] Antisense compounds may also include nucleobase (often referred to in the art as heterocyclic base or 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 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-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (--C.ident.C--CH.sub.3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., 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., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0085] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

Conjugates

[0086] Another modification of the antisense compounds of the invention involves chemically linking to the antisense compound one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of which are incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Antisense compounds of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0087] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

Chimeric Compounds

[0088] 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 incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

[0089] The present invention also includes antisense compounds which are chimeric compounds. "Chimeric" antisense compounds or "chimeras," in the context of this invention, are 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. Chimeric antisense oligonucleotides are thus a form of antisense compound. These 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, increased stability and/or increased 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 RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNAseL which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0090] 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 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. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 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.

G. Formulations

[0091] 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 other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 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; and 5,595,756, each of which is herein incorporated by reference.

[0092] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.

[0093] The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. For oligonucleotides, presently 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 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 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, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine. Sodium salts are presently believed to be more preferred.

[0094] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2'-O-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[0095] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0096] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0097] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.

[0098] Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 .mu.m in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0099] Formulations of the present invention include liposomal formulations. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0100] Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, 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 lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0101] The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0102] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0103] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0104] Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).

[0105] For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.

[0106] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. Nos. 09/108,673 (filed Jul. 1, 1998), 09/315,298 (filed May 20, 1999) and 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.

[0107] 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, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0108] Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0109] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

H. Dosing

[0110] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC.sub.50s found to be 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. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0111] 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. Each of the references, GenBank.RTM. accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.

EXAMPLES

Example 1

Design and Screening of Duplexed Antisense Compounds Targeting eIF4E-BP2

[0112] In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target eIF4E-BP2. The nucleobase sequence of the antisense strand of the duplex comprises at least a 8-nucleobase portion of an oligonucleotide in Table 1. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini. Overhangs can range from 2 to 6 nucleobases and these nucleobases may or may not be complementary to the target nucleic acid. In another embodiment, the duplexes may have an overhang on only one terminus.

[0113] For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 260) and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:

##STR00001##

[0114] In another embodiment, a duplex comprising an antisense strand having the same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 260) may be prepared with blunt ends (no single stranded overhang) as shown:

##STR00002##

[0115] The RNA duplex can be unimolecular or bimolecular; i.e., the two strands can be part of a single molecule or may be separate molecules.

[0116] RNA strands of the duplex can be synthesized by methods, disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands (or alternatively, the complementary portions of a single RNA strand in the case of a unimolecular duplex) are annealed. The single strands are aliquoted and diluted to a concentration of 50 uM. Once diluted, 30 uL of each strand is combined with 15 uL of a 5.times. solution of annealing buffer. The final concentration of said buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. This solution is incubated for 1 minute at 90.degree. C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37.degree. C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 uM. This solution can be stored frozen (-20.degree. C.) and freeze-thawed up to 5 times.

[0117] Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate eIF4E-BP2 expression.

[0118] When cells reached 80% confluency, they are treated with duplexed antisense compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 .mu.L OPTI-MEM.TM.-1 reduced-serum medium (Invitrogen Life Technologies, Carlsbad, Calif.) and then treated with 130 .mu.L of OPTI-MEM.TM.-1 containing 12 .mu.g/mL LIPOFECTIN.TM. (Invitrogen Life Technologies, Carlsbad, Calif.) per 200 nM of the desired duplex antisense compound. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by real-time PCR.

Example 2

Oligonucleotide Isolation

[0119] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55.degree. C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH.sub.4OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the -16 amu product (+/-32 +/-48). For some studies oligonucleotides were purified by HPLC, as 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.

Example 3

Oligonucleotide Synthesis

96 Well Plate Format

[0120] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 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 (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0121] Oligonucleotides were cleaved from support and deprotected with concentrated NH.sub.4OH at elevated temperature (55-60.degree. C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then 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 4

Oligonucleotide Analysis

96-Well Plate Format

[0122] 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 electrophoresis (CE) in either the 96-well format (Beckman P/ACE.TM. MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE.TM. 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing 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.

Example 5

Cell Culture and Oligonucleotide Treatment

[0123] 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 measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, 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 real-time PCR.

T-24 Cells:

[0124] 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 (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (e.g., Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 7000 cells/well for use in oligonucleotide transfection experiments and real-time PCR analysis.

[0125] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

A549 Cells:

[0126] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded onto 96-well plates (e.g., Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 5000 cells per well for use in oligonucleotide transfection experiments and real-time PCR analysis.

NHDF Cells:

[0127] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (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 recommended by the supplier.

HEK Cells:

[0128] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

b.END Cells:

[0129] The mouse brain endothelial cell line b.END was obtained from Dr. Werner Risau at the Max Plank Instititute (Bad Nauheim, Germany). b.END cells were routinely cultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (e.g., Falcon-Primaria #3872, BD Biosciences, Bedford, Mass.) at a density of approximately 3000 cells/well for use in oligonucleotide transfection experiments and real-time PCR analysis.

[0130] For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

A 10 Cells:

[0131] The rat aortic smooth muscle cell line A10 was obtained from the American Type Culture Collection (Manassas, Va.). A10 cells were routinely cultured in DMEM, high glucose (American Type Culture Collection, Manassas, Va.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 80% confluence. Cells were seeded into 96-well plates (e.g., Falcon-Primaria #3872, BD Biosciences, Bedford, Mass.) at a density of approximately 2500 cells/well for use in oligonucleotide transfection experiments and real-time PCR analysis.

[0132] For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

EMT-6 Cells:

[0133] The mouse mammary epithelial carcinoma cell line EMT-6 was obtained from American Type Culture Collection (Manassus, Va.). They were grown in serial monolayer culture in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum, (Invitrogen Life Technologies, Carlsbad, Calif.), 100 ug/ml penicillin and 100 ug/ml streptomycin (Invitrogen Life Technologies, Carlsbad, Calif.) in a humidified atmosphere of 90% air-10% CO.sub.2 at 37.degree. C. Cells were routinely passaged by trypsinization and dilution when they reached 85-90% confluencey. Cells were seeded into 96-well plates (e.g., Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 1000 cells/well for use in oligonucleotide transfection experiments and real-time PCR analysis.

Treatment with Antisense Compounds:

[0134] When cells reached 65-75% confluency, they were treated with oligonucleotide. Oligonucleotide was mixed with LIPOFECTIN.TM. (Invitrogen Life Technologies, Carlsbad, Calif.) in OPTI-MEM.TM.-1 reduced-serum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desired concentration of oligonucleotide and a concentration of 2.5 to 3 ug/mL LIPOFECTIN.TM. per 100 nM oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 .mu.L OPTI-MEM.TM.-1 reduced-serum medium and then treated with 130 .mu.L of the LIPOFECTIN.TM./oligonucleotide mixture. Cells are treated and data are obtained in duplicate or triplicate. After 4-7 hours of treatment at 37.degree. C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0135] The concentration of oligonucleotide used varies 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 selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2'-O-methoxyethyl gapmers (2'-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2'-O-methoxyethyl gapmer (2'-O-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-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is then utilized 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 c-H-ras, JNK2 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. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 6

Analysis of Oligonucleotide Inhibition of eIF4E-BP2 Expression

[0136] Antisense modulation of eIF4E-BP2 expression can be assayed in a variety of ways known in the art. For example, eIF4E-BP2 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR. Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM.TM. 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0137] Protein levels of eIF4E-BP2 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to eIF4E-BP2 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

Example 7

Design of Phenotypic Assays for the Use of eIF4E-BP2 Inhibitors

[0138] Once eIF4E-BP2 inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of eIF4E-BP2 in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).

[0139] In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with eIF4E-BP2 inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.

[0140] Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.

[0141] Measurement of the expression of one or more of the genes of the cell after treatment is also used as an indicator of the efficacy or potency of the eIF4E-BP2 inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

Example 8

RNA Isolation

[0142] Poly(A)+ mRNA Isolation

[0143] Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 .mu.L cold PBS. 60 .mu.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 well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 .mu.L of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 .mu.L of wash 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 .mu.L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70.degree. C., was added to each well, the plate was incubated on a 90.degree. C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0144] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Total RNA Isolation

[0145] Total RNA was isolated using an RNEASY 96.TM. kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 .mu.L cold PBS. 150 .mu.L Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 .mu.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 96.TM. well plate attached to a QIAVAC.TM. manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 .mu.L of Buffer RW1 was added to each well of the RNEASY 96.TM. plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 .mu.L of Buffer RW1 was added to each well of the RNEASY 96.TM. plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96.TM. plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC.TM. manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC.TM. manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 .mu.L of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0146] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). 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 steps are carried out.

Example 9

Real-Time Quantitative PCR Analysis of eIF4E-BP2 mRNA Levels

[0147] Quantitation of eIF4E-BP2 mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM.TM. 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This 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, 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., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5' end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) 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 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 sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM.TM. Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0148] Prior to 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 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, 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 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 multiplexable. Other methods of PCR are also known in the art.

[0149] Isolated RNA is subjected to a reverse transcriptase (RT) reaction, for the purpose of generating complementary DNA (cDNA), which is the substrate for the real-time PCR. Reverse transcriptase and real-time PCR reagents were obtained from Invitrogen Life Technologies, (Carlsbad, Calif.). The RT reaction and real-time PCR were carried out by adding 20 .mu.L PCR cocktail (2.5.times. PCR buffer minus MgCl.sub.2, 6.6 mM MgCl.sub.2, 375 .mu.M each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM.RTM. Taq, 5 Units MuLV reverse transcriptase, and 2.5.times. ROX dye) to 96-well plates containing 30 .mu.L total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48.degree. C. Following a 10 minute incubation at 95.degree. C. to activate the PLATINUM.RTM. Taq, 40 cycles of a two-step PCR protocol were carried out: 95.degree. C. for 15 seconds (denaturation) followed by 60.degree. C. for 1.5 minutes (annealing/extension).

[0150] Gene target quantities obtained by real-time PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen.TM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time real-time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen.TM. RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen.TM. are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0151] In this assay, 170 .mu.L of RiboGreen.TM. working reagent (RiboGreen.TM. reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 .mu.L purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

[0152] Probes and primers to human eIF4E-BP2 were designed to hybridize to a human eIF4E-BP2 sequence, using published sequence information (GenBank.RTM. accession number NM.sub.--004096.3, incorporated herein as SEQ ID NO: 4). For human eIF4E-BP2 the PCR primers were:

forward primer: CCTCTAGTTTTGGGTGTGCATGT (SEQ ID NO: 5) reverse primer: CCCATAGCAAGGCAGAATGG (SEQ ID NO: 6) and the PCR probe was: FAM-TGGAGTTTGTAGTGGGTGGTTTGTAAAACTGG-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 9) and the PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3' (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

[0153] Probes and primers to mouse eIF4E-BP2 were designed to hybridize to a mouse eIF4E-BP2 sequence, using published sequence information (GenBank.RTM. accession number NM.sub.--010124.1, incorporated herein as SEQ ID NO: 11). For mouse eIF4E-BP2 the PCR primers were:

forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 12) reverse primer: CGGACAGACGGACGATGAG (SEQ ID NO: 13) and the PCR probe was: FAM-CCTCCCAGGTCTCTCGCCCT-TAMRA (SEQ ID NO: 14) where FAM is the fluorescent reporter dye and TAMRA is the quencher dye. For mouse GAPDH the PCR primers were: forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 15) reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 16) and the PCR probe was: 5' JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3' (SEQ ID NO: 17) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

[0154] Probes and primers to rat eIF4E-BP2 were designed to hybridize to a rat eIF4E-BP2 sequence, using published sequence information (GenBank.RTM. accession number XM.sub.--215414.1, incorporated herein as SEQ ID NO: 18). For rat eIF4E-BP2 the PCR primers were:

forward primer: AGTGAACAACTTGAACAACCTGAACA (SEQ ID NO: 19) reverse primer: ACTGCAGCAGGGTCAGATGTC (SEQ ID NO: 20) and the PCR probe was: FAM-TCACGACAGGAAGCACGCAGTTGG-TAMRA (SEQ ID NO: 21) where FAM is the fluorescent reporter dye and TAMRA is the quencher dye. For rat GAPDH the PCR primers were: forward primer: TGTTCTAGAGACAGCCGCATCTT (SEQ ID NO: 22) reverse primer: CACCGACCTTCACCATCTTGT (SEQ ID NO: 23) and the PCR probe was: 5' JOE-TTGTGCAGTGCCAGCCTCGTCTCA-TAMRA 3' (SEQ ID NO: 24) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 10

Northern Blot Analysis of eIF4E-BP2 mRNA Levels

[0155] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL.TM. (TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND.TM.-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST "B" Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER.TM. UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB.TM. hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0156] To detect human eIF4E-BP2, a human eIF4E-BP2 specific probe was prepared by PCR using the forward primer CCTCTAGTTTTGGGTGTGCATGT (SEQ ID NO: 5) and the reverse primer CCCATAGCAAGGCAGAATGG (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0157] To detect mouse eIF4E-BP2, a mouse eIF4E-BP2 specific probe was prepared by PCR using the forward primer AGAGCAGCACAGGCTAAGACAGT (SEQ ID NO: 12) and the reverse primer CGGACAGACGGACGATGAG (SEQ ID NO: 13). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0158] To detect rat eIF4E-BP2, a rat eIF4E-BP2 specific probe was prepared by PCR using the forward primer AGTGAACAACTTGAACAACCTGAACA (SEQ ID NO: 19) and the reverse primer ACTGCAGCAGGGTCAGATGTC (SEQ ID NO: 20). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0159] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER.TM. and IMAGEQUANTT.TM. Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 11

Antisense Inhibition of Human eIF4E-BP2 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2'-MOE Wings and a Deoxy Gap

[0160] In accordance with the present invention, a series of antisense compounds was designed to target different regions of the human eIF4E-BP2 RNA, using published sequences (GenBank.RTM. accession number NM.sub.--004096.3, incorporated herein as SEQ ID NO: 4, nucleotides 20714677 to 20740000 of the sequence with GenBank.RTM. accession number NT.sub.--008583.16, incorporated herein as SEQ ID NO: 25, GenBank.RTM. accession number AK057643.1, incorporated herein as SEQ ID NO: 26, GenBank.RTM. accession number AK001936.1, incorporated herein as SEQ ID NO: 27, and GenBank.RTM. accession number BF686401.1, incorporated herein as SEQ ID NO: 28). The compounds are shown in Table 1. "Target site" indicates the first (5'-most) nucleotide number on the particular target sequence to which the compound 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 on both sides (5' and 3' directions) by five-nucleotide "wings". The wings are composed of 2'-O-methoxyethyl (2'-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P.dbd.S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human eIF4E-BP2 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which A549 cells were treated with 75 nM of the antisense oligonucleotides of the present invention. SEQ ID NO: 2 was used as the control oligonucleotide in this assay. If present, "N.D." indicates "no data".

TABLE-US-00001 TABLE 1 Inhibition of human eIF4E-BP2 mRNA levels by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap SEQ TARGET TARGET % ID ISIS # REGION SEQ ID NO SITE SEQUENCE INHIB NO 232773 Coding 4 420 gccatgggagaattgcgacg 68 29 232776 Coding 4 493 tttggagtcttcaattaagg 31 30 232777 Coding 4 498 tctactttggagtcttcaat 2 31 232828 3'UTR 4 1962 gtctgtagtcatcttaaaaa 52 32 322947 Coding 4 501 acttctactttggagtcttc 60 33 347546 Intron 1 25 1836 tagaccgcaggagctgcgaa 0 34 347547 Intron 1 25 8892 agtgattctcaaactgcaga 38 35 347548 Intron 1 25 11559 tcttctgatccatggccacc 52 36 347549 Intron 1 25 11918 tcagcactatctgttgaaaa 39 37 347550 Intron 1: Exon 2 25 16139 attcgagttcctggaaaaca 0 38 junction 347551 Exon 2: Intron 2 25 16324 ttctcttaccaactgcatgt 0 39 junction 347552 Intron 2: exon 3 25 17941 gcatcatcccctagttagga 27 40 junction 347553 5'UTR 4 146 cctcaggcggacggaaaagc 39 41 347554 Coding 4 332 cgggcgtggtgcaatagtca 63 42 347555 Coding 4 372 attcgagttcctcccggtgt 55 43 347556 Coding 4 392 gaaactttctgtcataaatg 0 44 347557 Coding 4 397 caacagaaactttctgtcat 15 45 347558 Coding 4 474 gtgccagggctagtgactcc 43 46 347559 Coding 4 526 attgttcaagttgttcaaat 0 47 347560 Coding 4 544 tgcatgtttcctgtcgtgat 59 48 347561 Coding 4 549 ccaactgcatgtttcctgtc 54 49 347562 Coding 4 558 gcatcatccccaactgcatg 54 50 347563 Coding 4 574 gtccatctcgaactgagcat 46 51 347564 Stop Codon 4 589 gcaggagagtcagatgtcca 47 52 347565 3'UTR 4 623 aagtatcagtgttgctgctt 45 53 347566 3'UTR 4 635 tcaggtgcacacaagtatca 43 54 347567 3'UTR 4 734 atcatttggcacccagagga 54 55 347568 3'UTR 4 747 agctcatcttcccatcattt 38 56 347569 3'UTR 4 772 acagggagaagaaatggtca 13 57 347570 3'UTR 4 803 taacctgtttaactgggaag 59 58 347571 3'UTR 4 829 cagaaatacagcaagggcct 70 59 347572 3'UTR 4 851 ctctaagggctgcttagctc 71 60 347573 3'UTR 4 868 agagttgaactgttttcctc 78 61 347574 3'UTR 4 921 caaaattacagggtatgagg 63 62 347575 3'UTR 4 1085 aagaccccaagcccagactc 9 63 347576 3'UTR 4 1105 atttccccctgctggtttta 62 64 347577 3'UTR 4 1130 aagggaaagcagctctcttt 70 65 347578 3'UTR 4 1180 agagttgcacaagctgtgct 40 66 347579 3'UTR 4 1217 agtggacctcaaaacagtgt 64 67 347580 3'UTR 4 1303 tctgcacaaatgcactaagt 65 68 347581 3'UTR 4 1350 aaaactggttaccaagggct 34 69 347582 3'UTR 4 1357 gaagagcaaaactggttacc 27 70 347583 3'UTR 4 1393 ccagcaacgagatgcaagca 65 71 347584 3'UTR 4 1410 agtacaagaggactctgcca 56 72 347585 3'UTR 4 1458 tggtatggacctgctctagg 51 73 347586 3'UTR 4 1472 gtgcctctattacttggtat 48 74 347587 3'UTR 4 1533 ttcttaggcattatctgaca 70 75 347588 3'UTR 4 1541 agcggtcattcttaggcatt 59 76 347589 3'UTR 4 1580 acgactgagaccgggtactc 67 77 347590 3'UTR 4 1614 acaactaccacaatgctcac 0 78 347591 3'UTR 4 1664 attctgaaaatcaacttcaa 0 79 347592 3'UTR 4 1724 tcccagcagccaaacaaagc 0 80 347593 3'UTR 4 1868 atttgaaaaatggcctggta 47 81 347594 3'UTR 4 1892 acacttcaggtatctttgat 6 82 347595 3'UTR 4 1900 agataccaacacttcaggta 49 83 347596 3'UTR 4 1912 acagatattctcagatacca 0 84 347597 3'UTR 4 2018 atgtttaattaaaaagttgc 0 85 347598 3'UTR 4 2028 acactggaagatgtttaatt 17 86 347599 3'UTR 4 2173 cagttttacaaaccacccac 0 87 347600 3'UTR 4 2218 aagaatgaggctttcttgaa 47 88 347601 3'UTR 4 2223 cagaaaagaatgaggctttc 34 89 347602 3'UTR 4 2246 tgaatgcaaaagcgaaaggg 0 90 347603 3'UTR 4 2301 tcccgggattattatgctgc 0 91 347604 3'UTR 4 2377 gaaattcccaggacaccagt 63 92 347605 3'UTR 4 2382 aaccagaaattcccaggaca 47 93 347606 3'UTR 4 2389 caaatccaaccagaaattcc 0 94 347607 3'UTR 4 2449 ccaaatggcctgttactctc 26 95 347608 3'UTR 4 2471 aacaaacaggtttctttctt 40 96 347609 3'UTR 4 2492 cttttcatagttcaaaagaa 19 97 347610 3'UTR 4 2536 cagacatccttcctctcttt 33 98 347611 3'UTR 4 2564 ttgtggcagaaaacagaaca 0 99 347612 3'UTR 4 2578 aactattcacatttttgtgg 62 100 347613 3'UTR 4 2632 tggagatccagcttattcct 49 101 347614 3'UTR 26 1189 aagaatgaaaagcttcattc 0 102 347615 3'UTR 26 1336 tttaaatccattcctcaccg 0 103 347616 3'UTR 26 2088 ataactaatacaggtggaag 41 104 347617 3'UTR 27 697 ggtcatctgaaatctctaaa 45 105 347618 3'UTR 28 464 gcctcccacccttagaaagg 2 106

[0161] As shown in Table 1, SEQ ID NOs 29, 30, 32, 33, 35, 36, 37, 40, 41, 42, 43, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 81, 83, 88, 89, 92, 93, 95, 96, 98, 100, 101, 104 and 105 demonstrated at least 25% inhibition of human eIF4E-BP2 expression in this assay and are therefore preferred. The target regions to which these preferred sequences are complementary are herein referred to as "preferred target segments" and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 5. These sequences are shown to contain thymine (T) but one of skill in the art will appreciate that thymine (T) is generally replaced by uracil (U) in RNA sequences. The sequences represent the reverse complement of the preferred antisense compounds disclosed herein. "Target site" indicates the first (5'-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 5 is the species in which each of the preferred target segments was found.

[0162] SEQ ID NOs 29, 30, 31 and 32 are cross species oligonucleotides which are also complementary to the mouse eIF4E-BP2 nucleic acid target. SEQ ID NOs 29 and 33 are cross species oligonucleotides which are also complementary to rat eIF4E-BP2.

Example 12

Antisense Inhibition of Mouse eIF4E-BP2 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2'-MOE Wings and a Deoxy Gap

[0163] In accordance with the present invention, a second series of antisense compounds was designed to target different regions of the mouse eIF4E-BP2 RNA, using published sequences (GenBank.RTM. accession number NM.sub.--010124.1, incorporated herein as SEQ ID NO: 11, GenBank.RTM. accession number BI696127.1, incorporated herein as SEQ ID NO: 107, and GenBank.RTM. accession number BE332409.1, incorporated herein as SEQ ID NO: 108). The compounds are shown in Table 2. "Target site" indicates the first (5'-most) nucleotide number on the particular target nucleic acid to which the compound 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 "wings". The wings are composed of 2'-O-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P.dbd.S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on mouse eIF4E-BP2 mRNA levels by quantitative real-time PCR as described in other examples herein. Data, shown in Table 2, are averages from two experiments in which b.END cells were treated with 150 nM of the antisense oligonucleotides of the present invention. SEQ ID NO: 2 was used as the control oligonucleotide in this assay. If present, "N.D." indicates "no data".

TABLE-US-00002 TABLE 2 Inhibition of mouse eIF4E-BP2 mRNA levels in b.END cells by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap SEQ TARGET TARGET % ID ISIS # REGION SEQ ID NO SITE SEQUENCE INHIB NO 232759 5'UTR 11 9 tctcaactcgcctgctctcg 92 109 232760 5'UTR 11 26 ggctcctcacgctcggctct 81 110 232761 5'UTR 11 86 tcgaggctttgtgcagcagc 64 111 232762 Coding 11 132 gctggtggctaccaccggcc 49 112 232763 Coding 11 137 gctgggctggtggctaccac 72 113 232764 Coding 11 179 gtcgctgatagccacggtgc 77 114 232765 Coding 11 201 agtcctgaggtagctgcgcg 79 115 232766 Coding 11 211 gtggtgcagtagtcctgagg 81 116 232767 Coding 11 264 cataaatgattcgtgttcct 73 117 232768 Coding 11 269 tcggtcataaatgattcgtg 86 118 232769 Coding 11 274 aactttcggtcataaatgat 52 119 232770 Coding 11 281 caacagaaactttcggtcat 72 120 232771 Coding 11 286 cggtccaacagaaactttcg 84 121 232772 Coding 11 299 gggagaattgcgacggtcca 80 122 232773 Coding 11 304 gccatgggagaattgcgacg 83 29 232774 Coding 11 309 tctgcgccatgggagaattg 66 123 232775 Coding 11 354 caggactggtgactccaggg 87 124 232776 Coding 11 377 tttggagtcttcaattaagg 24 30 232777 Coding 11 382 tctactttggagtcttcaat 69 31 232778 Coding 11 388 ttcacttctactttggagtc 71 125 232779 Coding 11 449 aaactgagcctcatccccaa 89 126 232780 Coding 11 454 atctcaaactgagcctcatc 85 127 232781 Coding 11 461 gatgtccatctcaaactgag 73 128 232782 Stop Codon 11 473 tggcagtagtcagatgtcca 91 129 232783 3'UTR 11 500 ggctgctccacgaggcctcc 90 130 232784 3'UTR 11 521 tgggccagtcaggtgcacac 77 131 232785 3'UTR 11 540 ctgtacactgtgttcctact 87 132 232786 3'UTR 11 607 atgtgatcagacagtgcaca 67 133 232787 3'UTR 11 614 cgggaagatgtgatcagaca 59 134 232788 3'UTR 11 696 ttcttctgtggactgtcagc 44 135 232789 3'UTR 11 787 gtgctgcttggagactgccc 54 136 232790 3'UTR 11 798 tacaagcagaggtgctgctt 47 137 232791 3'UTR 11 827 ggcactaaacctccttcacc 87 138 232792 3'UTR 11 835 acacaatgggcactaaacct 68 139 232793 3'UTR 11 845 gagcccaggaacacaatggg 61 140 232794 3'UTR 11 900 aatgtcccccacatccagcg 88 141 232795 3'UTR 11 909 ctgaggacaaatgtccccca 81 142 232796 3'UTR 11 927 caggactgtgctccagagct 78 143 232797 3'UTR 11 934 ggaggtacaggactgtgctc 69 144 232798 3'UTR 11 975 gaggctgctgtcacatgtcc 68 145 232799 3'UTR 11 998 aagccttcctcccagagaaa 81 146 232800 3'UTR 11 1020 tatcacacccaagacaagac 70 147 232801 3'UTR 11 1030 gatgatgagctatcacaccc 83 148 232802 3'UTR 11 1093 cccttcaggagggcttaaaa 70 149 232803 3'UTR 11 1127 cagacaggcaaagaccagct 85 150 232804 3'UTR 11 1156 tgcctacgggatgcaggtag 71 151 232805 3'UTR 11 1204 cttctgctctaaaagcagac 1 152 232806 3'UTR 11 1222 caggccaaggtgttggcact 57 153 232807 3'UTR 11 1250 gctgagagcaggctggactc 66 154 232808 3'UTR 11 1263 tctcaggcagaccgctgaga 54 155 232809 3'UTR 11 1276 gcccctgatgtattctcagg 72 156 232810 3'UTR 11 1282 tcagaggcccctgatgtatt 51 157 232811 3'UTR 11 1289 gtcctcttcagaggcccctg 89 158 232812 3'UTR 11 1303 tgcacggcggctcagtcctc 69 159 232813 3'UTR 11 1308 ctggctgcacggcggctcag 71 160 232814 3'UTR 11 1327 aaaaccatgacccccgaggc 92 161 232815 3'UTR 11 1340 tacacctggttttaaaacca 67 162 232816 3'UTR 11 1355 acacccaacgtaaggtacac 86 163 232817 3'UTR 11 1361 tgcaggacacccaacgtaag 85 164 232818 3'UTR 11 1381 aaactcaaggtatagtaacc 73 165 232819 3'UTR 11 1392 aagtcgactttaaactcaag 66 166 232820 3'UTR 11 1399 taagaggaagtcgactttaa 75 167 232821 3'UTR 11 1455 ctgtgctgctctctcagcag 21 168 232822 3'UTR 11 1467 cactgtcttagcctgtgctg 90 169 232823 3'UTR 11 1584 tggaaaatggcccggtggaa 82 170 232824 3'UTR 11 1619 tactaacatgggaggcatct 84 171 232825 3'UTR 11 1646 tgataaggagagactgatat 28 172 232826 3'UTR 11 1662 taaaaggtctctcctctgat 33 173 232827 3'UTR 11 1668 taaaaataaaaggtctctcc 24 174 232828 3'UTR 11 1682 gtctgtagtcatcttaaaaa 93 32 232829 3'UTR 11 1699 aacttatctaaaaataggtc 30 175 232830 3'UTR 11 1708 tgtactgaaaacttatctaa 74 176 232831 3'UTR 11 1749 atactggaagatgttttgtt 70 177 232832 3'UTR 11 1761 ataaccttcccaatactgga 82 178 232833 3'UTR 107 365 acagctcatggcaaggcaga 79 179 232834 3'UTR 107 437 aactgctcttctatgtgtgg 4 180 232835 3'UTR 107 454 tcgctgatagtctcttgaac 0 181 232836 5'UTR 108 36 ggctcttcacgctcggctct 73 182

[0164] As shown in Table 2, SEQ ID NOs 29, 31, 32, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 169, 170, 171, 176, 177, 178, 179 and 182 demonstrated at least 44% inhibition of mouse eIF4E-BP2 expression in this experiment and are therefore preferred. The target regions to which these preferred sequences are complementary are herein referred to as "preferred target segments" and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 4. These sequences are shown to contain thymine (T) but one of skill in the art will appreciate that thymine (T) is generally replaced by uracil (U) in RNA sequences. The sequences represent the reverse complement of the preferred antisense compounds disclosed herein. "Target site" indicates the first (5'-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 5 is the species in which each of the preferred target segments was found.

[0165] In a further embodiment, antisense oligonucleotides targeting mouse eIF4E-BP2 were tested in EMT-6 cells. The compounds were analyzed for their effect on mouse eIF4E-BP2 mRNA levels by quantitative real-time PCR as described in other examples herein. Data, shown in Table 3, are averages from two experiments in which EMT-6 cells were treated with 150 nM of the antisense oligonucleotides of the present invention. SEQ ID NO: 2 was used as the control oligonucleotide in this assay. If present, "N.D." indicates "no data".

TABLE-US-00003 TABLE 3 Inhibition of mouse eIF4E-BP2 mRNA levels in EMT-6 cells by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap SEQ TARGET TARGET % ID ISIS # REGION SEQ ID NO SITE SEQUENCE INHIB NO 232759 5'UTR 11 9 tctcaactcgcctgctctcg 95 109 232760 5'UTR 11 26 ggctcctcacgctcggctct 93 110 232761 5'UTR 11 86 tcgaggctttgtgcagcagc 96 111 232762 Coding 11 132 gctggtggctaccaccggcc 88 112 232763 Coding 11 137 gctgggctggtggctaccac 94 113 232764 Coding 11 179 gtcgctgatagccacggtgc 95 114 232765 Coding 11 201 agtcctgaggtagctgcgcg 97 115 232766 Coding 11 211 gtggtgcagtagtcctgagg 93 116 232767 Coding 11 264 cataaatgattcgtgttcct 92 117 232768 Coding 11 269 tcggtcataaatgattcgtg 98 118 232769 Coding 11 274 aactttcggtcataaatgat 80 119 232770 Coding 11 281 caacagaaactttcggtcat 84 120 232771 Coding 11 286 cggtccaacagaaactttcg 97 121 232772 Coding 11 299 gggagaattgcgacggtcca 95 122 232773 Coding 11 304 gccatgggagaattgcgacg 96 29 232774 Coding 11 309 tctgcgccatgggagaattg 93 123 232775 Coding 11 354 caggactggtgactccaggg 98 124 232776 Coding 11 377 tttggagtcttcaattaagg 73 30 232777 Coding 11 382 tctactttggagtcttcaat 85 31 232778 Coding 11 388 ttcacttctactttggagtc 93 125 232779 Coding 11 449 aaactgagcctcatccccaa 93 126 232780 Coding 11 454 atctcaaactgagcctcatc 92 127 232781 Coding 11 461 gatgtccatctcaaactgag 89 128 232782 Stop Codon 11 473 tggcagtagtcagatgtcca 95 129 232783 3'UTR 11 500 ggctgctccacgaggcctcc 98 130 232784 3'UTR 11 521 tgggccagtcaggtgcacac 95 131 232785 3'UTR 11 540 ctgtacactgtgttcctact 98 132 232786 3'UTR 11 607 atgtgatcagacagtgcaca 89 133 232787 3'UTR 11 614 cgggaagatgtgatcagaca 75 134 232788 3'UTR 11 696 ttcttctgtggactgtcagc 59 135 232789 3'UTR 11 787 gtgctgcttggagactgccc 77 136 232790 3'UTR 11 798 tacaagcagaggtgctgctt 87 137 232791 3'UTR 11 827 ggcactaaacctccttcacc 91 138 232792 3'UTR 11 835 acacaatgggcactaaacct 87 139 232793 3'UTR 11 845 gagcccaggaacacaatggg 89 140 232794 3'UTR 11 900 aatgtcccccacatccagcg 95 141 232795 3'UTR 11 909 ctgaggacaaatgtccccca 92 142 232796 3'UTR 11 927 caggactgtgctccagagct 95 143 232797 3'UTR 11 934 ggaggtacaggactgtgctc 91 144 232798 3'UTR 11 975 gaggctgctgtcacatgtcc 95 145 232799 3'UTR 11 998 aagccttcctcccagagaaa 83 146 232800 3'UTR 11 1020 tatcacacccaagacaagac 80 147 232801 3'UTR 11 1030 gatgatgagctatcacaccc 91 148 232802 3'UTR 11 1093 cccttcaggagggcttaaaa 85 149 232803 3'UTR 11 1127 cagacaggcaaagaccagct 94 150 232804 3'UTR 11 1156 tgcctacgggatgcaggtag 95 151 232805 3'UTR 11 1204 cttctgctctaaaagcagac 36 152 232806 3'UTR 11 1222 caggccaaggtgttggcact 83 153 232807 3'UTR 11 1250 gctgagagcaggctggactc 82 154 232808 3'UTR 11 1263 tctcaggcagaccgctgaga 74 155 232809 3'UTR 11 1276 gcccctgatgtattctcagg 93 156 232810 3'UTR 11 1282 tcagaggcccctgatgtatt 86 157 232811 3'UTR 11 1289 gtcctcttcagaggcccctg 95 158 232812 3'UTR 11 1303 tgcacggcggctcagtcctc 86 159 232813 3'UTR 11 1308 ctggctgcacggcggctcag 91 160 232814 3'UTR 11 1327 aaaaccatgacccccgaggc 96 161 232815 3'UTR 11 1340 tacacctggttttaaaacca 93 162 232816 3'UTR 11 1355 acacccaacgtaaggtacac 95 163 232817 3'UTR 11 1361 tgcaggacacccaacgtaag 97 164 232818 3'UTR 11 1381 aaactcaaggtatagtaacc 89 165 232819 3'UTR 11 1392 aagtcgactttaaactcaag 96 166 232820 3'UTR 11 1399 taagaggaagtcgactttaa 96 167 232821 3'UTR 11 1455 ctgtgctgctctctcagcag 79 168 232822 3'UTR 11 1467 cactgtcttagcctgtgctg 96 169 232823 3'UTR 11 1584 tggaaaatggcccggtggaa 96 170 232824 3'UTR 11 1619 tactaacatgggaggcatct 95 171 232825 3'UTR 11 1646 tgataaggagagactgatat 60 172 232826 3'UTR 11 1662 taaaaggtctctcctctgat 67 173 232827 3'UTR 11 1668 taaaaataaaaggtctctcc 23 174 232828 3'UTR 11 1682 gtctgtagtcatcttaaaaa 98 32 232829 3'UTR 11 1699 aacttatctaaaaataggtc 69 175 232830 3'UTR 11 1708 tgtactgaaaacttatctaa 97 176 232831 3'UTR 11 1749 atactggaagatgttttgtt 89 177 232832 3'UTR 11 1761 ataaccttcccaatactgga 95 178 232833 3'UTR 107 365 acagctcatggcaaggcaga 96 179 232834 3'UTR 107 437 aactgctcttctatgtgtgg 40 180 232835 3'UTR 107 454 tcgctgatagtctcttgaac 23 181 232836 5'UTR 108 36 ggctcttcacgctcggctct 88 182

[0166] As shown in Table 3, SEQ ID NOs 29, 30, 31, 32, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 176, 177, 178, 179 and 182 demonstrated at least 67% inhibition of mouse eIF4E-BP2 expression in this assay and are therefore preferred.

Example 13

Antisense Inhibition of Rat eIF4E-BP2 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2'-MOE Wings and a Deoxy Gap

[0167] In accordance with the present invention, a third series of antisense compounds was designed to target different regions of the rat eIF4E-BP2 RNA, using published sequences (GenBank.RTM. accession number XM.sub.--215414.1, incorporated herein as SEQ ID NO: 18). The compounds are shown in Table 4. "Target site" indicates the first (5'-most) nucleotide number on the particular target nucleic acid to which the compound binds. All compounds in Table 4 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 "wings". The wings are composed of 2'-O-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P.dbd.S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on rat eIF4E-BP2 mRNA levels by quantitative real-time PCR as described in other examples herein. Data, shown in Table 4, are averages from two experiments in which A 10 cells were treated with 50 nM of the antisense oligonucleotides of the present invention. SEQ ID NO: 2 was used as the control oligonucleotide in this assay. If present, "N.D." indicates "no data".

TABLE-US-00004 TABLE 4 Inhibition of rat eIF4E-BP2 mRNA levels by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap SEQ TARGET TARGET % ID ISIS # REGION SEQ ID NO SITE SEQUENCE INHIB NO 232773 Coding 11 304 gccatgggagaattgcgacg 90 29 322907 5'UTR 18 7 ggctcgtggctttgtgcagc 48 183 322908 Coding 18 48 tggtgtccaccaccggccga 49 184 322909 Coding 18 57 tggctgggctggtgtccacc 65 185 322910 Coding 18 59 tctggctgggctggtgtcca 50 186 322911 Coding 18 71 gaatggcgcggctctggctg 65 187 322912 Coding 18 93 ctaatagccacggtgcgtgt 65 188 322913 Coding 18 97 gtcgctaatagccacggtgc 83 189 322914 Coding 18 102 gctgcgtcgctaatagccac 80 190 322915 Coding 18 114 tgaggtagctgcgctgcgtc 62 191 322916 Coding 18 116 cctgaggtagctgcgctgcg 68 192 322917 Coding 18 120 tagtcctgaggtagctgcgc 77 193 322918 Coding 18 122 agtagtcctgaggtagctgc 75 194 322919 Coding 18 125 tgcagtagtcctgaggtagc 80 195 322920 Coding 18 127 ggtgcagtagtcctgaggta 85 196 322921 Coding 18 130 cgtggtgcagtagtcctgag 78 197 322922 Coding 18 132 ggcgtggtgcagtagtcctg 74 198 322923 Coding 18 159 ggtgttgtggagaacagcgt 35 199 322924 Coding 18 164 ctcccggtgttgtggagaac 48 200 322925 Coding 18 168 gttcctcccggtgttgtgga 78 201 322926 Coding 18 193 aaactttcggtcataaatga 53 202 322927 Coding 18 195 agaaactttcggtcataaat 41 203 322928 Coding 18 197 acagaaactttcggtcataa 65 204 322929 Coding 18 198 aacagaaactttcggtcata 79 205 322930 Coding 18 201 tccaacagaaactttcggtc 83 206 322931 Coding 18 203 gatccaacagaaactttcgg 83 207 322932 Coding 18 208 gcgacggtccaacagaaact 80 208 322933 Coding 18 210 ttgcgacggtccaacagaaa 76 209 322934 Coding 18 213 gaattgcgacggtccaacag 78 210 322935 Coding 18 215 gagaattgcgacggtccaac 75 211 322936 Coding 18 218 tgggagaattgcgacggtcc 36 212 322937 Coding 18 223 cgccatgggagaattgcgac 73 213 322938 Coding 18 225 tgcgccatgggagaattgcg 52 214 322939 Coding 18 228 gtctgcgccatgggagaatt 67 215 322940 Coding 18 250 attgggcagatggcaaggtg 33 216 322941 Coding 18 265 ggtgactccagggatattgg 35 217 322942 Coding 18 270 ggactggtgactccagggat 74 218 322943 Coding 18 275 cgccaggactggtgactcca 83 219 322944 Coding 18 292 ggagtcttccattaaggcgc 64 220 322945 Coding 18 294 ttggagtcttccattaaggc 66 221 322946 Coding 18 298 tactttggagtcttccatta 27 222 322947 Coding 18 303 acttctactttggagtcttc 68 33 322948 Coding 18 304 cacttctactttggagtctt 64 223 322949 Coding 18 308 tgttcacttctactttggag 87 224 322950 Coding 18 313 caagttgttcacttctactt 80 225 322951 Coding 18 316 gttcaagttgttcacttcta 82 226 322952 Coding 18 323 tcaggttgttcaagttgttc 83 227 322953 Coding 18 326 tgttcaggttgttcaagttg 84 228 322954 Coding 18 329 gattgttcaggttgttcaag 68 229 322955 Coding 18 332 cgtgattgttcaggttgttc 95 230 322956 Coding 18 335 tgtcgtgattgttcaggttg 95 231 322957 Coding 18 339 ttcctgtcgtgattgttcag 88 232 322958 Coding 18 341 gcttcctgtcgtgattgttc 95 233 322959 Coding 18 343 gtgcttcctgtcgtgattgt 92 234 322960 Coding 18 348 actgcgtgcttcctgtcgtg 97 235 322961 Coding 18 350 caactgcgtgcttcctgtcg 91 236 322962 Coding 18 353 ccccaactgcgtgcttcctg 85 237 322963 Coding 18 355 atccccaactgcgtgcttcc 48 238 322964 Coding 18 358 ctcatccccaactgcgtgct 83 239 322965 Coding 18 360 gcctcatccccaactgcgtg 90 240 322966 Coding 18 362 gagcctcatccccaactgcg 94 241 322967 Coding 18 364 ctgagcctcatccccaactg 89 242 322968 Coding 18 369 tcaaactgagcctcatcccc 50 243 322969 Stop Codon 18 390 cagcagggtcagatgtccat 81 244 322970 3'UTR 18 402 ccttcgacactgcagcaggg 88 245 322971 3'UTR 18 406 gccgccttcgacactgcagc 83 246 322972 3'UTR 18 428 gtgcacacgggccgtgtcag 76 247 322973 3'UTR 18 436 ccagtcaggtgcacacgggc 84 248 322974 3'UTR 18 439 ggtccagtcaggtgcacacg 86 249 322975 3'UTR 18 443 tactggtccagtcaggtgca 80 250 322976 3'UTR 18 446 tcctactggtccagtcaggt 72 251 322977 3'UTR 18 450 gtgttcctactggtccagtc 84 252 322978 3'UTR 18 454 cacggtgttcctactggtcc 83 253 322979 3'UTR 18 458 tgtacacggtgttcctactg 76 254 322980 3'UTR 18 462 tctctgtacacggtgttcct 89 255 322981 3'UTR 18 464 cttctctgtacacggtgttc 90 256 322982 3'UTR 18 469 tggagcttctctgtacacgg 90 257 322983 3'UTR 18 471 actggagcttctctgtacac 85 258

[0168] As shown in Table 4, SEQ ID NOs 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 and 258 demonstrated at least 48% inhibition of rat eIF4E-BP2 expression in this experiment and are therefore preferred. The target regions to which these preferred sequences are complementary are herein referred to as "preferred target segments" and are therefore preferred for targeting by compounds of the present invention. These sequences are shown to contain thymine (T) but one of skill in the art will appreciate that thymine (T) is generally replaced by uracil (U) in RNA sequences. The sequences represent the reverse complement of the preferred antisense compounds shown in tables above. "Target site" indicates the first (5'-most) nucleotide number on the particular target nucleic acid to which the compound binds.

[0169] "Preferred target segments," as described in Table 5 of U.S. Patent Application No. 60/538,752, filed Jan. 22, 2004, which is herein incorporated by reference in its entirety, have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these preferred target segments and consequently inhibit the expression of eIF4E-BP2.

[0170] According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, siRNAs, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds which hybridize to at least a portion of the target nucleic acid.

Example 14

Western Blot Analysis of eIF4E-BP2 Protein Levels

[0171] 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), 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 eIF4E-BP2 is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER.TM. (Molecular Dynamics, Sunnyvale Calif.).

Example 15

Reduction of Blood Glucose Levels in Ob/Ob Mice by Antisense Inhibition of eIF4E-BP2

[0172] Ob/ob mice have a mutation in the leptin gene which results in obesity and hyperglycemia. As such, these mice are a useful model for the investigation of obesity and diabetes and treatments designed to treat these conditions. In accordance with the present invention, compounds targeted to eIF4E-BP2 are tested in the ob/ob model of obesity and diabetes.

[0173] Seven-week old male C57B1/6J-Lepr ob/ob mice (Jackson Laboratory, Bar Harbor, Me.) are fed a diet with a fat content of 10-15% and are subcutaneously injected with oligonucleotides at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected animals, leptin wildtype littermates (i.e. lean littermates) and ob/ob mice fed a standard rodent diet serve as controls. After the treatment period, mice are sacrificed and target levels are evaluated in liver, brown adipose tissue (BAT) and white adipose tissue (WAT). RNA isolation and target mRNA expression level quantitation are performed as described by other examples herein.

[0174] To assess the physiological effects resulting from antisense inhibition of target mRNA, the ob/ob mice that receive antisense oligonucleotide treatment are further evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol, liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis, accumulation of lipids in the liver, is assessed by measuring the liver triglyceride content. Hepatic steatosis is assessed by routine histological analysis of frozen liver tissue sections stained with oil red O stain, which is commonly used to visualize lipid deposits, and counterstained with hematoxylin and eosin, to visualize nuclei and cytoplasm, respectively.

[0175] The effects of target inhibition on glucose and insulin metabolism are evaluated in the ob/ob mice treated with antisense oligonucleotides. Plasma glucose is measured at the start of the antisense oligonucleotide treatment and following two and four weeks of treatment. Both fed and fasted plasma glucose levels were measured. At start of study, the treatment groups of mice are chosen to have an average fed plasma glucose level of about 350 mg/dL. Plasma insulin is also measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose and insulin levels are measured before the insulin or glucose challenge and at 15, 20 or 30 minute intervals for up to 3 hours.

[0176] In mice treated with ISIS 232828 (SEQ ID NO: 32), an antisense inhibitor of eIF4E-BP2, fed plasma glucose levels were approximately 355 mg/dL at week 0, 295 mg/dL at week 2 and 210 mg/dL at week 4. In contrast, mice treated with saline alone had fed plasma glucose levels of approximately 365 mg/dL at week 0, 425 mg/dL at week 2 and 410 mg/dL at week 4. Mice treated with a positive control oligonucleotide, ISIS 116847 (CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 259), targeted to PTEN, had fed plasma glucose levels of approximately 360 mg/dL at week 0, 215 mg/dL at week 2 and 180 mg/dL at week 4.

[0177] Fasted plasma glucose was measured at week 3 of antisense treatment. Plasma glucose was approximately 330 mg/dL in saline treated mice, 245 mg/dL in mice treated with ISIS 232828 (inhibitor of eIF4E-BP2) and 195 mg/dL in mice treated with the positive control oligonucleotide, ISIS 116847.

[0178] At the end of the four week study, average liver weights were approximately 3.6 grams for saline treated mice, 3.2 grams for ISIS 232828-treated mice and 4.1 grams for positive control (ISIS 116847) treated mice. White adipose tissue weights were approximately 3.9 grams for saline treated mice, 3.8 grams for ISIS 232828-treated mice and 3.7 grams for positive control (ISIS 116847) treated mice.

[0179] At the end of the study, liver transaminases were found to be lower in mice treated with antisense to eIF4E-BP2 (ISIS 232828) than in mice treated with saline or the positive control oligonucleotide (ISIS 116847). AST levels were approximately 330 IU/L for saline treated mice, 110 IU/L for ISIS 232828-treated mice and 430 IU/L for ISIS 116847-treated mice. ALT levels were approximately 435 IU/L for saline treated mice, 140 IU/L for ISIS 232828-treated mice and 710 IU/L for ISIS 116847-treated mice.

[0180] Serum lipids were also measured at the end of the study. Cholesterol levels were approximately 230 mg/dL for saline treated mice, 210 mg/dL for ISIS 232828-treated mice and 260 mg/dL for ISIS 116847-treated mice. Triglycerides were approximately 135 mg/dL for saline treated mice, 80 mg/dL for ISIS 232828-treated mice and 110 mg/dL for ISIS 116847-treated mice.

[0181] eIF4E-BP2 mRNA levels in liver were measured at the end of study using RiboGreen.TM. RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) as taught in previous examples above. eIF4E-BP2 mRNA levels were reduced by approximately 90% in mice treated with ISIS 232828, compared to saline treatment. Target reduction in mice treated with ISIS 116847 was approximately 30%.

Sequence CWU 1

1

263120DNAArtificial SequenceAntisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20220DNAArtificial SequenceAntisense Oligonucleotide 2gtgcgcgcga gcccgaaatc 20320DNAArtificial SequenceAntisense Oligonucleotide 3atgcattctg cccccaagga 2042782DNAH. sapiensCDS(237)...(599) 4cgctgctgcc gctgctgttg ctcctgaggc tgctggctga ggccggagga tcgagcggcg 60gcggcggcgg cggctgagag ggcggcggcg ggagcggagc gggacgaggg aacgggagga 120agcgagcgag gagcgcgcag agcgcgcttt tccgtccgcc tgaggagccg aagcagcccc 180ggccccgccg ccgccgcctg cccgccggac aaagccgaga gcccgcgccc acagcc atg 239 Met 1tcc tcg tca gcc ggc agc ggc cac cag ccc agc cag agc cgc gcc atc 287Ser Ser Ser Ala Gly Ser Gly His Gln Pro Ser Gln Ser Arg Ala Ile 5 10 15ccc acc cgc acc gtg gcc atc agc gac gcc gcg cag cta cct cat gac 335Pro Thr Arg Thr Val Ala Ile Ser Asp Ala Ala Gln Leu Pro His Asp 20 25 30tat tgc acc acg ccc ggg ggg acg ctc ttc tcc acc aca ccg gga gga 383Tyr Cys Thr Thr Pro Gly Gly Thr Leu Phe Ser Thr Thr Pro Gly Gly 35 40 45act cga atc att tat gac aga aag ttt ctg ttg gat cgt cgc aat tct 431Thr Arg Ile Ile Tyr Asp Arg Lys Phe Leu Leu Asp Arg Arg Asn Ser50 55 60 65ccc atg gct cag acc cca ccc tgc cac ctg ccc aat atc cca gga gtc 479Pro Met Ala Gln Thr Pro Pro Cys His Leu Pro Asn Ile Pro Gly Val 70 75 80act agc cct ggc acc tta att gaa gac tcc aaa gta gaa gta aac aat 527Thr Ser Pro Gly Thr Leu Ile Glu Asp Ser Lys Val Glu Val Asn Asn 85 90 95ttg aac aac ttg aac aat cac gac agg aaa cat gca gtt ggg gat gat 575Leu Asn Asn Leu Asn Asn His Asp Arg Lys His Ala Val Gly Asp Asp 100 105 110gct cag ttc gag atg gac atc tga ctctcctgca aggattagaa gaaaagcagc 629Ala Gln Phe Glu Met Asp Ile 115 120aacactgata cttgtgtgca cctgatttgg ccaataggat caacagtgaa aagacagaag 689aggcaatacc agcagtcccc attacagtct ccacctcccc gtcttcctct gggtgccaaa 749tgatgggaag atgagcttca tctgaccatt tcttctccct gtctcctgtt ccccttccca 809gttaaacagg ttagattgaa ggcccttgct gtatttctgt agagctaagc agcccttaga 869ggaaaacagt tcaactctga ctttcctagt tgttttttta ttgagagcca ccctcatacc 929ctgtaatttt gtcccaaatc aaatatcaac ctaccaacaa ctgcctggct gggaagtctg 989gggaagggat acagagcttg gtgggcctaa caccattcat attccttacc ctctgtctct 1049cctccctgta tcccacctat ggttcagtgt tgcaagagtc tgggcttggg gtctttaaaa 1109ccagcagggg gaaatgataa aaagagagct gctttccctt ttaccttgag gtattcgtcc 1169ctcgggacag agcacagctt gtgcaactct ggtagcgtta ccctgtgaca ctgttttgag 1229gtccacttcc tttctttcct ctgggaggaa tgtcttctgt ctttggtatt atagttcatc 1289ttcccattct tttacttagt gcatttgtgc agatattttt aactctgtac atcagaagag 1349agcccttggt aaccagtttt gctcttcttc tgccactcct ccctgcttgc atctcgttgc 1409tggcagagtc ctcttgtact tcaagaaagc aaagtgattt tgtctgctcc tagagcaggt 1469ccataccaag taatagaggc actttagctt ccacttggtg ggtaaggcct gatcatagta 1529ttctgtcaga taatgcctaa gaatgaccgc tgaagaacgt tgacccattt gagtacccgg 1589tctcagtcgt catttttaag tccagtgagc attgtggtag ttgttcttag attgcagttt 1649cttatgtttt gagtttgaag ttgattttca gaatgttctt agaaaagaac tgcatttttt 1709tcctttgtgg atctgctttg tttggctgct gggatagata agcatgggct taaaaaatgt 1769gttcctccca gttttcttgc ctttcctgtt gtactctgaa tttctctccc tacctccctc 1829actttcttcc tctctccttc ctttccttcc tttttctcta ccaggccatt tttcaaattt 1889acatcaaaga tacctgaagt gttggtatct gagaatatct gtcactcctc ttatctgaga 1949agtgaccttt tatttttaag atgactacag acctattttt agatatgttt tcagtacaat 2009tttgaacagc aactttttaa ttaaacatct tccagtgtta ggaagttgag aaacgttcat 2069aggcaagtct gctgttctat gtcaccatct tttgtctccc ctagtccccc aggagctctt 2129tcctttcccc tctagttttg ggtgtgcatg tttggagttt gtagtgggtg gtttgtaaaa 2189ctggaccatt ctgccttgct atgggttgtt caagaaagcc tcattctttt ctgtgaccct 2249ttcgcttttg cattcaccct ccttcccacc tacctgtcct ggggctgttg agcagcataa 2309taatcccggg agaatgattc ccctcataga aagacaaaag catccatccc ctcatagtta 2369agtagccact ggtgtcctgg gaatttctgg ttggatttgg tgccctgaac ttttttatta 2429agaaatcaga tcccagggtg agagtaacag gccatttggc caagaaagaa acctgtttgt 2489ttttcttttg aactatgaaa agaccctgtt tgtgaatata ttttagaaag agaggaagga 2549tgtctgcaga actttgttct gttttctgcc acaaaaatgt gaatagttca gagtgaaaac 2609cttttgtgat ggttgatgtc tcaggaataa gctggatctc caatgttttg gggatgcttt 2669gagtctcaaa aaaaattgat aatcagaaaa gtaatttttg tttgtttgtt taatgtatcc 2729ctgttctgtt tttaattaaa ctccaagtct cattttaaaa aaaaaaaaaa aaa 2782523DNAArtificial SequencePCR Primer 5cctctagttt tgggtgtgca tgt 23620DNAArtificial SequencePCR Primer 6cccatagcaa ggcagaatgg 20732DNAArtificial SequencePCR Probe 7tggagtttgt agtgggtggt ttgtaaaact gg 32819DNAArtificial SequencePCR Primer 8gaaggtgaag gtcggagtc 19920DNAArtificial SequencePCR Primer 9gaagatggtg atgggatttc 201020DNAArtificial SequencePCR Probe 10caagcttccc gttctcagcc 20111781DNAM. musculusCDS(121)...(483) 11cgaccgggcg agagcaggcg agttgagagc cgagcgtgag gagccagagc cgcccggccc 60cgccgccgcc gccgccgccg ccgccgctgc tgcacaaagc ctcgagcccg cgtcggagcc 120atg tcc gcg tcg gcc ggt ggt agc cac cag ccc agc cag agc cgc gcc 168Met Ser Ala Ser Ala Gly Gly Ser His Gln Pro Ser Gln Ser Arg Ala1 5 10 15atc ccc acg cgc acc gtg gct atc agc gac gcc gcg cag cta cct cag 216Ile Pro Thr Arg Thr Val Ala Ile Ser Asp Ala Ala Gln Leu Pro Gln 20 25 30gac tac tgc acc acg ccc ggg ggg acg ctg ttc tcc aca acg ccg gga 264Asp Tyr Cys Thr Thr Pro Gly Gly Thr Leu Phe Ser Thr Thr Pro Gly 35 40 45gga aca cga atc att tat gac cga aag ttt ctg ttg gac cgt cgc aat 312Gly Thr Arg Ile Ile Tyr Asp Arg Lys Phe Leu Leu Asp Arg Arg Asn 50 55 60tct ccc atg gcg cag acc cca cct tgc cat ctg ccc aat atc cct gga 360Ser Pro Met Ala Gln Thr Pro Pro Cys His Leu Pro Asn Ile Pro Gly65 70 75 80gtc acc agt cct ggc gcc tta att gaa gac tcc aaa gta gaa gtg aac 408Val Thr Ser Pro Gly Ala Leu Ile Glu Asp Ser Lys Val Glu Val Asn 85 90 95aac tta aac aac ctg aac aat cat gac agg aag cat gca gtt ggg gat 456Asn Leu Asn Asn Leu Asn Asn His Asp Arg Lys His Ala Val Gly Asp 100 105 110gag gct cag ttt gag atg gac atc tga ctactgccat gtggaaggag 503Glu Ala Gln Phe Glu Met Asp Ile 115 120gcctcgtgga gcagcctgtg tgcacctgac tggcccagta ggaacacagt gtacagagaa 563gctcctgtcc ccctgtcccc tctgggtgcc aaataatggg agatgtgcac tgtctgatca 623catcttcccg tctcctgccc tctgcccagt taaggttagg ttgatgaata agcccttgga 683ttattctgtg gagctgacag tccacagaag aaagcagtcc ctgtagcttc cctggtcatt 743tcccaagaat cttcctgccc tgttgagact tgccccaagt ctagggcagt ctccaagcag 803cacctctgct tgtaggggtt gggggtgaag gaggtttagt gcccattgtg ttcctgggct 863ctccctgtcc ttccctacag accactactg gtggagcgct ggatgtgggg gacatttgtc 923ctcagctctg gagcacagtc ctgtacctcc tgcacctctg ctgcattcct gggacatgtg 983acagcagcct cccctttctc tgggaggaag gcttctgtct tgtcttgggt gtgatagctc 1043atcatccccc cccccccatt cctttaccca tttcattggc acgggtattt tttaagccct 1103cctgaaggga ccccttggtg accagctggt ctttgcctgt ctgacattct ttctacctgc 1163atcccgtagg cagagtctgc cctggcacac ccgtggctct gtctgctttt agagcagaag 1223tgccaacacc ttggcctgca cctggtgagt ccagcctgct ctcagcggtc tgcctgagaa 1283tacatcaggg gcctctgaag aggactgagc cgccgtgcag ccagcctcgg gggtcatggt 1343tttaaaacca ggtgtacctt acgttgggtg tcctgcaggt tactatacct tgagtttaaa 1403gtcgacttcc tcttacattt ctcccctgct ttggatctgc tttgtgcttg gctgctgaga 1463gagcagcaca ggctaagaca gtgtattcct cccaggtctc tcgcccttct catcgtccgt 1523ctgtccgtca gtccgtccgt ccttccctcc ctctcccctt aaattctttc cttctggttc 1583ttccaccggg ccattttcca catctgcatc agaagagatg cctcccatgt tagtatctga 1643taatatcagt ctctccttat cagaggagag accttttatt tttaagatga ctacagacct 1703atttttagat aagttttcag tacaattttg aactacaact tttttaacaa aacatcttcc 1763agtattggga aggttatt 17811223DNAArtificial SequencePCR Primer 12agagcagcac aggctaagac agt 231319DNAArtificial SequencePCR Primer 13cggacagacg gacgatgag 191420DNAArtificial SequencePCR Probe 14cctcccaggt ctctcgccct 201520DNAArtificial SequencePCR Primer 15ggcaaattca acggcacagt 201620DNAArtificial SequencePCR Primer 16gggtctcgct cctggaagat 201727DNAArtificial SequencePCR Probe 17aaggccgaga atgggaagct tgtcatc 2718497DNAR. norvegicusCDS(39)...(401) 18gccgccgctg cacaaagcca cgagcccgcc ccggagcc atg tcc gcg tcg gcc ggt 56 Met Ser Ala Ser Ala Gly 1 5ggt gga cac cag ccc agc cag agc cgc gcc att ccg aca cgc acc gtg 104Gly Gly His Gln Pro Ser Gln Ser Arg Ala Ile Pro Thr Arg Thr Val 10 15 20gct att agc gac gca gcg cag cta cct cag gac tac tgc acc acg ccc 152Ala Ile Ser Asp Ala Ala Gln Leu Pro Gln Asp Tyr Cys Thr Thr Pro 25 30 35ggg ggg acg ctg ttc tcc aca aca ccg gga gga aca cga atc att tat 200Gly Gly Thr Leu Phe Ser Thr Thr Pro Gly Gly Thr Arg Ile Ile Tyr 40 45 50gac cga aag ttt ctg ttg gac cgt cgc aat tct ccc atg gcg cag acc 248Asp Arg Lys Phe Leu Leu Asp Arg Arg Asn Ser Pro Met Ala Gln Thr55 60 65 70cca cct tgc cat ctg ccc aat atc cct gga gtc acc agt cct ggc gcc 296Pro Pro Cys His Leu Pro Asn Ile Pro Gly Val Thr Ser Pro Gly Ala 75 80 85tta atg gaa gac tcc aaa gta gaa gtg aac aac ttg aac aac ctg aac 344Leu Met Glu Asp Ser Lys Val Glu Val Asn Asn Leu Asn Asn Leu Asn 90 95 100aat cac gac agg aag cac gca gtt ggg gat gag gct cag ttt gag atg 392Asn His Asp Arg Lys His Ala Val Gly Asp Glu Ala Gln Phe Glu Met 105 110 115gac atc tga ccctgctgca gtgtcgaagg cggcccctga cacggcccgt gtgcacctga 451Asp Ile 120ctggaccagt aggaacaccg tgtacagaga agctccagtc cccctg 4971926DNAArtificial SequencePCR Primer 19agtgaacaac ttgaacaacc tgaaca 262021DNAArtificial SequencePCR Primer 20actgcagcag ggtcagatgt c 212124DNAArtificial SequencePCR Probe 21tcacgacagg aagcacgcag ttgg 242223DNAArtificial SequencePCR Primer 22tgttctagag acagccgcat ctt 232321DNAArtificial SequencePCR Primer 23caccgacctt caccatcttg t 212424DNAArtificial SequencePCR Probe 24ttgtgcagtg ccagcctcgt ctca 242525324DNAH. sapiens 25tttttaaaat acagtacatc gtgcatgtag tgcctgaatt aataatgaac aattaaaagt 60aaaattacat ttttaatgca taggaatgca tgagaaactc aagtctgctc aagtaaagtg 120gtgttaaccg acaatgacaa acaataagaa gaaagttttc cttgtccgcc ttcaccaggc 180tcacaaacac ttagctcgcg ctccctctgg ctcttctccc gctcgctggg gttaagccac 240tctcttcctc agccctgccc ctcgtccccg cccccttcaa caacttcagc cacgcccctc 300actgcctcgc cccgccccgc ggcgacgtca cctccggccg accagcttcc ccaactccct 360ctggctcccg ccttcgcccg cttccggtcg tcgtcgtcgc cgctgctgcc gctgctgttg 420ctcctgaggc tgctggctga ggccggagga tcgagcggcg gcggcggcgg cggctgagag 480ggcggcggcg ggagcggagc gggacgaggg aacgggagga agcgagcgag gagcgcgcag 540agcgcgcttt tccgtccgcc tgaggagccg aagcagcccc ggccccgccg ccgccgcctg 600cccgccggac aaagccgaga gcccgcgccc acagccatgt cctcgtcagc cggcagcggc 660caccagccca gccagagccg cgccatcccc acccgcaccg tggccatcag cgacgccgcg 720cagctacctc atgactattg caccacgccc ggggggacgc tcttctccac cacaccggga 780ggtgagcgcc ggccagccgt ccgccgcgcc cggtgtcccg ccgcggtcct ctaactcctc 840ggcgcctcgg tgcccggccg cttcgccccc gcccccagct ccaccgaagc cccggggacg 900ctgcccttgg gcccgcccga gcgttcggga ccctttactt cgtgttcgct cttgcccgca 960gctcgagtcg gcgcgcgccc cactcgggaa tgtggctgtc ctgtcgcgaa aaagagctct 1020tgtttccgct tcgtggcagg cttacgcatt cgacccagtt ctctctctcc tctctgcctc 1080cttcccgggc ggatttggct ccacttggcc ttgcattaca gtctgcattg cctgtcgtag 1140attgtgcaaa ttaatgcttg attttggagc tggctccggg gctttttaaa aaagaacttt 1200gggagaggaa ttcggccctg gcatcctgcg atggcttgtt tttgctgctt ttagaacacc 1260gggaggaggc tggaatgcgg agtctggaag cctcgcccag cgttatcccg ctttgacagc 1320attgtttact ttgctggacg aggccccacg ggtgagggga gtccccaggc cgggaggaga 1380gcgtgataaa ataaagctca agtaatagcc aagggaaagt aggtgggggt ggtagggtgc 1440tgacagcctt aaggtagggt gtctttcggc agcgacgcct ttggaaatgg attgaaggac 1500ctttgtcaag gacaccccag ttgggtgggg tggttgcttg atcctgtgga aggggctaga 1560gagaggcaat ccagagagag ggtggttccc tggcattgct tttcaaagca tgaccaggaa 1620ctgtttacaa ataagtaata ctggggtaga ggtgaagctt ggtcacggga agagagctca 1680aaggttgtct gtgccctaac tgggctgtcc tccagaggga ggagcctgga aagcgatttt 1740gaggagcctt attgaaggga acggggcctc ctttttaact tcagaacttt gtcttctttt 1800ggtgctgggg tggcctcttg ctagagggtg gggacttcgc agctcctgcg gtctagagga 1860ttgctagcct tgttcctgtg ggcagggctc aggagctgta ctacaaccaa tccgatgcag 1920ttaggcctgg accatcctta aatcagttgc acaatagcaa ggcctgtgga gtaaggagac 1980cttcttgcca acaccaaggg ataaaatcta ggagggagct ttacagagaa attcagccag 2040gccgctctgg gggctgggcg ggctgccttg aaaggctttt taaatgaccc aggcagaagt 2100tcagtaatat atatggagag ctgggtttaa ggaaatgtta actttgcaga atagtggagt 2160tcttaggtgg cttaactcat ggaagaaatc tccccccgat atgatcagtt caagaccaac 2220tcgtgtttga gcatggtaca gggtctcact ctgtcgccca ggctggagta cagtggcacg 2280acctcggctc actgcactct ctacttcctg ggctcaagcg atcctcccac ctcagcctcc 2340tgagtagctg gaccacaggc acgtgccacc acgcctggct aatttttttg tattttggta 2400gagtctgggt ttctccatgt tgcccaggct cgtcctgggc tcaagttatt cgccccgtca 2460gcctcccaaa gtgctgggat tacaggcgtg agccactgca cccggccaag atcttgcttt 2520ttaatcctga aaaattttgg cagaccagaa tctgctccat ataagagttg tcttgaactt 2580gaactgatag cttttaagaa atagatgctg ttttccgagg tgatggaaag ggatttataa 2640cttcttccag aattctttgt ggtattgctc agtaaatgcc tgtgcttcca gaagttcaga 2700acacgtcata gtgacaactg cactcagcta taatgtattt ggaaagggaa ctaaactttc 2760agctatatat taatcccctg agggaagagc cacctagaca cgtttttggc ttatgtcaaa 2820tagtcaagct accatacttt taaaaataag ggcatagtct ttaagcgttg cttcaaagat 2880agaagcctgt ctcatagcct ggattagttc ttaaagtgct tgcaaagagt ctgccagaaa 2940tactaattca ttacccctcc tcctaacaac actcaatcac tgttttctca aatatcactt 3000aacttccccc gacattgttt ctacacacag tttcttgttt tggatttaaa tacttgtgat 3060cttggctctt ctttactgtt gagcttgtat ttattgggag agcccatcat aatctttgga 3120tttatttgtt tatttattta tttatttttg gagacggagt ttcgctctgt agcctaggct 3180ggagtgcagt ggcgcgatct cggctcactg caagctctgc ctcctgggtt cacgccattc 3240tcctgcctca gcctcccgag tagctggggc tacaggcgtc caccaccacg cccagctaat 3300tttttgtatt tttagtagag atagggtttc accatgttag ccaggatggt cttttttttt 3360ttgttgttgt tgatcattct tgggtgtttc tcgcagaggg ggatttggca gggtcacagg 3420acaatagtgg agggaaggtc agcagataaa caagtgaaca aaggtctctg gttttcctag 3480gcagaggacc ctgtggcctt ccgcagtgtt tgtgtccatg ggtacttgag attagggagt 3540ggtgatgact cttaacgagc acgctgcctt caagcatctg tttaacaaag cacatcttgc 3600accaccctta atccattcaa ccctgagtgg acacagcaca tgtttcagag agcacagggt 3660tgggggtaag gtcacagatc aacaggatcc caaggcagaa gaatttttct tagtacagaa 3720caaaatgaaa agtctcccat gtccacctct ttctacacag acacggcaac catccgattt 3780ctcaatcttt tccccacctt tccccccttt ctattccaca aaaccgccgt tgtcatcatg 3840gcccgttctc aatgagctgt tgagtacacc tcccagatgg ggtggtggcc gggcagaggg 3900gctcctcact tcccagtagg agcggccggg cagaggcgcc gctcacctcc cgggcggggg 3960gctgaccccc cccccacctc cctcccagac ggggcggctg gccgggcaga ggggctcctc 4020acttcccagt aggggcggcc gggcagaggc acccctcacc tcccagatgg ggcggctggc 4080cgggcggggg gctgaccccc cacctccctc ccggatgggg cggctggccg ggcagagggg 4140ctcctcactt cccagtagga gcggccgggc agaggcgccc ctcacctcct ggacggggcg 4200gctggccggg cggggggctg acccccccac ctccctcccg gacggggcgg ctggccgggc 4260ggggggctga cccccccacc tccttcctgg acggggcggc tggccgggca gagggctcct 4320cacttcccag taggggcggc cgggcagagc gcccctcacc tcccggacgg ggcggctggc 4380caggcggggg gctgaccccc cccacctccc tcccggacgg ggcggctggc cgggcggggg 4440gctgaccccc ccacctcctt cctggacggg gcggctggcc gggcacaggg tctcctcact 4500tcccagtagg ggcggccggg cagaggcgcc cctcacctcc cggacggggc ggcttagcca 4560ggatggtctt aatcttctga cctcgtgatc tgcccgcctc ggcctcccaa agtgctggga 4620gtacaggcgt cagccactgc gcctggctgt aatcagtgga tttttaacat gagggcttat 4680ttaatatctt tataaggaca tggtgccgtt gcagttggat ttaatcacta ttacagcaat 4740gaatactgtt taggtgttct ttacttactg atttaacttc cccctctgac ggtttgtgta 4800tctctaatgc tacattcatc ttcctctcat catgtaattt tatttccctc aaccagggtg 4860caatagtttg ttatctggcc acctccagac tcctggatta atatggttag gtatcgttag 4920taaattttgt gtgcccaggg gagtggttcg cacaaaaggg gacaacagac aaaagtagga 4980aatacagtcc ttggtctgaa caggtttaaa ttcttgattt gggggtaggg gcagacagat 5040aagattttta gttctgaaag agtagagtca agtgctaaca tatatttcag acttgaaaac 5100atttagaaaa atgagtgata ggcttgtatt ggaaatctct atttagtggt aagggagggg

5160attaacattt cctgcctcaa acttctagaa atcttctgga gggtcacata catgtcagag 5220cattccagca taacaacagt tgatggaccg aatgccctaa atgatcagac tgagcaggga 5280cattcacagc atagcaaact tagagtttct cccccttgaa gagttcttta ttcattgggg 5340atcacaggag atacagctta cccttgagct ttctcaaaat tgtctttctg tggtttggga 5400agtgagaagt gggtgaaaac acacttgctg taacctcttg gatttctcat attgctggaa 5460gagtaccctt tctccttatg aaggaagagg aaactttttg ttgggcaaat tccaggccaa 5520aaaaaaagct cttggatttt attttttttt ctatctgcct gtggaggagt ggggatgata 5580tttggttctt ccattgagaa gctacctctg ccctcaaagg aaggtaaagt tactatagtt 5640ggctccactt ttaataacac tagtgaccta gaagaaacct aatgctttca ttttattatt 5700gagagaaatt gggacccaga gaagttaagt aacttgttag tattaaacag ctaattattg 5760tccgtgagtc cctcaaattt agtatcctga tcactattcc agttttttcc tctactgttt 5820tgagatgctt atcttactag gaaaggaagt ttgcaaacat ttaaagagac ttttctacct 5880ctggaatgct agctaatatg attctttccg ctaccttatt tcctttctag gtgctgaact 5940gccccctttt agaagagttg ttttgctctg cggaatgagc agaaacagag acacctcttt 6000taccctttct ctttgccttt cttttccttc acagagttct catcagccat ggaaaagtat 6060gctgttacca taacaagggc agcagagggg aactaactgt gatggtctga aattttgtct 6120gggttgtagc tcttctgcct cttggatagt tgatatgatt ggttcattgg gacagaattt 6180cataaaggtc attggatggg tttggaaaaa aggagaaagc aggaagaagg gaaaataatc 6240cactgggagt gaaagtgaac tataccaaaa taaagagtat tgggggtgaa gggagctgct 6300tttttttttc tttctttctt tgagacagag tttcactctt gttgcccagg ctggagtgca 6360atggcgccat cttggctcac cgcaacctcc gcctcctggg ttcaagtgat tctcctgcct 6420cagcctcctg agtagctggg attacgggcg cccaccacca cgcgtggctg atttttgtat 6480ttttagtaga gatggggttt taccatgttg gccaggctgg tctcgaactc ctgacctcaa 6540gtgatccgcc cacctcggcc tcccagagtg ctgggattga accaccatgc ccagccctgt 6600ttcttttgtt aagataaaaa ttgtctttgt tggtttatta gcacttcaga gatttgatgg 6660ttgtgcacag tgaatgtggt ttggccacca cctgtctccc tgaagaatac agagttaggc 6720agcttttagt ttcctctggg ttatttgcca tagagctttt caggagtctg tctcttcatc 6780cagagtctcc atgaagaaca gatgtttaaa actgaagttc attcatgaat gttctataac 6840tcagtcaata aaacatagac cttgttctac cttcctaagt tgaaagaata aaaagcagag 6900aaaacacttt ctttgttaag tcctttctgt tttttccttt cttattttcc cttatggggg 6960tgggagagag aaagacaggc ttagacttct tttctgagtg cattagaagc acttgctgct 7020tgttcatctt gtatctgttt ctcatctttt ggtgggccct tatgtgagac atagctggag 7080aattgtcaag aattcctagt agaaattgaa gttacatgcc aaatgctttg tttctttttt 7140taattcaatg tctaggcttg aaggatacct tcctcctcgt ggtccctgct gccctagcag 7200tgttggtttg tatgtatgta tttataagat attttgtaag catctttttt cttagttcct 7260tataatggtt ttttaataca ctatctcttg atgttttaaa catacataac aaaaatttcc 7320attttagtca tttttaattg tacagctcag tgggattaaa tatattcacc ttgttttgca 7380accatcacca ccatccatct tcagaacgtt tttcatcttc ctaaactgaa acctcatacc 7440aattaaacaa taacttccca ttaagcagta ccaccgcagc ctccggcagc taatcatcct 7500actttgtgtc ttcatgaatt tgactactct aggaatctca gttaaatgga accacgtagt 7560aattgtcctt tcgtaaatga acattcatca gtgttcatgc tttttttctt gccattttgc 7620tttttttttt tattattatt acattttttt aatacctatg aaagcaacaa agttgtaaaa 7680gtcatatagt cttactggcc acacaacaaa aagcaacatt ccgatgcccc gttccttcat 7740ctccatttcc actctcagag aaaccatttt aaactcattt agctaatgtg cactgcagcc 7800ttgacctcct gggctcagcc tcctgggatc ataggagtgc caccaagcac ggctaatttt 7860taaaattttt gtaaagatga tttctcacca tgtttcccaa gctggtctta aactcctggg 7920ctcagatgac cctcttgcct cagcctccca aagtgctgtg attataggtg tgagccccta 7980tgcccagcct atttcttagt ttgggatatt aattcatttt ctgctatgga agatgaggat 8040ttagttgtta tatacaccct tctcccaatg tatatacttc tgtctcctat cctcttaatt 8100taactttttt ttaccctttt tggtcaaact aatatgtatg taatctttac taaattttgg 8160taaatattga atgcagatgt ggctgacatt gttggtttcc tgctcaatag ctattccctc 8220ttcttgcttg ctgccagatt ccctcatttt ttaaatggca aggtgctaaa ccccaggata 8280tagactgtga cagctcttaa gtcagtcata gtttccccag tgtttggtct ggtgggcatc 8340tgaaccagtt ctggacaaat gagatgtaaa ggaagtctgc tgatggcttc tgagattttc 8400ccctccaacg gagaaagtcc caggaggaaa gccctgtttg acttcatgtt ctcctttcct 8460gcttggaact ctattttatg agggtgtggg tccatctcag cctttttgga gggcttgtgg 8520gcaagttact taatgtttcg ttctccaggc tgtctatttg cgtgagtaaa tggttaattc 8580taattctgga agcagactta gttaaacaga attttatgat ggcggccggg gggtgggggt 8640gggggctccc tgtaaaaata tagttaacaa ctaccctgta agttaaccat gttatagtgg 8700actttctctg tgtggtttaa tttcagctta cataatttct taactatata gcttaatgca 8760tggattattt atcatttaaa ctaaggtact tggtattgaa agaggccgtt acgcttgaat 8820gaccttgttt ctatactagc catcttggca agcataactt tgggctttat tcattgacct 8880tcttgttgtt ttctgcagtt tgagaatcac tggtttttag attcaaaggt agatagggtt 8940tttcccccct ctctgtcaaa gggactcagt tttactctca tatttcccta gtaatgttaa 9000atctagaaag tcctggatga aagtattaga tttatcctaa tatctggtca ctaagggatg 9060aaaaatttat aaatagctaa tgttaaccta gatctaaagc ttcctattct gaaatccaaa 9120catgaagact aagaaaaata tgtacatttt gaaacaaagc agaaaaatga aacttcaaca 9180atgtaaaaga gggtaaaatg ccagtagaca gccactttca gcagtttctg tttacagtgc 9240ttttgctggc taccattgtt gctgtaaata atatatgtat actgtgattt ctttaacatt 9300agacaatact gttaactccc cattatgaaa gatgaggaat ctaagatact tttattactt 9360tctgtctctt gtttctcatc ctctctccat ttactaattt ctgtaaattg tattatcatt 9420tttggctcct ctagtagtta tctttaaagc tctaaataat atgtgtctca acctgtcaac 9480tttagctctc cactgtataa aagtacacca ttcacctgtc ctcctgcctt tactttgctg 9540gtatgtaacc tcattatgtt tataatatca gggtttgtgg gcaggcttgg tggcgtacac 9600ctataatccc aacactttgt gaggccaagg caggagaatc gcttgagccc aggagtttga 9660gaccagtcag ggcaacgtaa tgagacccca tctctacaaa atataaaatt agttggactg 9720tagtcccagc tacttgggag gctaaggtgg gaggatcacc tgagcccagg aggtcaaggt 9780tgcagtaagc cgtgatcatg ccactgcact caagcctggg tgatagagca agaccctgac 9840tctcaaaaaa aaaaaaaaaa aaaaaagaaa tcagagtttg taacatttac gtactgttcc 9900ttaactgtta attcttctat gcttttacta tagtttgatt ataaacgttg aaaatcaaac 9960agcatataca atattgtgat tttttttcat aactttccaa tataagacta aggagcatga 10020ttattttcat agagaaggaa tggaaaattg ttttttaatt tttgtaacac tccatcaatt 10080gatcacaatc atgccccatt ttaatactgt gttatatgag ctatggattt cttgtgtagt 10140ctgttttcct tcttcagtag cttaaggtta acttgtttct agttacttat ggctgcattg 10200gttgacatta aattgagctg acatagcgtt ggagggagtc tgggtgaaat gtgaattgct 10260aggaagtcct tgcttcagaa aatgtacatc tgtgcatgca tatgtataag aaaccttaaa 10320tattttagat tttttaatag attgtcttgg aattgatgta agtttatagt aaacataata 10380ttcaataaag gaaataaagg gttttttcct gccattttat ccatggttga atcttaattt 10440tttttctttg aataattttg gttttttaaa aggtcagttt cttttgaatt acaccctaaa 10500atcattagtg attaacaagc tgattctgta tttttgcttt atacccacta caattaaatt 10560ttttttagct tgcagagatt actaaaattc aaaaacttat aaatactata ctttcatagt 10620catatttttc ctggctatat ttgagaacat tacagcatta agacgctagt tcttcagtgt 10680gtcttgttgt ctgaattatt tactgtgttg gcattcaaaa tcaaacatat cttaggaatg 10740cattaaaaaa actattagaa ataataggtg agtttgccaa ggttgcaaga taaaagagcc 10800atatagaaaa acctgttttt ggcccagcac agtggctcat gcctgtaatc ctagcacttt 10860gggaggccaa ggcttgcaga tcacttgagg tcaggagttc gagaccagcc tggccaacat 10920ggtgaaaccc catctctact aaaaatacaa aaattagctg ggcatggtag cgcatacctg 10980taatcccagc tatttgggag gctgaggcag aagaatcact tgaacccaga gggtggaggt 11040tgcagtgagc tgagatcaca ccctgcactc ccgcctgggc agcagaggga aaatccatct 11100caaaagcaaa aacaaagaaa actggtcacg cttgttatcc catcattttg gaaggctgag 11160gtgggcagat ggcttgagcc caggagttca agaccagcct gggcaacatg gtgaaacccc 11220gtctctacaa aaaatacaaa aattattttt ttctgggtga catgtgcctg tagtcccaac 11280tgctagggag gctgaggcag gaggaccgct tgagcccagg aggtgtaggt tgcagtgagc 11340caagattgca cttctgccct ctagcttgga cgacagagca agaccctctc tcgaaaaaaa 11400aaaaaaagaa aagaaaaaga aagcaatccc atttacaata gcatgaaaac aatttgatag 11460gaataacttt agtcacaaag cataaaactt ttactctgga aactacaaaa cattgttgaa 11520agaaattaaa gaagacccaa ataagtggaa agacatttgg tggccatgga tcagaagact 11580taatattgtt aagatggtag tactccacaa attgaccttc acattcagca cagtccctat 11640cataatctca gctggcttct ttgacaagct gacaaaattc atatggaact ttaagggacc 11700taaaatatcc agaacaatct tgaaaaagaa aaactaagtt ggaggactta cactttgtgg 11760tttcaaaact tattacaagt caagacagtg gggtactagc atatggatat acatatagat 11820caatggaata aaattgagag tccagaaata aacccagcaa taatggccca ttgatcttca 11880acaagggtgc caagacaatt ctgtggaaaa agaacagttt tcaacagata gtgctgatac 11940aactggatat gcacacagaa aaactgaagg tggaccagat ggctcaaaga cctaaatgtc 12000agagcaaaag ctattaatgt acaaccttta gaagaagaca gaggcaaatc tttatgacct 12060tggattaggc agtggcctga gatatgactc caaaagcaca aacaaaagaa aaaaaaaaaa 12120acatacattg gacatcatga aaattaagaa cttttgtgct tcgaagatca tgaagaaaat 12180gaaaagataa cccacagaat aggagactat atttgcagtc acataagaga tttatatcca 12240gaataaagaa ctatcataat ttagtaataa agacaaatca ttgaaaaatg ggtaaaggtt 12300ctgaatagac agtttcttca aaagaagata tgtggtggaa tggcctgtta agtacatgaa 12360aacagcatgt tcaacataat tagccatcag ggaaatgcaa attaagatca aaccacagtg 12420agaaaccact tcatatctgc taatgttggc tacaatatat aaaaattaga actcttacac 12480actgctgatg ggaatgtaaa atagtacaac cactttgaaa aacaggcagt tctggccggg 12540cgcggtggct cacgcctgta atcctaacac tttgggaggc cgagctgggc agatcacgag 12600gtcaagagat cgagaccatc ctagccgaca tggtgaaacc ctgtctctac tagaaataca 12660aaaattagcc gggcatggta gcatgcacct gtagtcccag gtacttggga ggctgaggca 12720ggagaatcgc ttgaacccag gaggcggagg ttgcagtgag ccaagatcat gcagctgtac 12780tccagcctgg tgacatagcg agactctgtc tcaaaaaaaa aaaaaaaaaa ccaggcaatt 12840tttcaaaggg ttaaaggtag agttaccata tgacccagta gttctacctt aattttttaa 12900tttttttttt ttttttttga gacagggtct cactctgtca tctaagctga gtgcagtggt 12960gcaatcatgg ctcactgtaa cctccatctc ctgggcttaa gtgatcctcc cacctcagcc 13020tccccagcag ctgggactat aggcatgcaa caccacgcct ggctaatttt tgtatttttt 13080tttaagagac gggattttgc catgttgccc aggctggtct tgaacttgtg aggtcaagta 13140atacacgttg gcctcccaaa gtgctgggat tacaggtgtg agccatgcct ggctaattcc 13200acttttttag tttggtatat acccaagaga atggaaatgt gttcacatga aaacttgtac 13260gtgaatgtta ttcataatag acaaaaagtg gaaacaaccc atttatcagt tgatggactt 13320gtttttatcg acttaccagt ccatcaactg ataaattgat aaggtgcgat atatacatac 13380aatgaaatat ttggtaatga aaaagaaatg aggtactgat aaatgctaca acataaatga 13440actttgaaga cattatgaaa gtgaaagaag cagtcacaaa agactgtatt gtatgattcc 13500atttatatga aatggccaga gtaggtaaaa ctatatagag acagaaagta gattagtggt 13560tgcctagggt cagagggtgt ggggagatca ggtggcatct aaggggttgg gggaggttat 13620gggggtaatg aaagggttct aaaattgatt atggtgatga ttgtacagtc ctgcgaatca 13680actaaaacta ttgaattata cactgtaagt ggatcaattg taaatgaatg ctatttcaat 13740aaacttgtaa aataccgtat cttatcagct ctttaaggtc atagaggttc ttcagagttt 13800ctcttatacc tcacacattt aggcagaatc tccactacct atatcctagt tggtttaatt 13860gcaattcttt aatgtatgcg ttgttgctga tacataattt agaaagcttg cttctgtgct 13920gtgctttttt caccacctca tctccacaat ttccaagctt gatgtactac agaagtaaga 13980ataatcttgt tccagtttgc aagttgactc atctgacagt gaaacctcac ccttctgaaa 14040tctcatcatg aacaaaagtg gggtaactca aacagatagg caaagtccat tgttaaagct 14100ccttcccttt gttcattgga aggccatcag accccctttc agcctatttg cccttatttt 14160acatagcatg ctaataagca agttgctgat ttccaaaccc accagactag aaacaccaag 14220ttagaaaggg tgttaagggt gtgctataca gattgaattg tgtctcccca aaattcttat 14280gttgaaacct taattcccaa catgatagta ttaggaggtg gtgcctttgg gagttaatta 14340ggctgagttg aggtcataaa ggtgggaccc tcatgatggg atttgtgccc ttataagatg 14400gaacaccaca gagttgaggc ccccttaccc ccatgtaagg gacacaaaga gaaggcagct 14460acctacaagc caggaagaga gccctcacca gaaaccatcc atgctggcat cttagtgttg 14520gacttctagc ctccagaact atgagacaat ttctgttgtt taagttgtgc agtctgtggt 14580attttgttat ggcagcctaa gctgactgag tcatagtagt agcctggaaa aaaggtagct 14640cttagtctaa ggggacaagg gcactgtggg ggaggggtgc accagcacaa ctttgaccat 14700ccgaactctg tatagtgatg ttcagctgta cagagcacca acttgatagg gcttagagac 14760ttcttaggtc acaacatagt ataataaaca attttcataa tactgtgatt catcaagagg 14820gttgaatcat tttctgttga aggccataaa gggatttaaa tctgtattag aagtattttc 14880attcaagata attgaaattg tttacccttt gaaagttgaa ctaagttttg tgctacccag 14940aatgcctcgt tcctcataat ttctagatgc ctttttgagt gctactctag tttttgtttt 15000tcaaagcaaa gaggttgaag gtaagtttct actttccttg tcctaaaatt atatttgatt 15060tacattttga ggcaggatct tgctctgttg cccaggctgg agtgcagtgg tgcagtcata 15120gctcactgta gcctcgaact cctggactca agctattctc ctggctcaac ccccgagtag 15180tagggacttc agaccaacag cacctcactt ggctcttttt aaaataattt ttttagagac 15240agggtctcac tatgtggccc agactagact cagactcctg ggctcaagca gtcctcctgc 15300cttggactcc caaactgttg gaattacagg catgagccac catgcccagg ctctcttgtc 15360tttttgataa tagccatcct aagagatgtg aagtgatatc tcattgtcgt tttgatttgc 15420atttccttga tgattagtga tgtcgagcac cttttaatgt acctgttgac catttgtaag 15480cagcacgttt ccataactgt ttatcagtta aagtgtactc ttcaaaacta ttgtttttaa 15540aacattcagg tagcacttaa ctggtaagca gattgttttg tagaatagag aggggctgcc 15600agtttcagct ttgactatag ctgtgagaag aatgttagtc tcagtccttc tttggaaagc 15660ctttaaaagg ggctgtttat tttttctcat acttgtttat tctttcattt aaaacttttt 15720ttttcttaaa gatgttaatg ttagggccta ggtcacatat aaataagaca tagccctgac 15780ataagaaact cagagtctat aaagagagac agatggataa acaggagtcc tgtaatacat 15840gatgtgtgta agatggagag gaagaacctt ttaaaaaagt gaggggtggg ggtggcttca 15900taaaggaagt gatggttgga gctgagtctt gaaggatcaa caggagtgtc cctggcagaa 15960gagaaagggc attccagatg gcagaagagc ctgtttacag gcagagaggt tagagattat 16020gctttcaact tgaattgtaa aatccttaaa agttaggatt acatgggatt taaattaaat 16080tcctgggtgg tattatatgt tgataacccc ttaaattgtt tcaaactctt tttaaccctg 16140ttttccagga actcgaatca tttatgacag aaagtttctg ttggatcgtc gcaattctcc 16200catggctcag accccaccct gccacctgcc caatatccca ggagtcacta gccctggcac 16260cttaattgaa gactccaaag tagaagtaaa caatttgaac aacttgaaca atcacgacag 16320gaaacatgca gttggtaaga gaatggcgat gttggagacc tagagcgtgg ctcttggaat 16380ttgaatgtct gtttgctgta ggttgagaag ctattgattc ttcagttttg ttttttgttt 16440tttgtttttt ttgagacaca gtctcattct gtcactgagg ctggagtgca gtggcacaat 16500ctcagcacac tgcaacctcc gcctcctggg ttcaagcgtt tctcctgcct cagcctcccg 16560agtagcgggg attacaggcg cctgccacca cgcctggcaa atttttgtat tttttagtag 16620agacggggtt tcgccatgtt gcttaggctg ttcttgaact cctgacctca ggtgatccac 16680ctgcctcagc ctcccaaagt gctaggatta caggtgtgag ccactgtgcc tggccaattc 16740ttcagttaat aactaatgtt cagttatatg tctctaaggt ttccctcctc ccccaaaggt 16800ttcttgaaat actctgaaaa ccctgaattc taaatcataa atctgatgta gttggtggga 16860gaaaagaatc cagaatcagg atgataaacc ccaaaaatga ctctttgaac catgccttaa 16920ggacttcctg gcctaggctt cattggacta aggataacac acacagaaaa caaaacaatt 16980tcatagcccc catttccatc accctcaggc aaatagacct tgtttctttt gttttgtttt 17040gttttttgag acggagtctt gctctgttgc ccaggctgga gtgcagtggc acgatctcgg 17100ctcactgcaa gctctggttc ccgggttcac gccattctcc tgcctcagcc tcccaagtag 17160ctggggctac aggcgcctgc caccacgccc ggctaatttt ttgtattttt tagtagagac 17220ggggtttcac catgttagcc aggatggtct cgatctcctg acctcgtgat ccgcccgcct 17280cagcctccca aagtgctgag attacaggtg tgagccactg cgcctggccg accttgtttc 17340ttatatgagt ttcttttata tggtgtgagg aaaagcggta tggtttattt cctctatttt 17400ccagaactgc gtttaaggtt tacctaaatt acccctcctc caacacattc tttttttctt 17460tatcactgta gaagcagaat ggtgcggtag gagtggccag ctcctgaaat attcctcagg 17520gactgcactt ggtgacccct agatgggggc cagcattccg ctttgtcata tttcagatga 17580gttttttaat ggacaaagtg ttagtttgga gcagggccaa agtccaaagc ctccagaaga 17640atgtgctccc ctgagagatg gcaaagagct ccccaagagc tgctgtttag tcatcctgaa 17700gacaaaggga caatggggat gttttcactt gtgtcctttt ccccaaaact tctccccatg 17760ggtgatggga ctgccattct tatttttcag attaagacca gccaaagcaa agcagagtaa 17820ttgttttaag gcagcagaga gtgggacaat ttgaattact ttttgtctct catttagagg 17880gaatgacagt taaaactgtt atgcttcttt gtgtacgtgc taaactcttc attttattgg 17940tcctaactag gggatgatgc tcagttcgag atggacatct gactctcctg caaggattag 18000aagaaaagca gcaacactga tacttgtgtg cacctgattt ggccaatagg atcaacagtg 18060aaaagacaga agaggcaata ccagcagtcc ccattacagt ctccacctcc ccgtcttcct 18120ctgggtgcca aatgatggga agatgagctt catctgacca tttcttctcc ctgtctcctg 18180ttccccttcc cagttaaaca ggttagattg aaggcccttg ctgtatttct gtagagctaa 18240gcagccctta gaggaaaaca gttcaactct gactttccta gttgtttttt tattgagagc 18300caccctcata ccctgtaatt ttgtcccaaa tcaaatatca acctaccaac aactgcctgg 18360ctgggaagtc tggggaaggg atacagagct tggtgggcct aacaccattc atattcctta 18420ccctctgtct ctcctccctg tatcccacct atggttcagt gttgcaagag tctgggcttg 18480gggtctttaa aaccagcagg gggaaatgat aaaaagagag ctgctttccc ttttaccttg 18540aggtattcgt ccctcgggac agagcacagc ttgtgcaact ctggtagcgt taccctgtga 18600cactgttttg aggtccactt cctttctttc ctctgggagg aatgtcttct gtctttggta 18660ttatagttca tcttcccatt cttttactta gtgcatttgt gcagatattt ttaactctgt 18720acatcagaag agagcccttg gtaaccagtt ttgctcttct tctgccactc ctccctgctt 18780gcatctcgtt gctggcagag tcctcttgta cttcaagaaa gcaaagtgat tttgtctgct 18840cctagagcag gtccatacca agtaatagag gcactttagc ttccacttgg tgggtaaggc 18900ctgatcatag tattctgtca gataatgcct aagaatgacc gctgaagaac gttgacccat 18960ttgagtaccc ggtctcagtc gtcattttta agtccagtga gcattgtggt agttgttctt 19020agattgcagt ttcttatgtt ttgagtttga agttgatttt cagaatgttc ttagaaaaga 19080actgcatttt tttcctttgt ggatctgctt tgtttggctg ctgggataga taagcatggg 19140cttaaaaaat gtgttcctcc cagttttctt gcctttcctg ttgtactctg aatttctctc 19200cctacctccc tcactttctt cctctctcct tcctttcctt cctttttctc taccaggcca 19260tttttcaaat ttacatcaaa gatacctgaa gtgttggtat ctgagaatat ctgtcactcc 19320tcttatctga gaagtgacct tttattttta agatgactac agacctattt ttagatatgt 19380tttcagtaca attttgaaca gcaacttttt aattaaacat cttccagtgt taggaagttg 19440agaaacgttc ataggcaagt ctgctgttct atgtcaccat cttttgtctc ccctagtccc 19500ccaggagctc tttcctttcc cctctagttt tgggtgtgca tgtttggagt ttgtagtggg 19560tggtttgtaa aactggacca ttctgccttg ctatgggttg ttcaagaaag cctcattctt 19620ttctgtgacc ctttcgcttt tgcattcacc ctccttccca cctacctgtc ctggggctgt 19680tgagcagcat aataatcccg ggagaatgat tcccctcata gaaagacaaa agcatccatc 19740ccctcatagt taagtagcca ctggtgtcct gggaatttct ggttggattt ggtgccctga 19800acttttttat taagaaatca gatcccaggg tgagagtaac aggccatttg gccaagaaag 19860aaacctgttt gtttttcttt tgaactatga aaagaccctg tttgtgaata tattttagaa 19920agagaggaag gatgtctgca gaactttgtt ctgttttctg ccacaaaaat gtgaatagtt 19980cagagtgaaa accttttgtg atggttgatg tctcaggaat aagctggatc tccaatgttt 20040tggggatgct ttgagtctca aaaaaaattg ataatcagaa aagtaatttt tgtttgtttg 20100tttaatgtat ccctgttctg tttttaatta aactccaagt ctcattttac atattcttgg 20160aaaaacctaa gttgctctgt aatttacata agaagcatgc tcaggacctc tttgtacccc

20220ggggagcctg attctttggg aatgaagctt ttcattcttc atacactggc cttggcatcc 20280tgtggaattt gacccaacta gcagctagtc agtctgtcag tgagcagaag agtgaactct 20340tcttgatctt tattgctatg tgtgaaaact tggcttcctc actgaacggt gaggaatgga 20400tttaaagcat gagctttagt agtatcaaga tgccattttc ctttttcttg ctgtcttggg 20460gagcttctgc atgtgacccc ctaatcagaa ggcatgtttt tagtatttct tgggagtgtc 20520agctgtataa tgcagcagct gttcaatccc ttacccttct ctgcaaggac ttccttacag 20580cttggtgcag ttctttccca gaggccacca ctactagaca gtctttcttt tatcttatgg 20640agataaattg gcatttaaaa aataatttca caaggcatga gataaacttt caatagatga 20700tacctttgtg tcatgcctca tggaattatt tttagaacaa gccagagtcc attgagtggt 20760ttacctctgc atgtttggag ggaacctcac agatgaaacc cttaatgaat aatgtgtccg 20820gggtttttta gagagaagga gcactcttaa gttaccactt tgagacagct cttaacatct 20880tagtgaccat ttgtagtttt ctttttatga ggaacccatg cttctatact tgggcggaca 20940atcgagctta atgagaagtg acttcccttc aaattccaac agcagacatg cattgtcatg 21000attctgtctt ctttttagtg tggtttattg agttcagcag ttctcatatt ctgtttaaat 21060aggtacagca ttttcaaggg cacagataca gagaagctgg ctttctaggt attgggcttc 21120caagccaaga gttttgtcct tccacctgta ttagttatct attgctgtgt aaaaaattac 21180cctaaattta gtatgttaaa atagtaaaca ttatctgtca gtttctgtgg gttaagaatt 21240tgggagtagt ttaacatgat ggttctggcc catggtatca tgaagttaca gtcaagattg 21300aacaggggct gcagtcagct ggagctggag aaaccacttc caagttctct ctttgtggcc 21360gttggcagga tgcctcagtt tcttcccatg tgggtctctg cgtagggcag catgagtgtc 21420ctcacaccat gacacctggc ttctccctga gcagctgatt ccagagatca tggggcaggg 21480ccaggagaaa gctgcagtgc cttttaagac atagtcttgg aaaggacaca ccgtcacttc 21540ttatcctagt tgttagaagc aagccactaa gttcagcctg cactcaagga gagaggaatt 21600acacatacct ctaccccaat ggagcagtag caaagaattt atggacatat cttaaaagca 21660ccacacccca actggggatg aaagtaggtc aacagggagg taggtttaat ctagataagc 21720tgaaagatag attgctatca aaaacagttc tccaagatgt gcatagccaa actgggatag 21780aaggcaaact ccccaaagct acctgctggt tttgagaggg gtggtaagac atggcaattc 21840ccaggagtag tagaaaataa tatgcctgac taccaacagc tcaagtatgc ttatttgcac 21900atcctagact tggtgtctgt aagactcagt taccactttt attttcctgt agctaggagt 21960tagcaaaagg aactggggcc ttccagccga gccactaaac ctgtcttatt tggaatgggg 22020attgtccagc aaagggagca aacatgaatt agatgttaag ctattgagct gaagaaaaga 22080aagcagttca catttaggtg aaatagatga tgttatcagg aagccaggtt cccaccagag 22140tcggtgcttg gtacctggtc tctccagtct caacagactc aggtcaggtc tctcacccag 22200gaagcaacca ctcaataaaa tagagaacat ctgagaatta caaatgtcta tgcttgattg 22260ctcctctaaa tccagtgcat aggttaaccc tgcatgccca tttcttcctg ggcttcttga 22320tggcaatgtg ttcttaaata actggtcttg tgttcatgct aaagacaaac ttacatgaag 22380tttttcagtt taagacattc tagtgaatgg ctgctatgtg tttctggcac tcattcctaa 22440ccaagtcttt agagatttca gatgacctta aagatgcaat atctttttct ttctttcttt 22500ctttcttttt ttctgagaca agagttgcgc cctgtcgccc aggctggggt gctggagtgc 22560agtggtgcga tctcagctca ctgcagcctc tgcttcccag gttcaagtgt ttctcctgcc 22620tcagccttcc gagtagctgg gattacaggc atgtaccact atgcctggct aatttttatt 22680tctatatttt tagtagagat ggggtttcat catgtttgcc aggctggtct cgaactcctg 22740gcctcaagtg atccgcccac ctcagcctcc caaagtgctg ggattacagg cgcgagccac 22800cgcgcctggc caaagatgca aattcttgtt tggatttatg ctctgcctct tcccagcatt 22860ttcttatctg tagccctgct tgcttgagag tatacttgga taagaagtat tgctgttgag 22920ggagctataa gaaaaggatt cttcttccag aagtaaagaa ctcatcttta gagtaccttt 22980aaatgaattt tgtttttctt tcttattttg aggtggattg gtcttctctt tttttgggtt 23040tccagctcac tgggactctc agaccttacc tttccagctc aaacaccatt agttaaattc 23100cttcattctc attagaatgc agcctgctga gtatgtgggt ttcactgccg gagtccatca 23160tttagccagt atacatagag gaactgcttc gaatcaaggc aactggtgaa gggcttagca 23220tgttggcagc aatatcccag agattgaatc tgtttgcatt ttcctcatct aggataacag 23280ctgcttgaag ccagggctct tagccctttg cattcccctt gagcgaggaa gccacactgc 23340ctttctgtgt ctggttcaga gctcttcctt cttggcatgt tttctggact acatgcacat 23400gggcagctat agattaatct gcaaaaccta gtcacttacc tacccataat atctgggaag 23460gtgtggtatt tgttttaaag aaacattgtt tctttgggag ggcagtttct gtctggactt 23520tgaggtggac ttagttatcc ctacagttct ttaactctca gcttttaata aaagatgaaa 23580tcagatattg atgcagttgg gtcacaattc tttagaatgc ttctacccca gggccgcttc 23640ctgttcctag tcatggtttt ccagtttagt agtggagttt cttgaggcta acttacagaa 23700atttctaact gaaaacttta agagttattg atacttgttt tttcagtcag tcacttacat 23760cacctagcct actctctgga atttaaattt atttctctag gctggtcctg gaagttgata 23820accttttggc aaagcttaga tttaggagaa ggcttgagtc cctgttcagc gggtctgtgg 23880attctctttg cttatggctc tctgcctgca gccctggcag accatactgt atgtcatgga 23940tacccagtgg aaatattact gagatgaaac acatttccaa gggtatttaa actctcactc 24000tgccaccttt ctaagggtgg gaggctggca gagatgctgc aatgcttgat aatcatttgg 24060ccacactgaa atttccaaag ggagctcttg ccggtgctta aaaccaaaac tcctggacac 24120ttagaaaatt ccatgaatct agcacaaaat atccattctt gcccaagtgt atcccctttc 24180tctccagctt aatctttttt tttttttttt ttttaaagcc caggccaagg gtacttttaa 24240ctggaaactg gggaggaggg aagaacacta gcagggagct aagaggcagg ttgctgggta 24300agccatcctg ctcctacctg gtgcctgtat ctacattgct gagtgctgtg cgccagtgcc 24360tttccttcat ctgcagatgg agcccatctc tttccacctg ggtgaggaga ccctctgcta 24420ctccaggggt aaaccttaaa gaaggtgtct tgaagagccc agaggacact cacgtgctaa 24480ggtgtccatt ttatgcatct ttaaaatatt ttatttaaaa aaaaaaatag ccctgccctg 24540tcttagtgcc actaacggcc cagttccatc cattctgaat ggaaaagcgg agactgccag 24600cactttcctt ggtcttccct ttgtctccca tgatgtgttg ttccctcatc cctcccatcc 24660atttcactgt gtgtggatgg atagcagagg gtaccacgca gtccttgagg cagtcctgtg 24720tgattccatg atcagttgtt tttgtatttt aactattctt ccaaaccagc agatgtttgg 24780aaattaagga aaaaattaaa ttctcatcaa tggttgctgt tatagttaaa tcagtaaaga 24840tcttgagtat caacttggtg ttttaatttt ttaaaaattt ctggtgaaat cctgctaagg 24900ttatttcaca tttcaggagt ttcagctggt gggggagatg ggcagaggta agaggcagtt 24960ggctctttat ctgtcagttc tccacacttg cggagcatgc actttgtcaa tgtggacctg 25020tgtatgcaaa ggagatggtg ggactctcag ggagcatgac cctggtcctg tgctcaggag 25080cttgcaggtg aacatgtata tgctgggctg acggcaccca agcatgtcct tctcttaagt 25140gccagccctg aggaagccca aacaactttt cctttctcag aagaggggct gcctgtgccc 25200ctgggagcac tggttagatg cccatcatgc ctgttacctc aaaccaagct gtgctgcatg 25260agcgtcagat tccctgctgt taactaatcc agcgggtttc atgtattagt cctgagaatg 25320agaa 25324263188DNAH. sapiens 26gttgattttc agaatgttct tagaaaagaa ctgcattttt ttcctttgtg gatctgcttt 60gtttggctgc tgggatagat aagcatgggc ttaaaaaatg tgttcctccc agttttcttg 120cctttcctgt tgtactctga atttctctcc ctacctccct cactttcttc ctctctcctt 180cctttccttc ctttttctct accaggccat ttttcaaatt tacatcaaag atacctgaag 240tgttggtatc tgagaatatc tgtcactcct cttatctgag aagtgacctt ttatttttaa 300gatgactaca gacctatttt tagatatgtt ttcagtacaa ttttgaacag caacttttta 360attaaacatc ttccagtgtt aggaagttga gaaacgttca taggcaagtc tgctgttcta 420tgtcaccatc ttttgtctcc cctagtcccc caggagctct ttcctttccc ctctagtttt 480gggtgtgcat gtttggagtt tgtagtgggt ggtttgtaaa actggaccat tctgccttgc 540tatgggttgt tcaagaaagc ctcattcttt tctgtgaccc tttcgctttt gcattcaccc 600tccttcccac ctacctgtcc tggggcagtt gagcagcata ataatcccgg gagaatgatt 660cccctcatag aaagacaaaa gcatccatcc cctcatagtt aagtagccac tggtgtcctg 720ggaatttctg gttggatttg gtgccctgaa cttttttatt aagaaatcag atcccagggt 780gagagtaaca ggccatttgg ccaagaaaga aacctgtttg tttttctttt gaactatgaa 840aagaccctgt ttgtgaatat attttagaaa gagaggaagg atgtctgcag aactttgttc 900tgttttctgc cacaaaaatg tgaatagttc agagtgaaaa ccttttgtga tggttgatgt 960ctcaggaata agctggatct ccaatgtttt ggggatgctt tgagtctcaa aaaaaattga 1020taatcagaaa agtaattttt gtttgtttgt ttaatgtacc cctgttctgt ttttaattaa 1080actccaagtc tcattttaca tattcttgga aaaacctaag ttgcgctgta atttacataa 1140gaagcatgct caggacctct ttgtaccccg gggagcctga ttctttggga atgaagcttt 1200tcattcttca tacactggcc ttggcatcct gtggaatttg acccaactag cagctagtca 1260gtctgtcagt gagcagaaga gtgaactctt cttgatcttt attgctatgt gtgaaaactt 1320ggcttcctca ctgaacggtg aggaatggat ttaaagcatg agctttagta gtatcaagat 1380gccattttcc tttttcttgc tgtcttgggg agcttctgca tgtgaccccc taatcagaag 1440gcatgttttt agtatttctt gggagtgtca gctgtataat gcagcagctg ttcaatccct 1500tacccttctc tgcaaggact tccttacagc ttggtgcagt tctttcccag aggccaccac 1560tactagacag tctttctttt atcttatgga gataaattgg catttaaaaa ataatttcac 1620aaggcatgag ataaactttc aatagatgat acctttgtgt catgcctcat ggaattattt 1680ttagaacaag ccagagtcca ttgagtggtt tacctctgca tgtttggagg gaacctcaca 1740gatgaaaccc ttaatgaata atgtgtccgg ggttttttag agagaaggag cactcttaag 1800ttaccacttt gagacagctc ttaacatctt agtgaccatt tgtagttttc tttttatgag 1860gaacccatgc ttctatactt gggcggacaa tcgagcttaa tgagaagtga cttcccttca 1920aattccaaca gcagacatgc attgtcatga ttctgtcttc tttttagtgt ggtttattga 1980gttcagcagt tctcatattc tgtttaaata ggtacagcat tttcaagggc acagatacag 2040agaagctggc tttctaggta ttgggcttcc aagccaagag ttttgtcctt ccacctgtat 2100tagttatcta ttgctgtgta aaaaattacc ctaaatttag tatgttaaaa tagtaaacat 2160tatctgtcag tttctgtggg ttaagaattt gggagtagtt taacatgatg gttctggccc 2220atggtatcat gaagttacag tcaagattga acaggggctg cagtcagctg gagctggaga 2280aaccacttcc aagttctctc tttgtggccg ttggcaggat gcctcagttt cttcccatgt 2340gggtctctgc gtagggcagc atgagtgtcc tcacaccatg acacctggct tctccctgag 2400cagctgattc cagagatcat ggggcagggc caggagaaag ctgcagtgcc ttttaagaca 2460tagtcttgga aaggacacac cgtcacttct tatcctagtt gttagaagca agccactaag 2520ttcagcctgc actcaaggag agaggaatta cacatacctc taccccaatg gagcagtagc 2580aaagaattta tggacatatc ttaaaagcac cacaccccaa ctggggatga aagtaggtca 2640acagggaggt aggtttaatc tagataagct gaaagataga ttgctatcaa aaacagttct 2700ccaagatgtg catagccaaa ctgggataga aggcaaactc cccaaagcta cctgctggtt 2760ttgagagggg tggtaagaca tggcaattcc caggagtagt agaaaataat atgcctgact 2820accaacagct caagtatgct tatttgcaca tcctagactt ggtgtctgta agactcagtt 2880accactttta ttttcctgta gctaggagtt agcaaaagga actggggcct tccagccgag 2940ccactaaacc tgtcttattt ggaatgggga ttgtccagca aagggagcaa acatgaatta 3000gatgttaagc tattgagctg aagaaaagaa agcagttcac atttaggtga aatagatgat 3060gttatcagga agccaggttc ccaccagagt cggtgcttgg tacctggtct ctccagtctc 3120aacagactca ggtcaggtct ctcacccagg aagcaaccac tcaataaaat agagaacatc 3180tgagaatt 3188271835DNAH. sapiens 27ctccaagatg tgcatagcca aactgggata gaaggcaaac tccccaaagc tacctgctgg 60ttttgagagg ggtggtaaga catggcaatt cccaggagta gtagaaaata atatgcctga 120ctaccaacag ctcaagtatg cttatttgca catcctagac ttggtgtctg taagactcag 180ttaccacttt tattttcctg tagctaggag ttagcaaaag gaactggggc cttccagccg 240agccactaaa cctgtcttat ttggaatggg gattgtccag cgaagggagc aaacatgaat 300tagatgttaa gctattgagc tgaagaaaag aaagcagttc acatttaggt gaaatagatg 360atgttatcag gaagccaggt tcccaccaga gtcggtgctt ggtacctggt ctctccagtc 420tcaacagact caggtcaggt ctctcaccca ggaagcaacc actcaataaa atagagaaca 480tctgagaatt acaaatgtct atgcttgatt gctcctctaa atccagtgca taggttaacc 540ctgcatgccc atttcttcct gggcttcttg atggcaatgt gttctaataa ctggtcttgt 600gttcatgcta aagacaaact tacatgaagt ttttcagttt aagacattct agtgaatggc 660tgctatgtgt ttctggcact cattcctaac caagtcttta gagatttcag atgaccttaa 720agatgcaata tctttttctt tctttctttc tttctttttt tctgagacaa gagttgcgcc 780ctgtcgccca gactggggtg ctggagtgca gtggtgcgat ctcagctcac tgcagcctct 840gcttcccagg ttcaagtgtt tctcctgcct cagccttccg agtagctgga attacaggca 900tgtaccacta tgcctggcta attttttatt tctatatttt tagtagagat ggggtttcat 960catgtttgcc aggctggtct cgaactcctg gcctcaagtg atccgcccac ctcagcctcc 1020caaagtgctg ggattacagg cgcgagccac cgcgcctggc caaagatgca aattcttgtt 1080tggatttatg ctctgcctct tcccagcatt ttcttatctg tagccctgct tgcttgagag 1140tatacttgga taagaagtat tgctgttgag ggagctataa gaaaaggatt cttcttccag 1200aagtaaagaa ctcatcttta gagtaccttt aaatgaattt tgtttttctt tcttattttg 1260aggtggattg gtcttctctt tttttgggtt tccagctcac tgggactctc agaccttacc 1320tttccagctc aaacaccatt agttaaattc cttcattctc attagaatgc agcctgctga 1380gtatgtgggt ttcactgccg gagtccatca tttagccagt atacatagag gaactgcttc 1440gaatcaaggc aactggtgaa gggcttagca tgttggcagc aatatcccag agattgaatc 1500tgtttgcatt ttcctcatct aggataacag ctgcttgaag ccagggctct tagccctttg 1560cattcccctt gagcgaggaa gccacactgc ctttctgtgt ctggttcaga gctcttcctt 1620cttggcatgt tttctggact acatgcacat gggcagctat agattaatct gcaaaaccta 1680gtcacttacc tacccataat atctgggaag gtgtggtatt tgttttaaag aaacattgtt 1740tctttgggag ggcagtttct gtctggactt tgaggtggac ttagttatcc ctacagttct 1800ttaactctca gctttttaat aaaagatgaa atcag 183528908DNAH. sapiensunsure754unknown 28gcctacagtt ctttaactct cagcttttaa taaaagatga aatcagatat gatgcagtgg 60gtcacaattc tttagaatgc ttctacccca gggccgcttc ctgttcctag tcatggtttt 120ccagtttagt agtggagttt cttgaggcta acttacagaa atttctaact gaaaacttta 180agagttattg atacttgttt tttcagtcag tcacttacat cacctagcct actctctgga 240atttaaattt atttctctag gctggtcctg gaagttgata acctttggca aagcttagat 300ttaggagaag gcttgagtcc ctgttcagcg ggtctgtgga ttctcttgct tatggctctc 360tgcctgcagc cctggcagac catactgtat gtcatggata cccagtggaa atattactga 420gatgaaacac atttccaagg gtatttaaac tctcactctg ccacctttct aagggtggga 480ggctggcaga gatgctgcaa tgcttgataa tcatttggcc acactgaaat ttccaaaggg 540agctcttgcc ggtgcttaaa accaaaactc ctggacactt agaaaattcc atgaatctag 600cacaaaatat ccattcttgc ccaagtgtat cccctttctc tccaggctta atcttttttt 660tttttttaaa gaccagggca gggtacttta actggaactg cgggggggag aaccttaggg 720agtcagaggc ggtgcggtag cactgtctac ctgngcccgt ttattgcgat gcgggcgggc 780ttcttattgg agggcatctc ccgggagaac cgtccggact aaggtgaaca ggacgcgctt 840ggttatttta acaacggtcg ggaagagttc ctagagctag cgtatctctg tgtggacact 900aattaacg 9082920DNAArtificial SequenceAntisense Oligonucleotide 29gccatgggag aattgcgacg 203020DNAArtificial SequenceAntisense Oligonucleotide 30tttggagtct tcaattaagg 203120DNAArtificial SequenceAntisense Oligonucleotide 31tctactttgg agtcttcaat 203220DNAArtificial SequenceAntisense Oligonucleotide 32gtctgtagtc atcttaaaaa 203320DNAArtificial SequenceAntisense Oligonucleotide 33acttctactt tggagtcttc 203420DNAArtificial SequenceAntisense Oligonucleotide 34tagaccgcag gagctgcgaa 203520DNAArtificial SequenceAntisense Oligonucleotide 35agtgattctc aaactgcaga 203620DNAArtificial SequenceAntisense Oligonucleotide 36tcttctgatc catggccacc 203720DNAArtificial SequenceAntisense Oligonucleotide 37tcagcactat ctgttgaaaa 203820DNAArtificial SequenceAntisense Oligonucleotide 38attcgagttc ctggaaaaca 203920DNAArtificial SequenceAntisense Oligonucleotide 39ttctcttacc aactgcatgt 204020DNAArtificial SequenceAntisense Oligonucleotide 40gcatcatccc ctagttagga 204120DNAArtificial SequenceAntisense Oligonucleotide 41cctcaggcgg acggaaaagc 204220DNAArtificial SequenceAntisense Oligonucleotide 42cgggcgtggt gcaatagtca 204320DNAArtificial SequenceAntisense Oligonucleotide 43attcgagttc ctcccggtgt 204420DNAArtificial SequenceAntisense Oligonucleotide 44gaaactttct gtcataaatg 204520DNAArtificial SequenceAntisense Oligonucleotide 45caacagaaac tttctgtcat 204620DNAArtificial SequenceAntisense Oligonucleotide 46gtgccagggc tagtgactcc 204720DNAArtificial SequenceAntisense Oligonucleotide 47attgttcaag ttgttcaaat 204820DNAArtificial SequenceAntisense Oligonucleotide 48tgcatgtttc ctgtcgtgat 204920DNAArtificial SequenceAntisense Oligonucleotide 49ccaactgcat gtttcctgtc 205020DNAArtificial SequenceAntisense Oligonucleotide 50gcatcatccc caactgcatg 205120DNAArtificial SequenceAntisense Oligonucleotide 51gtccatctcg aactgagcat 205220DNAArtificial SequenceAntisense Oligonucleotide 52gcaggagagt cagatgtcca 205320DNAArtificial SequenceAntisense Oligonucleotide 53aagtatcagt gttgctgctt 205420DNAArtificial SequenceAntisense Oligonucleotide 54tcaggtgcac acaagtatca 205520DNAArtificial SequenceAntisense Oligonucleotide 55atcatttggc acccagagga 205620DNAArtificial SequenceAntisense Oligonucleotide 56agctcatctt cccatcattt 205720DNAArtificial SequenceAntisense Oligonucleotide 57acagggagaa gaaatggtca 205820DNAArtificial SequenceAntisense Oligonucleotide 58taacctgttt aactgggaag 205920DNAArtificial SequenceAntisense Oligonucleotide 59cagaaataca gcaagggcct 206020DNAArtificial SequenceAntisense Oligonucleotide 60ctctaagggc

tgcttagctc 206120DNAArtificial SequenceAntisense Oligonucleotide 61agagttgaac tgttttcctc 206220DNAArtificial SequenceAntisense Oligonucleotide 62caaaattaca gggtatgagg 206320DNAArtificial SequenceAntisense Oligonucleotide 63aagaccccaa gcccagactc 206420DNAArtificial SequenceAntisense Oligonucleotide 64atttccccct gctggtttta 206520DNAArtificial SequenceAntisense Oligonucleotide 65aagggaaagc agctctcttt 206620DNAArtificial SequenceAntisense Oligonucleotide 66agagttgcac aagctgtgct 206720DNAArtificial SequenceAntisense Oligonucleotide 67agtggacctc aaaacagtgt 206820DNAArtificial SequenceAntisense Oligonucleotide 68tctgcacaaa tgcactaagt 206920DNAArtificial SequenceAntisense Oligonucleotide 69aaaactggtt accaagggct 207020DNAArtificial SequenceAntisense Oligonucleotide 70gaagagcaaa actggttacc 207120DNAArtificial SequenceAntisense Oligonucleotide 71ccagcaacga gatgcaagca 207220DNAArtificial SequenceAntisense Oligonucleotide 72agtacaagag gactctgcca 207320DNAArtificial SequenceAntisense Oligonucleotide 73tggtatggac ctgctctagg 207420DNAArtificial SequenceAntisense Oligonucleotide 74gtgcctctat tacttggtat 207520DNAArtificial SequenceAntisense Oligonucleotide 75ttcttaggca ttatctgaca 207620DNAArtificial SequenceAntisense Oligonucleotide 76agcggtcatt cttaggcatt 207720DNAArtificial SequenceAntisense Oligonucleotide 77acgactgaga ccgggtactc 207820DNAArtificial SequenceAntisense Oligonucleotide 78acaactacca caatgctcac 207920DNAArtificial SequenceAntisense Oligonucleotide 79attctgaaaa tcaacttcaa 208020DNAArtificial SequenceAntisense Oligonucleotide 80tcccagcagc caaacaaagc 208120DNAArtificial SequenceAntisense Oligonucleotide 81atttgaaaaa tggcctggta 208220DNAArtificial SequenceAntisense Oligonucleotide 82acacttcagg tatctttgat 208320DNAArtificial SequenceAntisense Oligonucleotide 83agataccaac acttcaggta 208420DNAArtificial SequenceAntisense Oligonucleotide 84acagatattc tcagatacca 208520DNAArtificial SequenceAntisense Oligonucleotide 85atgtttaatt aaaaagttgc 208620DNAArtificial SequenceAntisense Oligonucleotide 86acactggaag atgtttaatt 208720DNAArtificial SequenceAntisense Oligonucleotide 87cagttttaca aaccacccac 208820DNAArtificial SequenceAntisense Oligonucleotide 88aagaatgagg ctttcttgaa 208920DNAArtificial SequenceAntisense Oligonucleotide 89cagaaaagaa tgaggctttc 209020DNAArtificial SequenceAntisense Oligonucleotide 90tgaatgcaaa agcgaaaggg 209120DNAArtificial SequenceAntisense Oligonucleotide 91tcccgggatt attatgctgc 209220DNAArtificial SequenceAntisense Oligonucleotide 92gaaattccca ggacaccagt 209320DNAArtificial SequenceAntisense Oligonucleotide 93aaccagaaat tcccaggaca 209420DNAArtificial SequenceAntisense Oligonucleotide 94caaatccaac cagaaattcc 209520DNAArtificial SequenceAntisense Oligonucleotide 95ccaaatggcc tgttactctc 209620DNAArtificial SequenceAntisense Oligonucleotide 96aacaaacagg tttctttctt 209720DNAArtificial SequenceAntisense Oligonucleotide 97cttttcatag ttcaaaagaa 209820DNAArtificial SequenceAntisense Oligonucleotide 98cagacatcct tcctctcttt 209920DNAArtificial SequenceAntisense Oligonucleotide 99ttgtggcaga aaacagaaca 2010020DNAArtificial SequenceAntisense Oligonucleotide 100aactattcac atttttgtgg 2010120DNAArtificial SequenceAntisense Oligonucleotide 101tggagatcca gcttattcct 2010220DNAArtificial SequenceAntisense Oligonucleotide 102aagaatgaaa agcttcattc 2010320DNAArtificial SequenceAntisense Oligonucleotide 103tttaaatcca ttcctcaccg 2010420DNAArtificial SequenceAntisense Oligonucleotide 104ataactaata caggtggaag 2010520DNAArtificial SequenceAntisense Oligonucleotide 105ggtcatctga aatctctaaa 2010620DNAArtificial SequenceAntisense Oligonucleotide 106gcctcccacc cttagaaagg 20107598DNAM. musculus 107aaattctttc cttctggttc ttccaccggg ccattttcca catctgcatc agaagagatg 60cctcccatgt tagtatctga taatatcagt ctctccttat cagaggagag accttttatt 120tttaagatga ctacagacct atttttagat aagttttcag tacaattttg aactacaact 180tttttaacaa aacatcttcc agtattggga aggttatttt aaaaagaaaa aaaaacaatg 240tttgtaggca agtccactgc tgtcactgtc ctttgtctcc catagcccct tctgagctct 300cctgtgccct tgagctttgg ggctatttgg agtgtagaat gggtgttttg tgaaactgga 360ccagtctgcc ttgccatgag ctgttgaaga aaactccgtg tccctctcat ccgaaggtac 420acgatcacaa gctacgccac acatagaaga gcagttcaag agactatcag cgaaggaacg 480caacgcgcag ccacagaggc agcaagaaag gaagccgcac gaaaaaacac gagtgagaga 540gtgaagaata cgaagcacag gaaagtccat ggagaaaagg aacgagaaag acaaaagg 598108431DNAM. musculus 108cgcatcggga cgaccgggcg agagcaggcg agttgagagc cgagcgtgaa gagccgccgc 60cgccgccgct gctgcacaaa gcctcgagcc cgcgtcggag ccatgtccgc gtcggccggt 120ggtagccacc agcccagcca gagccgcgcc atccccacgc gcaccgtggc tatcagcgac 180gccgcgcagc tacctcagga ctactgcacc acgcccgggg ggacgctgtt ctccacaacg 240ccgggaggaa cacgaatcat ttatgaccga aagtttctgt tggaccgtcg caattctccc 300atggcgcaga ccccaccttg ccatctgccc aatatccctg gagtcaccag tcctggcgcc 360ttaattgaag actccaaaga gaagtgaaca acttaaacaa cctgaacaat catgacagga 420agcatgcagt t 43110920DNAArtificial SequenceAntisense Oligonucleotide 109tctcaactcg cctgctctcg 2011020DNAArtificial SequenceAntisense Oligonucleotide 110ggctcctcac gctcggctct 2011120DNAArtificial SequenceAntisense Oligonucleotide 111tcgaggcttt gtgcagcagc 2011220DNAArtificial SequenceAntisense Oligonucleotide 112gctggtggct accaccggcc 2011320DNAArtificial SequenceAntisense Oligonucleotide 113gctgggctgg tggctaccac 2011420DNAArtificial SequenceAntisense Oligonucleotide 114gtcgctgata gccacggtgc 2011520DNAArtificial SequenceAntisense Oligonucleotide 115agtcctgagg tagctgcgcg 2011620DNAArtificial SequenceAntisense Oligonucleotide 116gtggtgcagt agtcctgagg 2011720DNAArtificial SequenceAntisense Oligonucleotide 117cataaatgat tcgtgttcct 2011820DNAArtificial SequenceAntisense Oligonucleotide 118tcggtcataa atgattcgtg 2011920DNAArtificial SequenceAntisense Oligonucleotide 119aactttcggt cataaatgat 2012020DNAArtificial SequenceAntisense Oligonucleotide 120caacagaaac tttcggtcat 2012120DNAArtificial SequenceAntisense Oligonucleotide 121cggtccaaca gaaactttcg 2012220DNAArtificial SequenceAntisense Oligonucleotide 122gggagaattg cgacggtcca 2012320DNAArtificial SequenceAntisense Oligonucleotide 123tctgcgccat gggagaattg 2012420DNAArtificial SequenceAntisense Oligonucleotide 124caggactggt gactccaggg 2012520DNAArtificial SequenceAntisense Oligonucleotide 125ttcacttcta ctttggagtc 2012620DNAArtificial SequenceAntisense Oligonucleotide 126aaactgagcc tcatccccaa 2012720DNAArtificial SequenceAntisense Oligonucleotide 127atctcaaact gagcctcatc 2012820DNAArtificial SequenceAntisense Oligonucleotide 128gatgtccatc tcaaactgag 2012920DNAArtificial SequenceAntisense Oligonucleotide 129tggcagtagt cagatgtcca 2013020DNAArtificial SequenceAntisense Oligonucleotide 130ggctgctcca cgaggcctcc 2013120DNAArtificial SequenceAntisense Oligonucleotide 131tgggccagtc aggtgcacac 2013220DNAArtificial SequenceAntisense Oligonucleotide 132ctgtacactg tgttcctact 2013320DNAArtificial SequenceAntisense Oligonucleotide 133atgtgatcag acagtgcaca 2013420DNAArtificial SequenceAntisense Oligonucleotide 134cgggaagatg tgatcagaca 2013520DNAArtificial SequenceAntisense Oligonucleotide 135ttcttctgtg gactgtcagc 2013620DNAArtificial SequenceAntisense Oligonucleotide 136gtgctgcttg gagactgccc 2013720DNAArtificial SequenceAntisense Oligonucleotide 137tacaagcaga ggtgctgctt 2013820DNAArtificial SequenceAntisense Oligonucleotide 138ggcactaaac ctccttcacc 2013920DNAArtificial SequenceAntisense Oligonucleotide 139acacaatggg cactaaacct 2014020DNAArtificial SequenceAntisense Oligonucleotide 140gagcccagga acacaatggg 2014120DNAArtificial SequenceAntisense Oligonucleotide 141aatgtccccc acatccagcg 2014220DNAArtificial SequenceAntisense Oligonucleotide 142ctgaggacaa atgtccccca 2014320DNAArtificial SequenceAntisense Oligonucleotide 143caggactgtg ctccagagct 2014420DNAArtificial SequenceAntisense Oligonucleotide 144ggaggtacag gactgtgctc 2014520DNAArtificial SequenceAntisense Oligonucleotide 145gaggctgctg tcacatgtcc 2014620DNAArtificial SequenceAntisense Oligonucleotide 146aagccttcct cccagagaaa 2014720DNAArtificial SequenceAntisense Oligonucleotide 147tatcacaccc aagacaagac 2014820DNAArtificial SequenceAntisense Oligonucleotide 148gatgatgagc tatcacaccc 2014920DNAArtificial SequenceAntisense Oligonucleotide 149cccttcagga gggcttaaaa 2015020DNAArtificial SequenceAntisense Oligonucleotide 150cagacaggca aagaccagct 2015120DNAArtificial SequenceAntisense Oligonucleotide 151tgcctacggg atgcaggtag 2015220DNAArtificial SequenceAntisense Oligonucleotide 152cttctgctct aaaagcagac 2015320DNAArtificial SequenceAntisense Oligonucleotide 153caggccaagg tgttggcact 2015420DNAArtificial SequenceAntisense Oligonucleotide 154gctgagagca ggctggactc 2015520DNAArtificial SequenceAntisense Oligonucleotide 155tctcaggcag accgctgaga 2015620DNAArtificial SequenceAntisense Oligonucleotide 156gcccctgatg tattctcagg 2015720DNAArtificial SequenceAntisense Oligonucleotide 157tcagaggccc ctgatgtatt 2015820DNAArtificial SequenceAntisense Oligonucleotide 158gtcctcttca gaggcccctg 2015920DNAArtificial SequenceAntisense Oligonucleotide 159tgcacggcgg ctcagtcctc 2016020DNAArtificial SequenceAntisense Oligonucleotide 160ctggctgcac ggcggctcag 2016120DNAArtificial SequenceAntisense Oligonucleotide 161aaaaccatga cccccgaggc 2016220DNAArtificial SequenceAntisense Oligonucleotide 162tacacctggt tttaaaacca 2016320DNAArtificial SequenceAntisense Oligonucleotide 163acacccaacg taaggtacac 2016420DNAArtificial SequenceAntisense Oligonucleotide 164tgcaggacac ccaacgtaag 2016520DNAArtificial SequenceAntisense Oligonucleotide 165aaactcaagg tatagtaacc 2016620DNAArtificial SequenceAntisense Oligonucleotide 166aagtcgactt taaactcaag 2016720DNAArtificial SequenceAntisense Oligonucleotide 167taagaggaag tcgactttaa 2016820DNAArtificial SequenceAntisense Oligonucleotide 168ctgtgctgct ctctcagcag 2016920DNAArtificial SequenceAntisense Oligonucleotide 169cactgtctta gcctgtgctg 2017020DNAArtificial SequenceAntisense Oligonucleotide 170tggaaaatgg cccggtggaa 2017120DNAArtificial SequenceAntisense Oligonucleotide 171tactaacatg ggaggcatct 2017220DNAArtificial SequenceAntisense Oligonucleotide 172tgataaggag agactgatat 2017320DNAArtificial SequenceAntisense Oligonucleotide 173taaaaggtct ctcctctgat 2017420DNAArtificial SequenceAntisense Oligonucleotide 174taaaaataaa aggtctctcc 2017520DNAArtificial SequenceAntisense Oligonucleotide 175aacttatcta aaaataggtc 2017620DNAArtificial SequenceAntisense Oligonucleotide 176tgtactgaaa acttatctaa 2017720DNAArtificial SequenceAntisense Oligonucleotide 177atactggaag atgttttgtt 2017820DNAArtificial SequenceAntisense Oligonucleotide 178ataaccttcc caatactgga

2017920DNAArtificial SequenceAntisense Oligonucleotide 179acagctcatg gcaaggcaga 2018020DNAArtificial SequenceAntisense Oligonucleotide 180aactgctctt ctatgtgtgg 2018120DNAArtificial SequenceAntisense Oligonucleotide 181tcgctgatag tctcttgaac 2018220DNAArtificial SequenceAntisense Oligonucleotide 182ggctcttcac gctcggctct 2018320DNAArtificial SequenceAntisense Oligonucleotide 183ggctcgtggc tttgtgcagc 2018420DNAArtificial SequenceAntisense Oligonucleotide 184tggtgtccac caccggccga 2018520DNAArtificial SequenceAntisense Oligonucleotide 185tggctgggct ggtgtccacc 2018620DNAArtificial SequenceAntisense Oligonucleotide 186tctggctggg ctggtgtcca 2018720DNAArtificial SequenceAntisense Oligonucleotide 187gaatggcgcg gctctggctg 2018820DNAArtificial SequenceAntisense Oligonucleotide 188ctaatagcca cggtgcgtgt 2018920DNAArtificial SequenceAntisense Oligonucleotide 189gtcgctaata gccacggtgc 2019020DNAArtificial SequenceAntisense Oligonucleotide 190gctgcgtcgc taatagccac 2019120DNAArtificial SequenceAntisense Oligonucleotide 191tgaggtagct gcgctgcgtc 2019220DNAArtificial SequenceAntisense Oligonucleotide 192cctgaggtag ctgcgctgcg 2019320DNAArtificial SequenceAntisense Oligonucleotide 193tagtcctgag gtagctgcgc 2019420DNAArtificial SequenceAntisense Oligonucleotide 194agtagtcctg aggtagctgc 2019520DNAArtificial SequenceAntisense Oligonucleotide 195tgcagtagtc ctgaggtagc 2019620DNAArtificial SequenceAntisense Oligonucleotide 196ggtgcagtag tcctgaggta 2019720DNAArtificial SequenceAntisense Oligonucleotide 197cgtggtgcag tagtcctgag 2019820DNAArtificial SequenceAntisense Oligonucleotide 198ggcgtggtgc agtagtcctg 2019920DNAArtificial SequenceAntisense Oligonucleotide 199ggtgttgtgg agaacagcgt 2020020DNAArtificial SequenceAntisense Oligonucleotide 200ctcccggtgt tgtggagaac 2020120DNAArtificial SequenceAntisense Oligonucleotide 201gttcctcccg gtgttgtgga 2020220DNAArtificial SequenceAntisense Oligonucleotide 202aaactttcgg tcataaatga 2020320DNAArtificial SequenceAntisense Oligonucleotide 203agaaactttc ggtcataaat 2020420DNAArtificial SequenceAntisense Oligonucleotide 204acagaaactt tcggtcataa 2020520DNAArtificial SequenceAntisense Oligonucleotide 205aacagaaact ttcggtcata 2020620DNAArtificial SequenceAntisense Oligonucleotide 206tccaacagaa actttcggtc 2020720DNAArtificial SequenceAntisense Oligonucleotide 207ggtccaacag aaactttcgg 2020820DNAArtificial SequenceAntisense Oligonucleotide 208gcgacggtcc aacagaaact 2020920DNAArtificial SequenceAntisense Oligonucleotide 209ttgcgacggt ccaacagaaa 2021020DNAArtificial SequenceAntisense Oligonucleotide 210gaattgcgac ggtccaacag 2021120DNAArtificial SequenceAntisense Oligonucleotide 211gagaattgcg acggtccaac 2021220DNAArtificial SequenceAntisense Oligonucleotide 212tgggagaatt gcgacggtcc 2021320DNAArtificial SequenceAntisense Oligonucleotide 213cgccatggga gaattgcgac 2021420DNAArtificial SequenceAntisense Oligonucleotide 214tgcgccatgg gagaattgcg 2021520DNAArtificial SequenceAntisense Oligonucleotide 215gtctgcgcca tgggagaatt 2021620DNAArtificial SequenceAntisense Oligonucleotide 216attgggcaga tggcaaggtg 2021720DNAArtificial SequenceAntisense Oligonucleotide 217ggtgactcca gggatattgg 2021820DNAArtificial SequenceAntisense Oligonucleotide 218ggactggtga ctccagggat 2021920DNAArtificial SequenceAntisense Oligonucleotide 219cgccaggact ggtgactcca 2022020DNAArtificial SequenceAntisense Oligonucleotide 220ggagtcttcc attaaggcgc 2022120DNAArtificial SequenceAntisense Oligonucleotide 221ttggagtctt ccattaaggc 2022220DNAArtificial SequenceAntisense Oligonucleotide 222tactttggag tcttccatta 2022320DNAArtificial SequenceAntisense Oligonucleotide 223cacttctact ttggagtctt 2022420DNAArtificial SequenceAntisense Oligonucleotide 224tgttcacttc tactttggag 2022520DNAArtificial SequenceAntisense Oligonucleotide 225caagttgttc acttctactt 2022620DNAArtificial SequenceAntisense Oligonucleotide 226gttcaagttg ttcacttcta 2022720DNAArtificial SequenceAntisense Oligonucleotide 227tcaggttgtt caagttgttc 2022820DNAArtificial SequenceAntisense Oligonucleotide 228tgttcaggtt gttcaagttg 2022920DNAArtificial SequenceAntisense Oligonucleotide 229gattgttcag gttgttcaag 2023020DNAArtificial SequenceAntisense Oligonucleotide 230cgtgattgtt caggttgttc 2023120DNAArtificial SequenceAntisense Oligonucleotide 231tgtcgtgatt gttcaggttg 2023220DNAArtificial SequenceAntisense Oligonucleotide 232ttcctgtcgt gattgttcag 2023320DNAArtificial SequenceAntisense Oligonucleotide 233gcttcctgtc gtgattgttc 2023420DNAArtificial SequenceAntisense Oligonucleotide 234gtgcttcctg tcgtgattgt 2023520DNAArtificial SequenceAntisense Oligonucleotide 235actgcgtgct tcctgtcgtg 2023620DNAArtificial SequenceAntisense Oligonucleotide 236caactgcgtg cttcctgtcg 2023720DNAArtificial SequenceAntisense Oligonucleotide 237ccccaactgc gtgcttcctg 2023820DNAArtificial SequenceAntisense Oligonucleotide 238atccccaact gcgtgcttcc 2023920DNAArtificial SequenceAntisense Oligonucleotide 239ctcatcccca actgcgtgct 2024020DNAArtificial SequenceAntisense Oligonucleotide 240gcctcatccc caactgcgtg 2024120DNAArtificial SequenceAntisense Oligonucleotide 241gagcctcatc cccaactgcg 2024220DNAArtificial SequenceAntisense Oligonucleotide 242ctgagcctca tccccaactg 2024320DNAArtificial SequenceAntisense Oligonucleotide 243tcaaactgag cctcatcccc 2024420DNAArtificial SequenceAntisense Oligonucleotide 244cagcagggtc agatgtccat 2024520DNAArtificial SequenceAntisense Oligonucleotide 245ccttcgacac tgcagcaggg 2024620DNAArtificial SequenceAntisense Oligonucleotide 246gccgccttcg acactgcagc 2024720DNAArtificial SequenceAntisense Oligonucleotide 247gtgcacacgg gccgtgtcag 2024820DNAArtificial SequenceAntisense Oligonucleotide 248ccagtcaggt gcacacgggc 2024920DNAArtificial SequenceAntisense Oligonucleotide 249ggtccagtca ggtgcacacg 2025020DNAArtificial SequenceAntisense Oligonucleotide 250tactggtcca gtcaggtgca 2025120DNAArtificial SequenceAntisense Oligonucleotide 251tcctactggt ccagtcaggt 2025220DNAArtificial SequenceAntisense Oligonucleotide 252gtgttcctac tggtccagtc 2025320DNAArtificial SequenceAntisense Oligonucleotide 253cacggtgttc ctactggtcc 2025420DNAArtificial SequenceAntisense Oligonucleotide 254tgtacacggt gttcctactg 2025520DNAArtificial SequenceAntisense Oligonucleotide 255tctctgtaca cggtgttcct 2025620DNAArtificial SequenceAntisense Oligonucleotide 256cttctctgta cacggtgttc 2025720DNAArtificial SequenceAntisense Oligonucleotide 257tggagcttct ctgtacacgg 2025820DNAArtificial SequenceAntisense Oligonucleotide 258actggagctt ctctgtacac 2025920DNAArtificial SequenceAntisense Oligonucleotide 259ctgctagcct ctggatttga 2026019RNAArtificial SequenceSynthetic Oligonucleotide 260cgagaggcgg acgggaccg 1926121DNAArtificial SequenceSynthetic Oligonucleotide 261cgagaggcgg acgggaccgt t 2126221DNAArtificial SequenceSynthetic Oligonucleotide 262cggtcccgtc cgcctctcgt t 2126319DNAArtificial SequenceSynthetic Oligonucleotide 263cggtcccgtc cgcctctcg 19

Claims

1-40. (canceled)

41. A modified antisense compound 13 to 80 nucleobases in length targeted to a nucleic acid molecule encoding eIF4E-BP2 (SEQ ID NO:4), wherein said compound has at least an 8-nucleobase portion complementary within nucleotides 544-940, 420-439, 493-512, 1962-1981, 501-520, 8892-8911, 11559-11578, 11918-11937, 17941-17960, 146-165, 332-351, 372-391, 474-493, 1868-1887, 1900-1919, 2218-2237, 2223-2242, 2377-2396, 2382-2401, 2449-2468, 2471-2490, 2536-2555, 2578-2597, 2632-2651, 2088-2107, or 697-716 of SEQ ID NO:4 encoding eIF4E-BP2, and wherein said compound inhibits the expression of eIF4E-BP2 mRNA.

42. The antisense compound of claim 41 which is 13 to 50 nucleobases in length.

43. The antisense compound of claim 41 which is 15 to 30 nucleobases in length.

44. The antisense compound of claim 41 comprising an oligonucleotide.

45. The antisense compound of claim 41 comprising a DNA oligonucleotide.

46. The antisense compound of claim 41 comprising an RNA oligonucleotide.

47. The antisense compound of claim 41 which is a double-stranded oligonucleotide.

48. The antisense compound of claim 41 which is a single-stranded oligonucleotide.

49. The antisense compound of claim 41 comprising a chimeric oligonucleotide.

50. The antisense compound of claim 41 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.

51. The antisense compound of claim 41 having at least one modified internucleoside linkage, sugar moiety, or nucleobase.

52. The antisense compound of claim 41 having at least one 2'-O-methoxyethyl sugar moiety.

53. The antisense compound of claim 41 having at least one phosphorothioate internucleoside linkage.

54. The antisense compound of claim 41, wherein said antisense compound has at least an 8-nucleobase portion complementary within nucleotides 420-439 of SEQ ID NO:4 encoding eIF4E-BP2.

55. The antisense compound of claim 41, wherein said antisense compound has at least an 8-nucleobase portion complementary within nucleotides 501-520 of SEQ ID NO:4 encoding eIF4E-BP2.

56. The antisense compound of claim 41, wherein said antisense compound has at least an 8-nucleobase portion complementary within nucleotides 332-351 of SEQ ID NO:4 encoding eIF4E-BP2.

57. The antisense compound of claim 41, wherein said antisense compound has at least an 8-nucleobase portion complementary within nucleotides 829-848 of SEQ ID NO:4 encoding eIF4E-BP2.

58. The antisense compound of claim 41, wherein said antisense compound has at least an 8-nucleobase portion complementary within nucleotides 851-870 of SEQ ID NO:4 encoding eIF4E-BP2.

59. The antisense compound of claim 41, wherein said antisense compound has at least an 8-nucleobase portion complementary within nucleotides 868-887 of SEQ ID NO:4 encoding eIF4E-BP2.

60. The antisense compound of claim 41, wherein said antisense compound has at least an 8-nucleobase portion complementary within nucleotides 921-940 of SEQ ID NO:4 encoding eIF4E-BP2.

61. The antisense compound of claim 41, wherein said antisense compound has at least an 8-nucleobase portion complementary within nucleotides 2377-2396 of SEQ ID NO:4 encoding eIF4E-BP2.

62. The antisense compound of claim 41, wherein said antisense compound has at least an 8-nucleobase portion complementary within nucleotides 2578-2597 of SEQ ID NO:4 encoding eIF4E-BP2.