MODULATION OF STAT 6 EXPRESSION

Abstract

nd methods are provided for modulating the expression of STAT 6. The compositions comprise oligonucleotides, targeted to nucleic acid encoding STAT 6. Methods of using these compounds for modulation of STAT 6 expression and for diagnosis and treatment of disease associated with expression of STAT 6 are provided.

Description

RELATED APPLICATIONS

[0001]This application is a continuation of U.S. application Ser. No. 11/152,530, filed Jun. 14, 2005; which is a divisional of U.S. application Ser. No. 10/317,391, filed Dec. 11, 2002, now abandoned, each of which is incorporated herein by reference in its 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 PTS0010USC1SEQ.txt, created on Jul. 27, 2009 which is 80 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 STAT 6. In particular, this invention relates to compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding STAT 6. Such compounds are shown herein to modulate the expression of STAT 6.

BACKGROUND OF THE INVENTION

[0004]Cytokines function as protein mediators that play a critical role in host defense and serve as a communication link between cells of native and acquired immunity. Cells respond to cytokines with specific biological changes that are dependent on the activation of new gene expression.

[0005]Studies of the mechanism by which signals are signals are mediated from the receptor to the nucleus by the interferon cytokines have revealed the activation of latent cytoplasmic transcription factors that subsequently translocate to the nucleus (Bromberg, BioEssays, 2001, 23, 161-169).

[0006]The proteins of the STAT family (signal transducers and activators of transcription) are latent transcription factors that are abundantly expressed in many cell types. STATs are activated by phosphorylation on a single tyrosine in response to extracellular ligands. An active STAT dimer is formed through reciprocal interactions between the SH2 domain of one monomer and the phosphorylated tyrosine of the other monomer. The dimers accumulate in the nucleus, recognize specific DNA elements in the promoters of genes and activate transcription so that growth control and survival of normal cells in a developing or adult mammal are carefully balanced. Many of the signals that influence this balance are delivered by circulating polypeptides, whose binding to cell surface receptors governs gene-specific transcription. It has been shown that human cancer cells have lost control of these signaling mechanisms. In addition to persistent unregulated mitogenic signaling, the lack of suppressive signals is also critical in the development of cancers (Bromberg, BioEssays, 2001, 23, 161-169).

[0007]STAT 6 (also known as interleukin 4-STAT) was cloned and mapped to chromosome 12q13 (Leek et al., Cytogenet. Cell Genet., 1997, 79, 208-209; Quelle et al., Mol. Cell. Biol., 1995, 15, 3336-3343). Nucleic acid sequences encoding STAT 6 are disclosed and claimed in U.S. Pat. No. 5,710,266 (McKnight and Hou, 1998).

[0008]STAT 6 is primarily expressed as a 4 kb transcript in hematopoietic cells and expressed variably in other tissues (Quelle et al., Mol. Cell. Biol., 1995, 15, 3336-3343). A unique truncated isoform of STAT 6 is expressed in mast cells (Sherman, Immunol. Rev., 2001, 179, 48-56). Disclosed and claimed in PCT publication WO 99/10493 are nucleic acid sequences encoding variants of STAT 6 known as STAT 6b and STAT 6c as well as vectors comprising said nucleic acid sequences (Patel et al., 1999).

[0009]STAT 6 is an integral transcription factor involved in interleukin 4 and interleukin 13 signaling. Following activation of their respective receptors, interleukin 4 and interleukin 13 cause their common interleukin 4 receptor alpha chain to become phosphorylated by JAK3 and to subsequently bind to STAT 6. STAT 6 is then phosphorylated by JAK1, homodimerizes and translocates to the nucleus where it binds interleukin 4 response elements and initiates the transcription of a number of genes including IgE (Danahay et al., Inflamm. Res., 2000, 49, 692-699).

[0010]STAT 6 is involved in key pathological mechanisms in rheumatoid arthritis which operate in early and late stages of the disease (Muller-Ladner et al., J. Immunol., 2000, 164, 3894-3901).

[0011]Ghilardi et al. have found that STAT 6 interacts with an isoform of the leptin receptor (OB--R) and is thus, a potential mediator of the anti-obesity effects of leptin (Ghilardi et al., Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 6231-6235).

[0012]STAT 6 knockout mice are viable and develop normally with the exception that interleukin 4 functions are eliminated (Ihle, Curr. Opin. Cell Biol., 2001, 13, 211-217). Additionally, STAT 6 knockout mice fail to develop antigen-induced airway hyper-reactivity in a model of airway inflammation (Kuperman et al., J. Exp. Med., 1998, 187, 939-948).

[0013]Inhibition of STAT 6 is expected to attenuate the allergic response and thus, represents an attractive target for drug discovery strategies (Hill et al., Am. J. Respir. Cell Mol. Biol., 1999, 21, 728-737).

[0014]Small molecule inhibitors of STAT 6 are disclosed and claimed in PCT publication WO 00/27802 and Japanese Patent JP 2000229959 (Eyermann et al., 2000; Inoue et al., PCT, 2000, Abstract only). Disclosed and claimed in U.S. Pat. No. 6,207,391 are methods for screening modulators of STAT 6 binding to a STAT 6 receptor (Wu and McKinney, 2001).

[0015]Wang et al. have demonstrated targeted disruption of STAT 6 DNA-binding activity by a phosphorothioate cis-element decoy oligonucleotide (Wang et al., Blood, 2000, 95, 1249-1257).

[0016]Hill et al. have used a series of homologous human and murine antisense oligonucleotides targeting STAT 6 to interrupt interleukin 4 and interleukin 13 signaling and attenuate germline C-epsilon transcription in vitro (Hill et al., Am. J. Respir. Cell Mol. Biol., 1999, 21, 728-737). Subsequently, the in vitro and in vivo pharmacology of three of the antisense oligonucleotides used in the latter study was investigated. Although the oligonucleotides downregulated STAT 6 mRNA, their action was not sufficient to influence alterations in mRNA levels (Danahay et al., Inflamm. Res., 2000, 49, 692-699).

[0017]Currently, there are no known therapeutic agents that effectively inhibit the synthesis of STAT 6. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting STAT 6 function.

[0018]Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of STAT 6 expression.

[0019]The present invention provides compositions and methods for modulating STAT 6 expression.

SUMMARY OF THE INVENTION

[0020]The present invention is directed to compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding STAT 6, and which modulate the expression of STAT 6. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of STAT 6 and methods of modulating the expression of STAT 6 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 STAT 6 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

[0021]The present invention employs compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding STAT 6. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding STAT 6. As used herein, the terms "target nucleic acid" and "nucleic acid molecule encoding STAT 6" have been used for convenience to encompass DNA encoding STAT 6, 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.

[0022]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 STAT 6. 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.

[0023]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.

[0024]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.

[0025]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.

[0026]"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.

[0027]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% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise 90% sequence complementarity and even more preferably comprise 95% 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).

B. Compounds of the Invention

[0028]According to the present invention, compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, 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.

[0029]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.

[0030]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.

[0031]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).

[0032]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).

[0033]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.

[0034]While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.

[0035]The 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.

[0036]In one preferred embodiment, the compounds of the invention are 12 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases in length.

[0037]In another preferred embodiment, the 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.

[0038]Particularly preferred compounds are oligonucleotides from about 12 to about 50 nucleobases, even more preferably those comprising from about 15 to about 30 nucleobases.

[0039]Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0040]Exemplary preferred antisense compounds include oligonucleotide sequences that comprise at least the 8 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 8 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). 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

[0041]"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 STAT 6.

[0042]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.

[0043]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 STAT 6, 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).

[0044]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.

[0045]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.

[0046]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.

[0047]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.

[0048]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.

[0049]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.

[0050]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.

[0051]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.

[0052]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.

[0053]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.

[0054]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). 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.

[0055]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.

D. Screening and Target Validation

[0056]In a further embodiment, the "preferred target segments" identified herein may be employed in a screen for additional compounds that modulate the expression of STAT 6. "Modulators" are those compounds that decrease or increase the expression of a nucleic acid molecule encoding STAT 6 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 STAT 6 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 STAT 6. 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 STAT 6, the modulator may then be employed in further investigative studies of the function of STAT 6, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

[0057]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.

[0058]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).

[0059]The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between STAT 6 and a disease state, phenotype, or condition. These methods include detecting or modulating STAT 6 comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of STAT 6 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

[0060]The 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.

[0061]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.

[0062]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.

[0063]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).

[0064]The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding STAT 6. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective STAT 6 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 STAT 6 and in the amplification of said nucleic acid molecules for detection or for use in further studies of STAT 6. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding STAT 6 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 STAT 6 in a sample may also be prepared.

[0065]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.

[0066]For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of STAT 6 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 STAT 6 inhibitor. The STAT 6 inhibitors of the present invention effectively inhibit the activity of the STAT 6 protein or inhibit the expression of the STAT 6 protein. In one embodiment, the activity or expression of STAT 6 in an animal is inhibited by about 10%. Preferably, the activity or expression of STAT 6 in an animal is inhibited by about 30%. More preferably, the activity or expression of STAT 6 in an animal is inhibited by 50% or more.

[0067]For example, the reduction of the expression of STAT 6 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 STAT 6 protein and/or the STAT 6 protein itself.

[0068]The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.

F. Modifications

[0069]As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to 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)

[0070]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.

[0071]Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, 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 a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0072]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.

[0073]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 CH₂ component parts.

[0074]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

[0075]In other preferred 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.

[0076]Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH₂--NH--O--CH₂--, --CH₂--N(CH₃)--O--CH₂-- [known as a methylene (methylimino) or MMI backbone], --CH₂--O--N(CH₃)--CH₂--, --CH₂--N(CH₃)--N(CH₃)--CH₂-- and --O--N(CH₃)--CH₂--CH₂-- [wherein the native phosphodiester backbone is represented as --O--P--O--CH₂--] of the above referenced 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

[0077]Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise 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 C₁ to C₁0 alkyl or C₂ to C₁0 alkenyl and alkynyl. Particularly preferred are O[(CH₂)_nO]_mCH₃, O(CH₂)_nOCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_nON[(CH₂)_nCH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C₁ to C₁0 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy (2'-O--CH₂CH₂OCH₃, 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(CH₂)₂ON(CH₃)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--CH₂--O--CH₂--N(CH₃)₂, also described in examples hereinbelow.

[0078]Other preferred modifications include 2'-methoxy (2'-O--CH₃), 2'-aminopropoxy (2'-OCH₂CH₂CH₂NH₂), 2'-allyl (2'-CH₂--CH═CH₂), 2'-O-allyl (2'-O--CH₂--CH═CH₂) 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. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures 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.

[0079]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 (--CH₂--)_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

[0080]Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases 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≡C--CH₃) 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-deazaadenine, 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° C. and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0081]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

[0082]Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. 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. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, 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.

[0083]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

[0084]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.

[0085]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. 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.

[0086]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

[0087]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.

[0088]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. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. The term "prodrug" indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0089]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.

[0090]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.

[0091]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.

[0092]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.

[0093]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.

[0094]Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm 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.

[0095]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.

[0096]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.

[0097]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.

[0098]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.

[0099]One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0100]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).

[0101]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.

[0102]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.

[0103]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.

[0104]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 chemo-therapeutic 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.

[0105]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

[0106]The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 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.

[0107]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.

EXAMPLES

Example 1

Synthesis of Nucleoside Phosphoramidites

[0108]The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5'-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5'-O-Dimethoxytrityl-2'-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5'-O-Dimethoxytrityl-2'-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-deoxy-N⁴-benzoyl-5-methylcy- tidin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2'-Fluorodeoxyadenosine, 2'-Fluorodeoxyguanosine, 2'-Fluorouridine, 2'-Fluorodeoxycytidine, 2'-O-(2-Methoxyethyl) modified amidites, 2'-O-(2-methoxyethyl)-5-methyluridine intermediate, 5'-O-DMT-2'-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-5-methyluridi- n-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5'-O-Dimethoxytrityl-2'-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5'-O-dimethoxytrityl-2'-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytid- ine penultimate intermediate, [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N⁴-benzo- yl-5-methylcytidin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N⁶-benzo- yladenosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.su- p.4-isobutyrylguanosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidit- e (MOE G amidite), 2'-O-(Aminooxyethyl) nucleoside amidites and 2'-O-(dimethylaminooxyethyl) nucleoside amidites, 2'-(Dimethylaminooxyethoxy) nucleoside amidites, 5'-O-tert-Butyldiphenylsilyl-O²-2'-anhydro-5-methyluridine, 5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine, 2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine, 5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluri- dine, 5'-O-tert-Butyldiphenylsilyl-2'-O--[N,N-dimethylaminooxyethyl]-5-met- hyluridine, 2'-O-(dimethylaminooxyethyl)-5-methyluridine, 5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine, 5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoe- thyl)-N,N-diisopropylphosphoramidite], 2'-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(- 4,4'-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphora- midite], 2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside amidites, 2'-O--[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine, 5'-O-dimethoxytrityl-2'-O--[2(2-N,N-dimethyl-aminoethoxy)-ethyl)]-5-methy- l uridine and 5'-O-Dimethoxytrityl-2'-O--[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2

Oligonucleotide and Oligonucleoside Synthesis

[0109]The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0110]Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0111]Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0112]3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporated by reference.

[0113]Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0114]Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0115]3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0116]Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0117]Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

[0118]Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0119]Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0120]Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3

RNA Synthesis

[0121]In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5'-hydroxyl in combination with an acid-labile orthoester protecting group on the 2'-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2' hydroxyl.

[0122]Following this procedure for the sequential protection of the 5'-hydroxyl in combination with protection of the 2'-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.

[0123]RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3'- to 5'-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3'-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5'-end of the first nucleoside. The support is washed and any unreacted 5'-hydroxyl groups are capped with acetic anhydride to yield 5'-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5'-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.

[0124]Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2'-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.

[0125]The 2'-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.

[0126]Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand. 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedron Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).

[0127]RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 μl of each of the complementary strands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and 15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid.

Example 4

Synthesis of Chimeric Oligonucleotides

[0128]Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the "gap" segment of linked nucleosides is positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open end" type wherein the "gap" segment is located at either the 3' or the 5' terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as "gapmers" or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as "hemimers" or "wingmers".

[2'-O-Me]-[2'-deoxy]-[2'-O-Me] Chimeric Phosphorothioate Oligonucleotides

[0129]Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and 2'-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for 5' and 3' wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[2'-O-(2-Methoxyethyl)]-[2'-deoxy]-[2'-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0130][2'-O-(2-methoxyethyl)]-[2'-deoxy]-[2'-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2'-O-methyl chimeric oligonucleotide, with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites.

[2'-O-(2-Methoxyethyl)Phosphodiester]-[2'-deoxy Phosphorothioate]-[2'-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides

[0131][2'-O-(2-methoxyethyl phosphodiester]-[2'-deoxy phosphorothioate]-[2'-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2'-O-methyl chimeric oligonucleotide with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0132]Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5

Design and Screening of Duplexed Antisense Compounds Targeting STAT 6

[0133]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 STAT 6. The nucleobase sequence of the antisense strand of the duplex comprises at least a 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.

[0134]For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:

##STR00001##

[0135]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 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× 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° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° 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° C.) and freeze-thawed up to 5 times.

[0136]Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate STAT 6 expression.

[0137]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 μL OPTI-MEM-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at a final concentration of 200 nM. 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 RT-PCR.

Example 6

Oligonucleotide Isolation

[0138]After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc 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 7

Oligonucleotide Synthesis

96 Well Plate Format

[0139]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.

[0140]Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° 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 8

Oligonucleotide Analysis

96-Well Plate Format

[0141]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® MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE® 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 9

Cell Culture and Oligonucleotide Treatment

[0142]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 RT-PCR.

T-24 Cells:

[0143]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 Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0144]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:

[0145]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 Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

NHDF Cells:

[0146]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:

[0147]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.

Treatment with Antisense Compounds:

[0148]When cells reached 65-75% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM®-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM®-1 containing 3.75 μg/mL LIPOFECTIN® (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. Cells are treated and data are obtained in triplicate. After 4-7 hours of treatment at 37° C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0149]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 10

Analysis of Oligonucleotide Inhibition of STAT 6 Expression

[0150]Antisense modulation of STAT 6 expression can be assayed in a variety of ways known in the art. For example, STAT 6 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR(RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. 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® 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0151]Protein levels of STAT 6 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 STAT 6 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 11

Design of Phenotypic Assays and In Vivo Studies for the Use of STAT 6 Inhibitors

Phenotypic Assays

[0152]Once STAT 6 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 STAT 6 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.).

[0153]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 STAT 6 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.

[0154]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.

[0155]Analysis of the geneotype of the cell (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 STAT 6 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.

In Vivo studies

[0156]The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.

[0157]The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study. To account for the psychological effects of receiving treatments, volunteers are randomly given placebo or STAT 6 inhibitor. Furthermore, to prevent the doctors from being biased in treatments, they are not informed as to whether the medication they are administering is a STAT 6 inhibitor or a placebo. Using this randomization approach, each volunteer has the same chance of being given either the new treatment or the placebo.

[0158]Volunteers receive either the STAT 6 inhibitor or placebo for eight week period with biological parameters associated with the indicated disease state or condition being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements include the levels of nucleic acid molecules encoding STAT 6 or STAT 6 protein levels in body fluids, tissues or organs compared to pre-treatment levels. Other measurements include, but are not limited to, indices of the disease state or condition being treated, body weight, blood pressure, serum titers of pharmacologic indicators of disease or toxicity as well as ADME (absorption, distribution, metabolism and excretion) measurements.

[0159]Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for the indicated disease or condition.

[0160]Volunteers taking part in this study are healthy adults (age 18 to 65 years) and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and STAT 6 inhibitor treatment. In general, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with the STAT 6 inhibitor show positive trends in their disease state or condition index at the conclusion of the study.

Example 12

RNA Isolation

[0161]Poly(A)+ mRNA Isolation

[0162]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 μL cold PBS. 60 μL 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 μL 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 μL 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 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0163]Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Total RNA Isolation

[0164]Total RNA was isolated using an RNEASY 96® 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 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL 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® well plate attached to a QIAVAC® manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96® plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96® plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96® 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® manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC® manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0165]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 13

Real-Time Quantitative PCR Analysis of STAT 6 mRNA Levels

[0166]Quantitation of STAT 6 mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM® 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® 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.

[0167]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.

[0168]PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM 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® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0169]Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen® (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen® are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0170]In this assay, 170 μL of RiboGreen® working reagent (RiboGreen® reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

[0171]Probes and primers to human STAT 6 were designed to hybridize to a human STAT 6 sequence, using published sequence information (GenBank accession number NM_--003153.1, incorporated herein as SEQ ID NO:4). For human STAT 6 the PCR primers were:

forward primer: CCAAACGCTGTCTCCGGA (SEQ ID NO: 5)reverse primer: GCTAGTAACGTACTGTTTGCTGATGAA (SEQ ID NO: 6) and the PCR probe was: FAM-CTACTGGTCTGACCGGCTGATCATTGG-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.

Example 14

Northern Blot Analysis of STAT 6 mRNA Levels

[0172]Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL® (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®-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® UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB® hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0173]To detect human STAT 6, a human STAT 6 specific probe was prepared by PCR using the forward primer CCAAACGCTGTCTCCGGA (SEQ ID NO: 5) and the reverse primer GCTAGTAACGTACTGTTTGCTGATGAA (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.).

[0174]Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER® and IMAGEQUANT® Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

Antisense Inhibition of Human STAT 6 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2'-MOE Wings and a Deoxy Gap

[0175]In accordance with the present invention, a series of antisense compounds were designed to target different regions of the human STAT 6 RNA, using published sequences (GenBank accession number NM_--003153.1, incorporated herein as SEQ ID NO: 4; GenBank accession number BC005823.1, incorporated herein as SEQ ID NO: 11; a genomic sequence of human STAT 6 represented by the complement of residues 157501-174000 of GenBank accession number AC018673.4, incorporated herein as SEQ ID NO: 12; GenBank accession number BE972840.1, the complement of which is incorporated herein as SEQ ID NO: 13; and GenBank accession number BF902909.1, the complement of which is incorporated herein as SEQ ID NO: 14). 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'-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human STAT 6 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments in which T-24 cells were treated with the oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, "N.D." indicates "no data".

TABLE-US-00001 TABLE 1 Inhibition of human STAT 6 mRNA levels by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap TAR- GET CON- SEQ TAR- TROL ID GET % SEQ SEQ ISIS # REGION NO SITE SEQUENCE INHIB ID NO ID NO 153765 Coding 4 1951 agtgagcgaatggacaggtc 96 15 2 153766 Coding 4 2483 cgctgtcactggctggctca 83 16 2 153767 Coding 4 1171 ttgatgatttctccagtgct 92 17 2 153768 Coding 4 940 aggacttcatccagccggcc 50 18 2 153769 Coding 4 1054 cccaggaacctcaagcccaa 68 19 2 153770 Coding 4 242 gtcacccagaagatgccgca 53 20 2 153771 Coding 4 2100 tttccacggtcatcttgatg 59 21 2 153772 Coding 4 379 aagatggtgctcccctcccc 34 22 2 153773 Coding 4 765 gccgtttccaaatctggatc 76 23 2 153774 Coding 4 729 ctttggctgcctctagctct 89 24 2 153775 Coding 4 1725 gtttggtgaggtccaggaca 89 25 2 153776 Coding 4 2321 catctgcaggtgaggctcct 85 26 2 153777 Coding 4 2676 tggcccttaggtccatgtgg 76 27 2 153778 Coding 4 1905 ctatctgtggagagccatcc 72 28 2 153779 Coding 4 1805 attgagaagaaggctagtaa 83 29 2 153780 Coding 4 392 gctgatgtgttgcaagatgg 78 30 2 153781 3'UTR 4 3019 gccccatcaccctcagagag 80 31 2 153782 Coding 4 417 ccctctgatatatgctctca 73 32 2 153783 Coding 4 1797 gaaggctagtaacgtactgt 84 33 2 153784 Coding 4 1819 gttccgtcgggctcattgag 92 34 2 153785 Coding 4 2479 gtcactggctggctcaggca 87 35 2 153786 Coding 4 1513 ttcagagtttcacacatctt 83 36 2 153787 5'UTR 4 79 caggccccataggtctgtag 88 37 2 153788 Coding 4 644 tatcaagctgtgcagagaca 80 38 2 153789 Coding 4 272 caggaactcccagggctggc 74 39 2 153790 3'UTR 4 3000 gctctgtatgtgtgtgtgcg 90 40 2 153791 Coding 4 1972 agatcccggattcggtcccc 88 41 2 153792 Coding 4 794 cggtgcgccattccctgcca 94 42 2 153793 Coding 4 1997 gggatagagatttttgagct 53 43 2 153794 Start 4 146 gatctgggacttggaggttg 71 44 2 Codon 153795 Coding 4 2165 tccaaggtcataagaaggca 88 45 2 153796 Coding 4 1762 atgatcagccggtcagacca 84 46 2 153797 Coding 4 1205 cccaggaatgctgttctcca 88 47 2 153798 Coding 4 944 tctcaggacttcatccagcc 6 48 2 153799 Coding 4 1671 ccagcaggatctccttgttg 82 49 2 153800 Coding 4 1160 tccagtgctttctgctccag 87 50 2 153801 Coding 4 328 acagtgtctgaaagtagggc 50 51 2 195427 5'UTR 4 39 gctggccctgctagcacctc 68 52 2 195428 Start 4 158 ccacagagacatgatctggg 79 53 2 Codon 195429 Coding 4 541 gtcttaaacttgagttcttc 53 54 2 195430 Coding 4 718 tctagctctccagtggtctc 78 55 2 195431 Coding 4 1085 ggccctgaccagcggaggct 72 56 2 195432 Coding 4 1290 cctctgtgacagactcagtg 72 57 2 195433 Coding 4 1615 tccatactgaggctgttgtc 20 58 2 195434 Coding 4 1887 cctggccccggatgacatgg 53 59 2 195435 Coding 4 2152 gaaggcaccatggtaggcat 55 60 2 195436 Coding 4 2506 ccaatccaagtgccctgagg 70 61 2 195437 Stop 4 2699 cagctgggatcaccaactgg 49 62 2 Codon 195438 3'UTR 4 2944 gtgtctcagagcctgaactt 77 63 2 195439 5'UTR 11 23 taagcagtggctgccccagc 51 64 2 195440 5'UTR 11 38 cctccctcttcagtgtaagc 65 65 2 195441 3'UTR 11 3185 agaagccttccatgccctaa 83 66 2 195442 3'UTR 11 3230 tatgttcctgcctatccgtc 76 67 2 195443 3'UTR 11 3531 caactaaggtgccagctata 86 68 2 195444 3'UTR 11 3539 tggtcatgcaactaaggtgc 84 69 2 195445 3'UTR 11 3585 atttgtgttgtcacgtaggc 84 70 2 195446 3'UTR 11 3599 tctcaccctcccaaatttgt 48 71 2 195447 3'UTR 11 3629 agcacacttgctgctgtctt 74 72 2 195448 3'UTR 11 3779 gccaggcctggacccagact 60 73 2 195449 3'UTR 11 3835 gggcaacagaaaagatgcag 50 74 2 195450 Intron 12 2812 aatgtcagcttttaatctgt 67 75 2 195451 Intron 12 3082 gagtcaatgcctgagatggg 50 76 2 195452 Intron: 12 6200 caggaagcaactgggagtga 7 77 2 Exon Junction 195453 Exon: 12 8677 ccatctcagagaaggcattg 81 78 2 Intron Junction 195454 Intron 12 10476 tgcacatgtccctgtgggat 64 79 2 195455 Exon: 12 11486 gggactcaccggtcagacca 26 80 2 Intron Junction 195456 Intron: 12 12582 agtggttggtccctggagga 71 81 2 Exon Junction 195457 Exon: 12 12691 agctccttacaccatatctg 0 82 2 Intron Junction 195458 Genomic 13 9 caaagtgtggaagtgaaagg 0 83 2 195459 Genomic 13 148 ctctggtggccacggtggga 0 84 2 195460 Genomic 13 654 ggtgtatggctgctcagact 67 85 2 195461 Genomic 14 66 aggaggtacatgtgactgac 23 86 2

[0176]As shown in Table 1, SEQ ID NOs: 15, 16, 17, 19, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 45, 46, 47, 49, 50, 52, 53, 55, 56, 57, 61, 63, 65, 66, 67, 68, 69, 70, 72, 73, 75, 78, 79, 81 and 85 demonstrated at least 60% inhibition of human STAT 6 expression in this assay and are therefore preferred. More preferred are SEQ ID NOs: 15, 34 and 42. 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 2. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. "Target site" indicates the first (5'-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 2 is the species in which each of the preferred target segments was found.

TABLE-US-00002 TABLE 2 Sequence and position of preferred target segments identified in STAT 6. TAR- GET TAR- REV SEQ GET COMP OF ACTIVE SEQ ID SITEID ID NO SITE SEQUENCE SEQ ID IN NO 69306 4 1951 gacctgtccattcgctcact 15 H. sapiens 87 69307 4 2483 tgagccagccagtgacagcg 16 H. sapiens 88 69308 4 1171 agcactggagaaatcatcaa 17 H. sapiens 89 69310 4 1054 ttgggcttgaggttcctggg 19 H. sapiens 90 69314 4 765 gatccagatttggaaacggc 23 H. sapiens 91 69315 4 729 agagctagaggcagccaaag 24 H. sapiens 92 69316 4 1725 tgtcctggacctcaccaaac 25 H. sapiens 93 69317 4 2321 aggagcctcacctgcagatg 26 H. sapiens 94 69318 4 2676 ccacatggacctaagggcca 27 H. sapiens 95 69319 4 1905 ggatggctctccacagatag 28 H. sapiens 96 69320 4 1805 ttactagccttcttctcaat 29 H. sapiens 97 69321 4 392 ccatcttgcaacacatcagc 30 H. sapiens 98 69322 4 3019 ctctctgagggtgatggggc 31 H. sapiens 99 69323 4 417 tgagagcatatatcagaggg 32 H. sapiens 100 69324 4 1797 acagtacgttactagccttc 33 H. sapiens 101 69325 4 1819 ctcaatgagcccgacggaac 34 H. sapiens 102 69326 4 2479 tgcctgagccagccagtgac 35 H. sapiens 103 69327 4 1513 aagatgtgtgaaactctgaa 36 H. sapiens 104 69328 4 79 ctacagacctatggggcctg 37 H. sapiens 105 69329 4 644 tgtctctgcacagcttgata 38 H. sapiens 106 69330 4 272 gccagccctgggagttcctg 39 H. sapiens 107 69331 4 3000 cgcacacacacatacagagc 40 H. sapiens 108 69332 4 1972 ggggaccgaatccgggatct 41 H. sapiens 109 69333 4 794 tggcagggaatggcgcaccg 42 H. sapiens 110 69335 4 146 caacctccaagtcccagatc 44 H. sapiens 111 69336 4 2165 tgccttcttatgaccttgga 45 H. sapiens 112 69337 4 1762 tggtctgaccggctgatcat 46 H. sapiens 113 69338 4 1205 tggagaacagcattcctggg 47 H. sapiens 114 69340 4 1671 caacaaggagatcctgctgg 49 H. sapiens 115 69341 4 1160 ctggagcagaaagcactgga 50 H. sapiens 116 113659 4 39 gaggtgctagcagggccagc 52 H. sapiens 117 113660 4 158 cccagatcatgtctctgtgg 53 H. sapiens 118 113662 4 718 gagaccactggagagctaga 55 H. sapiens 119 113663 4 1085 agcctccgctggtcagggcc 56 H. sapiens 120 113664 4 1290 cactgagtctgtcacagagg 57 H. sapiens 121 113668 4 2506 cctcagggcacttggattgg 61 H. sapiens 122 113670 4 2944 aagttcaggctctgagacac 63 H. sapiens 123 113672 11 38 gcttacactgaagagggagg 65 H. sapiens 124 113673 11 3185 ttagggcatggaaggcttct 66 H. sapiens 125 113674 11 3230 gacggataggcaggaacata 67 H. sapiens 126 113675 11 3531 tatagctggcaccttagttg 68 H. sapiens 127 113676 11 3539 gcaccttagttgcatgacca 69 H. sapiens 128 113677 11 3585 gcctacgtgacaacacaaat 70 H. sapiens 129 113679 11 3629 aagacagcagcaagtgtgct 72 H. sapiens 130 113680 11 3779 agtctgggtccaggcctggc 73 H. sapiens 131 113682 12 2812 acagattaaaagctgacatt 75 H. sapiens 132 113685 12 8677 caatgccttctctgagatgg 78 H. sapiens 133 113686 12 10476 atcccacagggacatgtgca 79 H. sapiens 134 113688 12 12582 tcctccagggaccaaccact 81 H. sapiens 135 113692 13 654 agtctgagcagccatacacc 85 H. sapiens 136

[0177]As these "preferred target segments" 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 STAT 6.

[0178]According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, 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 16

Western Blot Analysis of STAT 6 Protein Levels

[0179]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 STAT 6 is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER® (Molecular Dynamics, Sunnyvale Calif.).

Example 17

Targeting of Individual Oligonucleotides to Specific Variants of STAT 6

[0180]It is advantageous to selectively inhibit the expression of one or more variants of STAT 6. Consequently, in one embodiment of the present invention are oligonucleotides that selectively target, hybridize to, and specifically inhibit one or more, but fewer than all of the variants of STAT 6. A summary of the target sites of the variants is shown in Table 3 and includes GenBank accession number BC005823.1, representing STAT 6 main mRNA (represented in Table 3 as STAT 6), incorporated herein as SEQ ID NO: 11; GenBank accession number BE972840.1, representing STAT 6d, incorporated herein as SEQ ID NO: 13; GenBank accession number BF902909.1, representing STAT 6e, incorporated herein as SEQ ID NO: 14; GenBank accession number AR204914.1, representing STAT 6b, incorporated herein as SEQ ID NO: 137; and GenBank accession number AR204915.1, representing STAT 6c, incorporated herein as SEQ ID NO: 138.

TABLE-US-00003 TABLE 3 Targeting of individual oligonucleotides to specific variants of STAT 6 OLIGO SEQ TARGET VARIANT ISIS # ID NO. SITE VARIANT SEQ ID NO. 153765 15 2066 STAT 6 11 153765 15 1932 STAT 6c 137 153765 15 1741 STAT 6b 138 153766 16 2597 STAT 6 11 153766 16 2464 STAT 6c 137 153766 16 2273 STAT 6b 138 153767 17 1286 STAT 6 11 153767 17 1236 STAT 6c 137 153767 17 961 STAT 6b 138 153768 18 1055 STAT 6 11 153768 18 1005 STAT 6c 137 153768 18 730 STAT 6b 138 153769 19 1169 STAT 6 11 153769 19 1119 STAT 6c 137 153769 19 844 STAT 6b 138 153770 20 357 STAT 6 11 153770 20 307 STAT 6c 137 153770 20 171 STAT 6b 138 153771 21 2215 STAT 6 11 153771 21 2081 STAT 6c 137 153771 21 1890 STAT 6b 138 153772 22 494 STAT 6 11 153772 22 444 STAT 6c 137 153773 23 880 STAT 6 11 153773 23 830 STAT 6c 137 153773 23 555 STAT 6b 138 153774 24 844 STAT 6 11 153774 24 794 STAT 6c 137 153774 24 519 STAT 6b 138 153775 25 1840 STAT 6 11 153775 25 1790 STAT 6c 137 153775 25 1515 STAT 6b 138 153776 26 2435 STAT 6 11 153776 26 298 STAT 6e 14 153776 26 2302 STAT 6c 137 153776 26 2111 STAT 6b 138 153777 27 2790 STAT 6 11 153777 27 2657 STAT 6c 137 153777 27 2466 STAT 6b 138 153778 28 2020 STAT 6 11 153778 28 1886 STAT 6c 137 153778 28 1695 STAT 6b 138 153779 29 1920 STAT 6 11 153779 29 1595 STAT 6b 138 153780 30 507 STAT 6 11 153780 30 457 STAT 6c 137 153781 31 3133 STAT 6 11 153781 31 3000 STAT 6c 137 153781 31 2809 STAT 6b 138 153782 32 532 STAT 6 11 153782 32 482 STAT 6c 137 153783 33 1912 STAT 6 11 153783 33 1587 STAT 6b 138 153784 34 1934 STAT 6 11 153784 34 1609 STAT 6b 138 153785 35 2593 STAT 6 11 153785 35 2460 STAT 6c 137 153785 35 2269 STAT 6b 138 153786 36 1628 STAT 6 11 153786 36 377 STAT 6d 13 153786 36 1578 STAT 6c 137 153786 36 1303 STAT 6b 138 153787 37 194 STAT 6 11 153787 37 76 STAT 6c 137 153787 37 8 STAT 6b 138 153788 38 759 STAT 6 11 153788 38 709 STAT 6c 137 153788 38 434 STAT 6b 138 153789 39 387 STAT 6 11 153789 39 337 STAT 6c 137 153790 40 3114 STAT 6 11 153790 40 2981 STAT 6c 137 153790 40 2790 STAT 6b 138 153791 41 2087 STAT 6 11 153791 41 1953 STAT 6c 137 153791 41 1762 STAT 6b 138 153792 42 909 STAT 6 11 153792 42 859 STAT 6c 137 153792 42 584 STAT 6b 138 153793 43 2112 STAT 6 11 153793 43 1978 STAT 6c 137 153793 43 1787 STAT 6b 138 153794 44 261 STAT 6 11 153794 44 211 STAT 6c 137 153794 44 75 STAT 6b 138 153795 45 2280 STAT 6 11 153795 45 142 STAT 6e 14 153795 45 2146 STAT 6c 137 153795 45 1955 STAT 6b 138 153796 46 1877 STAT 6 11 153796 46 1552 STAT 6b 138 153797 47 1320 STAT 6 11 153797 47 1270 STAT 6c 137 153797 47 995 STAT 6b 138 153798 48 1059 STAT 6 11 153798 48 1009 STAT 6c 137 153798 48 734 STAT 6b 138 153799 49 1786 STAT 6 11 153799 49 535 STAT 6d 13 153799 49 1736 STAT 6c 137 153799 49 1461 STAT 6b 138 153800 50 1275 STAT 6 11 153800 50 1225 STAT 6c 137 153800 50 950 STAT 6b 138 153801 51 443 STAT 6 11 153801 51 393 STAT 6c 137 195427 52 154 STAT 6 11 195427 52 36 STAT 6c 137 195428 53 273 STAT 6 11 195428 53 223 STAT 6c 137 195428 53 87 STAT 6b 138 195429 54 656 STAT 6 11 195429 54 606 STAT 6c 137 195429 54 331 STAT 6b 138 195430 55 833 STAT 6 11 195430 55 783 STAT 6c 137 195430 55 508 STAT 6b 138 195431 56 1200 STAT 6 11 195431 56 1150 STAT 6c 137 195431 56 875 STAT 6b 138 195432 57 1405 STAT 6 11 195432 57 1355 STAT 6c 137 195432 57 1080 STAT 6b 138 195433 58 1730 STAT 6 11 195433 58 479 STAT 6d 13 195433 58 1680 STAT 6c 137 195433 58 1405 STAT 6b 138 195434 59 2002 STAT 6 11 195434 59 1868 STAT 6c 137 195434 59 1677 STAT 6b 138 195435 60 2267 STAT 6 11 195435 60 129 STAT 6e 14 195435 60 2133 STAT 6c 137 195435 60 1942 STAT 6b 138 195436 61 2620 STAT 6 11 195436 61 2487 STAT 6c 137 195436 61 2296 STAT 6b 138 195437 62 2813 STAT 6 11 195437 62 2680 STAT 6c 137 195437 62 2489 STAT 6b 138 195438 63 3058 STAT 6 11 195438 63 2925 STAT 6c 137 195438 63 2734 STAT 6b 138 195439 64 23 STAT 6 11 195440 65 38 STAT 6 11 195441 66 3185 STAT 6 11 195441 66 3052 STAT 6c 137 195441 66 2861 STAT 6b 138 195442 67 3230 STAT 6 11 195442 67 3097 STAT 6c 137 195442 67 2906 STAT 6b 138 195443 68 3531 STAT 6 11 195443 68 3398 STAT 6c 137 195443 68 3207 STAT 6b 138 195444 69 3539 STAT 6 11 195444 69 3406 STAT 6c 137 195444 69 3215 STAT 6b 138 195445 70 3585 STAT 6 11 195445 70 3452 STAT 6c 137 195445 70 3261 STAT 6b 138 195446 71 3599 STAT 6 11 195446 71 3466 STAT 6c 137 195446 71 3275 STAT 6b 138 195447 72 3629 STAT 6 11 195447 72 3496 STAT 6c 137 195447 72 3305 STAT 6b 138 195448 73 3779 STAT 6 11 195448 73 3646 STAT 6c 137 195448 73 3455 STAT 6b 138 195449 74 3835 STAT 6 11 195449 74 3702 STAT 6c 137 195449 74 3511 STAT 6b 138 195453 78 1567 STAT 6 11 195453 78 1517 STAT 6c 137 195453 78 1242 STAT 6b 138 195455 80 624 STAT 6d 13 195456 81 90 STAT 6e 14 195458 83 9 STAT 6d 13 195459 84 148 STAT 6d 13 195460 85 654 STAT 6d 13 195461 86 66 STAT 6e 14

Sequence CWU 1

141120DNAArtificial SequenceAntisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20220DNAArtificial SequenceAntisense Oligonucleotide 2gtgcgcgcga gcccgaaatc 20320DNAArtificial SequenceAntisense Oligonucleotide 3atgcattctg cccccaagga 2043046DNAH. sapiensCDS(166)...(2709) 4atcttatttt tctttttggt ggtggtggtg gaagggggga ggtgctagca gggccagcct 60tgaactcgct ggacagagct acagacctat ggggcctgga agtgcccgct gagaaaggga 120gaagacagca gaggggttgc cgaggcaacc tccaagtccc agatc atg tct ctg tgg 177Met Ser Leu Trp1ggt ctg gtc tcc aag atg ccc cca gaa aaa gtg cag cgg ctc tat gtc 225Gly Leu Val Ser Lys Met Pro Pro Glu Lys Val Gln Arg Leu Tyr Val5 10 15 20gac ttt ccc caa cac ctg cgg cat ctt ctg ggt gac tgg ctg gag agc 273Asp Phe Pro Gln His Leu Arg His Leu Leu Gly Asp Trp Leu Glu Ser25 30 35cag ccc tgg gag ttc ctg gtc ggc tcc gac gcc ttc tgc tgc aac ttg 321Gln Pro Trp Glu Phe Leu Val Gly Ser Asp Ala Phe Cys Cys Asn Leu40 45 50gct agt gcc cta ctt tca gac act gtc cag cac ctt cag gcc tcg gtg 369Ala Ser Ala Leu Leu Ser Asp Thr Val Gln His Leu Gln Ala Ser Val55 60 65gga gag cag ggg gag ggg agc acc atc ttg caa cac atc agc acc ctt 417Gly Glu Gln Gly Glu Gly Ser Thr Ile Leu Gln His Ile Ser Thr Leu70 75 80gag agc ata tat cag agg gac ccc ctg aag ctg gtg gcc act ttc aga 465Glu Ser Ile Tyr Gln Arg Asp Pro Leu Lys Leu Val Ala Thr Phe Arg85 90 95 100caa ata ctt caa gga gag aaa aaa gct gtt atg gaa cag ttc cgc cac 513Gln Ile Leu Gln Gly Glu Lys Lys Ala Val Met Glu Gln Phe Arg His105 110 115ttg cca atg cct ttc cac tgg aag cag gaa gaa ctc aag ttt aag aca 561Leu Pro Met Pro Phe His Trp Lys Gln Glu Glu Leu Lys Phe Lys Thr120 125 130ggc ttg cgg agg ctg cag cac cga gta ggg gag atc cac ctt ctc cga 609Gly Leu Arg Arg Leu Gln His Arg Val Gly Glu Ile His Leu Leu Arg135 140 145gaa gcc ctg cag aag ggg gct gag gct ggc caa gtg tct ctg cac agc 657Glu Ala Leu Gln Lys Gly Ala Glu Ala Gly Gln Val Ser Leu His Ser150 155 160ttg ata gaa act cct gct aat ggg act ggg cca agt gag gcc ctg gcc 705Leu Ile Glu Thr Pro Ala Asn Gly Thr Gly Pro Ser Glu Ala Leu Ala165 170 175 180atg cta ctg cag gag acc act gga gag cta gag gca gcc aaa gcc cta 753Met Leu Leu Gln Glu Thr Thr Gly Glu Leu Glu Ala Ala Lys Ala Leu185 190 195gtg ctg aag agg atc cag att tgg aaa cgg cag cag cag ctg gca ggg 801Val Leu Lys Arg Ile Gln Ile Trp Lys Arg Gln Gln Gln Leu Ala Gly200 205 210aat ggc gca ccg ttt gag gag agc ctg gcc cca ctc cag gag agg tgt 849Asn Gly Ala Pro Phe Glu Glu Ser Leu Ala Pro Leu Gln Glu Arg Cys215 220 225gaa agc ctg gtg gac att tat tcc cag cta cag cag gag gta ggg gcg 897Glu Ser Leu Val Asp Ile Tyr Ser Gln Leu Gln Gln Glu Val Gly Ala230 235 240gct ggt ggg gag ctt gag ccc aag acc cgg gca tcg ctg act ggc cgg 945Ala Gly Gly Glu Leu Glu Pro Lys Thr Arg Ala Ser Leu Thr Gly Arg245 250 255 260ctg gat gaa gtc ctg aga acc ctc gtc acc agt tgc ttc ctg gtg gag 993Leu Asp Glu Val Leu Arg Thr Leu Val Thr Ser Cys Phe Leu Val Glu265 270 275aag cag ccc ccc cag gta ctg aag act cag acc aag ttc cag gct gga 1041Lys Gln Pro Pro Gln Val Leu Lys Thr Gln Thr Lys Phe Gln Ala Gly280 285 290gtt cga ttc ctg ttg ggc ttg agg ttc ctg ggg gcc cca gcc aag cct 1089Val Arg Phe Leu Leu Gly Leu Arg Phe Leu Gly Ala Pro Ala Lys Pro295 300 305ccg ctg gtc agg gcc gac atg gtg aca gag aag cag gcg cgg gag ctg 1137Pro Leu Val Arg Ala Asp Met Val Thr Glu Lys Gln Ala Arg Glu Leu310 315 320agt gtg cct cag ggt cct ggg gct gga gca gaa agc act gga gaa atc 1185Ser Val Pro Gln Gly Pro Gly Ala Gly Ala Glu Ser Thr Gly Glu Ile325 330 335 340atc aac aac act gtg ccc ttg gag aac agc att cct ggg aac tgc tgc 1233Ile Asn Asn Thr Val Pro Leu Glu Asn Ser Ile Pro Gly Asn Cys Cys345 350 355tct gcc ctg ttc aag aac ctg ctt ctc aag aag atc aag cgg tgt gag 1281Ser Ala Leu Phe Lys Asn Leu Leu Leu Lys Lys Ile Lys Arg Cys Glu360 365 370cgg aag ggc act gag tct gtc aca gag gag aag tgc gct gtg ctc ttc 1329Arg Lys Gly Thr Glu Ser Val Thr Glu Glu Lys Cys Ala Val Leu Phe375 380 385tct gcc agc ttc aca ctt ggc ccc ggc aaa ctc ccc atc cag ctc cag 1377Ser Ala Ser Phe Thr Leu Gly Pro Gly Lys Leu Pro Ile Gln Leu Gln390 395 400gcc ctg tct ctg ccc ctg gtg gtc atc gtc cat ggc aac caa gac aac 1425Ala Leu Ser Leu Pro Leu Val Val Ile Val His Gly Asn Gln Asp Asn405 410 415 420aat gcc aaa gcc act atc ctg tgg gac aat gcc ttc tct gag atg gac 1473Asn Ala Lys Ala Thr Ile Leu Trp Asp Asn Ala Phe Ser Glu Met Asp425 430 435cgc gtg ccc ttt gtg gtg gct gag cgg gtg ccc tgg gag aag atg tgt 1521Arg Val Pro Phe Val Val Ala Glu Arg Val Pro Trp Glu Lys Met Cys440 445 450gaa act ctg aac ctg aag ttc atg gct gag gtg ggg acc aac cgg ggg 1569Glu Thr Leu Asn Leu Lys Phe Met Ala Glu Val Gly Thr Asn Arg Gly455 460 465ctg ctc cca gag cac ttc ctc ttc ctg gcc cag aag atc ttc aat gac 1617Leu Leu Pro Glu His Phe Leu Phe Leu Ala Gln Lys Ile Phe Asn Asp470 475 480aac agc ctc agt atg gag gcc ttc cag cac cgt tct gtg tcc tgg tcg 1665Asn Ser Leu Ser Met Glu Ala Phe Gln His Arg Ser Val Ser Trp Ser485 490 495 500cag ttc aac aag gag atc ctg ctg ggc cgt ggc ttc acc ttt tgg cag 1713Gln Phe Asn Lys Glu Ile Leu Leu Gly Arg Gly Phe Thr Phe Trp Gln505 510 515tgg ttt gat ggt gtc ctg gac ctc acc aaa cgc tgt ctc cgg agc tac 1761Trp Phe Asp Gly Val Leu Asp Leu Thr Lys Arg Cys Leu Arg Ser Tyr520 525 530tgg tct gac cgg ctg atc att ggc ttc atc agc aaa cag tac gtt act 1809Trp Ser Asp Arg Leu Ile Ile Gly Phe Ile Ser Lys Gln Tyr Val Thr535 540 545agc ctt ctt ctc aat gag ccc gac gga acc ttt ctc ctc cgc ttc agc 1857Ser Leu Leu Leu Asn Glu Pro Asp Gly Thr Phe Leu Leu Arg Phe Ser550 555 560gac tca gag att ggg ggc atc acc att gcc cat gtc atc cgg ggc cag 1905Asp Ser Glu Ile Gly Gly Ile Thr Ile Ala His Val Ile Arg Gly Gln565 570 575 580gat ggc tct cca cag ata gag aac atc cag cca ttc tct gcc aaa gac 1953Asp Gly Ser Pro Gln Ile Glu Asn Ile Gln Pro Phe Ser Ala Lys Asp585 590 595ctg tcc att cgc tca ctg ggg gac cga atc cgg gat ctt gct cag ctc 2001Leu Ser Ile Arg Ser Leu Gly Asp Arg Ile Arg Asp Leu Ala Gln Leu600 605 610aaa aat ctc tat ccc aag aag ccc aag gat gag gct ttc cgg agc cac 2049Lys Asn Leu Tyr Pro Lys Lys Pro Lys Asp Glu Ala Phe Arg Ser His615 620 625tac aag cct gaa cag atg ggt aag gat ggc agg ggt tat gtc cca gct 2097Tyr Lys Pro Glu Gln Met Gly Lys Asp Gly Arg Gly Tyr Val Pro Ala630 635 640acc atc aag atg acc gtg gaa agg gac caa cca ctt cct acc cca gag 2145Thr Ile Lys Met Thr Val Glu Arg Asp Gln Pro Leu Pro Thr Pro Glu645 650 655 660ctc cag atg cct acc atg gtg cct tct tat gac ctt gga atg gcc cct 2193Leu Gln Met Pro Thr Met Val Pro Ser Tyr Asp Leu Gly Met Ala Pro665 670 675gat tcc tcc atg agc atg cag ctt ggc cca gat atg gtg ccc cag gtg 2241Asp Ser Ser Met Ser Met Gln Leu Gly Pro Asp Met Val Pro Gln Val680 685 690tac cca cca cac tct cac tcc atc ccc ccg tat caa ggc ctc tcc cca 2289Tyr Pro Pro His Ser His Ser Ile Pro Pro Tyr Gln Gly Leu Ser Pro695 700 705gaa gaa tca gtc aac gtg ttg tca gcc ttc cag gag cct cac ctg cag 2337Glu Glu Ser Val Asn Val Leu Ser Ala Phe Gln Glu Pro His Leu Gln710 715 720atg ccc ccc agc ctg ggc cag atg agc ctg ccc ttt gac cag cct cac 2385Met Pro Pro Ser Leu Gly Gln Met Ser Leu Pro Phe Asp Gln Pro His725 730 735 740ccc cag ggc ctg ctg ccg tgc cag cct cag gag cat gct gtg tcc agc 2433Pro Gln Gly Leu Leu Pro Cys Gln Pro Gln Glu His Ala Val Ser Ser745 750 755cct gac ccc ctg ctc tgc tca gat gtg acc atg gtg gaa gac agc tgc 2481Pro Asp Pro Leu Leu Cys Ser Asp Val Thr Met Val Glu Asp Ser Cys760 765 770ctg agc cag cca gtg aca gcg ttt cct cag ggc act tgg att ggt gaa 2529Leu Ser Gln Pro Val Thr Ala Phe Pro Gln Gly Thr Trp Ile Gly Glu775 780 785gac ata ttc cct cct ctg ctg cct ccc act gaa cag gac ctc act aag 2577Asp Ile Phe Pro Pro Leu Leu Pro Pro Thr Glu Gln Asp Leu Thr Lys790 795 800ctt ctc ctg gag ggg caa ggg gag tcg ggg gga ggg tcc ttg ggg gca 2625Leu Leu Leu Glu Gly Gln Gly Glu Ser Gly Gly Gly Ser Leu Gly Ala805 810 815 820cag ccc ctc ctg cag ccc tcc cac tat ggg caa tct ggg atc tca atg 2673Gln Pro Leu Leu Gln Pro Ser His Tyr Gly Gln Ser Gly Ile Ser Met825 830 835tcc cac atg gac cta agg gcc aac ccc agt tgg tga tcccagctgg 2719Ser His Met Asp Leu Arg Ala Asn Pro Ser Trp840 845agggagaacc caaagagaca gctcttctac tacccccaca gacctgctct ggacacttgc 2779tcatgccctg ccaagcagca gatggggagg gtgccctcct atccccacct actcctgggt 2839caggaggaaa agactaacag gagaatgcac agtgggtgga gccaatccac tccttccttt 2899ctatcattcc cctgcccacc tccttccagc actgactgga agggaagttc aggctctgag 2959acacgcccca acatgcctgc acctgcagcg cgcacacgca cgcacacaca catacagagc 3019tctctgaggg tgatggggct gagcagg 3046518DNAArtificial SequencePCR Primer 5ccaaacgctg tctccgga 18627DNAArtificial SequencePCR Primer 6gctagtaacg tactgtttgc tgatgaa 27727DNAArtificial SequencePCR Probe 7ctactggtct gaccggctga tcattgg 27819DNAArtificial SequencePCR Primer 8gaaggtgaag gtcggagtc 19920DNAArtificial SequencePCR Primer 9gaagatggtg atgggatttc 201020DNAArtificial SequencePCR Probe 10caagcttccc gttctcagcc 20113971DNAH. sapiensCDS(281)...(2320) 11ggcacgaggc cggaaacagc gggctggggc agccactgct tacactgaag agggaggacg 60ggagaggagt gtgtgtgtgt gtgtgtgtgt gtgtgtgtat gtatgtgtgt gctttatctt 120atttttcttt ttggtggtgg tggtggaagg ggggaggtgc tagcagggcc agccttgaac 180tcgctggaca gagctacaga cctatggggc ctggaagtgc ccgctgagaa agggagaaga 240cagcagaggg gttgccgagg caacctccaa gtcccagatc atg tct ctg tgg ggt 295Met Ser Leu Trp Gly1 5ctg gtc tcc aag atg ccc cca gaa aaa gtg cag cgg ctc tat gtc gac 343Leu Val Ser Lys Met Pro Pro Glu Lys Val Gln Arg Leu Tyr Val Asp10 15 20ttt ccc caa cac ctg cgg cat ctt ctg ggt gac tgg ctg gag agc cag 391Phe Pro Gln His Leu Arg His Leu Leu Gly Asp Trp Leu Glu Ser Gln25 30 35ccc tgg gag ttc ctg gtc ggc tcc gac gcc ttc tgc tgc aac ttg gct 439Pro Trp Glu Phe Leu Val Gly Ser Asp Ala Phe Cys Cys Asn Leu Ala40 45 50agt gcc cta ctt tca gac act gtc cag cac ctt cag gcc tcg gtg gga 487Ser Ala Leu Leu Ser Asp Thr Val Gln His Leu Gln Ala Ser Val Gly55 60 65gag cag ggg gag ggg agc acc atc ttg caa cac atc agc acc ctt gag 535Glu Gln Gly Glu Gly Ser Thr Ile Leu Gln His Ile Ser Thr Leu Glu70 75 80 85agc ata tat cag agg gac ccc ctg aag ctg gtg gcc act ttc aga caa 583Ser Ile Tyr Gln Arg Asp Pro Leu Lys Leu Val Ala Thr Phe Arg Gln90 95 100ata ctt caa gga gag aaa aaa gct gtt atg gaa cag ttc cgc cac ttg 631Ile Leu Gln Gly Glu Lys Lys Ala Val Met Glu Gln Phe Arg His Leu105 110 115cca atg cct ttc cac tgg aag cag gaa gaa ctc aag ttt aag aca ggc 679Pro Met Pro Phe His Trp Lys Gln Glu Glu Leu Lys Phe Lys Thr Gly120 125 130ttg cgg agg ctg cag cac cga gta ggg gag atc cac ctt ctc cga gaa 727Leu Arg Arg Leu Gln His Arg Val Gly Glu Ile His Leu Leu Arg Glu135 140 145gcc ctg cag aag ggg gct gag gct ggc caa gtg tct ctg cac agc ttg 775Ala Leu Gln Lys Gly Ala Glu Ala Gly Gln Val Ser Leu His Ser Leu150 155 160 165ata gaa act cct gct aat ggg act ggg cca agt gag gcc ctg gcc atg 823Ile Glu Thr Pro Ala Asn Gly Thr Gly Pro Ser Glu Ala Leu Ala Met170 175 180cta ctg cag gag acc act gga gag cta gag gca gcc aaa gcc cta gtg 871Leu Leu Gln Glu Thr Thr Gly Glu Leu Glu Ala Ala Lys Ala Leu Val185 190 195ctg aag agg atc cag att tgg aaa cgg cag cag cag ctg gca ggg aat 919Leu Lys Arg Ile Gln Ile Trp Lys Arg Gln Gln Gln Leu Ala Gly Asn200 205 210ggc gca ccg ttt gag gag agc ctg gcc cca ctc cag gag agg tgt gaa 967Gly Ala Pro Phe Glu Glu Ser Leu Ala Pro Leu Gln Glu Arg Cys Glu215 220 225agc ctg gtg gac att tat tcc cag cta cag cag gag gta ggg gcg gct 1015Ser Leu Val Asp Ile Tyr Ser Gln Leu Gln Gln Glu Val Gly Ala Ala230 235 240 245ggt ggg gag ctt gag ccc aag acc cgg gca tcg ctg act ggc cgg ctg 1063Gly Gly Glu Leu Glu Pro Lys Thr Arg Ala Ser Leu Thr Gly Arg Leu250 255 260gat gaa gtc ctg aga acc ctc gtc acc agt tgc ttc ctg gtg gag aag 1111Asp Glu Val Leu Arg Thr Leu Val Thr Ser Cys Phe Leu Val Glu Lys265 270 275cag ccc ccc cag gta ctg aag act cag acc aag ttc cag gct gga gtt 1159Gln Pro Pro Gln Val Leu Lys Thr Gln Thr Lys Phe Gln Ala Gly Val280 285 290cga ttc ctg ttg ggc ttg agg ttc ctg ggg gcc cca gcc aag cct ccg 1207Arg Phe Leu Leu Gly Leu Arg Phe Leu Gly Ala Pro Ala Lys Pro Pro295 300 305ctg gtc agg gcc gac atg gtg aca gag aag cag gcg cgg gag ctg agt 1255Leu Val Arg Ala Asp Met Val Thr Glu Lys Gln Ala Arg Glu Leu Ser310 315 320 325gtg cct cag ggt cct ggg gct gga gca gaa agc act gga gaa atc atc 1303Val Pro Gln Gly Pro Gly Ala Gly Ala Glu Ser Thr Gly Glu Ile Ile330 335 340aac aac act gtg ccc ttg gag aac agc att cct ggg aac tgc tgc tct 1351Asn Asn Thr Val Pro Leu Glu Asn Ser Ile Pro Gly Asn Cys Cys Ser345 350 355gcc ctg ttc aag aac ctg ctt ctc aag aag atc aag cgg tgt gag cgg 1399Ala Leu Phe Lys Asn Leu Leu Leu Lys Lys Ile Lys Arg Cys Glu Arg360 365 370aag ggc act gag tct gtc aca gag gag aag tgc gct gtg ctc ttc tct 1447Lys Gly Thr Glu Ser Val Thr Glu Glu Lys Cys Ala Val Leu Phe Ser375 380 385gcc agc ttc aca ctt ggc ccc ggc aaa ctc ccc atc cag ctc cag gcc 1495Ala Ser Phe Thr Leu Gly Pro Gly Lys Leu Pro Ile Gln Leu Gln Ala390 395 400 405ctg tct ctg ccc ctg gtg gtc atc gtc cat ggc aac caa gac aac aat 1543Leu Ser Leu Pro Leu Val Val Ile Val His Gly Asn Gln Asp Asn Asn410 415 420gcc aaa gcc act atc ctg tgg tac aat gcc ttc tct gag atg gac cgc 1591Ala Lys Ala Thr Ile Leu Trp Tyr Asn Ala Phe Ser Glu Met Asp Arg425 430 435gtg ccc ttt gtg gtg gct gag cgg gtg ccc tgg gag aag atg tgt gaa 1639Val Pro Phe Val Val Ala Glu Arg Val Pro Trp Glu Lys Met Cys Glu440 445 450act ctg aac ctg aag ttc atg gct gag gtg ggg acc aac cgg ggg ctg 1687Thr Leu Asn Leu Lys Phe Met Ala Glu Val Gly Thr Asn Arg Gly Leu455 460 465ctc cca gag cac ttc ctc ttc ctg gcc cag aag atc ttc aat gac aac 1735Leu Pro Glu His Phe Leu Phe Leu Ala Gln Lys Ile Phe Asn Asp Asn470 475 480 485agc ctc agt atg gag gcc ttc cag cac cgt tct gtg tcc tgg tcg cag 1783Ser Leu Ser Met Glu Ala Phe Gln His Arg Ser Val Ser Trp Ser Gln490 495 500ttc aac aag gag atc ctg ctg ggc cgt ggc ttc acc ttt tgg cag tgg 1831Phe Asn Lys Glu Ile Leu Leu Gly Arg Gly Phe Thr Phe Trp Gln Trp505 510 515ttt gat ggt gtc ctg gac ctc acc aaa cgc tgt ctc cgg agc tac tgg 1879Phe Asp Gly Val Leu Asp Leu Thr Lys Arg Cys Leu Arg Ser Tyr Trp520 525 530tct gac cgg ctg atc att ggc ttc atc agc aaa cag tac gtt act agc 1927Ser Asp Arg Leu Ile Ile Gly Phe Ile Ser Lys Gln Tyr Val Thr Ser535 540 545ctt ctt ctc aat gag ccc gac gga acc ttt ctc ctc cgc ttc agc gac 1975Leu Leu Leu Asn Glu Pro Asp Gly Thr Phe Leu Leu Arg Phe Ser Asp550 555 560 565tca gag att ggg ggc atc acc

att gcc cat gtc atc cgg ggc cag gat 2023Ser Glu Ile Gly Gly Ile Thr Ile Ala His Val Ile Arg Gly Gln Asp570 575 580ggc tct cca cag ata gag aac atc cag cca ttc tct gcc aaa gac ctg 2071Gly Ser Pro Gln Ile Glu Asn Ile Gln Pro Phe Ser Ala Lys Asp Leu585 590 595tcc att cgc tca ctg ggg gac cga atc cgg gat ctt gct cag ctc aaa 2119Ser Ile Arg Ser Leu Gly Asp Arg Ile Arg Asp Leu Ala Gln Leu Lys600 605 610aat ctc tat ccc aag aag ccc aag gat gag gct ttc cgg agc cac tac 2167Asn Leu Tyr Pro Lys Lys Pro Lys Asp Glu Ala Phe Arg Ser His Tyr615 620 625aag cct gaa cag atg ggt aag gat ggc agg ggt tat gtc cca gct acc 2215Lys Pro Glu Gln Met Gly Lys Asp Gly Arg Gly Tyr Val Pro Ala Thr630 635 640 645atc aag atg acc gtg gaa agg gac caa cca ctt cct acc cca gag ctc 2263Ile Lys Met Thr Val Glu Arg Asp Gln Pro Leu Pro Thr Pro Glu Leu650 655 660cag atg cct acc atg gtg cct tct tat gac ctt gga atg gcc ctg att 2311Gln Met Pro Thr Met Val Pro Ser Tyr Asp Leu Gly Met Ala Leu Ile665 670 675cct cca tga gcatgcagct tggcccagat atggtgcccc aggtgtaccc accacactct 2370Pro Procactccatcc ccccgtatca aggcctctcc ccagaagaat cagtcaacgt gttgtcagcc 2430ttccaggagc ctcacctgca gatgcccccc agcctgggcc agatgagcct gccctttgac 2490cagcctcacc cccagggcct gctgccgtgc cagcctcagg agcatgctgt gtccagccct 2550gaccccctgc tctgctcaga tgtgaccatg gtggaagaca gctgcctgag ccagccagtg 2610acagcgtttc ctcagggcac ttggattggt gaagacatat tccctcctct gctgcctccc 2670actgaacagg acctcactaa gcttctcctg gaggggcaag gggagtcggg gggagggtcc 2730ttgggggcac agcccctcct gcagccctcc cactatgggc aatctgggat ctcaatgtcc 2790cacatggacc taagggccaa ccccagttgg tgatcccagc tggagggaga acccaaagag 2850acagctcttc tactaccccc acagacctgc tctggacact tgctcatgcc ctgccaagca 2910gcagatgggg agggtgccct cctatcccca cctactcctg ggtcaggagg aaaagactaa 2970caggagaatg cacagtgggt ggagccaatc cactccttcc tttctatcat tcccctgccc 3030acctccttcc agcactgact ggaagggaag ttcaggctct gagacacgcc ccaacatgcc 3090tgcacctgca gcgcgcacac gcacgcacac acacatacag agctctctga gggtgatggg 3150gctgagcagg aggggggctg ggtaagagca caggttaggg catggaaggc ttctccgccc 3210attctgaccc agggcctagg acggataggc aggaacatac agacacattt acactagagg 3270ccagggatag aggatattgg gtctcagccc taggggaatg ggaagcagct caagggaccc 3330tgggtgggag cataggaggg gtctggacat gtggttacta gtacaggttt tgccctgatt 3390aaaaaatctc ccaaagcccc aaattcctgt tagccaggtg gaggcttctg atacgtgtat 3450gagactatgc aaaagtacaa gggctgagat tcttcgtgta tagctgtgtg aacgtgtatg 3510tacctaggat atgttaaatg tatagctggc accttagttg catgaccaca tagaacatgt 3570gtctatctgc ttttgcctac gtgacaacac aaatttggga gggtgagaca ctgcacagaa 3630gacagcagca agtgtgctgg cctctctgac atatgctaac ccccaaatac tctgaatttg 3690gagtctgact gtgcccaagt gggtccaagt ggctgtgaca tctacgtatg gctccacacc 3750tccaatgctg cctgggagcc agggtgagag tctgggtcca ggcctggcca tgtggccctc 3810cagtgtatga gagggccctg cctgctgcat cttttctgtt gccccatcca ccgccagctt 3870cccttcactc ccctatccca ttctccctct caaggcaggg gtcatagatc ctaagccata 3930aaataaattt tattccaaaa taaaaaaaaa aaaaaaaaaa a 39711216500DNAH. sapiens 12tccccccaag cctggctcca aggcctggac cccagtcctg atcccccacg tgttccccca 60ctcggcacag gaggcacaca tattcacccc actttcttcc tcttcctcct ccagcccact 120ttctcttctc tgtgtcgtca gagctccagg gagggacctg ggtagaagga gaagccggaa 180acagcgggct ggggcagcca ctgcttacac tgaagaggga ggacgggaga ggagtgtgtg 240tgtgtgtgtg tgtgtgtgta tgtatgtgtg tgctttatct tatttttctt tttggtggtg 300gtggtggaag gggggaggtg ctagcagggc cagccttgaa ctcgctggac agagctacag 360acctatgggg cctggaagtg cccgctgaga aagggagaag acagcagagg ggttgccgag 420gtgaggggtt gcctccgagg tgggtgcggg ggcctctatg agtgcatggg ggtggattcg 480tggggggagc tctcgggatc ctcccctggc tgggtggatg gtccccaaga gatggtttca 540gctagtgttg gtggctggtg gcactgggtt ttagcagttt cgaactcctg gaggaatctg 600ggagggtcca ggcctcagta ctcccctccc ccatgggtca cgttttcaca gcctcacccc 660tgcaccccca gggccatgga aagtcaggga aaggaggtga aggagtgccc ctctgccctg 720agtcggggga agtggccgcc cctccctgga aggttgatgc cagagggcag tggatccttg 780ttaaacccct atcctgccct ccactaaagg ttcctgttca agggtgtggc tggggcgtga 840gcaagcccca gatgtagacc tcatggtggc ccagacgagg gggaatttcc ccctcaaaac 900tgctccacgc ttggctgctg tagacgctga gatttcccag cggcggcgcc gagttaaccc 960tcctcgtgct gaactggctc cacctccccg cctgccccca ccgccacatt cacgcattgg 1020gcaactcaga gaagctgttt taactttcga tcctgtggtc ccacaatcag aggactcggg 1080cagatagggg ttgagataag cgagtttagg ccaccaagcg ggcggacgag gatcccagac 1140cttgcgcttc ccttctgagt ttgggaggta acactggccc cgcccctcac gccgtggctc 1200ctccctccct tccccttcaa ggggctgaag acaaaaggtg cccctgtcct ggtcaagcca 1260atcgacccag ccttgttatg ggttggggtg gggagaaatg tgtcctcctg atggctgggg 1320aagaagaggg gttggatatt tctagccagg gccatgccag gaggctggtc actctgcaag 1380gggatgcaga ggaaagcggt gcccactcac tccagaggac ctttctctct tgggctagag 1440aaaggcctat tggaggaacc tgagcaggag gggtaaggat tctgccttga ggagaaaaga 1500gctggggtaa gtgggcactg gaggaaagag gggcatgaag gtcttggagc agaaacatcc 1560agagaaggga cctctccatt ttccatccct ctgagaggcc tgggagaggt gagaggctga 1620acgtgcaaca ggaggacttg gggttactgg gtttggggag acctggggag ttgtcatccc 1680atcctctccc tcatctctgg gagagggata ttatgagaaa cgtgaactga gaggcccctg 1740ggaaaccact ggttacccag tcctccctga acctggaaat ggggatgcaa ccccctcttc 1800tacttccctg tcccctcctc tcctttctac ctgttttcgt ctctcatctt tgccttctag 1860ccctccagct tcctctctct tctaggctct ttcctcctag cttactaaac ccgccttttt 1920tccagtctct tccatcctct tccttagttc tctctacttt ccttttccac ctctcctcct 1980tcaagtctcc tcccaccttc ccccacttct taggatgatc agatttgccc ctggaaggga 2040tcctaacaac acagtgcgat ggttaatccc cactcagatt caaagcctgc tttccaaact 2100cacttactga gtggccttgg gcagagtaga gaaactcctt aagcctcagt ttcttcatct 2160ataaaatggg atattatata ttttaaaaag tgtcgtgagg cctgaaggag ataatacact 2220gagtgtaatg cctcatacac agtaagtgct taacaaatag tagctgttat tactctccca 2280tcctcttcat catctagcct tgtggttttc atttttattt tatttcattt atttatttat 2340ttattttgag acagagtctc tctctgtcgc ccaggctgga gtgcagtggc tcgatctctg 2400ctcactgcaa gctccgcccc ccaggttcac gccattctgt cacctcagcc tccccagtag 2460ctgggactac aggcgctcgc caccacgccc tgctaatttt gtttttgtat ttttagtaga 2520gatggggttt cactgtgtta gccaggatgg tcttgatctc ctgacctcgt gatctgcccg 2580cctcggcctc ccaaagcgct gggattacag gcatgagcca ctgcgcctgg ccgagccttg 2640tggttttcaa attatctcat ggagtcctag aattttgaga ggtttgtcta gggatgcctt 2700tggcgtcagg aggtggggag agggaagtag aagcagtcga gtttcaggct ttccatgctt 2760gctttcaaca gggcatcttc ggtttcgtac cttttatgta attgagattc cacagattaa 2820aagctgacat tgcctaccgc tttaaaaagt ttggaaagtt ttccactcat ctaacactca 2880tattttatag atgagaagat cgaagcccac aaagggaagg ctctttgccc acagaaccag 2940agccaggtct agagctgcaa ctaaatcctc tgccactcta agagagctct cgctctactg 3000ccctgtctcc ctttgcctcc ccatccctct ggctacagct cagctcttcc cacccctgtg 3060tctatcactg aaggagttac ccccatctca ggcattgact caggatgccc ctggtttaag 3120gtggtctggc catgagtggt ggtggggaca gtccctagga gggctatcta tgggaggtcc 3180ctggctgccc caggagatag gccaagtttc ttgggcaccc ctcagagtgg ccttattttt 3240ctcctccagg caacctccaa gtcccagatc atgtctctgt ggggtctggt ctccaagatg 3300cccccagaaa aagtgcagcg gctctatgtc gactttcccc aacacctgcg gcatcttctg 3360ggtgactggc tggagagcca gccctggtga gtcctggctg ctccctgctg gtcccccaag 3420tcttccctaa ctcatcttcc ttctccttag atttttctcc cctcacccat ggattcagaa 3480cttgagacct gttattccat gtgtagtgac ctagatttag cagggagtct gtgccccatc 3540aagaccaggc tatgaatgtt gacagatgga gaccccatct cttaggaggc tgagccgaag 3600aggagggggg tttgggctgg gacaaaggca cttctcataa cagctagaag actgggaaac 3660aaggcgcatg ggtgaaagct acagagggcc tagatggaga ataaggagcg agaaaggaac 3720tgctgagctt ttggctgtgg ggtaaagggt caggagagct gaggaagccc tggcctgagg 3780tagcctcatc ctgatcttcc tgcagggagt tcctggtcgg ctccgacgcc ttctgctgca 3840acttggctag tgccctactt tcagacactg tccagcacct tcaggcctcg gtgggagagc 3900agggggaggg gagcaccatc ttgcaacaca tcagcaccct tgaggtgggg caggagggga 3960ggggacaagg ctgggtgggg ctgaggttga actgggttga gcattgggcc ctggaagaaa 4020attggttgga tgctggaagc aaattggtgt tcctgtggtt aactgctagc tagcaggcaa 4080attagatttt aaaagcatgc aaatgcacaa aaacttctgg agtctacagt tgtgcttcct 4140tatagtatat gtgtgaatgc aggcctgggg attggaggga ttgaaggaca tgggtaagag 4200caaagctcac tgtttaccac cctcatttct gtagagcata tatcagaggg accccctgaa 4260gctggtggcc actttcagac aaatacttca aggagagaaa aaagctgtta tggaacaggt 4320attgtgatat tccacctccc accccaactc aatcccctga gactttggcc tgagccatga 4380caaactagaa agaatttgaa cctcagtaaa ggctcagtgt tctaggccca ggaatgacca 4440aaggaggttc ctagggtcag agtgaacccc aagtcaagct cagggaatct ttctatgagg 4500gactgaaggt aagaggccgg ggagaacaga gcaagggata aggagctgat tctgctagga 4560gcaaggtctt atctccacga tattccaaaa ggtcaggaag aactgccaaa ggggagaggg 4620gaacaagaaa acgctatctg cagagcagag agtggaggcc aggtatagag ggatgagcag 4680agtgtttcac ttcttggcat ctgtccttcc tgtgtagttc cgccacttgc caatgccttt 4740ccactggaag caggaagaac tcaagtttaa gacaggcttg cggaggctgc agcaccgagt 4800aggggagatc caccttctcc gagaagccct gcagaagggg gctgaggctg gccaaggtgg 4860gggccagggt ggttctgggg agtgtgtagg agtggttgcc tcttggatct caaccttatc 4920tgaacctcta atctgtctgc acccttgatt tctgccccca accctcagtg tctctgcaca 4980gcttgataga aactcctgct aatgggactg ggccaagtga ggtgagtaat gggctgacag 5040gtggagacct tggtcaaagt gcagctggag ggatggaagc tagacctcag aaagacacag 5100gctgaagtag ggcaagggaa tgccagagga gtgagaaaaa gagccgtatc ccaggagctg 5160ggtgtggagg cagcgtgagg ccctggctca ggcccctctc tgcccatagg ccctggccat 5220gctactgcag gagaccactg gagagctaga ggcagccaaa gccctagtgc tgaagaggat 5280ccagatttgg aaacggcagc agcagctggc agggaatggc gcaccgtttg aggagagcct 5340ggccccactc caggagaggt tgggctaggg ctgatgggga agagggggca agctgggggt 5400gggcagctga ccctgctgaa ggccctacag gtgagagaaa gaagccaggc gggagggcct 5460tggagtggac caagatgcat aaaagccagt tccagcgggg ctgtgcacac tgtcgttcag 5520gtcgcatcct gtacaagtgg gcctagtgga ggggcacaag cggggactca tccaacccag 5580gcttctctcc tcaagcccca tgcctagagg aataggaggg cttttccatt tggtttattg 5640ggtgggaaca ctttccaatt tgccacaaag cactgtaagt ggtggcagtt gtcctgggtg 5700caagagccgt cgggggagag gcagctgggt ttccacaggg ggtgtaggca ctgagaatga 5760acctcccacc cagaccctag gccaacagat cacagaaccc ccttcagccc aggtgccttg 5820cagccacacc cactacccac cccacttctc cacacatgat agcctttctc cctgggtata 5880ggggaagggg gtctgggccg gagcaagcag ccttaatcct gtgccccctg accactgtcc 5940tggccccagg tgtgaaagcc tggtggacat ttattcccag ctacagcagg aggtaggggc 6000ggctggtggg gagcttgagc ccaagacccg ggcatcgctg actggccggc tggatgaagt 6060cctgagaacc ctcgtcacca ggtattcccc gggagctccc agtctggcct agaacagacc 6120tcgggaagaa aagaaggggg ctagagctgt ggggagggca ccagcaggga cctagccccc 6180aactcccctt gtgtcctcct cactcccagt tgcttcctgg tggagaagca gcccccccag 6240gtactgaaga ctcagaccaa gttccaggct ggagttcgat tcctgttggg cttgaggttc 6300ctgggggccc cagccaagcc tccgctggtc agggccgaca tggtgacaga gaagcaggcg 6360cgggagctga gtgtgcctca gggtcctggg gctggagcgt aagctgggat tggacctggg 6420gttggagaag ggctgttagg gtgatggagg cagcctggag ggctggcact gaaaagagca 6480agggatgggg agggagggcc atgggatgtg gagaccctga atggtcaagg cagaggaaag 6540ggagggaccc atttagggct ggaatggggt gggggcatca tgatttggcc aagatgggga 6600ctcctccctt aagaacccaa acagagacat ggagatttag ggctggtgac agtgggtagt 6660ctacactcac ccatgcactc gccacacctg acgacagtga gatgagctcg ttcacactct 6720gacctcccct ggcagagaaa gcactggaga aatcatcaac aacactgtgc ccttggagaa 6780cagcattcct gggaactgct gctctgccct gttcaagaac ctggtgaggg gctttggggt 6840gcagtgaggg gggcaccact aggagactgt gggactctcc ttggagagga tgtcaggaag 6900cccaggagga gcggtctctg tcctcatgac ctcgcccttg ctctccctca ccccacccac 6960agcttctcaa gaagatcaag cggtgtgagc ggaagggcac tgagtctgtc acagaggaga 7020agtgcgctgt gctcttctct gccagcttca cacttggccc cggcaaactc cccatccagc 7080tccaggtgaa ccgtggccca gccctgcccc aatctgggac cccgagtcct cctccaatgc 7140cacacacaag ggccctggac cctcacctct tgtgactgcc ccatacccca tgtgtctggg 7200attcatgcac actggggccc gggtgagtgg gggtgagcaa gagcatggag tgcacagggc 7260agggaatggt agtggatagc agcaaacact tcggaagcac ttcctataga ccagggcact 7320ctattaaatg atacatacgc acatgcgtgc cagcacacac acgtctggtt ttcacaataa 7380cattatgagg taggcagtat tatcagcctc attttataga taaggacatt gagacagaga 7440gtttaagtag tttgtcccag tcacacagct aagtgttgga gctggtattt gaaacctgga 7500ggtctggttc catagcgatg actaataacc acttctctac ggtgaggccc tgattgagct 7560tcagaacgca tttaataaca tggcatgagc tttttgatta tgatgtgtga gtccaataac 7620ttctctgagt gctcagagcc agtcccctga ggaaacttct tgcttcacta agaaacccct 7680gtccggctgg gcatggtggc tcaagcctgt aatcccagca ctttgggagg ccgaggtggg 7740tagatcacaa ggtcaggagt tcaagaccag cctggccaat atggtgaaac cccgtctcca 7800ctaaaaatac aaaaattagc tgggcgtggt ggtgcaggcc tgtagtccca gctgctcggg 7860aggctaagca ggagaatcgc ttgaacccag gaggcggagg ttgcagtgag ccaagattgc 7920gccactgccc ttcagcctgg gcgacagagc aagactatgt ctcaaaaaca aaacaaaaca 7980actcagcact ttgggaggcc aaggtaggag gatcgcttga gcctgcaagt ttaagaccag 8040cctgggctac atagggagat ccaatctcta caaaaaataa aaaattggcc gggcatggtg 8100gctcacgcct gtaatcccag cactttggga ggccaaggcg ggcggatcat gaggtcagga 8160aatcgagacc atcctggcta acacggtgaa acctcgtatc tactaaaaat acaaaaaatt 8220agccaggcat ggtggcgggc gcctgagtcc cagctactcg ggaggctgaa gcaggagaat 8280ggcgtgaacc tgggagggag agcttgcagt gagccaagat cgcgccgctg cactccagcc 8340tgagtgacag agcgagactc tgtctcaaaa ataaataaat aaataattag ctggattagg 8400tggtacattt ctgtagttcc agctattcag gaggctgagg tggaaggatc acttgagccc 8460tgaaggctga ggctgcagtg agctgagatt gcactactgc actccagcct gggcaacaga 8520gtgagatact atctaaaaaa aaaaaaaaaa aaaaaaaagg aaagaaagaa agaaaagaaa 8580cccctgtcct caccctcttc aggccctgtc tctgcccctg gtggtcatcg tccatggcaa 8640ccaagacaac aatgccaaag ccactatcct gtgggacaat gccttctctg agatggtgag 8700gaaagtcctg gagttggagg gaacaggggc agggtgggtt ctaacatggg cagtggtgca 8760ggcctgctga tggggtggtg ggcatgttta aatgggtgtg accttaacac tttctcatgg 8820gcctgctttc gtgcttctga cctcttttca ccccagtctt aacaactatc aggccacagc 8880actgtaacct agaaaaaaca gcatgtttgt gagcgatatc aggggctgtg gaggggtagg 8940ccacaggcag gtgggaggga tgaaggccgg cccgaggaat aacaagacgg tagcctgcag 9000tgctctcttc ttcccccttc tccccaggac cgcgtgccct ttgtggtggc tgagcgggtg 9060ccctgggaga agatgtgtga aactctgaac ctgaagttca tggctgaggt ggggaccaac 9120cgggggctgc tcccagagca cttcctcttc ctggcccaga agatcttcaa tgacaacagc 9180ctcagtatgg aggccttcca gcaccgttct gtgtcctggt cgcagttcaa caaggtcatt 9240ctcctgccct ttggacctcc cacccccaag ctcttcatcc ctggggcact cagggcctgc 9300tcagcctcca tgcagggacc ttccactgga ttctccacag tgccccctca ggtcctttag 9360gaaggcctgt catggaccag ggaggaaaaa ccccaggcct gggggttggc tctggagatg 9420cgttctctga catccctgag gttttggtct gggggccatc tgtccttcct ctttaccagt 9480gacttgcatg actcacccag gttgtgtgta aacagagctc tgattcaaag tgactttgac 9540ctgttggaaa aatagttcct ggccgggcac agcggctcat gcctgtaatc ccagtctttg 9600acatgccggg gtgggtggat cacctgaggt caggagtttg agaccagcct ggccaacatg 9660gtgaaactcc atctctacta aaaatacaaa aattagccag ttgcggtggc acatgcctgt 9720aatcccagct acatgggagg ctgacgcagg agaattgctt gaacccagga ggtggaggtt 9780gcagtgagct gagatcatac cactgcactc aagcctgggt gacagagcaa gactctgtct 9840caaaaaaaaa aaaaaaaaaa ggccaggcat ggtggttcat gcctgtaatc ccagcacttt 9900gggaggccga gacggataga tcacctgagg tcaggagttc gagaccagcc tggccaacat 9960ggcaaaaccc cgtctctact aaaaacaaaa aaatagccag gagtggtcgt ttgcgtctgt 10020aatcccagct actcggctga ggcaggaggt gaacccagga ggtagaggct gcagggaaga 10080tgaaaccatt gcactccagc ctgggcaaga ctctgtatca aaaaaaaaaa aaaaaaaggc 10140taggtgtggt ggctcacacc tgtaatccca gcactttggg aggctgaggc gggcggatca 10200caaggtcaag agatcgagac catcctgacc aacatggtga aaccccgtct ctactaaaaa 10260tacaaaaatt acctgggcat ggtggcgcat gcctgtagtc ccaactactc gggaggctga 10320ggcaggagaa tcacttgaac ctgggaggca gaggttgcag cgagccaaga ttgtgccact 10380gcactccagc ctgccaacag aatgagattc tgtctaaaaa aaaaaagaaa gaaagaaaga 10440aagaaaaaga attcctgttg caaaaactga acaaaatccc acagggacat gtgcagtaat 10500accagctacc acgtgttgac agcttatatg ccaggcgctg tgcttaacac cttatgtatg 10560ttatctcact taatcctccc aacatctctt tgaggtagat actattatta tccccatttt 10620acagatgagg aatctgatgc tcagagggtt atgtagtttg ttcaagttcc caaagcaggt 10680gagtgccatg gctaggagag aaccacatat ttctgactct tgctctttta ttttatgtta 10740tattatgtta ttttatgttt tggttttttt ttcttttctt tctttctttc tttctttctt 10800tctttctctt tctttctttc tttctttctt tctttctttc tttctttctt tctctttctc 10860tttctctctt tttctttctt ttgtgtgaga cagaatcttt aaagagaaga aagaaatgct 10920catgtgacca gagggtgtgt tagctaaagg gagcaagaca gtcacaccca gcaggttacc 10980ttcctttggg cgtcacctct gccacacctc cttagggaga gggtgtagca tagtagttaa 11040gaggggctcc agggccagaa tgcctgggtt taaatcctag ctctgcctct taccagctat 11100gtagacctgg gcaagtcatt cgacgttttt ggacttccat ttcttcatct gtaagatgga 11160attattataa tccctacttc catagcctgg taaagagcaa ataaatatat ggaaaggctt 11220gaaatagtgg ctggcacgtg taagcattag gattggtcgt tgtcattgat ggagtctcag 11280gttcggtctg atcctcagcc ctgtgattct gtcgtgaggg cactcacagc tcactgcctg 11340ccctaaacag gctccagctc tggccctccc tcggctcaca cctttccccc tctcccccta 11400ggagatcctg ctgggccgtg gcttcacctt ttggcagtgg tttgatggtg tcctggacct 11460caccaaacgc tgtctccgga gctactggtc tgaccggtga gtccccaccc tgggtagtct 11520gagcagccat acaccagtca cctccatact cactgcccat gccccatcct ctccttcatc 11580ccggccaggc tgatcattgg cttcatcagc aaacagtacg ttactagcct tcttctcaat 11640gagcccgacg gaacctttct cctccgcttc agcgactcag agattggggg catcaccatt 11700gcccatgtca tccggggcca ggatggtgag gccaccccag ccagtcctct gtctctgtgc 11760ctgtgccctc tggggtttct tctgggaatg aaatgtcctg accttcctga tgccgatcct 11820gatcttcagg aagttcttcc agcttctctt cttccttctg tggtctaaat gttcaccttc 11880tcactgtgag ctctgtggga acggagacta gtgggtctct ctccctcagg agccccaccc 11940taggtcctct ctcccttgcc ttggtggagt gagaacaggt cttatggtag gggttgggga 12000aggggaagaa agtccggaca gagggatctc agggtctcct tcctaccata ggctctccac 12060agatagagaa catccagcca ttctctgcca aagacctgtc cattcgctca ctgggggacc 12120gaatccggga tcttgctcag ctcaaaaatc tctatcccaa gaagcccaag gatgaggctt 12180tccggagcca ctacaagcgt gagctggaac tggcagctct gattccttcc tgtcacccac 12240ttcctccctg ctccccgctg ccctcctctc cctgcccgtg tgtcatcctg atgtcactcc 12300ctatttcata gctgtgcttc tcttacttcc ccatgatcca

tgcccacctt ttccacctcc 12360cttcctccct aaccccagag cactccatgg ctgtcttttc cttctcacaa cagctgaaca 12420gatgggtaag gatggcaggg gttatgtccc agctaccatc aagatgaccg tggaaaggtg 12480agtgtggtgg tatggacagt gggtaggtca ggggcttagt gcttatctgc aggaaggagg 12540ggtggcatca acccttggtc agtcacatgt acctccttcc ctcctccagg gaccaaccac 12600ttcctacccc agagctccag atgcctacca tggtgccttc ttatgacctt ggaatggccc 12660ctgattcctc catgagcatg cagcttggcc cagatatggt gtaaggagct ggaaagacag 12720gaatgggagt ggtctgtgca gatgggctaa tcttagcatg ggcagctggg agagctggca 12780ctgggggctg aacagggaat cttcctttcc atgagaggga cacctgttca aaagcagggt 12840gtggtggtgt ccaggagaag ggctggcatc agggggtctg ttttctttcc ccaggcccca 12900ggtgtaccca ccacactctc actccatccc cccgtatcaa ggcctctccc cagaagaatc 12960agtcaacgtg ttgtcagcct tccaggagta agtgaaaaac ctcatgggga taccatccca 13020ctctaagggg gtgggcattt gaattgttag aagaggctct tctgtgagaa aggagcagca 13080aatgctaaca gcctgtcttc ttctcttctg tccactctaa tgagggggta gtagttaaga 13140tctggactgc ctaggtttga attctagctc caccacttac tggtttgggg caaattactt 13200agcctttggt gccttatctg cacaatgggg gataataatg ctaataataa taacctacct 13260cactgcatta ttgtggagat taaatgagtt cataacactt aaaaagctga gcatagtgca 13320tggctcatag caaaagctgt gtaagtccag tcgtggatca cttaatgaag gagcattttc 13380tgtctttggc agtttcataa ttatgcgaat accattgagt ataattacac aaacctagat 13440ggtatagact actatacact gaggctatat tgtgtagcct attgatccta gctttaaacc 13500cgagcagcat gatactgttc tgaatagtat aaggaaatag taacataatg gtaaatattt 13560gtgtgatagg aattttcagc ttgattataa tttttttttt ttgagacagg gtctcactca 13620ctggagtgca gtggtgcgat cttagctccc tgcaacctcc gcctcttggg ctcgagcaat 13680cctcctgctg tagtgcacca cgacactcgg ctaattcttt tttaagattt ttctgcagac 13740aaggtctcac ttactgccca agctggtctc aaactcctgg gcttaagtga tcctcccacc 13800tcggcctccc aaagcgttag gattacaggc gtgagtcact ctgcctggcc ttgattataa 13860tcttatggga ccactgtggt ctgtagttga cagaaatgtc gttaatgtgg tgcatgactg 13920ttattattat tttctgtcct gcccctgaga gccactgtca cttctctgct gtattggttt 13980ttgtttactc atctgttttg gccttgaaat ggcctagaca tttttcttcc cgaagtatga 14040cactcgggtg cttattaact tagtcaagac acaacatctc ccttcccaga aggtgaggcg 14100ggagtgagga cttggggact taagaactac caaagttcag agtccaaaga aacattagaa 14160attggctaat ccacccccat aacacgcaca ttttacagat gagaagactg agctcagagc 14220atagaaatag cttgcccagg ccatgactaa gtcaggataa ggagctggag cttgtttcct 14280cactcagtgg tcctgacttt gcaccactct gcatttgcct agcctgcctt cctctaactg 14340tgctctccct acttccaggc ctcacctgca gatgcccccc agcctgggcc agatgagcct 14400gccctttgac cagcctcacc cccagtgagt gacaaagccc ctcctgaccc catgtgcctc 14460ttctttcctg gccttgcccc gctctcctta tttccattgc tggttcctgg caggggcctg 14520ctgccgtgcc agcctcagga gcatgctgtg tccagccctg accccctgct ctgctcagat 14580gtgaccatgg tggaagacag ctgcctgagc cagccagtga cagcgtttcc tcagggcact 14640tggtgagtgg cagcttggga gtggaggctg ggtggcatct aggggagtgg gcgccatgcc 14700tactccactg cttctcccat ctccttgcag gattggtgaa gacatattcc ctcctctgct 14760gcctcccact gaacaggacc tcactaagct tctcctggag gggcaagggg agtcgggggg 14820agggtccttg ggggcacagc ccctcctgca gccctcccac tatgggcaat ctgggatctc 14880aatgtcccac atggacctaa gggccaaccc cagttggtga tcccagctgg agggagaacc 14940caaagagaca gctcttctac tacccccaca gacctgctct ggacacttgc tcatgccctg 15000ccaagcagca gatggggagg gtgccctcct atccccacct actcctgggt caggaggaaa 15060agactaacag gagaatgcac agtgggtgga gccaatccac tccttccttt ctatcattcc 15120cctgcccacc tccttccagc actgactgga agggaagttc aggctctgag acacgcccca 15180acatgcctgc acctgcagcg cgcacacgca cgcacacaca catacagagc tctctgaggg 15240tgatggggct gagcaggagg ggggctgggt aagagcacag gttagggcat ggaaggcttc 15300tccgcccatt ctgacccagg gcctaggacg gataggcagg aacatacaga cacatttaca 15360ctagaggcca gggatagagg atattgggtc tcagccctag gggaatggga agcagctcaa 15420gggaccctgg gtgggagcat aggaggggtc tggacatgtg gttactagta caggttttgc 15480cctgattaaa aaatctccca aagccccaaa ttcctgttag ccaggtggag gcttctgata 15540cgtgtatgag actatgcaaa agtacaaggg ctgagattct tcgtgtatag ctgtgtgaac 15600gtgtatgtac ctaggatatg ttaaatatat agctggcacc ttagttgcat gaccacatag 15660aacatgtgtc tatctgcttt tgcctacgtg acaacacaaa tttgggaggg tgagacactg 15720cacagaagac agcagcaagt gtgctggcct ctctgacata tgctaacccc caaatactct 15780gaatttggag tctgactgtg cccaagtggg tccaagtggc tgtgacatct acgtatggct 15840ccacacctcc aatgctgcct gggagccagg gtgagagtct gggtccaggc ctggccatgt 15900ggccctccag tgtatgagag ggccctgcct gctgcatctt ttctgttgcc ccatccaccg 15960ccagcttccc ttcactcccc tatcccattc tccctctcaa ggcaggggtc atagatccta 16020agccataaaa taaattttat tccaaaataa caaaataaat aatctactgt acacaatctg 16080aaaagaaaga cgctctaact gctcagatag gtgctgcggt ccagccccca gctggaggag 16140accctgagtc caacccaggc ctcccgaggg ggccagtgaa gggatcccac acccaccgcc 16200cctatgtagg gcagggaaga aattgcaaag gacttggggg atagatggga atgggagggc 16260aaactgcagc acttgttaaa ttaattaaag aaacaaacca gaagcacaaa aacggggaag 16320gagaggggag aaggagcagg tccagtgttc ccaggccccc aattctgggg gcaaatgttg 16380ccacttttag ctggaccttc ccagggaagt ccccctttcc cccttgtcca aactgagtcc 16440aactgctcac accactggtg caaacctaaa gagaatggga gtgtgttgtg tgagggaggg 1650013697DNAH. sapiens 13ttctcttccc tttcacttcc acactttgtc cctcccccca aattttttat ttttttgtcc 60acgccccaac aatttttttt gttttttttt tttaaaagaa tccaccccct ttcctgagct 120ccctgactgg gatttcactt cttcacctcc caccgtggcc accagagtta aaaacctatc 180ttataatata aaataaaaaa ggaaagaaag aaagaaaaga aaccctgtcc tcaccctctt 240caggccctgg tctctgcccc tggtggtcat cgtccatggc aaccaagaca acatgccaaa 300ccactatcct gtgggacatg ccttctctga gatggaccgc gtgccctttg tggtggctga 360gcgggtgccc tgggagaaga tgtgtgaaac tctgaacctg aagttcatgg ctgaggtggg 420gaccaaccgg gggctgctcc cagagcactt cctcttcctg gcccagaaga tcttcaatga 480caacagcctc agtatggagg ccttccagca ccgttctgtg tcctggtcgc agttcaacaa 540ggagatcctg ctggccgtgg cttcaccttt tggcagtggt ttgatggtgt cctggacctc 600accaacgctg tctccggagc tactggtctg accggtgagt ccccaccctg ggtagtctga 660gcagccatac accagtcacc tccatactca ctgccca 69714423DNAH. sapiensunsure58unknown 14tggacagtgg gtaggtcagg ggcttagtgc ttatctgcag gaaggagggg tggcatcnac 60ccttggtcag tcacatgtac ctccttccct cctccaggga ccaaccactt cctaccccag 120agctccagat gcctaccatg gtgccttctt atgaccttgg aatggcccct gattcctcca 180tgagcatgca gcttggccca gatatggtgc cccaggtgta cccaccacac tctcactcca 240tccccccgta tcaaggcctc tccccagaag aatcagtcaa cgtgttgtca gccttccagg 300agcctcacct gcagatgccc cccagcctgg gccagatgag cctgcccttt gaccagcctc 360acccccaggg cctgctgtcg tgccagcctc tggagcatgc tgtgtccagc cctgaccccc 420tgc 4231520DNAArtificial SequenceAntisense Oligonucleotide 15agtgagcgaa tggacaggtc 201620DNAArtificial SequenceAntisense Oligonucleotide 16cgctgtcact ggctggctca 201720DNAArtificial SequenceAntisense Oligonucleotide 17ttgatgattt ctccagtgct 201820DNAArtificial SequenceAntisense Oligonucleotide 18aggacttcat ccagccggcc 201920DNAArtificial SequenceAntisense Oligonucleotide 19cccaggaacc tcaagcccaa 202020DNAArtificial SequenceAntisense Oligonucleotide 20gtcacccaga agatgccgca 202120DNAArtificial SequenceAntisense Oligonucleotide 21tttccacggt catcttgatg 202220DNAArtificial SequenceAntisense Oligonucleotide 22aagatggtgc tcccctcccc 202320DNAArtificial SequenceAntisense Oligonucleotide 23gccgtttcca aatctggatc 202420DNAArtificial SequenceAntisense Oligonucleotide 24ctttggctgc ctctagctct 202520DNAArtificial SequenceAntisense Oligonucleotide 25gtttggtgag gtccaggaca 202620DNAArtificial SequenceAntisense Oligonucleotide 26catctgcagg tgaggctcct 202720DNAArtificial SequenceAntisense Oligonucleotide 27tggcccttag gtccatgtgg 202820DNAArtificial SequenceAntisense Oligonucleotide 28ctatctgtgg agagccatcc 202920DNAArtificial SequenceAntisense Oligonucleotide 29attgagaaga aggctagtaa 203020DNAArtificial SequenceAntisense Oligonucleotide 30gctgatgtgt tgcaagatgg 203120DNAArtificial SequenceAntisense Oligonucleotide 31gccccatcac cctcagagag 203220DNAArtificial SequenceAntisense Oligonucleotide 32ccctctgata tatgctctca 203320DNAArtificial SequenceAntisense Oligonucleotide 33gaaggctagt aacgtactgt 203420DNAArtificial SequenceAntisense Oligonucleotide 34gttccgtcgg gctcattgag 203520DNAArtificial SequenceAntisense Oligonucleotide 35gtcactggct ggctcaggca 203620DNAArtificial SequenceAntisense Oligonucleotide 36ttcagagttt cacacatctt 203720DNAArtificial SequenceAntisense Oligonucleotide 37caggccccat aggtctgtag 203820DNAArtificial SequenceAntisense Oligonucleotide 38tatcaagctg tgcagagaca 203920DNAArtificial SequenceAntisense Oligonucleotide 39caggaactcc cagggctggc 204020DNAArtificial SequenceAntisense Oligonucleotide 40gctctgtatg tgtgtgtgcg 204120DNAArtificial SequenceAntisense Oligonucleotide 41agatcccgga ttcggtcccc 204220DNAArtificial SequenceAntisense Oligonucleotide 42cggtgcgcca ttccctgcca 204320DNAArtificial SequenceAntisense Oligonucleotide 43gggatagaga tttttgagct 204420DNAArtificial SequenceAntisense Oligonucleotide 44gatctgggac ttggaggttg 204520DNAArtificial SequenceAntisense Oligonucleotide 45tccaaggtca taagaaggca 204620DNAArtificial SequenceAntisense Oligonucleotide 46atgatcagcc ggtcagacca 204720DNAArtificial SequenceAntisense Oligonucleotide 47cccaggaatg ctgttctcca 204820DNAArtificial SequenceAntisense Oligonucleotide 48tctcaggact tcatccagcc 204920DNAArtificial SequenceAntisense Oligonucleotide 49ccagcaggat ctccttgttg 205020DNAArtificial SequenceAntisense Oligonucleotide 50tccagtgctt tctgctccag 205120DNAArtificial SequenceAntisense Oligonucleotide 51acagtgtctg aaagtagggc 205220DNAArtificial SequenceAntisense Oligonucleotide 52gctggccctg ctagcacctc 205320DNAArtificial SequenceAntisense Oligonucleotide 53ccacagagac atgatctggg 205420DNAArtificial SequenceAntisense Oligonucleotide 54gtcttaaact tgagttcttc 205520DNAArtificial SequenceAntisense Oligonucleotide 55tctagctctc cagtggtctc 205620DNAArtificial SequenceAntisense Oligonucleotide 56ggccctgacc agcggaggct 205720DNAArtificial SequenceAntisense Oligonucleotide 57cctctgtgac agactcagtg 205820DNAArtificial SequenceAntisense Oligonucleotide 58tccatactga ggctgttgtc 205920DNAArtificial SequenceAntisense Oligonucleotide 59cctggccccg gatgacatgg 206020DNAArtificial SequenceAntisense Oligonucleotide 60gaaggcacca tggtaggcat 206120DNAArtificial SequenceAntisense Oligonucleotide 61ccaatccaag tgccctgagg 206220DNAArtificial SequenceAntisense Oligonucleotide 62cagctgggat caccaactgg 206320DNAArtificial SequenceAntisense Oligonucleotide 63gtgtctcaga gcctgaactt 206420DNAArtificial SequenceAntisense Oligonucleotide 64taagcagtgg ctgccccagc 206520DNAArtificial SequenceAntisense Oligonucleotide 65cctccctctt cagtgtaagc 206620DNAArtificial SequenceAntisense Oligonucleotide 66agaagccttc catgccctaa 206720DNAArtificial SequenceAntisense Oligonucleotide 67tatgttcctg cctatccgtc 206820DNAArtificial SequenceAntisense Oligonucleotide 68caactaaggt gccagctata 206920DNAArtificial SequenceAntisense Oligonucleotide 69tggtcatgca actaaggtgc 207020DNAArtificial SequenceAntisense Oligonucleotide 70atttgtgttg tcacgtaggc 207120DNAArtificial SequenceAntisense Oligonucleotide 71tctcaccctc ccaaatttgt 207220DNAArtificial SequenceAntisense Oligonucleotide 72agcacacttg ctgctgtctt 207320DNAArtificial SequenceAntisense Oligonucleotide 73gccaggcctg gacccagact 207420DNAArtificial SequenceAntisense Oligonucleotide 74gggcaacaga aaagatgcag 207520DNAArtificial SequenceAntisense Oligonucleotide 75aatgtcagct tttaatctgt 207620DNAArtificial SequenceAntisense Oligonucleotide 76gagtcaatgc ctgagatggg 207720DNAArtificial SequenceAntisense Oligonucleotide 77caggaagcaa ctgggagtga 207820DNAArtificial SequenceAntisense Oligonucleotide 78ccatctcaga gaaggcattg 207920DNAArtificial SequenceAntisense Oligonucleotide 79tgcacatgtc cctgtgggat 208020DNAArtificial SequenceAntisense Oligonucleotide 80gggactcacc ggtcagacca 208120DNAArtificial SequenceAntisense Oligonucleotide 81agtggttggt ccctggagga 208220DNAArtificial SequenceAntisense Oligonucleotide 82agctccttac accatatctg 208320DNAArtificial SequenceAntisense Oligonucleotide 83caaagtgtgg aagtgaaagg 208420DNAArtificial SequenceAntisense Oligonucleotide 84ctctggtggc cacggtggga 208520DNAArtificial SequenceAntisense Oligonucleotide 85ggtgtatggc tgctcagact 208620DNAArtificial SequenceAntisense Oligonucleotide 86aggaggtaca tgtgactgac 208720DNAHomo sapiens 87gacctgtcca ttcgctcact 208820DNAHomo sapiens 88tgagccagcc agtgacagcg 208920DNAHomo sapiens 89agcactggag aaatcatcaa 209020DNAHomo sapiens 90ttgggcttga ggttcctggg 209120DNAHomo sapiens 91gatccagatt tggaaacggc 209220DNAHomo sapiens 92agagctagag gcagccaaag 209320DNAHomo sapiens 93tgtcctggac ctcaccaaac 209420DNAHomo sapiens 94aggagcctca cctgcagatg 209520DNAHomo sapiens 95ccacatggac ctaagggcca 209620DNAHomo sapiens 96ggatggctct ccacagatag 209720DNAHomo sapiens 97ttactagcct tcttctcaat 209820DNAHomo sapiens 98ccatcttgca acacatcagc 209920DNAHomo sapiens 99ctctctgagg gtgatggggc

2010020DNAHomo sapiens 100tgagagcata tatcagaggg 2010120DNAHomo sapiens 101acagtacgtt actagccttc 2010220DNAHomo sapiens 102ctcaatgagc ccgacggaac 2010320DNAHomo sapiens 103tgcctgagcc agccagtgac 2010420DNAHomo sapiens 104aagatgtgtg aaactctgaa 2010520DNAHomo sapiens 105ctacagacct atggggcctg 2010620DNAHomo sapiens 106tgtctctgca cagcttgata 2010720DNAHomo sapiens 107gccagccctg ggagttcctg 2010820DNAHomo sapiens 108cgcacacaca catacagagc 2010920DNAHomo sapiens 109ggggaccgaa tccgggatct 2011020DNAHomo sapiens 110tggcagggaa tggcgcaccg 2011120DNAHomo sapiens 111caacctccaa gtcccagatc 2011220DNAHomo sapiens 112tgccttctta tgaccttgga 2011320DNAHomo sapiens 113tggtctgacc ggctgatcat 2011420DNAHomo sapiens 114tggagaacag cattcctggg 2011520DNAHomo sapiens 115caacaaggag atcctgctgg 2011620DNAHomo sapiens 116ctggagcaga aagcactgga 2011720DNAHomo sapiens 117gaggtgctag cagggccagc 2011820DNAHomo sapiens 118cccagatcat gtctctgtgg 2011920DNAHomo sapiens 119gagaccactg gagagctaga 2012020DNAHomo sapiens 120agcctccgct ggtcagggcc 2012120DNAHomo sapiens 121cactgagtct gtcacagagg 2012220DNAHomo sapiens 122cctcagggca cttggattgg 2012320DNAHomo sapiens 123aagttcaggc tctgagacac 2012420DNAHomo sapiens 124gcttacactg aagagggagg 2012520DNAHomo sapiens 125ttagggcatg gaaggcttct 2012620DNAHomo sapiens 126gacggatagg caggaacata 2012720DNAHomo sapiens 127tatagctggc accttagttg 2012820DNAHomo sapiens 128gcaccttagt tgcatgacca 2012920DNAHomo sapiens 129gcctacgtga caacacaaat 2013020DNAHomo sapiens 130aagacagcag caagtgtgct 2013120DNAHomo sapiens 131agtctgggtc caggcctggc 2013220DNAHomo sapiens 132acagattaaa agctgacatt 2013320DNAHomo sapiens 133caatgccttc tctgagatgg 2013420DNAHomo sapiens 134atcccacagg gacatgtgca 2013520DNAHomo sapiens 135tcctccaggg accaaccact 2013620DNAHomo sapiens 136agtctgagca gccatacacc 201373790DNAHomo sapiens 137gctgagaaag ggagaagaca gcagaggggt tgccgagaga aaggcctatt ggaggaacct 60gagcaggagg ggtaaggatt ctgccttgag gagaaaagag ctggggcaac ctccaagtcc 120cagatcatgt ctctgtgggg tctggtctcc aagatgcccc cagaaaaagt gcagcggctc 180tatgtcgact ttccccaaca cctgcggcat cttctgggtg actggctgga gagccagccc 240tgggagttcc tggtcggctc cgacgccttc tgctgcaact tggctagtgc cctactttca 300gacactgtcc agcaccttca ggcctcggtg ggagagcagg gggaggggag caccatcttg 360caacacatca gcacccttga gagcatatat cagagggacc ccctgaagct ggtggccact 420ttcagacaaa tacttcaagg agagaaaaaa gctgttatgg aacagttccg ccacttgcca 480atgcctttcc actggaagca ggaagaactc aagtttaaga caggcttgcg gaggctgcag 540caccgagtag gggagatcca ccttctccga gaagccctgc agaagggggc tgaggctggc 600caagtgtctc tgcacagctt gatagaaact cctgctaatg ggactgggcc aagtgaggcc 660ctggccatgc tactgcagga gaccactgga gagctagagg cagccaaagc cctagtgctg 720aagaggatcc agatttggaa acggcagcag cagctggcag ggaatggcgc accgtttgag 780gagagcctgg ccccactcca ggagaggtgt gaaagcctgg tggacattta ttcccagcta 840cagcaggagg taggggcggc tggtggggag cttgagccca agacccgggc atcgctgact 900ggccggctgg atgaagtcct gagaaccctc gtcaccagtt gcttcctggt ggagaagcag 960cccccccagg tactgaagac tcagaccaag ttccaggctg gagttcgatt cctgttgggc 1020ttgaggttcc tgggggcccc agccaagcct ccgctggtca gggccgacat ggtgacagag 1080aagcaggcgc gggagctgag tgtgcctcag ggtcctgggg ctggagcaga aagcactgga 1140gaaatcatca acaacactgt gcccttggag aacagcattc ctgggaactg ctgctctgcc 1200ctgttcaaga acctgcttct caagaagatc aagcggtgtg agcggaaggg cactgagtct 1260gtcacagagg agaagtgcgc tgtgctcttc tctgccagct tcacacttgg ccccggcaaa 1320ctccccatcc agctccaggc cctgtctctg cccctggtgg tcatcgtcca tggcaaccaa 1380gacaacaatg ccaaagccac tatcctgtgg gacaatgcct tctctgagat ggaccgcgtg 1440ccctttgtgg tggctgagcg ggtgccctgg gagaagatgt gtgaaactct gaacctgaag 1500ttcatggctg aggtggggac caaccggggg ctgctcccag agcacttcct cttcctggcc 1560cagaagatct tcaatgacaa cagcctcagt atggaggcct tccagcaccg ttctgtgtcc 1620tggtcgcagt tcaacaagga gatcctgctg ggccgtggct tcaccttttg gcagtggttt 1680gatggtgtcc tggacctcac caaacgctgt ctccggagct actggtctga ccgcgactca 1740gagattgggg gcatcaccat tgcccatgtc atccggggcc aggatggctc tccacagata 1800gagaacatcc agccattctc tgccaaagac ctgtccattc gctcactggg ggaccgaatc 1860cgggatcttg ctcagctcaa aaatctctat cccaagaagc ccaaggatga ggctttccgg 1920agccactaca agcctgaaca gatgggtaag gatggcaggg gttatgtccc agctaccatc 1980aagatgaccg tggaaaggga ccaaccactt cctaccccag agctccagat gcctaccatg 2040gtgccttctt atgaccttgg aatggcccct gattcctcca tgagcatgca gcttggccca 2100gatatggtgc cccaggtgta cccaccacac tctcactcca tccccccgta tcaaggcctc 2160tccccagaag aatcagtcaa cgtgttgtca gccttccagg agcctcacct gcagatgccc 2220cccagcctgg gccagatgag cctgcccttt gaccagcctc acccccaggg cctgctgccg 2280tgccagcctc aggagcatgc tgtgtccagc cctgaccccc tgctctgctc agatgtgacc 2340atggtggaag acagctgcct gagccagcca gtgacagcgt ttcctcaggg cacttggatt 2400ggtgaagaca tattccctcc tctgctgcct cccactgaac aggacctcac taagcttctc 2460ctggaggggc aaggggagtc ggggggaggg tccttggggg cacagcccct cctgcagccc 2520tcccactatg ggcaatctgg gatctcaatg tcccacatgg acctaagggc caaccccagt 2580tggtgatccc agctggaggg agaacccaaa gagacagctc ttctactacc cccacagacc 2640tgctctggac acttgctcat gccctgccaa gcagcagatg gggagggtgc cctcctatcc 2700ccacctactc ctgggtcagg aggaaaagac taacaggaga atgcacagtg ggtggagcca 2760atccactcct tcctttctat cattcccctg cccacctcct tccagcactg actggaaggg 2820aagttcaggc tctgagacac gccccaacat gcctgcacct gcagcgcgca cacgcacgca 2880cacacacata cagagctctc tgagggtgat ggggctgagc aggagggggg ctgggtaaga 2940gcacaggtta gggcatggaa ggcttctccg cccattctga cccagggcct aggacggata 3000ggcaggaaca tacagacaca tttacactag aggccaggga tagaggatat tgggtctcag 3060ccctagggga atgggaagca gctcaaggga ccctgggtgg gagcatagga ggagtctgga 3120catgtggtta ctagtacagg ttttgccctg attaaaaaat ctcccaaagc cccaaattcc 3180tgttagccag gtggaggctt ctgatacgtg tatgagacta tgcaaaagta caagggctga 3240gattcttcgt gtatagctgt gtgaacgtgt atgtacctag gatatgttaa atatatagct 3300ggcaccttag ttgcatgacc acatagaaca tgtgtctatc tgcttttgcc tacgtgacaa 3360cacaaatttg ggagggtgag acactgcaca gaagacagca gcaagtgtgc tggcctctct 3420gacatatgct aacccccaaa tactctgaat ttggagtctg actgtgccca agtgggtcca 3480agtggctgtg acatctacgt atggctccac acctccaatg ctgcctggga gccagggtga 3540gagtctgggt ccaggcctgg ccatgtggcc ctccagtgta tgagagggcc ctgcctgctg 3600catcttttct gttgccccat ccaccgccag cttcccttca ctcccctatc ccattctccc 3660tctcaaggca ggggtcatag atcctaagcc ataaaataaa ttttattcca aaataacaaa 3720ataaataatc tactgtacac aatctgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3780aaaaaaaaaa 37901383667DNAHomo sapiens 138gacagagcta cagacctatg gggcctggaa gtgcccgctg agaaagggag aagacagcag 60aggggttgcc gaggcaacct ccaagtccca gatcatgtct ctgtggggtc tggtctccaa 120gatgccccca gaaaaagtgc agcggctcta tgtcgacttt ccccaacacc tgcggcatct 180tctgggtgac tggctggaga gccagccctg agcatatatc agagggaccc cctgaagctg 240gtggccactt tcagacaaat acttcaagga gagaaaaaag ctgttatgga acagttccgc 300cacttgccaa tgcctttcca ctggaagcag gaagaactca agtttaagac aggcttgcgg 360aggctgcagc accgagtagg ggagatccac cttctccgag aagccctgca gaagggggct 420gaggctggcc aagtgtctct gcacagcttg atagaaactc ctgctaatgg gactgggcca 480agtgaggccc tggccatgct actgcaggag accactggag agctagaggc agccaaagcc 540ctagtgctga agaggatcca gatttggaaa cggcagcagc agctggcagg gaatggcgca 600ccgtttgagg agagcctggc cccactccag gagaggtgtg aaagcctggt ggacatttat 660tcccagctac agcaggaggt aggggcggct ggtggggagc ttgagcccaa gacccgggca 720tcgctgactg gccggctgga tgaagtcctg agaaccctcg tcaccagttg cttcctggtg 780gagaagcagc ccccccaggt actgaagact cagaccaagt tccaggctgg agttcgattc 840ctgttgggct tgaggttcct gggggcccca gccaagcctc cgctggtcag ggccgacatg 900gtgacagaga agcaggcgcg ggagctgagt gtgcctcagg gtcctggggc tggagcagaa 960agcactggag aaatcatcaa caacactgtg cccttggaga acagcattcc tgggaactgc 1020tgctctgccc tgttcaagaa cctgcttctc aagaagatca agcggtgtga gcggaagggc 1080actgagtctg tcacagagga gaagtgcgct gtgctcttct ctgccagctt cacacttggc 1140cccggcaaac tccccatcca gctccaggcc ctgtctctgc ccctggtggt catcgtccat 1200ggcaaccaag acaacaatgc caaagccact atcctgtggg acaatgcctt ctctgagatg 1260gaccgcgtgc cctttgtggt ggctgagcgg gtgccctggg agaagatgtg tgaaactctg 1320aacctgaagt tcatggctga ggtggggacc aaccgggggc tgctcccaga gcacttcctc 1380ttcctggccc agaagatctt caatgacaac agcctcagta tggaggcctt ccagcaccgt 1440tctgtgtcct ggtcgcagtt caacaaggag atcctgctgg gccgtggctt caccttttgg 1500cagtggtttg atggtgtcct ggacctcacc aaacgctgtc tccggagcta ctggtctgac 1560cggctgatca ttggcttcat cagcaaacag tacgttacta gccttcttct caatgagccc 1620gacggaacct ttctcctccg cttcagcgac tcagagattg ggggcatcac cattgcccat 1680gtcatccggg gccaggatgg ctctccacag atagagaaca tccagccatt ctctgccaaa 1740gacctgtcca ttcgctcact gggggaccga atccgggatc ttgctcagct caaaaatctc 1800tatcccaaga agcccaagga tgaggctttc cggagccact acaagcctga acagatgggt 1860aaggatggca ggggttatgt cccagctacc atcaagatga ccgtggaaag ggaccaacca 1920cttcctaccc cagagctcca gatgcctacc atggtgcctt cttatgacct tggaatggcc 1980cctgattcct ccatgagcat gcagcttggc ccagatatgg tgccccaggt gtacccacca 2040cactctcact ccatcccccc gtatcaaggc ctctccccag aagaatcagt caacgtgttg 2100tcagccttcc aggagcctca cctgcagatg ccccccagcc tgggccagat gagcctgccc 2160tttgaccagc ctcaccccca gggcctgctg ccgtgccagc ctcaggagca tgctgtgtcc 2220agccctgacc ccctgctctg ctcagatgtg accatggtgg aagacagctg cctgagccag 2280ccagtgacag cgtttcctca gggcacttgg attggtgaag acatattccc tcctctgctg 2340cctcccactg aacaggacct cactaagctt ctcctggagg ggcaagggga gtcgggggga 2400gggtccttgg gggcacagcc cctcctgcag ccctcccact atgggcaatc tgggatctca 2460atgtcccaca tggacctaag ggccaacccc agttggtgat cccagctgga gggagaaccc 2520aaagagacag ctcttctact acccccacag acctgctctg gacacttgct catgccctgc 2580caagcagcag atggggaggg tgccctccta tccccaccta ctcctgggtc aggaggaaaa 2640gactaacagg agaatgcaca gtgggtggag ccaatccact ccttcctttc tatcattccc 2700ctgcccacct ccttccagca ctgactggaa gggaagttca ggctctgaga cacgccccaa 2760catgcctgca cctgcagcgc gcacacgcac gcacacacac atacagagct ctctgagggt 2820gatggggctg agcaggaggg gggctgggta agagcacagg ttagggcatg gaaggcttct 2880ccgcccattc tgacccaggg cctaggacgg ataggcagga acatacagac acatttacac 2940tagaggccag ggatagagga tattgggtct cagccctagg ggaatgggaa gcagctcaag 3000ggaccctggg tgggagcata ggaggagtct ggacatgtgg ttactagtac aggttttgcc 3060ctgattaaaa aatctcccaa agccccaaat tcctgttagc caggtggagg cttctgatac 3120gtgtatgaga ctatgcaaaa gtacaagggc tgagattctt cgtgtatagc tgtgtgaacg 3180tgtatgtacc taggatatgt taaatatata gctggcacct tagttgcatg accacataga 3240acatgtgtct atctgctttt gcctacgtga caacacaaat ttgggagggt gagacactgc 3300acagaagaca gcagcaagtg tgctggcctc tctgacatat gctaaccccc aaatactctg 3360aatttggagt ctgactgtgc ccaagtgggt ccaagtggct gtgacatcta cgtatggctc 3420cacacctcca atgctgcctg ggagccaggg tgagagtctg ggtccaggcc tggccatgtg 3480gccctccagt gtatgagagg gccctgcctg ctgcatcttt tctgttgccc catccaccgc 3540cagcttccct tcactcccct atcccattct ccctctcaag gcaggggtca tagatcctaa 3600gccataaaat aaattttatt ccaaaataaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3660aaaaaaa 366713919RNAArtificial SequenceAntisense Oligonucleotide 139cgagaggcgg acgggaccg 1914021DNAArtificial SequenceAntisense Oligonucleotide 140cgagaggcgg acgggaccgt t 2114121DNAArtificial SequenceAntisense Oligonucleotide 141cggtcccgtc cgcctctcgt t 21

Claims

1. A compound 8 to 80 nucleobases in length targeted to a nucleic acid molecule encoding STAT 6, wherein said compound specifically hybridizes with at least an 8 nucleobase portion of nucleobases 1762-1838 of said nucleic acid molecule encoding STAT 6 (SEQ ID NO: 4) and inhibits the expression of STAT 6.

2. The compound of claim 1, comprising 12 to 50 nucleobases in length.

3. The compound of claim 2, comprising 15 to 30 nucleobases in length.

4. The compound of claim 1, comprising an oligonucleotide.

5. The compound of claim 4, comprising an antisense oligonucleotide.

6. The compound of claim 4, comprising a DNA oligonucleotide.

7. The compound of claim 4, comprising an RNA oligonucleotide.

8. The compound of claim 4, comprising a chimeric oligonucleotide.

9. The compound of claim 4, wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.

10. The compound of claim 1, having at least 90% complementarity with a nucleic acid molecule encoding STAT 6 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of STAT 6.

11. The compound of claim 1, having at least 95% complementarity with a nucleic acid molecule encoding STAT 6 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of STAT 6.

12. The compound of claim 1, having at least one modified internucleoside linkage, sugar moiety, or nucleobase.

13. The compound of claim 1, having at least one 2'-O-(2-methoxyethyl) (2'-MOE) sugar moiety.

14. The compound of claim 1, having at least one phosphorothioate internucleoside linkage.

15. The compound of claim 1, having at least one 5-methylcytosine.

16. A method of inhibiting the expression of STAT 6 in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of STAT 6 is inhibited.

17. A kit or assay device comprising the compound of claim 1.

18. A method of treating an animal having a disease or condition associated with STAT 6 comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of STAT 6 is inhibited.

19. The method of claim 18, wherein the disease or condition is an autoimmune disorder.

20. The compound of claim 1 wherein the compound comprises SEQ ID NO: 29, 33, 34 or 46.

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