Patent Publication Number: US-2003235911-A1

Title: Antisense modulation of PRL-3 expression

Description:
FIELD OF THE INVENTION  
       [0001] The present invention provides compositions and methods for modulating the expression of PRL-3. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding PRL-3. Such compounds have been shown to modulate the expression of PRL-3.  
       BACKGROUND OF THE INVENTION  
       [0002] The process of phosphorylation, defined as the attachment of a phosphate moiety to a biological molecule through the action of enzymes called kinases, represents one course by which intracellular signals are propagated resulting finally in a cellular response. Within the cell, proteins can be phosphorylated on serine, threonine or tyrosine residues and the extent of phosphorylation is regulated by the opposing action of phosphatases, which remove the phosphate moieties. While the majority of protein phosphorylation within the cell is on serine and threonine residues, tyrosine phosphorylation is modulated to the greatest extent during oncogenic transformation and growth factor stimulation (Zhang,  Crit. Rev. Biochem. Mol. Biol.,  1998, 33, 1-52).  
       [0003] Because phosphorylation is such a ubiquitous process within cells and because cellular phenotypes are largely influenced by the activity of these pathways, it is currently believed that a number of disease states and/or disorders are a result of either aberrant activation of, or functional mutations in, kinases and phosphatases. Consequently, considerable attention has been devoted recently to the characterization of tyrosine kinases and tyrosine phosphatases.  
       [0004] Human PRL-3 (also known as protein tyrosine phosphatase type IVA, member 3; PTPIVA3 and potentially prenylated protein tyrosine phosphatase) was recently identified by database searching and screening of a heart cDNA library (Matter et al.,  Biochem. Biophys. Res. Commun.,  2001, 283, 1061-1068). The gene was originally discovered in the mouse as the third member of a group of three prenylated protein-tyrosine phosphatases (Zeng et al.,  Biochem. Biophys. Res. Commun.,  1998, 244, 421-427; Zeng et al.,  J. Biol. Chem.,  2000, 275, 21444-21452). Human PRL-3 is expressed as an approximately 2.3-kb PRL-3 transcript predominantly in heart and skeletal muscle, with lower expression in pancreas. Overexpression of PRL-3 in HEK293 cells resulted in increased cell growth (Matter et al.,  Biochem. Biophys. Res. Commun.,  2001, 283, 1061-1068).  
       [0005] Nucleic acid sequences encoding PRL-3 are disclosed in U.S. Pat. No. 6,258,582 (Acton, 2001). PRL-3 variants exist and are known as PRL-3 variant 1 and PRL-3 variant 2.  
       [0006] To gain insights into the molecular basis for metastasis, Saha et al. compared the global gene expression profile of metastatic colorectal cancer with that of primary cancers, benign colorectal tumors, and normal colorectal epithelium. PRL-3 was expressed at high levels in each of 18 cancer metastases studied but at lower levels in non-metastatic tumors and normal colorectal epithelium. In 3 of 12 metastases examined, multiple copies of the PRL-3 gene were found within a small amplicon located at chromosome 8q24.3. Saha et al. concluded that the PRL-3 gene is important for colorectal cancer metastasis (Saha et al.,  Science,  2001, 294, 1343-1346).  
       [0007] Small molecule inhibitors of tyrosine phosphatases are well known in the art. For example, disclosed and claimed in U.S. Pat. No. 6,169,087 are small molecule inhibitors of protein tyrosine phosphatases for the treatment of type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, obesity, and a number of other diseases (Andersen et al., 2001).  
       [0008] Vanadate tyrosine phosphatase inhibitors were employed in an investigation of PRL-3 activity in HEK293 cells and potassium bisperoxo (bipyridine) oxovanadate V was found to be the most potent inhibitor. Matter et al. subsequently suggested that the development of selective inhibitors against PRL-3 may allow investigators to determine whether pharmacologic intervention against PRL-3 will be sufficient by itself or in conjunction with other therapies to arrest the progression of cardiac hypertrophy and heart failure (Matter et al.,  Biochem. Biophys. Res. Commun.,  2001, 283, 1061-1068). Likewise, Saha et al. proposed that enzymes such as PRL-3, whose expression is elevated in cancer cells, provide excellent targets for drug discovery (Saha et al.,  Science,  2001, 294, 1343-1346).  
       [0009] Currently, there are no known therapeutic agents that effectively inhibit the synthesis of PRL-3. To date, investigative strategies aimed at modulating PRL-3 expression have involved the use of vanadate inhibitors. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting PRL-3 function.  
       [0010] 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 expression of PRL-3.  
       [0011] The present invention provides compositions and methods for modulating expression of PRL-3, including modulation of variants of PRL-3.  
       SUMMARY OF THE INVENTION  
       [0012] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding PRL-3, and which modulate the expression of PRL-3. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of PRL-3 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of PRL-3 by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.  
       DETAILED DESCRIPTION OF THE INVENTION  
       [0013] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding PRL-3, ultimately modulating the amount of PRL-3 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding PRL-3. As used herein, the terms “target nucleic acid” and “nucleic acid encoding PRL-3” encompass DNA encoding PRL-3, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the 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 mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of PRL-3. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.  
       [0014] It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding PRL-3. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation 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 molecule transcribed from a gene encoding PRL-3, regardless of the sequence(s) of such codons.  
       [0015] 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). 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.  
       [0016] 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. 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 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. The 5′ cap region may also be a preferred target region.  
       [0017] 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. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It has also been found that introns can be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.  
       [0018] 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 extronic regions.  
       [0019] 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.  
       [0020] 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.  
       [0021] Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.  
       [0022] In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.  
       [0023] An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. It is preferred that the antisense compounds of the present invention comprise at least 80% sequence complementarity to a target region within the target nucleic acid, moreover that they comprise 90% sequence complementarity and even more 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, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al.,  J. Mol. Biol.,  1990, 215, 403-410; Zhang and Madden,  Genome Res.,  1997, 7, 649-656).  
       [0024] Antisense and other compounds of the invention, which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The sites to which these preferred antisense compounds are specifically hybridizable are hereinbelow referred to as “preferred target regions” and are therefore preferred sites for targeting. As used herein the term “preferred target region” 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 regions represent regions of the target nucleic acid which are accessible for hybridization.  
       [0025] While the specific sequences of particular preferred target regions are set forth below, 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 regions may be identified by one having ordinary skill.  
       [0026] Target regions 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target regions are considered to be suitable preferred target regions as well.  
       [0027] Exemplary good preferred target regions include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred target regions (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly good preferred target regions 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 regions (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art, once armed with the empirically-derived preferred target regions illustrated herein will be able, without undue experimentation, to identify further preferred target regions. In addition, one having ordinary skill in the art will also be able to identify additional compounds, including oligonucleotide probes and primers, that specifically hybridize to these preferred target regions using techniques available to the ordinary practitioner in the art.  
       [0028] Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.  
       [0029] For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in combination with other antisense 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.  
       [0030] 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.  
       [0031] 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 (reviewed in To,  Comb. Chem. High Throughput Screen,  2000, 3, 235-41).  
       [0032] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.  
       [0033] In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. 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 nucleic acid target and increased stability in the presence of nucleases.  
       [0034] While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides from about 8 to about 50 nucleobases, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.  
       [0035] 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.  
       [0036] Exemplary preferred antisense compounds include DNA or RNA 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 DNA or RNA beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds 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 antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art, once armed with the empirically-derived preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.  
       [0037] Antisense and other compounds of the invention, which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are herein identified as preferred embodiments of the invention. While specific sequences of the antisense compounds 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 antisense compounds may be identified by one having ordinary skill.  
       [0038] 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 structure can be further joined to form a circular structure, however, open linear structures are generally preferred. In addition, linear structures may also have internal nucleobase complementarity and may therefore fold in a manner as to produce a double stranded structure. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.  
       [0039] 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.  
       [0040] Preferred modified oligonucleotide backbones 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 abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.  
       [0041] 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.  
       [0042] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2  component parts.  
       [0043] 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.  
       [0044] 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 base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The 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.  
       [0045] Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH 2 —NH—O—CH 2 —, —CH 2 —N(CH 3 )—O—CH 2 -[known as a methylene (methylimino) or MMI backbone], —CH 2 —O—N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 — and —O—N(CH 3 )—CH 2 —CH 2 -[wherein the native phosphodiester backbone is represented as —O—P—O—CH 2 —] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.  
       [0046] 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 C 1  to C 10  alkyl or C 2  to C 10  alkenyl and alkynyl. Particularly preferred are O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 ] 2 , where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C 1  to C 10  lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , 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—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al.,  Helv. Chim. Acta,  1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2  group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH 2 —O—CH 2 —N(CH 3 ) 2 , also described in examples hereinbelow.  
       [0047] Other preferred modifications include 2′-methoxy (2′-O—CH 3 ), 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 ), 2′-allyl (2′-CH 2 —CH═CH 2 ), 2′-O-allyl (2′-O—CH 2 —CH═CH 2 ) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. 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.  
       [0048] A further preferred modification 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 methelyne (—CH 2 —) 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.  
       [0049] 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—CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in  The Concise Encyclopedia Of Polymer Science And Engineering,  pages 858-859, Kroschwitz, J. I., ed. John Wiley &amp; 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 oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 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. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds.,  Antisense Research and Applications,  CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.  
       [0050] 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.  
       [0051] 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. The compounds of the invention 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 oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al.,  Proc. Natl. Acad. Sci. USA,  1989, 86, 6553-6556), cholic acid (Manoharan et al.,  Bioorg. Med. Chem. Let.,  1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,  Ann. N.Y. Acad. Sci.,  1992, 660, 306-309; Manoharan et al.,  Bioorg. Med. Chem. Let.,  1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,  Nucl. Acids Res.,  1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,  EMBO J.,  1991, 10, 1111-1118; Kabanov et al.,  FEBS Lett.,  1990, 259, 327-330; Svinarchuk et al.,  Biochimie,  1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,  Tetrahedron Lett.,  1995, 36, 3651-3654; Shea et al.,  Nucl. Acids Res.,  1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al.,  Nucleosides  &amp;  Nucleotides,  1995, 14, 969-973), or adamantane acetic acid (Manoharan et al.,  Tetrahedron Lett.,  1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,  Biochim. Biophys. Acta,  1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al.,  J. Pharmacol. Exp. Ther.,  1996, 277, 923-937). 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 United States patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.  
       [0052] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. No. 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.  
       [0053] 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. 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 inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as interferon-induced RNAseL which cleaves both cellular and viral RNA. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.  
       [0054] 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. No. 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.  
       [0055] 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.  
       [0056] 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.  
       [0057] 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.  
       [0058] 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.  
       [0059] 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.  
       [0060] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of  Pharma Sci.,  1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.  
       [0061] For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.  
       [0062] The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of PRL-3 is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.  
       [0063] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding PRL-3, enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding PRL-3 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 PRL-3 in a sample may also be prepared.  
       [0064] 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.  
       [0065] 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. Preferred topical formulations 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). 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 include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C 1-10  alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. 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.  
       [0066] 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 include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). 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 include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673 (filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624 (filed May 21, 1998) and 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.  
       [0067] 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.  
       [0068] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.  
       [0069] 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.  
       [0070] 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.  
       [0071] In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention. Emulsions The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in  Remington&#39;s Pharmaceutical Sciences,  Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.  
       [0072] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).  
       [0073] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).  
       [0074] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.  
       [0075] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).  
       [0076] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.  
       [0077] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.  
       [0078] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.  
       [0079] In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in:  Controlled Release of Drugs:  Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in  Remington&#39;s Pharmaceutical Sciences,  Mack Publishing Co., Easton, Pa., 1985, p. 271).  
       [0080] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.  
       [0081] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous,solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C 8 -C 12 ) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C 8 -C 10  glycerides, vegetable oils and silicone oil.  
       [0082] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al.,  Pharmaceutical Research,  1994, 11, 1385-1390; Ritschel,  Meth. Find. Exp. Clin. Pharmacol.,  1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al.,  Pharmaceutical Research,  1994, 11, 1385; Ho et al.,  J. Pharm. Sci.,  1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.  
       [0083] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, p. 92). Each of these classes has been discussed above.  
       [0084] Liposomes  
       [0085] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.  
       [0086] Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.  
       [0087] In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.  
       [0088] Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.  
       [0089] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.  
       [0090] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.  
       [0091] Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.  
       [0092] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al.,  Biochem. Biophys. Res. Commun.,  1987, 147, 980-985).  
       [0093] Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al.,  Journal of Controlled Release,  1992, 19, 269-274).  
       [0094] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.  
       [0095] Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al.,  Journal of Drug Targeting,  1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al.,  Antiviral Research,  1992, 18, 259-265).  
       [0096] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al.  S.T.P.Pharma. Sci.,  1994, 4, 6, 466).  
       [0097] 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 (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al.,  FEBS Letters,  1987, 223, 42; Wu et al.,  Cancer Research,  1993, 53, 3765).  
       [0098] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. ( Ann. N.Y. Acad. Sci.,  1987, 507, 64) reported the ability of monosialoganglioside G M1 , galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. ( Proc. Natl. Acad. Sci. U.S.A.,  1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G M1  or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).  
       [0099] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. ( Bull. Chem. Soc. Jpn.,  1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C 12 15G, that contains a PEG moiety. Illum et al. ( FEBS Lett.,  1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. ( FEBS Lett.,  1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. ( Biochimica et Biophysica Acta,  1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.  
       [0100] A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. Wo 97/04787 to Love et al. discloses liposomes comprising antisense oligonucleotides targeted to the raf gene.  
       [0101] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.  
       [0102] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).  
       [0103] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.  
       [0104] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.  
       [0105] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.  
       [0106] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.  
       [0107] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in  Pharmaceutical Dosage Forms,  Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).  
       [0108] Penetration Enhancers  
       [0109] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.  
       [0110] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.  
       [0111] Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,  J. Pharm. Pharmacol.,  1988, 40, 252).  
       [0112] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C 1-10  alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, p.92; Muranishi,  Critical Reviews in Therapeutic Drug Carrier Systems,  1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).  
       [0113] Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman &amp; Gilman&#39;s  The Pharmacological Basis of Therapeutics,  9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, page 92; Swinyard, Chapter 39 In:  Remington&#39;s Pharmaceutical Sciences,  18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi,  Critical Reviews in Therapeutic Drug Carrier Systems,  1990, 7, 1-33; Yamamoto et al.,  J. Pharm. Exp. Ther.,  1992, 263, 25; Yamashita et al.,  J. Pharm. Sci.,  1990, 79, 579-583).  
       [0114] Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett,  J. Chromatogr.,  1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, page 92; Muranishi,  Critical Reviews in Therapeutic Drug Carrier Systems,  1990, 7, 1-33; Buur et al.,  J. Control Rel.,  1990, 14, 43-51).  
       [0115] Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi,  Critical Reviews in Therapeutic Drug Carrier Systems,  1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,  J. Pharm. Pharmacol.,  1987, 39, 621-626).  
       [0116] Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.  
       [0117] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.  
       [0118] Carriers  
       [0119] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,  Antisense Res. Dev.,  1995, 5, 115-121; Takakura et al.,  Antisense &amp; Nucl. Acid Drug Dev.,  1996, 6, 177-183).  
       [0120] Excipients  
       [0121] In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).  
       [0122] Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.  
       [0123] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.  
       [0124] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.  
       [0125] Other Components  
       [0126] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.  
       [0127] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.  
       [0128] Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to 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). See, generally,  The Merck Manual of Diagnosis and Therapy,  15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally,  The Merck Manual of Diagnosis and Therapy,  15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.  
       [0129] 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. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.  
       [0130] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC 50 s 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.  
       [0131] 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  
     [0132] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy amidites  
     [0133] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2′-alkoxy amidites, optimized synthesis cycles were developed that incorporate multiple steps coupling longer wait times relative to standard synthesis cycles.  
     [0134] The following abbreviations are used in the text: thin layer chromatography (TLC), melting point (MP), high pressure liquid chromatography (HPLC), Nuclear Magnetic Resonance (NMR), argon (Ar), methanol (MeOH), dichloromethane (CH 2 Cl 2 ), triethylamine (TEA), dimethyl formamide (DMF), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF).  
     [0135] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-dC) nucleotides were synthesized according to published methods (Sanghvi, et. al.,  Nucleic Acids Research,  1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.) or prepared as follows:  
     [0136] Preparation of 5′-O-Dimethoxytrityl-thymidine Intermediate for 5-methyl dC Amidite  
     [0137] To a 50 L glass reactor equipped with air stirrer and Ar gas line was added thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 L) at ambient temperature. Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol, 1.05 eq) was added as a solid in four portions over 1 h. After 30 min, TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent and by-products and 2% 3′,5′-bis DMT product (Rf in EtOAc 0.45, 0.05, 0.98, 0.95 respectively). Saturated sodium bicarbonate (4 L) and CH 2 Cl 2  were added with stirring (pH of the aqueous layer 7.5). An additional 18 L of water was added, the mixture was stirred, the phases were separated, and the organic layer was transferred to a second 50 L vessel. The aqueous layer was extracted with additional CH 2 Cl 2  (2×2 L). The combined organic layer was washed with water (10 L) and then concentrated in a rotary evaporator to approx. 3.6 kg total weight. This was redissolved in CH 2 Cl 2  (3.5 L), added to the reactor followed by water (6 L) and hexanes (13 L). The mixture was vigorously stirred and seeded to give a fine white suspended solid starting at the interface. After stirring for 1 h, the suspension was removed by suction through a ½″ diameter teflon tube into a 20 L suction flask, poured onto a 25 cm Coors Buchner funnel, washed with water (2×3 L) and a mixture of hexanes—CH 2 Cl 2  (4:1, 2×3 L) and allowed to air dry overnight in pans (1″ deep). This was further dried in a vacuum oven (75° C., 0.1 mm Hg, 48 h) to a constant weight of 2072 g (93%) of a white solid, (mp 122-124° C.). TLC indicated a trace contamination of the bis DMT product. NMR spectroscopy also indicated that 1-2 mole percent pyridine and about 5 mole percent of hexanes was still present.  
     [0138] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine Intermediate for 5-methyl-dC Amidite  
     [0139] To a 50 L Schott glass-lined steel reactor equipped with an electric stirrer, reagent addition pump (connected to an addition funnel), heating/cooling system, internal thermometer and an Ar gas line was added 5′-O-dimethoxytrityl-thymidine (3.00 kg, 5.51 mol), anhydrous acetonitrile (25 L) and TEA (12.3 L, 88.4 mol, 16 eq). The mixture was chilled with stirring to −10° C. internal temperature (external −20° C.). Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30 minutes while maintaining the internal temperature below −5° C., followed by a wash of anhydrous acetonitrile (1 L). Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition. The reaction was allowed to warm to 0° C. and the reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; R f  0.43 to 0.84 of starting material and silyl product, respectively). Upon completion, triazole (3.05 kg, 44 mol, 8.0 eq) was added the reaction was cooled to −20° C. internal temperature (external −30° C.). Phosphorous oxychloride (1035 mL, 11.1 mol, 2.01 eq) was added over 60 min so as to maintain the temperature between −20° C. and −10° C. during the strongly exothermic process, followed by a wash of anhydrous acetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1 h. TLC indicated a complete conversion to the triazole product (R f  0.83 to 0.34 with the product spot glowing in long wavelength UV light). The reaction mixture was a peach-colored thick suspension, which turned darker red upon warming without apparent decomposition. The reaction was cooled to −15° C. internal temperature and water (5 L) was slowly added at a rate to maintain the temperature below +10° C. in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic). Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2×8 L). The combined water layers were back-extracted with EtOAc (6 L). The water layer was discarded and the organic layers were concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam). The second half of the reaction was treated in the same way. Each residue was dissolved in dioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight (although the reaction is complete within 1 h).  
     [0140] TLC indicated a complete reaction (product R f  0.35 in EtOAc-MeOH 4:1). The reaction solution was concentrated on a rotary evaporator to a dense foam. Each foam was slowly redissolved in warm EtOAc (4 L; 50° C.), combined in a 50 L glass reactor vessel, and extracted with water (2×4L) to remove the triazole by-product. The water was back-extracted with EtOAc (2 L). The organic layers were combined and concentrated to about 8 kg total weight, cooled to 0° C. and seeded with crystalline product. After 24 hours, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc (3×3L) until a white powder was left and then washed with ethyl ether (2×3L). The solid was put in pans (1″ deep) and allowed to air dry overnight. The filtrate was concentrated to an oil, then redissolved in EtOAc (2 L), cooled and seeded as before. The second crop was collected and washed as before (with proportional solvents) and the filtrate was first extracted with water (2×1L) and then concentrated to an oil. The residue was dissolved in EtOAc (1 L) and yielded a third crop which was treated as above except that more washing was required to remove a yellow oily layer.  
     [0141] After air-drying, the three crops were dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g, respectively) and combined to afford 2550 g (85%) of a white crystalline product (MP 215-217° C.) when TLC and NMR spectroscopy indicated purity. The mother liquor still contained mostly product (as determined by TLC) and a small amount of triazole (as determined by NMR spectroscopy), bis DMT product and unidentified minor impurities. If desired, the mother liquor can be purified by silica gel chromatography using a gradient of MeOH (0-25%) in EtOAc to further increase the yield.  
     [0142] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine Penultimate Intermediate for 5-methyl dC Amidite  
     [0143] Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000 g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambient temperature in a 50 L glass reactor vessel equipped with an air stirrer and argon line. Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86 mol, 1.05 eq) was added and the reaction was stirred at ambient temperature for 8 h. TLC (CH 2 Cl 2 -EtOAc; CH 2 Cl 2 -EtOAc 4:1; R f  0.25) indicated approx. 92% complete reaction. An additional amount of benzoic anhydride (44 g, 0.19 mol) was added. After a total of 18 h, TLC indicated approx. 96% reaction completion. The solution was diluted with EtOAc (20 L), TEA (1020 mL, 7.36 mol, ca 2.0 eq) was added with stirring, and the mixture was extracted with water (15 L, then 2×10 L). The aqueous layer was removed (no back-extraction was needed) and the organic layer was concentrated in 2×20 L rotary evaporator flasks until a foam began to form. The residues were coevaporated with acetonitrile (1.5 L each) and dried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a dense foam. High pressure liquid chromatography (HPLC) revealed a contamination of 6.3% of N4, 3′-O-dibenzoyl product, but very little other impurities.  
     [0144] THe product was purified by Biotage column chromatography (5 kg Biotage) prepared with 65:35:1 hexanes-EtOAc-TEA (4L). The crude product (800 g),dissolved in CH 2 Cl 2  (2 L), was applied to the column. The column was washed with the 65:35:1 solvent mixture (20 kg), then 20:80:1 solvent mixture (10 kg), then 99:1 EtOAc:TEA (17 kg). The fractions containing the product were collected, and any fractions containing the product and impurities were retained to be resubjected to column chromatography. The column was re-equilibrated with the original 65:35:1 solvent mixture (17 kg). A second batch of crude product (840 g) was applied to the column as before. The column was washed with the following solvent gradients: 65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1 (10 kg), and 99:1 EtOAc:TEA(15 kg). The column was reequilibrated as above, and a third batch of the crude product (850 g) plus impure fractions recycled from the two previous columns (28 g) was purified following the procedure for the second batch. The fractions containing pure product combined and concentrated on a 20L rotary evaporator, co-evaporated with acetontirile (3 L) and dried (0.1 mm Hg, 48 h, 25° C.) to a constant weight of 2023 g (85%) of white foam and 20 g of slightly contaminated product from the third run. HPLC indicated a purity of 99.8% with the balance as the diBenzoyl product.  
     [0145] [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N 4 -benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC Amidite)  
     [0146] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N 4 -benzoyl-5-methylcytidine (998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (300 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (15 ml) was added and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2.5 L) and water (600 ml), and extracted with hexane (3×3 L). The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (7.5 L) and hexane (6 L). The two layers were separated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L) and water (3×2 L), and the phases were separated. The organic layer was dried (Na 2 SO 4 ), filtered and rotary evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried to a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 g an off-white foam solid (96%).  
     [0147] 2′-Fluoro Amidites  
     [0148] 2′-Fluorodeoxyadenosine Amidites  
     [0149] 2′-fluoro oligonucleotides were synthesized as described previously [Kawasaki, et. al.,  J. Med. Chem.,  1993, 36, 831-841] and U.S. Pat. No. 5,670,633, herein incorporated by reference. The preparation of 2′-fluoropyrimidines containing a 5-methyl substitution are described in U.S. Pat. No. 5,861,493. Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and whereby the 2′-alpha-fluoro atom is introduced by a SN2-displacement of a 2′-beta-triflate group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates. 2′-Fluorodeoxyguanosine The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate isobutyryl-arabinofuranosylguanosine. Alternatively, isobutyryl-arabinofuranosylguanosine was prepared as described by Ross et al., ( Nucleosides  &amp;  Nucleosides,  16, 1645, 1997). Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give isobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.  
     [0150] 2′-Fluorouridine  
     [0151] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a literature procedure in which 2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.  
     [0152] 2′-Fluorodeoxycytidine  
     [0153] 2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.  
     [0154] 2′-O-(2-Methoxyethyl) Modified Amidites  
     [0155] 2′-O-Methoxyethyl-substituted nucleoside amidites (otherwise known as MOE amidites) are prepared as follows, or alternatively, as per the methods of Martin, P., (Helvetica Chimica Acta, 1995, 78, 486-504).  
     [0156] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate  
     [0157] 2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol), tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60 g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12 L three necked flask and heated to 130° C. (internal temp) at atmospheric pressure, under an argon atmosphere with stirring for 21 h. TLC indicated a complete reaction. The solvent was removed under reduced pressure until a sticky gum formed (50-85° C. bath temp and 100-11 mm Hg) and the residue was redissolved in water (3 L) and heated to boiling for 30 min in order the hydrolyze the borate esters. The water was removed under reduced pressure until a foam began to form and then the process was repeated. HPLC indicated about 77% product, 15% dimer (5′ of product attached to 2′ of starting material) and unknown derivatives, and the balance was a single unresolved early eluting peak.  
     [0158] The gum was redissolved in brine (3 L), and the flask was rinsed with additional brine (3 L). The combined aqueous solutions were extracted with chloroform (20 L) in a heavier-than continuous extractor for 70 h. The chloroform layer was concentrated by rotary evaporation in a 20 L flask to a sticky foam (2400 g). This was coevaporated with MeOH (400 mL) and EtOAc (8 L) at 75° C. and 0.65 atm until the foam dissolved at which point the vacuum was lowered to about 0.5 atm. After 2.5 L of distillate was collected a precipitate began to form and the flask was removed from the rotary evaporator and stirred until the suspension reached ambient temperature. EtOAc (2 L) was added and the slurry was filtered on a 25 cm table top Buchner funnel and the product was washed with EtOAc (3×2 L). The bright white solid was air dried in pans for 24 h then further dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) to afford 1649 g of a white crystalline solid (mp 115.5-116.5° C.).  
     [0159] The brine layer in the 20 L continuous extractor was further extracted for 72 h with recycled chloroform. The chloroform was concentrated to 120 g of oil and this was combined with the mother liquor from the above filtration (225 g), dissolved in brine (250 mL) and extracted once with chloroform (250 mL). The brine solution was continuously extracted and the product was crystallized as described above to afford an additional 178 g of crystalline product containing about 2% of thymine. The combined yield was 1827 g (69.4%). HPLC indicated about 99.5% purity with the balance being the dimer.  
     [0160] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine Penultimate Intermediate  
     [0161] In a 50 L glass-lined steel reactor, 2′-O-(2-methoxyethyl)-5-methyl-uridine (MOE-T, 1500 g, 4.738 mol), lutidine (1015 g, 9.476 mol) were dissolved in anhydrous acetonitrile (15 L). The solution was stirred rapidly and chilled to −10° C. (internal temperature). Dimethoxytriphenylmethyl chloride (1765.7 g, 5.21 mol) was added as a solid in one portion. The reaction was allowed to warm to −2° C. over 1 h. (Note: The reaction was monitored closely by TLC (EtOAc) to determine when to stop the reaction so as to not generate the undesired bis-DMT substituted side product). The reaction was allowed to warm from −2 to 3° C. over 25 min. then quenched by adding MeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L). The solution was transferred to a clear 50 L vessel with a bottom outlet, vigorously stirred for 1 minute, and the layers separated. The aqueous layer was removed and the organic layer was washed successively with 10% aqueous citric acid (8 L) and water (12 L). The product was then extracted into the aqueous phase by washing the toluene solution with aqueous sodium hydroxide (0.5N, 16 L and 8 L). The combined aqueous layer was overlayed with toluene (12 L) and solid citric acid (8 moles, 1270 g) was added with vigorous stirring to lower the pH of the aqueous layer to 5.5 and extract the product into the toluene. The organic layer was washed with water (10 L) and TLC of the organic layer indicated a trace of DMT-O-Me, bis DMT and dimer DMT.  
     [0162] The toluene solution was applied to a silica gel column (6 L sintered glass funnel containing approx. 2 kg of silica gel slurried with toluene (2 L) and TEA(25 mL)) and the fractions were eluted with toluene (12 L) and EtOAc (3×4 L) using vacuum applied to a filter flask placed below the column. The first EtOAc fraction containing both the desired product and impurities were resubjected to column chromatography as above. The clean fractions were combined, rotary evaporated to a foam, coevaporated with acetonitrile (6 L) and dried in a vacuum oven (0.1 mm Hg, 40 h, 40° C.) to afford 2850 g of a white crisp foam. NMR spectroscopy indicated a 0.25 mole % remainder of acetonitrile (calculates to be approx. 47 g) to give a true dry weight of 2803 g (96%). HPLC indicated that the product was 99.41% pure, with the remainder being 0.06 DMT-O-Me, 0.10 unknown, 0.44 bis DMT, and no detectable dimer DMT or 3′-O-DMT.  
     [0163] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite) 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridine (1237 g, 2.0 mol) was dissolved in anhydrous DMF (2.5 L). The solution was co-evaporated with toluene (200 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g, 1.0 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (20 ml) was added and the solution was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (3.5 L) and water (600 ml) and extracted with hexane (3×3L). The mixture was diluted with water (1.6 L) and extracted with the mixture of toluene (12 L) and hexanes (9 L). The upper layer was washed with DMF-water (7:3 v/v, 3×3 L) and water (3×3 L). The organic layer was dried (Na 2 SO 4 ), filtered and evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1526 g of an off-white foamy solid (95%).  
     [0164] Preparation of 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate  
     [0165] To a 50 L Schott glass-lined steel reactor equipped with an electric stirrer, reagent addition pump (connected to an addition funnel), heating/cooling system, internal thermometer and argon gas line was added 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-uridine (2.616 kg, 4.23 mol, purified by base extraction only and no scrub column), anhydrous acetonitrile (20 L), and TEA (9.5 L, 67.7 mol, 16 eq). The mixture was chilled with stirring to −10° C. internal temperature (external −20° C.). Trimethylsilylchloride (1.60 L, 12.7 mol, 3.0 eq) was added over 30 min. while maintaining the internal temperature below −5° C., followed by a wash of anhydrous acetonitrile (1 L). (Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition). The reaction was allowed to warm to 0° C. and the reaction progress was confirmed by TLC (EtOAc, Rf 0.68 and 0.87 for starting material and silyl product, respectively). Upon completion, triazole (2.34 kg, 33.8 mol, 8.0 eq) was added the reaction was cooled to −20° C. internal temperature (external −30° C.). Phosphorous oxychloride (793 mL, 8.51 mol, 2.01 eq) was added slowly over 60 min so as to maintain the temperature between −20° C. and −10° C. (note: strongly exothermic), followed by a wash of anhydrous acetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1 h, at which point it was an off-white thick suspension. TLC indicated a complete conversion to the triazole product (EtOAc, Rf 0.87 to 0.75 with the product spot glowing in long wavelength UV light). The reaction was cooled to −15° C. and water (5 L) was slowly added at a rate to maintain the temperature below +10° C. in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic). Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2×8 L). The second half of the reaction was treated in the same way. The combined aqueous layers were back-extracted with EtOAc (8 L) The organic layers were combined and concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam). The residue was dissolved in dioxane (2 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight  
     [0166] TLC indicated a complete reaction (CH 2 Cl 2 -acetone-MeOH, 20:5:3, R f  0.51). The reaction solution was concentrated on a rotary evaporator to a dense foam and slowly redissolved in warm CH 2 Cl 2  (4 L, 40° C.) and transferred to a 20 L glass extraction vessel equipped with a air-powered stirrer. The organic layer was extracted with water (2×6 L) to remove the triazole by-product. (Note: In the first extraction an emulsion formed which took about 2 h to resolve). The water layer was back-extracted with CH 2 Cl 2  (2×2 L), which in turn was washed with water (3 L). The combined organic layer was concentrated in 2×20 L flasks to a gum and then recrystallized from EtOAc seeded with crystalline product. After sitting overnight, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc until a white free-flowing powder was left (about 3×3 L). The filtrate was concentrated to an oil recrystallized from EtOAc, and collected as above. The solid was air-dried in pans for 48 h, then further dried in a vacuum oven (50° C., 0.1 mm Hg, 17 h) to afford 2248 g of a bright white, dense solid (86%). An HPLC analysis indicated both crops to be 99.4% pure and NMR spectroscopy indicated only a faint trace of EtOAc remained.  
     [0167] Preparation of 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N-4-benzoyl-5-methyl-cytidine Penultimate Intermediate:  
     [0168] Crystalline 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-cytidine (1000 g, 1.62 mol) was suspended in anhydrous DMF (3 kg) at ambient temperature and stirred under an Ar atmosphere. Benzoic anhydride (439.3 g, 1.94 mol) was added in one portion. The solution clarified after 5 hours and was stirred for 16 h. HPLC indicated 0.45% starting material remained (as well as 0.32% N4, 3′-O-bis Benzoyl). An additional amount of benzoic anhydride (6.0 g, 0.0265 mol) was added and after 17 h, HPLC indicated no starting material was present. TEA (450 mL, 3.24 mol) and toluene (6 L) were added with stirring for 1 minute. The solution was washed with water (4×4 L), and brine (2×4 L). The organic layer was partially evaporated on a 20 L rotary evaporator to remove 4 L of toluene and traces of water. HPLC indicated that the bis benzoyl side product was present as a 6% impurity. The residue was diluted with toluene (7 L) and anhydrous DMSO (200 mL, 2.82 mol) and sodium hydride (60% in oil, 70 g, 1.75 mol) was added in one portion with stirring at ambient temperature over 1 h. The reaction was quenched by slowly adding then washing with aqueous citric acid (10%, 100 mL over 10 min, then 2×4 L), followed by aqueous sodium bicarbonate (2%, 2 L), water (2×4 L) and brine (4 L). The organic layer was concentrated on a 20 L rotary evaporator to about 2 L total volume. The residue was purified by silica gel column chromatography (6 L Buchner funnel containing 1.5 kg of silica gel wetted with a solution of EtOAc-hexanes-TEA (70:29:1)). The product was eluted with the same solvent (30 L) followed by straight EtOAc (6 L). The fractions containing the product were combined, concentrated on a rotary evaporator to a foam and then dried in a vacuum oven (50° C., 0.2 mm Hg, 8 h) to afford 1155 g of a crisp, white foam (98%). HPLC indicated a purity of &gt;99.7%.  
     [0169] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 4 -benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C Amidite)  
     [0170] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 4 -benzoyl-5-methylcytidine (1082 g, 1.5 mol) was dissolved in anhydrous DMF (2 L) and co-evaporated with toluene (300 ml) at 50° C. under reduced pressure. The mixture was cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (1 L) and water (400 ml) and extracted with hexane (3×3 L). The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (9 L) and hexanes (6 L). The two layers were separated and the upper layer was washed with DMF-water (60:40 v/v, 3×3 L) and water (3×2 L). The organic layer was dried (Na 2 SO 4 ), filtered and evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1336 g of an off-white foam (97%).  
     [0171] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 6 -benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A Amdite)  
     [0172] b  5 ′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 6 -benzoyladenosine (purchased from Reliable Biopharmaceutical, St. Lois, MO), 1098 g, 1.5 mol) was dissolved in anhydrous DMF (3 L) and co-evaporated with toluene (300 ml) at 50° C. The mixture was cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (78.8 g, 1.24 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (1 L) and water (400 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (1.4 L) and extracted with the mixture of toluene (9 L) and hexanes (6 L). The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L) and water (3×2 L). The organic layer was dried (Na 2 SO 4 ), filtered and evaporated to a sticky foam. The residue was co-evaporated with acetonitrile (2.5 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1350 g of an off-white foam solid (96%).  
     [0173] Prepartion of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 4 -isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G Amidite)  
     [0174] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 4 -isobutyrlguanosine (purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (200 ml) at 50° C., cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2 L) and water (600 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (2 L) and extracted with a mixture of toluene (10 L) and hexanes (5 L). The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and the solution was washed with water (3×4 L). The organic layer was dried (Na 2 SO 4 ), filtered and evaporated to approx. 4 kg. Hexane (4 L) was added, the mixture was shaken for 10 min, and the supernatant liquid was decanted. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1660 g of an off-white foamy solid (91%).  
     [0175] 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) Nucleoside Amidites  
     [0176] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites  
     [0177] 2′-(Dimethylaminooxyethoxy) nucleoside amidites (also known in the art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine. 5′-O-tert-Butyldiphenylsilyl-0 2 -2′-anhydro-5-methyluridine O 2 -2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, l.leq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22, EtOAc) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between CH 2 Cl 2  (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of EtOAc and ethyl ether (600 mL) and cooling the solution to −10° C. afforded a white crystalline solid which was collected by filtration, washed with ethyl ether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to afford 149 g of white solid (74.8%). TLC and NMR spectroscopy were consistent with pure product.  
     [0178] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine  
     [0179] In the fume hood, ethylene glycol (350 mL, excess) was added cautiously with manual stirring to a 2 L stainless steel pressure reactor containing borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). (Caution: evolves hydrogen gas). 5′-O-tert-Butyldiphenylsilyl-O 2 -2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160° C. was reached and then maintained for 16 h (pressure &lt;100 psig). The reaction vessel was cooled to ambient temperature and opened. TLC (EtOAc, Rf 0.67 for desired product and Rf 0.82 for ara-T side product) indicated about 70% conversion to the product. The solution was concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol. (Alternatively, once the THF has evaporated the solution can be diluted with water and the product extracted into EtOAc). The residue was purified by column chromatography (2 kg silica gel, EtOAc-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, evaporated and dried to afford 84 g of a white crisp foam (50%), contaminated starting material (17.4 g, 12% recovery) and pure reusable starting material (20 g, 13% recovery). TLC and NMR spectroscopy were consistent with 99% pure product.  
     [0180] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine  
     [0181] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol) and dried over P 2 O 5  under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle). Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture with the rate of addition maintained such that the resulting deep red coloration is just discharged before adding the next drop. The reaction mixture was stirred for 4 hrs., after which time TLC (EtOAc:hexane, 60:40) indicated that the reaction was complete. The solvent was evaporated in vacuuo and the residue purified by flash column chromatography (eluted with 60:40 EtOAc:hexane), to yield 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%) upon rotary evaporation.  
     [0182] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine  
     [0183] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH 2 Cl 2  (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h the mixture was filtered, the filtrate washed with ice cold CH 2 Cl 2 , and the combined organic phase was washed with water and brine and dried (anhydrous Na 2 SO 4 ). The solution was filtered and evaporated to afford 2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). Formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was stirred for 1 h. The solvent was removed under vacuum and the residue was purified by column chromatography to yield 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%) upon rotary evaporation.  
     [0184] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine  
     [0185] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL) and cooled to 10° C. under inert atmosphere. Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and the reaction mixture was stirred. After 10 minutes the reaction was warmed to room temperature and stirred for 2 h. while the progress of the reaction was monitored by TLC (5% MeOH in CH 2 Cl 2 ). Aqueous NaHCO 3  solution (5%, 10 mL) was added and the product was extracted with EtOAc (2×20 mL). The organic phase was dried over anhydrous Na 2 SO4, filtered, and evaporated to dryness. This entire procedure was repeated with the resulting residue, with the exception that formaldehyde (20% w/w, 30 mL, 3.37 mol) was added upon dissolution of the residue in the PPTS/MeOH solution. After the extraction and evaporation, the residue was purified by flash column chromatography and (eluted with 5% MeOH in CH 2 Cl 2 ) to afford 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%) upon rotary evaporation. 2′-O-(dimethylaminooxyethyl)-5-methyluridine Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and TEA (1.67 mL, 12 mmol, dry, stored over KOH) and added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol). The reaction was stirred at room temperature for 24 hrs and monitored by TLC (5% MeOH in CH 2 Cl 2 ). The solvent was removed under vacuum and the residue purified by flash column chromatography (eluted with 10% MeOH in CH 2 Cl 2 ) to afford 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%) upon rotary evaporation of the solvent.  
     [0186] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0187] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P 2 O 5  under high vacuum overnight at 40° C., co-evaporated with anhydrous pyridine (20 mL), and dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol) and 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) were added to the pyridine solution and the reaction mixture was stirred at room temperature until all of the starting material had reacted. Pyridine was removed under vacuum and the residue was purified by column chromatography (eluted with 10% MeOH in CH 2 Cl 2  containing a few drops of pyridine) to yield 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%) upon rotary evaporation.  
     [0188] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0189] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried over P 2 O 5  under high vacuum overnight at 40° C. This was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N 1 ,N 1 -tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 h under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, then the residue was dissolved in EtOAc (70 mL) and washed with 5% aqueous NaHCO 3  (40 mL). The EtOAc layer was dried over anhydrous Na 2 SO 4 , filtered, and concentrated. The residue obtained was purified by column chromatography (EtOAc as eluent) to afford 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%) upon rotary evaporation.  
     [0190] 2′-(Aminooxyethoxy) Nucleoside Amidites  
     [0191] 2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as 2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.  
     [0192] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0193] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside along with aminor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may be phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].  
     [0194] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites  
     [0195] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH 2 —O—CH 2 —N(CH 2 ) 2 , or 2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside amidites are prepared similarly.  
     [0196] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine  
     [0197] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) was slowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. (Caution: Hydrogen gas evolves as the solid dissolves). O 2 -,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) were added and the bomb was sealed, placed in an oil bath and heated to 155° C. for 26 h. then cooled to room temperature. The crude solution was concentrated, the residue was diluted with water (200 mL) and extracted with hexanes (200 mL). The product was extracted from the aqueous layer with EtOAc (3×200 mL) and the combined organic layers were washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (eluted with 5:100:2 MeOH/CH 2 Cl 2 /TEA) as the eluent. The appropriate fractions were combined and evaporated to afford the product as a white solid.  
     [0198] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyl Uridine  
     [0199] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. The reaction mixture was poured into water (200 mL) and extracted with CH 2 Cl 2  (2×200 mL). The combined CH 2 Cl 2  layers were washed with saturated NaHCO 3  solution, followed by saturated NaCl solution, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography (eluted with 5:100:1 MeOH/CH 2 Cl 2 /TEA) to afford the product.  
     [0200] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite  
     [0201] Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) were added to a solution of 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH 2 Cl 2  (20 mL) under an atmosphere of argon. The reaction mixture was stirred overnight and the solvent evaporated. The resulting residue was purified by silica gel column chromatography with EtOAc as the eluent to afford the title compound.  
     Example 2  
     [0202] Oligonucleotide Synthesis  
     [0203] 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.  
     [0204] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3H-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 &gt;3 volumes of ethanol from a 1 M NH 4 OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.  
     [0205] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.  
     [0206] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.  
     [0207] Phosphoramidite oligonucleotides are prepared as described in U.S. Patent, 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.  
     [0208] 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.  
     [0209] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.  
     [0210] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.  
     [0211] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.  
     Example 3  
     [0212] Oligonucleoside Synthesis  
     [0213] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethyl-hydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligo-nucleosides, also identified as amide-4 linked oligonucleo-sides, 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.  
     [0214] 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.  
     [0215] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.  
     Example 4  
     [0216] PNA Synthesis  
     [0217] Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications,  Bioorganic  &amp;  Medicinal Chemistry,  1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.  
     Example 5  
     [0218] Synthesis of Chimeric Oligonucleotides  
     [0219] 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”.  
     [0220] [2′-O—Me]-[2′-deoxy]-[2′-O—Me] Chimeric Phosphorothioate Oligonucleotides  
     [0221] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligo-nucleotide 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-phosphor-amidite 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 (NH 4 OH) 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 spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.  
     [0222] [2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides  
     [0223] [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.  
     [0224] [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides  
     [0225] [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.  
     [0226] 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 6  
     [0227] Oligonucleotide Isolation  
     [0228] 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 NH 4 OAc with &gt;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  
     [0229] oligonucleotide Synthesis—96 Well Plate Format  
     [0230] 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.  
     [0231] Oligonucleotides were cleaved from support and deprotected with concentrated NH 4 OH 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  
     [0232] Oligonucleotide Analysis—96-Well Plate Format  
     [0233] 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  
     [0234] Cell Culture and Oligonucleotide Treatment  
     [0235] 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.  
     [0236] T-24 Cells:  
     [0237] 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&#39;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 #3872) at a density of 7000 cells/well for use in RT-PCR analysis.  
     [0238] 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.  
     [0239] A549 Cells:  
     [0240] 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.  
     [0241] NHDF Cells:  
     [0242] 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.  
     [0243] HEK Cells:  
     [0244] 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.  
     [0245] HEPA 1-6 Cells:  
     [0246] The mouse hepatoma cell line HEPA 1-6 is a derivative of the BW7756 mouse hepatoma that arose in a C57/L mouse and is supplied by the American Type Culture Collection (Manassas, Va.). The cells are propagated in Dulbecco&#39;s minimal essential medium with 10% fetal bovine serum. Cells are subcultured by removing the medium, adding fresh 0.25% trypsin, 0.03% EDTA solution and letting the culture sit at room temperature for 3 minutes. Trypsin is then removed and the culture allowed to sit an additional 5 minutes until the cells begin to detach, at which point, fresh medium is added.  
     [0247] Treatment with Antisense Compounds:  
     [0248] When cells reached 70% 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. After 4-7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.  
     [0249] 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-Ha-ras (for ISIS 13920) 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 H-ras 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  
     [0250] Analysis of Oligonucleotide Inhibition of PRL-3 Expression  
     [0251] Antisense modulation of PRL-3 expression can be assayed in a variety of ways known in the art. For example, PRL-3 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 taught in, for example, Ausubel, F. M. et al.,  Current Protocols in Molecular Biology,  Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley &amp; Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al.,  Current Protocols in Molecular Biology,  Volume 1, pp. 4.2.1-4.2.9, John Wiley &amp; Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer&#39;s instructions.  
     [0252] Protein levels of PRL-3 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to PRL-3 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 antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology,  Volume 2, pp. 11.12.1-11.12.9, John Wiley &amp; Sons, Inc., 1997). Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology,  Volume 2, pp. 11.4.1-11.11.5, John Wiley &amp; Sons, Inc., 1997).  
     [0253] Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology,  Volume 2, pp. 10.16.1-10.16.11, John Wiley &amp; Sons, Inc., 1998). Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology,  Volume 2, pp. 10.8.1-10.8.21, John Wiley &amp; Sons, Inc., 1997). Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology,  Volume 2, pp. 11.2.1-11.2.22, John Wiley &amp; Sons, Inc., 1991).  
     Example 11  
     [0254] Poly(A)+ mRNA Isolation  
     [0255] Poly(A)+mRNA was isolated according to Miura et al., ( Clin. Chem.,  1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology,  Volume 1, pp. 4.5.1-4.5.3, John Wiley &amp; Sons, Inc., 1993). 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.  
     [0256] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.  
     Example 12  
     [0257] Total RNA Isolation  
     [0258] Total RNA was isolated using an RNEASY 96 ™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer&#39;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 QIAVACTM 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 S96™ 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 QIAVACTM manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 170 μL water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.  
     [0259] 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  
     [0260] Real-Time Quantitative PCR Analysis of PRL-3 mRNA Levels  
     [0261] Quantitation of PRL-3 mRNA levels was determined by real-time quantitative PCR using the ABI PRISMTM 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer&#39;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, CA or Integrated DNA Technologies Inc., Coralville, IA) 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, CA 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™ 7700 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.  
     [0262] 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.  
     [0263] 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 (—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. 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).  
     [0264] 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 from Molecular Probes. Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).  
     [0265] 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 480 nm and emission at 520 nm.  
     [0266] Probes and primers to human PRL-3 were designed to hybridize to a human PRL-3 sequence, using published sequence information (GenBank accession number NM — 007079.2, incorporated herein as SEQ ID NO:4). For human PRL-3 the PCR primers were:  
     [0267] forward primer: ACGCTCAGCACCTTCATTGA (SEQ ID NO: 5)  
     [0268] reverse primer: CCAGCGGCGTTTTGTCATA (SEQ ID NO: 6) and the PCR probe was: FAM-CTACCACTGTGGTGCGTGTGTGTGAAGTG-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were:  
     [0269] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8)  
     [0270] reverse primer: GAAGATGGTGATGGGATTTC GGGTCTCGCTCCTGGAAGAT(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.  
     [0271] Probes and primers to mouse PRL-3 were designed to hybridize to a mouse PRL-3 sequence, using published sequence information (GenBank accession number AK014601.1, incorporated herein as SEQ ID NO:11). For mouse PRL-3 the PCR primers were:  
     [0272] forward primer: TTTTCCAGCACCTTTGTTACCA (SEQ ID NO:12)  
     [0273] reverse primer: CCCCTGCTCTATAACACTCTTCACA (SEQ ID NO: 13) and the PCR probe was: FAM-TGGACTCTCAAGGCAATAAATCAGGAGCTG-TAMRA (SEQ ID NO: 14) where FAM is the fluorescent reporter dye and TAMRA is the quencher dye. For mouse GAPDH the PCR primers were: forward primer: GGCAAATTCAACGGCACAGT(SEQ ID NO:15) reverse primer: GGGTCTCGCTCCTGGAAGAT(SEQ ID NO:16) and the PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 17) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.  
     Example 14  
     [0274] Northern Blot Analysis of PRL-3 mRNA Levels  
     [0275] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOLTM (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer&#39;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, TX). 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&#39;s recommendations for stringent conditions.  
     [0276] To detect human PRL-3, a human PRL-3 specific probe was prepared by PCR using the forward primer ACGCTCAGCACCTTCATTGA (SEQ ID NO: 5) and the reverse primer CCAGCGGCGTTTTGTCATA (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.).  
     [0277] To detect mouse PRL-3, a mouse PRL-3 specific probe was prepared by PCR using the forward primer TTTTCCAGCACCTTTGTTACCA (SEQ ID NO: 12) and the reverse primer CCCCTGCTCTATAACACTCTTCACA (SEQ ID NO: 13). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).  
     [0278] 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  
     [0279] Antisense Inhibition of Human PRL-3 Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap  
     [0280] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human PRL-3 RNA, using published sequences (GenBank accession number NM — 007079.2, representing PRL-3 variant 1, incorporated herein as SEQ ID NO: 4, GenBank accession number NM — 032611.1, representing PRL-3 variant 2, incorporated herein as SEQ ID NO: 18, GenBank accession number AL525371.1, representing a 5′-extension of SEQ ID NO: 18, incorporated herein as SEQ ID NO: 19, and GenBank accession number BI489974.1, representing a 5′-extension of SEQ ID NO: 19, incorporated herein as SEQ ID NO: 20). The oligonucleotides are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked 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 PRL-3 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which A549 cells were treated with 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 1                              Inhibition of human PRL-3 mRNA levels by chimeric               phosphorothioate oligonucleotides having 2′-MOE wings and a           deoxy gap                                                                     TARGET                       CONTROL                       SEQ ID       TARGET               SEQ ID       SEQ ID           ISIS #       REGION       NO       SITE       SEQUENCE       % INHIB       NO       NO                                                           217393   5′UTR   19   695   cacaaatattgtgcaaatac   58   21   1           217394   5′UTR   19   700   ccccgcacaaatattgtgca   82   22   1       217398   Coding   4   437   ccgtacttcttcaggtcctc   94   23   1       217399   Coding   4   442   tagccccgtacttcttcagg   96   24   1       217400   Coding   4   447   agtggtagccccgtacttct   97   25   1       217401   Coding   4   452   accacagtggtagccccgta   0   26   1       217402   Coding   4   476   tcataggtcacttcacacac   79   27   1       217404   Coding   18   695   tcgtacttcatcccgctctc   90   28   1       217405   Coding   18   701   gcgtcctcgtacttcatccc   94   29   1       217406   Coding   4   677   agctgcttgctgttgatggc   89   30   1       217407   Coding   4   682   aggtgagctgcttgctgttg   86   31   1       217408   Coding   4   687   caggtaggtgagctgcttgc   83   32   1       217409   Coding   4   692   ttctccaggtaggtgagctg   83   33   1       217410   Coding   4   735   cgtgtgtgggtctttgaacc   90   34   1       217455   5′UTR   18   8   ctcccgtctctcagagctgg   94   35   1       217456   5′UTR   18   14   tccaaactcccgtctctcag   81   36   1       217457   5′UTR   18   21   gggcaactccaaactcccgt   92   37   1       217458   5′UTR   18   26   aaagcgggcaactccaaact   66   38   1       217459   5′UTR   18   33   ccaaagtaaagcgggcaact   88   39   1       217460   5′UTR   18   116   ccactgtttggataattaaa   82   40   1       217461   5′UTR   18   129   gggaggaagctgcccactgt   79   41   1       217462   5′UTR   18   168   cataccccgcacaaatattg   49   42   1       217463   5′UTR   18   201   gtccaagagaaacgagattt   88   43   1       217464   5′UTR   18   219   aacgagatccctgtgcttgt   91   44   1       217465   5′UTR   18   256   cctgagaagtccccacacac   62   45   1       217466   5′UTR   18   287   gaagggactgcagagaaggc   84   46   1       217467   Start   18   330   gttcatccgagccatggcgc   90   47   1           Codon       217468   Start   18   334   ggcggttcatccgagccatg   81   48   1           Codon       217469   Coding   18   360   tttgtagctcacctccaccg   85   49   1       217470   Coding   18   370   agcgcatgtgtttgtagctc   81   50   1       217471   Coding   18   426   caggtcctcaatgaaggtgc   92   51   1       217472   Coding   18   470   gtcacttcacacacacgcac   86   52   1       217473   Coding   18   507   ggtgatgccatccttctcca   89   53   1       217474   Coding   18   514   ccacaacggtgatgccatcc   88   54   1       217475   Coding   18   518   cagtccacaacggtgatgcc   85   55   1       217476   Coding   18   525   aaacggccagtccacaacgg   92   56   1       217477   Coding   18   573   gctcagccagtcttccacta   95   57   1       217478   Coding   18   594   acagaacttggccttcacca   92   58   1       217479   Coding   18   623   tgcacagccacgcagctgcc   92   59   1       217480   Coding   18   638   aggcccgccacgcagtgcac   90   60   1       217481   Coding   18   654   gactggagcccggcccaggc   48   61   1       217482   Coding   18   658   caaggactggagcccggccc   83   62   1       217483   Coding   18   723   cttctggcggatgaactgga   86   63   1       217484   Coding   18   779   ttgggccggtatttctccag   91   64   1       217485   Coding   18   784   tctgtttgggccggtatttc   85   65   1       217486   Coding   18   797   ttgaaccgcagcctctgttt   90   66   1       217487   Coding   18   823   accgggtcttgtgcgtgtgt   81   67   1       217488   Coding   18   832   taacgcagcaccgggtcttg   92   68   1       217489   Stop   18   837   ctacataacgcagcaccggg   88   69   1           Codon       217490   Stop   18   845   gtcctgagctacataacgca   90   70   1           Codon       217491   3′UTR   18   862   gaccaggcccagccaaggtc   89   71   1       217492   3′UTR   18   867   atgacgaccaggcccagcca   83   72   1       217493   3′UTR   18   879   gtcctgacctacatgacgac   95   73   1       217494   3′UTR   18   886   agccaaggtcctgacctaca   93   74   1       217495   3′UTR   18   938   tggagcccctgctgggctgg   67   75   1       217497   3′UTR   18   951   gccagccaaggcctggagcc   93   76   1       217500   3′UTR   18   992   acaagtgcacggaggtgtcg   79   77   1       217502   3′UTR   18   1004   cgctcctcggacacaagtgc   91   78   1       217504   3′UTR   18   1011   ggctcctcgctcctcggaca   89   79   1       217506   3′UTR   18   1103   acccacctcagagagacaga   78   80   1       217509   3′UTR   18   1145   ccagcctggctggtgtggga   35   81   1       217511   3′UTR   18   1159   caggctagaggagaccagcc   62   82   1       217514   3′UTR   18   1192   tggttacaaaatataccccc   87   83   1       217516   3′UTR   18   1198   gcccagtggttacaaaatat   82   84   1       217518   3′UTR   18   1247   aggtgccgagaacaggtcag   91   85   1       217521   3′UTR   18   1259   tctaataatttaaggtgccg   91   86   1       217524   3′UTR   18   1286   tgtccggagcacctgactgc   92   87   1       217526   3′UTR   18   1298   attgccttcgggtgtccgga   82   88   1       217529   3′UTR   18   1306   cctgttttattgccttcggg   82   89   1       217531   3′UTR   18   1314   tcacggctcctgttttattg   81   90   1       217534   5′UTR   4   115   cactgtttggataattaaaa   74   91   1       217536   5′UTR   19   234   tgtgtcacgttaccccatcg   94   92   1       217538   5′UTR   19   318   cttgaactgaaccaactcca   92   93   1       217541   5′UTR   19   324   aatgaacttgaactgaacca   79   94   1       217544   5′UTR   19   373   gcccccttcactcagaggtg   90   95   1       217546   5′UTR   19   394   acattggtggatgggcagac   56   96   1       217549   5′UTR   19   442   cgccaagcagtgtccagagc   89   97   1       217551   5′UTR   20   38   ctggcggtgcccacggtgct   76   98   1                  
 
     [0281] As shown in Table 1, SEQ ID NOs 22, 23, 24, 25, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 43, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 78, 79, 83, 84, 85, 86, 87, 88, 89, 90, 92, 93, 95 and 97 demonstrated at least 80% inhibition of human PRL-3 expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “preferred target regions” and are therefore preferred sites for targeting by compounds of the present invention. These preferred target regions are shown in Table 3. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number of the corresponding target nucleic acid. Also shown in Table 3 is the species in which each of the preferred target regions was found.  
     Example 16  
     [0282] Antisense Inhibition of Mouse PRL-3 Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap.  
     [0283] In accordance with the present invention, a second series of oligonucleotides were designed to target different regions of the mouse PRL-3 RNA, using published sequences (GenBank accession number AK014601.1, incorporated herein as SEQ ID NO: 11, and GenBank accession number AK004562.1, incorporated herein as SEQ ID NO: 99). The oligonucleotides are shown in Table 2. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “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 mouse PRL-3 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which HEPA 1-6 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 2                              Inhibition of mouse PRL-3 mRNA levels by chimeric               phosphorothioate oligonucleotides having 2′-MOE wings and a           deoxy gap                                                             TARGET                       CONTROL                       SEQ ID       TARGET             SEQ ID     SEQ ID           ISIS #       REGION       NO       SITE       SEQUENCE       % INHIB       NO       NO                                                           217377   5′UTR   11   18   ggccgcctccatgcgccagg   0   100   3           217378   5′UTR   11   70   catcaatcccaccctggtct   2   101   3       217379   5′UTR   11   128   ggcagcacccacggtgttgg   32   102   3       217380   5′UTR   11   159   tgagggccagaggtgtacag   0   103   3       217381   5′UTR   11   222   ggctcaggcagcagtgctgc   0   104   3       217382   5′UTR   11   280   tgatccggtctgtcacgtta   35   105   3       217383   5′UTR   11   329   aactcaaccaaactctagcg   31   106   3       217384   5′UTR   11   360   agaagtcaggaccaaaggga   0   107   3       217385   5′UTR   11   382   cctttggtcagagttgggcc   37   108   3       217386   5′UTR   11   411   tctctgcatgggtggacgga   26   109   3       217387   5′UTR   11   421   ggagacaccctctctgcatg   24   110   3       217388   5′UTR   11   456   ggccgcagagcagtgtcctc   46   111   3       217389   5′UTR   11   476   aacttcgggttggtgactcc   41   112   3       217390   5′UTR   11   552   tcctgtccctcagagtggag   21   113   3       217391   5′UTR   11   603   aaacaagcacacttctcccc   46   114   3       217392   5′UTR   11   649   cctcgtggcggataattcca   47   115   3       217395   5′UTR   11   702   cctacgccccgcacaaatat   20   116   3       217396   5′UTR   11   723   aagacttcttaaaacctcgc   35   117   3       217397   Start   11   841   catgcgggccatgcctcctc   0   118   3           Codon       217403   Coding   11   1108   gtagaacttggccttcagca   0   119   3       217411   Stop   11   1360   gggcctgagctacatgacgc   23   120   3           Codon       217412   3′UTR   11   1446   acagccgtgggtacagatgg   0   121   3       217413   3′UTR   11   1460   acagcttcagaaagacagcc   0   122   3       217414   3′UTR   11   1552   aactcccacctgcaatacac   0   123   3       217415   3′UTR   11   1645   gccttgagagtccagagcac   80   124   3       217416   3′UTR   11   1658   ctcctgatttattgccttga   66   125   3       217417   3′UTR   11   1689   gctctataacactcttcaca   0   126   3       217418   3′UTR   11   1728   ggagaccatggcctcccggg   0   127   3       217419   3′UTR   11   1775   cgccactgcagcagcagtag   13   128   3       217420   3′UTR   11   1785   catggaggcacgccactgca   0   129   3       217421   3′UTR   11   1848   ttgcgtcaagaacttgtaga   4   130   3       217422   3′UTR   11   1855   ctgatttttgcgtcaagaac   25   131   3       217423   3′UTR   11   1875   cgtcaccagtccaagctgtg   0   132   3       217424   3′UTR   11   1884   gttacaaaccgtcaccagtc   43   133   3       217425   3′UTR   11   1914   ctgcagaggtgatcagtctc   3   134   3       217426   3′UTR   11   1923   tactctgtcctgcagaggtg   0   135   3       217427   3′UTR   11   1945   ctgaaagttccaagtgaccc   11   136   3       217428   3′UTR   11   1955   gtggaggccactgaaagttc   0   137   3       217429   3′UTR   11   2033   ggcagacccatgtcaccaaa   16   138   3       217430   3′UTR   11   2041   aggtcctaggcagacccatg   16   139   3       217431   3′UTR   11   2049   gcaggatgaggtcctaggca   20   140   3       217432   3′UTR   11   2055   ttataagcaggatgaggtcc   0   141   3       217433   3′UTR   11   2067   actgctcagaacttataagc   33   142   3       217434   3′UTR   11   2093   tcctcagctaagggaagagt   50   143   3       217435   3′UTR   11   2461   actgacacattgtcactgtt   39   144   3       217436   3′UTR   11   2470   cacatccacactgacacatt   0   145   3               217437   3′UTR   11   2477   cagccagcacatccacactg   0   146   3       217438   3′UTR   11   2488   gtgctataagacagccagca   0   147   3       217439   3′UTR   11   2504   cgtcctaagtatgtcagtgc   0   148   3       217440   3′UTR   11   2527   ggtctgcaaaggcctgagga   0   149   3       217441   3′UTR   11   2557   ggatctctgttctgggcaca   0   150   3       217442   3′UTR   11   2563   cgccctggatctctgttctg   0   151   3       217443   3′UTR   11   2586   aggcagcaggtaccagtgag   0   152   3       217444   3′UTR   11   2596   tgatgcaagcaggcagcagg   0   153   3       217445   3′UTR   11   2604   agagggaatgatgcaagcag   7   154   3       217446   3′UTR   11   2612   gaacttgaagagggaatgat   21   155   3       217447   3′UTR   11   2621   agctaggtggaacttgaaga   1   156   3       217448   3′UTR   11   2642   ggtttctgggacctagtcca   0   157   3       217449   3′UTR   11   2691   ctaggacaggcaagaacagg   16   158   3       217450   3′UTR   11   2713   ggctacaccttgtcactccc   0   159   3       217451   3′UTR   11   2729   caccattactgagtttggct   0   160   3       217452   3′UTR   11   2788   aaccagtgactttgtggaaa   39   161   3       217453   Genomic   99   39   Cacaatctggcggtccgggc   0   162   3       217454   Genomic   99   49   Cttcagaaggcacaatctgg   38   163   3                  
 
     [0284] As shown in Table 2, SEQ ID NOs 105, 108, 111, 112, 114, 115, 117, 124, 125, 133, 143, 144, 161 and 163 demonstrated at least 35% inhibition of mouse PRL-3 expression in this experiment and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “preferred target regions” and are therefore preferred sites for targeting by compounds of the present invention. These preferred target regions are shown in Table 3. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number of the corresponding target nucleic acid. Also shown in Table 3 is the species in which each of the preferred target regions was found.  
                     TABLE 3                              Sequence and position of preferred target regions identified               in PRL-3.                                                     TARGET               REV COMP                           SEQ ID       TARGET           OF SEQ           SEQ ID           SITEID       NO       SITE       SEQUENCE       ID       ACTIVE IN       NO                                                       134110   19   700   tgcacaatatttgtgcgggg   22     H. sapiens     164           134114   4   437   gaggacctgaagaagtacgg   23     H. sapiens     165       134115   4   442   cctgaagaagtacggggcta   24     H. sapiens     166       134116   4   447   agaagtacggggctaccact   25     H. sapiens     167       134120   18   695   gagagcgggatgaagtacga   28     H. sapiens     168       134121   18   701   gggatgaagtacgaggacgc   29     H. sapiens     169       134122   4   677   gccatcaacagcaagcagct   30     H. sapiens     170       134123   4   682   caacagcaagcagctcacct   31     H. sapiens     171       134124   4   687   gcaagcagctcacctacctg   32     H. sapiens     172       134125   4   692   cagctcacctacctggagaa   33     H. sapiens     173       134126   4   735   ggttcaaagacccacacacg   34     H. sapiens     174       134171   18   8   ccagctctgagagacgggag   35     H. sapiens     175       134172   18   14   ctgagagacgggagtttgga   36     H. sapiens     176       134173   18   21   acgggagtttggagttgccc   37     H. sapiens     177       134175   18   33   agttgcccgctttactttgg   39     H. sapiens     178       134176   18   116   tttaattatccaaacagtgg   40     H. sapiens     179       134179   18   201   aaatctcgtttctcttggac   43     H. sapiens     180       134180   18   219   acaagcacagggatctcgtt   44     H. sapiens     181       134182   18   287   gccttctctgcagtcccttc   46     H. sapiens     182       134183   18   330   gcgccatggctcggatgaac   47     H. sapiens     183       134184   18   334   catggctcggatgaaccgcc   48     H. sapiens     184       134185   18   360   cggtggaggtgagctacaaa   49     H. sapiens     185       134186   18   370   gagctacaaacacatgcgct   50     H. sapiens     186       134187   18   426   gcaccttcattgaggacctg   51     H. sapiens     187       134188   18   470   gtgcgtgtgtgtgaagtgac   52     H. sapiens     188       134189   18   507   tggagaaggatggcatcacc   53     H. sapiens     189       134190   18   514   ggatggcatcaccgttgtgg   54     H. sapiens     190       134191   18   518   ggcatcaccgttgtggactg   55     H. sapiens     191       134192   18   525   ccgttgtggactggccgttt   56     H. sapiens     192       134193   18   573   tagtggaagactggctgagc   57     H. sapiens     193       134194   18   594   tggtgaaggccaagttctgt   58     H. sapiens     194       134195   18   623   ggcagctgcgtggctgtgca   59     H. sapiens     195       134196   18   638   gtgcactgcgtggcgggcct   60     H. sapiens     196       134198   18   658   gggccgggctccagtccttg   62     H. sapiens     197       134199   18   723   tccagttcatccgccagaag   63     H. sapiens     198       134200   18   779   ctggagaaataccggcccaa   64     H. sapiens     199       134201   18   784   gaaataccggcccaaacaga   65     H. sapiens     200       134202   18   797   aaacagaggctgcggttcaa   66     H. sapiens     201       134203   18   823   acacacgcacaagacccggt   67     H. sapiens     202       134204   18   832   caagacccggtgctgcgtta   68     H. sapiens     203       134205   18   837   cccggtgctgcgttatgtag   69     H. sapiens     204       134206   18   845   tgcgttatgtagctcaggac   70     H. sapiens     205       134207   18   862   gaccttggctgggcctggtc   71     H. sapiens     206       134208   18   867   tggctgggcctggtcgtcat   72     H. sapiens     207       134209   18   879   gtcgtcatgtaggtcaggac   73     H. sapiens     208       134210   18   886   tgtaggtcaggaccttggct   74     H. sapiens     209       134212   18   951   ggctccaggccttggctggc   76     H. sapiens     210       134214   18   1004   gcacttgtgtccgagqagcg   78     H. sapiens     211       134215   18   1011   tgtccgaggagcgaggagcc   79     H. sapiens     212       134219   18   1192   gggggtatattttgtaacca   83     H. sapiens     213       134220   18   1198   atattttgtaaccactgggc   84     H. sapiens     214       134221   18   1247   ctgacctgttctcggcacct   85     H. sapiens     215       134222   18   1259   cggcaccttaaattattaga   86     H. sapiens     216       134223   18   1286   gcagtcaggtgctccggaca   87     H. sapiens     217       134224   18   1298   tccggacacccgaaggcaat   88     H. sapiens     218       134225   18   1306   cccgaaggcaataaaacagg   89     H. sapiens     219       134226   18   1314   caataaaacaggagccgtga   90     H. sapiens     220       134228   19   234   cgatggggtaacgtgacaca   92     H. sapiens     221       134229   19   318   tggagttggttcagttcaag   93     H. sapiens     222       134231   19   373   cacctctgagtgaagggggc   95     H. sapiens     223       134233   19   442   gctctggacactgcttggcg   97     H. sapiens     224       134098   11   280   taacgtgacagaccggatca   105     M. musculus     225       134101   11   382   ggcccaactctgaccaaagg   108     M. musculus     226       134104   11   456   gaggacactgctctgcggcc   111     M. musculus     227       134105   11   476   ggagtcaccaacccgaagtt   112     M. musculus     228       134107   11   603   ggggagaagtgtgcttgttt   114     M. musculus     229       134108   11   649   tggaattatccgccacgagg   115     M. musculus     230       134112   11   723   gcgaggttttaagaagtctt   117     M. musculus     231       134131   11   1645   gtgctctggactctcaaggc   124     M. musculus     232       134132   11   1658   tcaaggcaataaatcaggag   125     M. musculus     233       134140   11   1884   gactggtgacggtttgtaac   133     M. musculus     234       134150   11   2093   actcttcccttagctgagga   143     M. musculus     235       134151   11   2461   aacagtgacaatgtgtcagt   144     M. musculus     236       134168   11   2788   tttccacaaagtcactggtt   161     M. musculus     237       134170   99   49   ccagattgtgccttctgaag 163     M. musculus     238                  
 
     [0285] As these “preferred target regions” 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 sites and consequently inhibit the expression of PRL-3.  
     [0286] In one embodiment, the “preferred target region” may be employed in screening candidate antisense compounds. “Candidate antisense compounds” are those that inhibit the expression of a nucleic acid molecule encoding PRL-3 and which comprise at least an 8-nucleobase portion which is complementary to a preferred target region. The method comprises the steps of contacting a preferred target region of a nucleic acid molecule encoding PRL-3 with one or more candidate antisense compounds, and selecting for one or more candidate antisense compounds which inhibit the expression of a nucleic acid molecule encoding PRL-3. Once it is shown that the candidate antisense compound or compounds are capable of inhibiting the expression of a nucleic acid molecule encoding PRL-3, the candidate antisense compound may be employed as an antisense compound in accordance with the present invention.  
     [0287] According to the present invention, antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.  
     Example 17  
     [0288] Western Blot Analysis of PRL-3 Protein Levels  
     [0289] 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 PRL-3 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 18  
     [0290] Targeting of Individual Oligonucleotides to Specific Variants of PRL-3  
     [0291] It is advantageous to selectively inhibit the expression of one or more variants of PRL-3. 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 PRL-3. A summary of the target sites of the variants is shown in Table 4 and includes Genbank accession number NM — 007079.2, representing PRL-3 variant 2, incorporated herein as SEQ ID NO: 4; Genbank accession number NM — 032611.1, representing PRL-3 variant 1, incorporated herein as SEQ ID NO: 18; and GenBank accession number NM — 007079.1, representing the main mRNA of PRL-3, incorporated herein as SEQ ID NO: 239.  
               TABLE 4                          Targeting of individual oligonucleotides to specific variants       of PRL-3                                     OLIGO SEQ   TARGET       VARIANT       ISIS #   ID NO.   SITE   VARIANT   SEQ ID NO.                                         217404   28   695   PRL-3 variant 1   18       217404   28   598   PRL-3   239       217405   29   701   PRL-3 variant 1   18       217405   29   604   PRL-3   239       217406   30   677   PRL-3 variant 2   4       217406   30   752   PRL-3 variant 1   18       217455   35   8   PRL-3 variant 2   4       217455   35   8   PRL-3 variant 1   18       217456   36   14   PRL-3 variant 2   4       217456   36   14   PRL-3 variant 1   18       217457   37   21   PRL-3 variant 2   4       217457   37   21   PRL-3 variant 1   18       217458   38   26   PRL-3 variant 2   4       217458   38   26   PRL-3 variant 1   18       217459   39   33   PRL-3 variant 2   4       217459   39   33   PRL-3 variant 1   18       217481   61   654   PRL-3 variant 1   18       217482   62   658   PRL-3 variant 1   18       217483   63   723   PRL-3 variant 1   18       217483   63   626   PRL-3   239       217506   80   1028   PRL-3 variant 2   4       217506   80   1103   PRL-3 variant 1   18       217509   81   1070   PRL-3 variant 2   4       217509   81   1145   PRL-3 variant 1   18       217511   82   1084   PRL-3 variant 2   4       217511   82   1159   PRL-3 variant 1   18       217514   83   1117   PRL-3 variant 2   4       217514   83   1192   PRL-3 variant 1   18       217516   84   1123   PRL-3 variant 2   4       217516   84   1198   PRL-3 variant 1   18       217518   85   1172   PRL-3 variant 2   4       217518   85   1247   PRL-3 variant 1   18       217521   86   1184   PRL-3 variant 2   4       217521   86   1259   PRL-3 variant 1   18       217524   87   1211   PRL-3 variant 2   4       217524   87   1286   PRL-3 variant 1   18       217526   88   1223   PRL-3 variant 2   4       217526   88   1298   PRL-3 variant 1   18       217529   89   1231   PRL-3 variant 2   4       217529   89   1306   PRL-3 variant 1   18       217531   90   1239   PRL-3 variant 2   4       217531   90   1314   PRL-3 variant 1   18                  
 
     [0292] 
    
     
       
         1 
         
           
             239  
           
           
             1  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            1 

tccgtcatcg ctcctcaggg                                                 20 

 
           
             2  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            2 

gtgcgcgcga gcccgaaatc                                                 20 

 
           
             3  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            3 

atgcattctg cccccaagga                                                 20 

 
           
             4  
             1321  
             DNA  
             H. sapiens  
             
 
             
               CDS  
               (335)...(781)  
             
           
            4 

tgactatcca gctctgagag acgggagttt ggagttgccc gctttacttt ggttgggttg     60 

gggggggcgg cgggctgttt tgttcctttt cttttttaag agttgggttt tcttttttaa    120 

ttatccaaac agtgggcagc ttcctccccc acacccaagt atttgcacaa tatttgtgcg    180 

gggtatgggg gtgggttttt aaatctcgtt tctcttggac aagcacaggg atctcgttct    240 

cctcattttt tgggggtgtg tggggacttc tcaggtcgtg tccccagcct tctctgcagt    300 

cccttctgcc ctgccgggcc cgtcgggagg cgcc atg gct cgg atg aac cgc ccg    355 
                                      Met Ala Arg Met Asn Arg Pro 
                                        1               5 

gcc ccg gtg gag gtg agc tac aaa cac atg cgc ttc ctc atc acc cac      403 
Ala Pro Val Glu Val Ser Tyr Lys His Met Arg Phe Leu Ile Thr His 
         10                  15                  20 

aac ccc acc aac gcc acg ctc agc acc ttc att gag gac ctg aag aag      451 
Asn Pro Thr Asn Ala Thr Leu Ser Thr Phe Ile Glu Asp Leu Lys Lys 
     25                  30                  35 

tac ggg gct acc act gtg gtg cgt gtg tgt gaa gtg acc tat gac aaa      499 
Tyr Gly Ala Thr Thr Val Val Arg Val Cys Glu Val Thr Tyr Asp Lys 
 40                  45                  50                  55 

acg ccg ctg gag aag gat ggc atc acc gtt gtg gac tgg ccg ttt gac      547 
Thr Pro Leu Glu Lys Asp Gly Ile Thr Val Val Asp Trp Pro Phe Asp 
                 60                  65                  70 

gat ggg gcg ccc ccg ccc ggc aag gta gtg gaa gac tgg ctg agc ctg      595 
Asp Gly Ala Pro Pro Pro Gly Lys Val Val Glu Asp Trp Leu Ser Leu 
             75                  80                  85 

gtg aag gcc aag ttc tgt gag gcc ccc ggc agc tgc gtg gct gtg cac      643 
Val Lys Ala Lys Phe Cys Glu Ala Pro Gly Ser Cys Val Ala Val His 
         90                  95                 100 

tgc gtg gcg ggc ctg ggc cgg aag cgc cgc gga gcc atc aac agc aag      691 
Cys Val Ala Gly Leu Gly Arg Lys Arg Arg Gly Ala Ile Asn Ser Lys 
    105                 110                 115 

cag ctc acc tac ctg gag aaa tac cgg ccc aaa cag agg ctg cgg ttc      739 
Gln Leu Thr Tyr Leu Glu Lys Tyr Arg Pro Lys Gln Arg Leu Arg Phe 
120                 125                 130                 135 

aaa gac cca cac acg cac aag acc cgg tgc tgc gtt atg tag ctcaggacct   791 
Lys Asp Pro His Thr His Lys Thr Arg Cys Cys Val Met 
                140                 145 

tggctgggcc tggtcgtcat gtaggtcagg accttggctg gacctggagg ccctgcccag    851 

ccctgctctg cccagcccag caggggctcc aggccttggc tggccccaca tcgccttttc    911 

ctccccgaca cctccgtgca cttgtgtccg aggagcgagg agcccctcgg gccctgggtg    971 

gcctctgggc cctttctcct gtctccgcca ctccctctgg cggcgctggc cgtggctctg   1031 

tctctctgag gtgggtcggg cgccctctgc ccgccccctc ccacaccagc caggctggtc   1091 

tcctctagcc tgtttgttgt ggggtggggg tatattttgt aaccactggg cccccagccc   1151 

ctcttttgcg accccttgtc ctgacctgtt ctcggcacct taaattatta gaccccgggg   1211 

cagtcaggtg ctccggacac ccgaaggcaa taaaacagga gccgtgaaaa aaaaaaaaaa   1271 

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa              1321 

 
           
             5  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            5 

acgctcagca ccttcattga                                                 20 

 
           
             6  
             19  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            6 

ccagcggcgt tttgtcata                                                  19 

 
           
             7  
             29  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            7 

ctaccactgt ggtgcgtgtg tgtgaagtg                                       29 

 
           
             8  
             19  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            8 

gaaggtgaag gtcggagtc                                                  19 

 
           
             9  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            9 

gaagatggtg atgggatttc                                                 20 

 
           
             10  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            10 

caagcttccc gttctcagcc                                                 20 

 
           
             11  
             3222  
             DNA  
             M. musculus  
             
 
             
               CDS  
               (849)...(1370)  
             
           
            11 

ggtcccggcc cggccggcct ggcgcatgga ggcggccgca cgcctgcggg cgcggattgt     60 

gccttctgaa gaccagggtg ggattgatgg agaagcccca ggagggcagc tctgactact    120 

gccgttccca acaccgtggg tgctgccgct gaggagacct gtacacctct ggccctcacc    180 

attgtccttg cctcccaatg gcctctgctg ccaggccgga ggcagcactg ctgcctgagc    240 

ccgctgcccc tcttcaggac ttgccgttcc ctgatggggt aacgtgacag accggatcag    300 

aggctgcctg cccaccacgg cccagggccg ctagagtttg gttgagttca agttcatttt    360 

ccctttggtc ctgacttctg gggcccaact ctgaccaaag gggacactcc tccgtccacc    420 

catgcagaga gggtgtctcc agcgacggcc ccatagagga cactgctctg cggccggagt    480 

caccaacccg aagttctctc tgctcagttt tttttggttg ttgttatttt tattttgact    540 

ccttgtaact tctccactct gagggacagg actttggcgc tgcccgtctt gctgcggggt    600 

ggggggagaa gtgtgcttgt ttcttttctt ttttagaatt ggtttttttg gaattatccg    660 

ccacgaggca gcttcctctc cctctcccag gtatttgcac aatatttgtg cggggcgtag    720 

gggcgaggtt ttaagaagtc ttttctttgt ggacaagcac ggggatctca ctggacttgg    780 

tgtggggggc tgggggaccc cccgtgcagc ccttgctggc tagtcccctc tgggtccccg    840 

gaggaggc atg gcc cgc atg aac cgg cct gcg cct gtg gag gtg agc tac     890 
         Met Ala Arg Met Asn Arg Pro Ala Pro Val Glu Val Ser Tyr 
           1               5                  10 

cgg cac atg cgc ttc ctc atc acc cac aac ccc agc aat gcc acc ctc      938 
Arg His Met Arg Phe Leu Ile Thr His Asn Pro Ser Asn Ala Thr Leu 
 15                  20                  25                  30 

agc acg ttc atc gag gac ctg aag aag tac ggg gct acc act gtg gtg      986 
Ser Thr Phe Ile Glu Asp Leu Lys Lys Tyr Gly Ala Thr Thr Val Val 
                 35                  40                  45 

cgc gtg tgt gaa gtg acc tat gac aag acc ccc ctg gag aag gac ggc     1034 
Arg Val Cys Glu Val Thr Tyr Asp Lys Thr Pro Leu Glu Lys Asp Gly 
             50                  55                  60 

atc act gtt gtg gac tgg ccc ttt gat gat gga gcg ccc cct cct ggc     1082 
Ile Thr Val Val Asp Trp Pro Phe Asp Asp Gly Ala Pro Pro Pro Gly 
         65                  70                  75 

aaa gtg gta gag gac tgg ctg agc ctg ctg aag gcc aag ttc tac aat     1130 
Lys Val Val Glu Asp Trp Leu Ser Leu Leu Lys Ala Lys Phe Tyr Asn 
     80                  85                  90 

gac ccg gga agc tgc gta gct gtg cac tgt gtg gcg ggc ctg gga agg     1178 
Asp Pro Gly Ser Cys Val Ala Val His Cys Val Ala Gly Leu Gly Arg 
 95                 100                 105                 110 

gcc cca gtg ctc gtg gct ctc gcc ctc atc gag agc ggg atg aag tac     1226 
Ala Pro Val Leu Val Ala Leu Ala Leu Ile Glu Ser Gly Met Lys Tyr 
                115                 120                 125 

gag gac gcc atc cag ttc atc cga cag aag cgc cgt ggg gcc atc aac     1274 
Glu Asp Ala Ile Gln Phe Ile Arg Gln Lys Arg Arg Gly Ala Ile Asn 
            130                 135                 140 

agc aag cag ctc acc tac ctg gag aag tac cgg cct aag cag aga ctg     1322 
Ser Lys Gln Leu Thr Tyr Leu Glu Lys Tyr Arg Pro Lys Gln Arg Leu 
        145                 150                 155 

agg ttc aaa gac cca cac acg cac aag acc aga tgc tgc gtc atg tag     1370 
Arg Phe Lys Asp Pro His Thr His Lys Thr Arg Cys Cys Val Met 
    160                 165                 170 

ctcaggccct ggccctgtac ctcattacat ctgtgtctaa ggagtccaac ggctatgtgc   1430 

gtccctgctc tgtccccatc tgtacccacg gctgtctttc tgaagctgtc cctggaccct   1490 

ctgccagtcc tgtccaaccc ctgtccctca ccccccactg cccaggcctt ccccctggcc   1550 

tgtgtattgc aggtgggagt ttttaaacca ctgggcccaa tgcctcagcg gcgtggccct   1610 

caccctaacc tttttccagc acctttgtta ccaggtgctc tggactctca aggcaataaa   1670 

tcaggagctg tggatgtgtg tgaagagtgt tatagagcag gggaataggg tgtggatccc   1730 

gggaggccat ggtctccatc tcttctgtcc tattgctgct gctgctactg ctgctgcagt   1790 

ggcgtgcctc catggcgccc tctggtggcc atcccttgct ctgcctccct gacttgttct   1850 

acaagttctt gacgcaaaaa tcagcacagc ttggactggt gacggtttgt aacattagga   1910 

ggtgagactg atcacctctg caggacagag tagggggtca cttggaactt tcagtggcct   1970 

ccacccccga ccttcatgca accagaggtg tgggttgcag gtagatctga gtgtagatgg   2030 

cctttggtga catgggtctg cctaggacct catcctgctt ataagttctg agcagtgggc   2090 

tgactcttcc cttagctgag gaaagggtat catgagggac agggctggct atatgtgtgt   2150 

cttagccagg gttttatggc tgtgaacaga caccgggacc aaggcaactc ttacaaggac   2210 

aacatttagt tggggctgcc ttacaggttc agaggttcag tctgtcatca aggtgggagc   2270 

atggcagcat ccatgcaggc atggggcagc tgtagctgag agctctacac cttcatctga   2330 

aggctactag tggaagactg atttccaggg agctagaatg agggccttaa agcccatgcc   2390 

cacagtgaca cacctactcc aacaaggtca gacctccaaa cggtgccact ccccaggcca   2450 

gtcgtattta aacagtgaca atgtgtcagt gtggatgtgc tggctgtctt atagcactga   2510 

catacttagg acgtgatcct caggcctttg cagacccagt cccctctgtg cccagaacag   2570 

agatccaggg cgcctctcac tggtacctgc tgcctgcttg catcattccc tcttcaagtt   2630 

ccacctagct ctggactagg tcccagaaac catccgggcc cacgtagact cccagcagtc   2690 

cctgttcttg cctgtcctag tagggagtga caaggtgtag ccaaactcag taatggtgac   2750 

cttgtgtggg ctggaaactc actaccccgg tgccatattt ccacaaagtc actggttttt   2810 

gtttttgttt tagtctgcca agcttttttt ttttttaaaa gatttattta ttttatgtat   2870 

atgaatatac tgtagctgta cagatggttg tgagccttca tgtggttgtc gggaattgaa   2930 

tttaggacct ctgcttgctc tggtcaactc cgctcactcc ggttcgccaa tctctctcag   2990 

tccttgctag cttcggccca aagatttatt tattatacat aagtacactg ttgctgtctt   3050 

cagacatacc agaagaggac gccagatctc attaccggtg gttgtgagcc accatgtggt   3110 

tgctgagatt tgaactcagg actttcggaa gagccatctg accagcccca agctttttta   3170 

tttttatttt ttaaatcttt aaatttaaaa aaaattatta tttgtttgct gt           3222 

 
           
             12  
             22  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            12 

ttttccagca cctttgttac ca                                              22 

 
           
             13  
             25  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            13 

cccctgctct ataacactct tcaca                                           25 

 
           
             14  
             30  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            14 

tggactctca aggcaataaa tcaggagctg                                      30 

 
           
             15  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            15 

ggcaaattca acggcacagt                                                 20 

 
           
             16  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            16 

gggtctcgct cctggaagat                                                 20 

 
           
             17  
             27  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            17 

aaggccgaga atgggaagct tgtcatc                                         27 

 
           
             18  
             1396  
             DNA  
             H. sapiens  
             
 
             
               CDS  
               (335)...(856)  
             
           
            18 

tgactatcca gctctgagag acgggagttt ggagttgccc gctttacttt ggttgggttg     60 

gggggggcgg cgggctgttt tgttcctttt cttttttaag agttgggttt tcttttttaa    120 

ttatccaaac agtgggcagc ttcctccccc acacccaagt atttgcacaa tatttgtgcg    180 

gggtatgggg gtgggttttt aaatctcgtt tctcttggac aagcacaggg atctcgttct    240 

cctcattttt tgggggtgtg tggggacttc tcaggtcgtg tccccagcct tctctgcagt    300 

cccttctgcc ctgccgggcc cgtcgggagg cgcc atg gct cgg atg aac cgc ccg    355 
                                      Met Ala Arg Met Asn Arg Pro 
                                        1               5 

gcc ccg gtg gag gtg agc tac aaa cac atg cgc ttc ctc atc acc cac      403 
Ala Pro Val Glu Val Ser Tyr Lys His Met Arg Phe Leu Ile Thr His 
         10                  15                  20 

aac ccc acc aac gcc acg ctc agc acc ttc att gag gac ctg aag aag      451 
Asn Pro Thr Asn Ala Thr Leu Ser Thr Phe Ile Glu Asp Leu Lys Lys 
     25                  30                  35 

tac ggg gct acc act gtg gtg cgt gtg tgt gaa gtg acc tat gac aaa      499 
Tyr Gly Ala Thr Thr Val Val Arg Val Cys Glu Val Thr Tyr Asp Lys 
 40                  45                  50                  55 

acg ccg ctg gag aag gat ggc atc acc gtt gtg gac tgg ccg ttt gac      547 
Thr Pro Leu Glu Lys Asp Gly Ile Thr Val Val Asp Trp Pro Phe Asp 
                 60                  65                  70 

gat ggg gcg ccc ccg ccc ggc aag gta gtg gaa gac tgg ctg agc ctg      595 
Asp Gly Ala Pro Pro Pro Gly Lys Val Val Glu Asp Trp Leu Ser Leu 
             75                  80                  85 

gtg aag gcc aag ttc tgt gag gcc ccc ggc agc tgc gtg gct gtg cac      643 
Val Lys Ala Lys Phe Cys Glu Ala Pro Gly Ser Cys Val Ala Val His 
         90                  95                 100 

tgc gtg gcg ggc ctg ggc cgg gct cca gtc ctt gtg gcg ctg gcg ctt      691 
Cys Val Ala Gly Leu Gly Arg Ala Pro Val Leu Val Ala Leu Ala Leu 
    105                 110                 115 

att gag agc ggg atg aag tac gag gac gcc atc cag ttc atc cgc cag      739 
Ile Glu Ser Gly Met Lys Tyr Glu Asp Ala Ile Gln Phe Ile Arg Gln 
120                 125                 130                 135 

aag cgc cgc gga gcc atc aac agc aag cag ctc acc tac ctg gag aaa      787 
Lys Arg Arg Gly Ala Ile Asn Ser Lys Gln Leu Thr Tyr Leu Glu Lys 
                140                 145                 150 

tac cgg ccc aaa cag agg ctg cgg ttc aaa gac cca cac acg cac aag      835 
Tyr Arg Pro Lys Gln Arg Leu Arg Phe Lys Asp Pro His Thr His Lys 
            155                 160                 165 

acc cgg tgc tgc gtt atg tag ctcaggacct tggctgggcc tggtcgtcat         886 
Thr Arg Cys Cys Val Met 
        170 

gtaggtcagg accttggctg gacctggagg ccctgcccag ccctgctctg cccagcccag    946 

caggggctcc aggccttggc tggccccaca tcgccttttc ctccccgaca cctccgtgca   1006 

cttgtgtccg aggagcgagg agcccctcgg gccctgggtg gcctctgggc cctttctcct   1066 

gtctccgcca ctccctctgg cggcgctggc cgtggctctg tctctctgag gtgggtcggg   1126 

cgccctctgc ccgccccctc ccacaccagc caggctggtc tcctctagcc tgtttgttgt   1186 

ggggtggggg tatattttgt aaccactggg cccccagccc ctcttttgcg accccttgtc   1246 

ctgacctgtt ctcggcacct taaattatta gaccccgggg cagtcaggtg ctccggacac   1306 

ccgaaggcaa taaaacagga gccgtgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa   1366 

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa                                    1396 

 
           
             19  
             909  
             DNA  
             H. sapiens  
             
 
           
            19 

gccgcccgga ccgccagakg ctgtgtgctg tggacccacc tggggttcat ggagtgggcc     60 

acggggccca gccctaagca ctgctgcgcc cagggtcgcc gcgcctcctg ctgaggggtc    120 

cccgtgccac tggctctcac cattgccctc gcctgccgat ggcctctgct gcccagcctg    180 

gggccagctc taccgcctga gccccctgcc ccactccagg actcaccgta ccccgatggg    240 

gtaacgtgac acaggcccca cacgtcagag gccgctgtcc ccacggccac tgcccgtgac    300 

ccctggccca aggcagctgg agttggttca gttcaagttc attcttcctc tggcccttgg    360 

gggcttgggg cccacctctg agtgaagggg gctgtctgcc catccaccaa tgtggagagg    420 

gcgcccccgg tgtggggtcc agctctggac actgcttggc ggccgggttc actttgagtt    480 

tttaagtttt ctttgctgag cttttttggt tgttcttttt attttttgcc tctttatgac    540 

tatccagctc tgagagacgg gagtatggag ttgcccgctt tactttggtt gggttggggg    600 

gggcggcggg ctgttttgtt ccttttcttt tttaagagtt gggttttctt ttttaattat    660 

ccaaacagtg ggcagcttcc tcccccacac ccaagtattt gcacaatatt tgtgcggggt    720 

atgggggtgg gtttttaaat ctcgtttctc ttggacaagc acagggatct cgttctcctc    780 

attttttggg ggtgtgtggg gacttctcag gtcgtgtccc cagccttctc tgcagtycct    840 

tctgccctgc cgggcccgtc gggaggcgcc atggctcgga tgaaccgccc ggccccggtg    900 

gaggtgarc                                                            909 

 
           
             20  
             671  
             DNA  
             H. sapiens  
             
 
           
            20 

ggctccgcgg cggaggggcg gcgccccgac ccaggccagc accgtgggca ccgccaggcc     60 

ggcgcgtatg gaggcggtgg gacgcctgcg gcgcggatgc tgtgtgctgt ggacccacct    120 

ggggttcatg gagtgggcca cggggcccag ccctaagcac tgctgcgccc agggtcgccg    180 

cgcctcctgc tgaggggtcc ccgtgccact ggctctcacc attgccctcg cctgccgatg    240 

gcctctgctg cccagcctgg ggccagctct accgcctgag ccccctgccc cactccagga    300 

ctcaccgtac cccgatgggg taacgtgaca caggccccac acgtcagagg ccgctgtccc    360 

cacggccact gcccgtgacc cctggcccaa ggcagctgga gttggttcag ttcaagttca    420 

ttcttcctct ggcccttggg ggcttggggc ccacctctga gtgaaggggg ctgtctgccc    480 

atccaccaat gtggagaggg cgcccccggt gtggggtcca gctctggaca ctgcttggcg    540 

gccgggttca ctttgagttt ttaagttttc tttgctgagc tttttaggtt gttcttttta    600 

ttttttgcct ctttatgact atccagctct gagagacggg agtttggagt tgcccgcttt    660 

actttggttg g                                                         671 

 
           
             21  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            21 

cacaaatatt gtgcaaatac                                                 20 

 
           
             22  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            22 

ccccgcacaa atattgtgca                                                 20 

 
           
             23  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            23 

ccgtacttct tcaggtcctc                                                 20 

 
           
             24  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            24 

tagccccgta cttcttcagg                                                 20 

 
           
             25  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            25 

agtggtagcc ccgtacttct                                                 20 

 
           
             26  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            26 

accacagtgg tagccccgta                                                 20 

 
           
             27  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            27 

tcataggtca cttcacacac                                                 20 

 
           
             28  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            28 

tcgtacttca tcccgctctc                                                 20 

 
           
             29  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            29 

gcgtcctcgt acttcatccc                                                 20 

 
           
             30  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            30 

agctgcttgc tgttgatggc                                                 20 

 
           
             31  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            31 

aggtgagctg cttgctgttg                                                 20 

 
           
             32  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            32 

caggtaggtg agctgcttgc                                                 20 

 
           
             33  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            33 

ttctccaggt aggtgagctg                                                 20 

 
           
             34  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            34 

cgtgtgtggg tctttgaacc                                                 20 

 
           
             35  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            35 

ctcccgtctc tcagagctgg                                                 20 

 
           
             36  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            36 

tccaaactcc cgtctctcag                                                 20 

 
           
             37  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            37 

gggcaactcc aaactcccgt                                                 20 

 
           
             38  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            38 

aaagcgggca actccaaact                                                 20 

 
           
             39  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            39 

ccaaagtaaa gcgggcaact                                                 20 

 
           
             40  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            40 

ccactgtttg gataattaaa                                                 20 

 
           
             41  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            41 

gggaggaagc tgcccactgt                                                 20 

 
           
             42  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            42 

cataccccgc acaaatattg                                                 20 

 
           
             43  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            43 

gtccaagaga aacgagattt                                                 20 

 
           
             44  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            44 

aacgagatcc ctgtgcttgt                                                 20 

 
           
             45  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            45 

cctgagaagt ccccacacac                                                 20 

 
           
             46  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            46 

gaagggactg cagagaaggc                                                 20 

 
           
             47  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            47 

gttcatccga gccatggcgc                                                 20 

 
           
             48  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            48 

ggcggttcat ccgagccatg                                                 20 

 
           
             49  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            49 

tttgtagctc acctccaccg                                                 20 

 
           
             50  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            50 

agcgcatgtg tttgtagctc                                                 20 

 
           
             51  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            51 

caggtcctca atgaaggtgc                                                 20 

 
           
             52  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            52 

gtcacttcac acacacgcac                                                 20 

 
           
             53  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            53 

ggtgatgcca tccttctcca                                                 20 

 
           
             54  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            54 

ccacaacggt gatgccatcc                                                 20 

 
           
             55  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            55 

cagtccacaa cggtgatgcc                                                 20 

 
           
             56  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            56 

aaacggccag tccacaacgg                                                 20 

 
           
             57  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            57 

gctcagccag tcttccacta                                                 20 

 
           
             58  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            58 

acagaacttg gccttcacca                                                 20 

 
           
             59  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            59 

tgcacagcca cgcagctgcc                                                 20 

 
           
             60  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            60 

aggcccgcca cgcagtgcac                                                 20 

 
           
             61  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            61 

gactggagcc cggcccaggc                                                 20 

 
           
             62  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            62 

caaggactgg agcccggccc                                                 20 

 
           
             63  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            63 

cttctggcgg atgaactgga                                                 20 

 
           
             64  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            64 

ttgggccggt atttctccag                                                 20 

 
           
             65  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            65 

tctgtttggg ccggtatttc                                                 20 

 
           
             66  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            66 

ttgaaccgca gcctctgttt                                                 20 

 
           
             67  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            67 

accgggtctt gtgcgtgtgt                                                 20 

 
           
             68  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            68 

taacgcagca ccgggtcttg                                                 20 

 
           
             69  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            69 

ctacataacg cagcaccggg                                                 20 

 
           
             70  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            70 

gtcctgagct acataacgca                                                 20 

 
           
             71  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            71 

gaccaggccc agccaaggtc                                                 20 

 
           
             72  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            72 

atgacgacca ggcccagcca                                                 20 

 
           
             73  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            73 

gtcctgacct acatgacgac                                                 20 

 
           
             74  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            74 

agccaaggtc ctgacctaca                                                 20 

 
           
             75  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            75 

tggagcccct gctgggctgg                                                 20 

 
           
             76  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            76 

gccagccaag gcctggagcc                                                 20 

 
           
             77  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            77 

acaagtgcac ggaggtgtcg                                                 20 

 
           
             78  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            78 

cgctcctcgg acacaagtgc                                                 20 

 
           
             79  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            79 

ggctcctcgc tcctcggaca                                                 20 

 
           
             80  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            80 

acccacctca gagagacaga                                                 20 

 
           
             81  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            81 

ccagcctggc tggtgtggga                                                 20 

 
           
             82  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            82 

caggctagag gagaccagcc                                                 20 

 
           
             83  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            83 

tggttacaaa atataccccc                                                 20 

 
           
             84  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            84 

gcccagtggt tacaaaatat                                                 20 

 
           
             85  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            85 

aggtgccgag aacaggtcag                                                 20 

 
           
             86  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            86 

tctaataatt taaggtgccg                                                 20 

 
           
             87  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            87 

tgtccggagc acctgactgc                                                 20 

 
           
             88  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            88 

attgccttcg ggtgtccgga                                                 20 

 
           
             89  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            89 

cctgttttat tgccttcggg                                                 20 

 
           
             90  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            90 

tcacggctcc tgttttattg                                                 20 

 
           
             91  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            91 

cactgtttgg ataattaaaa                                                 20 

 
           
             92  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            92 

tgtgtcacgt taccccatcg                                                 20 

 
           
             93  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            93 

cttgaactga accaactcca                                                 20 

 
           
             94  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            94 

aatgaacttg aactgaacca                                                 20 

 
           
             95  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            95 

gcccccttca ctcagaggtg                                                 20 

 
           
             96  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            96 

acattggtgg atgggcagac                                                 20 

 
           
             97  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            97 

cgccaagcag tgtccagagc                                                 20 

 
           
             98  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            98 

ctggcggtgc ccacggtgct                                                 20 

 
           
             99  
             1678  
             DNA  
             M. musculus  
             
               unsure  
               220  
               unknown  
             
           
            99 

ggcgcgtgcc gagccctccg cgtcgtgccg gcgccggcgc ccggaccgcc agattgtgcc     60 

ttctgaagac cagggtggga ttgatggaga agccccaaga gggcagctct gactactgcc    120 

gttcccaaca ccgtgggtgc tgccgctgag gagacctgta cacctctggc cctcaccatt    180 

gtccttgcct cccaatggcc tctgctgcca ggccggagcn gncactgctg cctgagcccg    240 

ctgcccctct tcaggacttg ccgttccctg atggggtaac gtgacagacc ggatcagagg    300 

ctgcctgccc accacggccc agggccgcta gagtttggtt gagttcaagt tcattttccc    360 

tttggtcctg acttctgggg cccaactctg accaaagggg acactcctcc gtccacccat    420 

gcagagaggg tgtctccagc gacggcccca tagaggacac tgctctgcgg ccggagtcac    480 

caacccgaag ttctctctgc tcagtttttt ttttggttgt tgttattttt attttgactc    540 

cttgtaactt ctccactctg agggacagga ctttggcgct gcccgtcttg ctgcggggtg    600 

gggggagaag tgtgcttgtt tcttttcttt tttagaattg gtttttttgg aattatccgc    660 

cacgaggcag cttcctctcc ctctcccagg tatttgcaca atatttgtgc ggggcgtagg    720 

ggcgaggttt taagaagtct tttctttgtg gacaagcacg gggatctcac tggacttggt    780 

gtgtggggct gggggacccc cgtgcagcct tgctggctag tccctctggg tccccggagg    840 

aggcatggcc cgcatgaacc ggcctgcgcc tgtggaggtg agctaccggc acatgcgctt    900 

cctcatcacc cacaacccca gcaatgccac cctcagcacg ttcatcgagg acctgaagaa    960 

gtacggggct accactgtgg tgcgcgtgtg tgaagtgacc tatgacaaga cccccctgga   1020 

gaaggacggc atcactgttg tggactggcc ctttgatgat ggagcgcccc ctcctggcaa   1080 

agtggtagag gactggctga gcctgctgaa ggccaagttc tacaatgacc cgggaagctg   1140 

cgtagctgtg cactgtgtgg cgggcctggg aagggcccca gtgctcgtgc tctcgccctc   1200 

atcgagagcg ggatgaagta cgaggacgcc atccagttca tccgacagaa gcgccgtggg   1260 

gccatcaaca gcaagcagct cacctacctg gagaagtacc ggcctaagca gagactgagg   1320 

ttcaaagacc cacacacgca caagaccaga tgctgcgtca tgtagctcag gccctggccc   1380 

tgtacctcat tacatctgtg tctaaggagt ccaacggcta tgtgcgtccc tgctctgtcc   1440 

ccatctgtac ccacggctgt ctttctgaag ctgtccctgg accctctgcc agtcctgtcc   1500 

aacccctgtc cctcaccccc cactgcccag gccttccccc tggcctgtgt attgcaggtg   1560 

ggagttttta aaccactggg cccaatgcct cagcggcgtg gccctcaccc taaccttttt   1620 

ccagcacctt tgttaccagg tgctctggac tctcaaggca ataaatcagg agctgtgg     1678  
           
             100  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            100 

ggccgcctcc atgcgccagg                                                 20 

 
           
             101  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            101 

catcaatccc accctggtct                                                 20 

 
           
             102  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            102 

ggcagcaccc acggtgttgg                                                 20 

 
           
             103  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            103 

tgagggccag aggtgtacag                                                 20 

 
           
             104  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            104 

ggctcaggca gcagtgctgc                                                 20 

 
           
             105  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            105 

tgatccggtc tgtcacgtta                                                 20 

 
           
             106  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            106 

aactcaacca aactctagcg                                                 20 

 
           
             107  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            107 

agaagtcagg accaaaggga                                                 20 

 
           
             108  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            108 

cctttggtca gagttgggcc                                                 20 

 
           
             109  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            109 

tctctgcatg ggtggacgga                                                 20  
           
             110  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            110 

ggagacaccc tctctgcatg                                                 20 

 
           
             111  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            111 

ggccgcagag cagtgtcctc                                                 20 

 
           
             112  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            112 

aacttcgggt tggtgactcc                                                 20 

 
           
             113  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            113 

tcctgtccct cagagtggag                                                 20 

 
           
             114  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            114 

aaacaagcac acttctcccc                                                 20 

 
           
             115  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            115 

cctcgtggcg gataattcca                                                 20 

 
           
             116  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            116 

cctacgcccc gcacaaatat                                                 20 

 
           
             117  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            117 

aagacttctt aaaacctcgc                                                 20 

 
           
             118  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            118 

catgcgggcc atgcctcctc                                                 20 

 
           
             119  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            119 

gtagaacttg gccttcagca                                                 20 

 
           
             120  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            120 

gggcctgagc tacatgacgc                                                 20 

 
           
             121  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            121 

acagccgtgg gtacagatgg                                                 20 

 
           
             122  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            122 

acagcttcag aaagacagcc                                                 20 

 
           
             123  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            123 

aactcccacc tgcaatacac                                                 20 

 
           
             124  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            124 

gccttgagag tccagagcac                                                 20 

 
           
             125  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            125 

ctcctgattt attgccttga                                                 20 

 
           
             126  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            126 

gctctataac actcttcaca                                                 20 

 
           
             127  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            127 

ggagaccatg gcctcccggg                                                 20 

 
           
             128  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            128 

cgccactgca gcagcagtag                                                 20 

 
           
             129  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            129 

catggaggca cgccactgca                                                 20 

 
           
             130  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            130 

ttgcgtcaag aacttgtaga                                                 20 

 
           
             131  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            131 

ctgatttttg cgtcaagaac                                                 20 

 
           
             132  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            132 

cgtcaccagt ccaagctgtg                                                 20 

 
           
             133  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            133 

gttacaaacc gtcaccagtc                                                 20 

 
           
             134  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            134 

ctgcagaggt gatcagtctc                                                 20 

 
           
             135  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            135 

tactctgtcc tgcagaggtg                                                 20 

 
           
             136  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            136 

ctgaaagttc caagtgaccc                                                 20 

 
           
             137  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            137 

gtggaggcca ctgaaagttc                                                 20 

 
           
             138  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            138 

ggcagaccca tgtcaccaaa                                                 20 

 
           
             139  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            139 

aggtcctagg cagacccatg                                                 20 

 
           
             140  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            140 

gcaggatgag gtcctaggca                                                 20 

 
           
             141  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            141 

ttataagcag gatgaggtcc                                                 20 

 
           
             142  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            142 

actgctcaga acttataagc                                                 20 

 
           
             143  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            143 

tcctcagcta agggaagagt                                                 20 

 
           
             144  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            144 

actgacacat tgtcactgtt                                                 20 

 
           
             145  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            145 

cacatccaca ctgacacatt                                                 20 

 
           
             146  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            146 

cagccagcac atccacactg                                                 20 

 
           
             147  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            147 

gtgctataag acagccagca                                                 20 

 
           
             148  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            148 

cgtcctaagt atgtcagtgc                                                 20 

 
           
             149  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            149 

ggtctgcaaa ggcctgagga                                                 20 

 
           
             150  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            150 

ggatctctgt tctgggcaca                                                 20 

 
           
             151  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            151 

cgccctggat ctctgttctg                                                 20 

 
           
             152  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            152 

aggcagcagg taccagtgag                                                 20 

 
           
             153  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            153 

tgatgcaagc aggcagcagg                                                 20 

 
           
             154  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            154 

agagggaatg atgcaagcag                                                 20 

 
           
             155  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            155 

gaacttgaag agggaatgat                                                 20 

 
           
             156  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            156 

agctaggtgg aacttgaaga                                                 20 

 
           
             157  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            157 

ggtttctggg acctagtcca                                                 20 

 
           
             158  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            158 

ctaggacagg caagaacagg                                                 20 

 
           
             159  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            159 

ggctacacct tgtcactccc                                                 20 

 
           
             160  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            160 

caccattact gagtttggct                                                 20 

 
           
             161  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            161 

aaccagtgac tttgtggaaa                                                 20 

 
           
             162  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            162 

cacaatctgg cggtccgggc                                                 20 

 
           
             163  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            163 

cttcagaagg cacaatctgg                                                 20 

 
           
             164  
             20  
             DNA  
             H. sapiens  
             
 
           
            164 

tgcacaatat ttgtgcgggg                                                 20 

 
           
             165  
             20  
             DNA  
             H. sapiens  
             
 
           
            165 

gaggacctga agaagtacgg                                                 20 

 
           
             166  
             20  
             DNA  
             H. sapiens  
             
 
           
            166 

cctgaagaag tacggggcta                                                 20 

 
           
             167  
             20  
             DNA  
             H. sapiens  
             
 
           
            167 

agaagtacgg ggctaccact                                                 20 

 
           
             168  
             20  
             DNA  
             H. sapiens  
             
 
           
            168 

gagagcggga tgaagtacga                                                 20 

 
           
             169  
             20  
             DNA  
             H. sapiens  
             
 
           
            169 

gggatgaagt acgaggacgc                                                 20 

 
           
             170  
             20  
             DNA  
             H. sapiens  
             
 
           
            170 

gccatcaaca gcaagcagct                                                 20 

 
           
             171  
             20  
             DNA  
             H. sapiens  
             
 
           
            171 

caacagcaag cagctcacct                                                 20 

 
           
             172  
             20  
             DNA  
             H. sapiens  
             
 
           
            172 

gcaagcagct cacctacctg                                                 20 

 
           
             173  
             20  
             DNA  
             H. sapiens  
             
 
           
            173 

cagctcacct acctggagaa                                                 20 

 
           
             174  
             20  
             DNA  
             H. sapiens  
             
 
           
            174 

ggttcaaaga cccacacacg                                                 20 

 
           
             175  
             20  
             DNA  
             H. sapiens  
             
 
           
            175 

ccagctctga gagacgggag                                                 20 

 
           
             176  
             20  
             DNA  
             H. sapiens  
             
 
           
            176 

ctgagagacg ggagtttgga                                                 20 

 
           
             177  
             20  
             DNA  
             H. sapiens  
             
 
           
            177 

acgggagttt ggagttgccc                                                 20 

 
           
             178  
             20  
             DNA  
             H. sapiens  
             
 
           
            178 

agttgcccgc tttactttgg                                                 20 

 
           
             179  
             20  
             DNA  
             H. sapiens  
             
 
           
            179 

tttaattatc caaacagtgg                                                 20 

 
           
             180  
             20  
             DNA  
             H. sapiens  
             
 
           
            180 

aaatctcgtt tctcttggac                                                 20 

 
           
             181  
             20  
             DNA  
             H. sapiens  
             
 
           
            181 

acaagcacag ggatctcgtt                                                 20 

 
           
             182  
             20  
             DNA  
             H. sapiens  
             
 
           
            182 

gccttctctg cagtcccttc                                                 20 

 
           
             183  
             20  
             DNA  
             H. sapiens  
             
 
           
            183 

gcgccatggc tcggatgaac                                                 20 

 
           
             184  
             20  
             DNA  
             H. sapiens  
             
 
           
            184 

catggctcgg atgaaccgcc                                                 20 

 
           
             185  
             20  
             DNA  
             H. sapiens  
             
 
           
            185 

cggtggaggt gagctacaaa                                                 20 

 
           
             186  
             20  
             DNA  
             H. sapiens  
             
 
           
            186 

gagctacaaa cacatgcgct                                                 20 

 
           
             187  
             20  
             DNA  
             H. sapiens  
             
 
           
            187 

gcaccttcat tgaggacctg                                                 20 

 
           
             188  
             20  
             DNA  
             H. sapiens  
             
 
           
            188 

gtgcgtgtgt gtgaagtgac                                                 20 

 
           
             189  
             20  
             DNA  
             H. sapiens  
             
 
           
            189 

tggagaagga tggcatcacc                                                 20 

 
           
             190  
             20  
             DNA  
             H. sapiens  
             
 
           
            190 

ggatggcatc accgttgtgg                                                 20 

 
           
             191  
             20  
             DNA  
             H. sapiens  
             
 
           
            191 

ggcatcaccg ttgtggactg                                                 20 

 
           
             192  
             20  
             DNA  
             H. sapiens  
             
 
           
            192 

ccgttgtgga ctggccgttt                                                 20 

 
           
             193  
             20  
             DNA  
             H. sapiens  
             
 
           
            193 

tagtggaaga ctggctgagc                                                 20 

 
           
             194  
             20  
             DNA  
             H. sapiens  
             
 
           
            194 

tggtgaaggc caagttctgt                                                 20 

 
           
             195  
             20  
             DNA  
             H. sapiens  
             
 
           
            195 

ggcagctgcg tggctgtgca                                                 20 

 
           
             196  
             20  
             DNA  
             H. sapiens  
             
 
           
            196 

gtgcactgcg tggcgggcct                                                 20 

 
           
             197  
             20  
             DNA  
             H. sapiens  
             
 
           
            197 

gggccgggct ccagtccttg                                                 20 

 
           
             198  
             20  
             DNA  
             H. sapiens  
             
 
           
            198 

tccagttcat ccgccagaag                                                 20 

 
           
             199  
             20  
             DNA  
             H. sapiens  
             
 
           
            199 

ctggagaaat accggcccaa                                                 20 

 
           
             200  
             20  
             DNA  
             H. sapiens  
             
 
           
            200 

gaaataccgg cccaaacaga                                                 20 

 
           
             201  
             20  
             DNA  
             H. sapiens  
             
 
           
            201 

aaacagaggc tgcggttcaa                                                 20 

 
           
             202  
             20  
             DNA  
             H. sapiens  
             
 
           
            202 

acacacgcac aagacccggt                                                 20 

 
           
             203  
             20  
             DNA  
             H. sapiens  
             
 
           
            203 

caagacccgg tgctgcgtta                                                 20 

 
           
             204  
             20  
             DNA  
             H. sapiens  
             
 
           
            204 

cccggtgctg cgttatgtag                                                 20 

 
           
             205  
             20  
             DNA  
             H. sapiens  
             
 
           
            205 

tgcgttatgt agctcaggac                                                 20 

 
           
             206  
             20  
             DNA  
             H. sapiens  
             
 
           
            206 

gaccttggct gggcctggtc                                                 20 

 
           
             207  
             20  
             DNA  
             H. sapiens  
             
 
           
            207 

tggctgggcc tggtcgtcat                                                 20 

 
           
             208  
             20  
             DNA  
             H. sapiens  
             
 
           
            208 

gtcgtcatgt aggtcaggac                                                 20 

 
           
             209  
             20  
             DNA  
             H. sapiens  
             
 
           
            209 

tgtaggtcag gaccttggct                                                 20 

 
           
             210  
             20  
             DNA  
             H. sapiens  
             
 
           
            210 

ggctccaggc cttggctggc                                                 20 

 
           
             211  
             20  
             DNA  
             H. sapiens  
             
 
           
            211 

gcacttgtgt ccgaggagcg                                                 20 

 
           
             212  
             20  
             DNA  
             H. sapiens  
             
 
           
            212 

tgtccgagga gcgaggagcc                                                 20 

 
           
             213  
             20  
             DNA  
             H. sapiens  
             
 
           
            213 

gggggtatat tttgtaacca                                                 20 

 
           
             214  
             20  
             DNA  
             H. sapiens  
             
 
           
            214 

atattttgta accactgggc                                                 20 

 
           
             215  
             20  
             DNA  
             H. sapiens  
             
 
           
            215 

ctgacctgtt ctcggcacct                                                 20 

 
           
             216  
             20  
             DNA  
             H. sapiens  
             
 
           
            216 

cggcacctta aattattaga                                                 20 

 
           
             217  
             20  
             DNA  
             H. sapiens  
             
 
           
            217 

gcagtcaggt gctccggaca                                                 20 

 
           
             218  
             20  
             DNA  
             H. sapiens  
             
 
           
            218 

tccggacacc cgaaggcaat                                                 20 

 
           
             219  
             20  
             DNA  
             H. sapiens  
             
 
           
            219 

cccgaaggca ataaaacagg                                                 20 

 
           
             220  
             20  
             DNA  
             H. sapiens  
             
 
           
            220 

caataaaaca ggagccgtga                                                 20 

 
           
             221  
             20  
             DNA  
             H. sapiens  
             
 
           
            221 

cgatggggta acgtgacaca                                                 20 

 
           
             222  
             20  
             DNA  
             H. sapiens  
             
 
           
            222 

tggagttggt tcagttcaag                                                 20 

 
           
             223  
             20  
             DNA  
             H. sapiens  
             
 
           
            223 

cacctctgag tgaagggggc                                                 20 

 
           
             224  
             20  
             DNA  
             H. sapiens  
             
 
           
            224 

gctctggaca ctgcttggcg                                                 20 

 
           
             225  
             20  
             DNA  
             M. musculus  
             
 
           
            225 

taacgtgaca gaccggatca                                                 20 

 
           
             226  
             20  
             DNA  
             M. musculus  
             
 
           
            226 

ggcccaactc tgaccaaagg                                                 20 

 
           
             227  
             20  
             DNA  
             M. musculus  
             
 
           
            227 

gaggacactg ctctgcggcc                                                 20 

 
           
             228  
             20  
             DNA  
             M. musculus  
             
 
           
            228 

ggagtcacca acccgaagtt                                                 20 

 
           
             229  
             20  
             DNA  
             M. musculus  
             
 
           
            229 

ggggagaagt gtgcttgttt                                                 20 

 
           
             230  
             20  
             DNA  
             M. musculus  
             
 
           
            230 

tggaattatc cgccacgagg                                                 20 

 
           
             231  
             20  
             DNA  
             M. musculus  
             
 
           
            231 

gcgaggtttt aagaagtctt                                                 20 

 
           
             232  
             20  
             DNA  
             M. musculus  
             
 
           
            232 

gtgctctgga ctctcaaggc                                                 20 

 
           
             233  
             20  
             DNA  
             M. musculus  
             
 
           
            233 

tcaaggcaat aaatcaggag                                                 20 

 
           
             234  
             20  
             DNA  
             M. musculus  
             
 
           
            234 

gactggtgac ggtttgtaac                                                 20 

 
           
             235  
             20  
             DNA  
             M. musculus  
             
 
           
            235 

actcttccct tagctgagga                                                 20 

 
           
             236  
             20  
             DNA  
             M. musculus  
             
 
           
            236 

aacagtgaca atgtgtcagt                                                 20 

 
           
             237  
             20  
             DNA  
             M. musculus  
             
 
           
            237 

tttccacaaa gtcactggtt                                                 20 

 
           
             238  
             20  
             DNA  
             M. musculus  
             
 
           
            238 

ccagattgtg ccttctgaag                                                 20 

 
           
             239  
             1006  
             DNA  
             Homo sapiens  
             
               CDS  
               (238)...(759)  
             
           
            239 

aagagttggg ttttcttttt taattatcca aacagtgggc agcttcctcc cccacaccca     60 

agtatttgca caatatttgt gcggggtatg ggggtgggtt tttaaatctc gtttctcttg    120 

gacaagcaca gggatctcgt tctcctcatt ttttgggggt gtgtggggac ttctcaggtc    180 

gtgtccccag ccttctctgc agtcccttct gccctgccgg gcccgtcggg aggcgcc atg   240 
                                                               Met 
                                                                1 

gct cgg atg aac cgc ccg gcc ccg gtg gag gtg agc tac aaa cac atg      288 
Ala Arg Met Asn Arg Pro Ala Pro Val Glu Val Ser Tyr Lys His Met 
             5                   10                  15 

cgc ttc ctc atc acc cac aac ccc acc aac gcc acg ctc agc acc ttc      336 
Arg Phe Leu Ile Thr His Asn Pro Thr Asn Ala Thr Leu Ser Thr Phe 
         20                  25                  30 

att gag gac ctg aag aag tac ggg gct acc act gtg gtg cgt gtg tgt      384 
Ile Glu Asp Leu Lys Lys Tyr Gly Ala Thr Thr Val Val Arg Val Cys 
     35                  40                  45 

gaa gtg acc tat gac aaa acg ccg ctg gag aag gat ggc atc acc gtt      432 
Glu Val Thr Tyr Asp Lys Thr Pro Leu Glu Lys Asp Gly Ile Thr Val 
 50                  55                  60                  65 

gtg gac tgg ccg ttt gac gat ggg gcg ccc ccg cct ggc aag gta gtg      480 
Val Asp Trp Pro Phe Asp Asp Gly Ala Pro Pro Pro Gly Lys Val Val 
                 70                  75                  80 

gaa gac tgg ctg agc ctg gtg aag gcc aag ttc tgt gag gcc ccc ggc      528 
Glu Asp Trp Leu Ser Leu Val Lys Ala Lys Phe Cys Glu Ala Pro Gly 
             85                  90                  95 

agc tgc gtg gct gtg cac tgc gtg gcg ggc ctg ggg cgg gct cca gtc      576 
Ser Cys Val Ala Val His Cys Val Ala Gly Leu Gly Arg Ala Pro Val 
        100                 105                 110 

ctt gtg gcg ctg gcg ctt att gag agc ggg atg aag tac gag gac gcc      624 
Leu Val Ala Leu Ala Leu Ile Glu Ser Gly Met Lys Tyr Glu Asp Ala 
    115                 120                 125 

atc cag ttc atc cgc cag aag cgc cgc gga cgc atc aac agc aag cag      672 
Ile Gln Phe Ile Arg Gln Lys Arg Arg Gly Arg Ile Asn Ser Lys Gln 
130                 135                 140                 145 

ctc acc tac ctg gag aaa tac cgg ccc aaa cag agg ctg cgg ttc aaa      720 
Leu Thr Tyr Leu Glu Lys Tyr Arg Pro Lys Gln Arg Leu Arg Phe Lys 
                150                 155                 160 

gac cca cac acg cac aag acc cgg tgc tgc gtt atg tag ctcaggacct       769 
Asp Pro His Thr His Lys Thr Arg Cys Cys Val Met  * 
            165                 170 

tggctgggcc tggtcgtcat gtaggtcagg accttggctg gacctggagg ccctgccagc    829 

cctgctctgc ccagcccagc agggctccag gccttggctg gccccacatc gccttttcct    889 

ccccgacacc tccgtgcact tgtgtccgag gagcgaggag cccctcggcg ccttgggtgg    949 

cttctgggcc ctttctcctg tctccgtact ccctctggcg gcgctggcgt ggctctg      1006