Patent Publication Number: US-2003224511-A1

Title: Antisense modulation of cathepsin Z expression

Description:
FIELD OF THE INVENTION  
       [0001] The present invention provides compositions and methods for modulating the expression of cathepsin Z. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding cathepsin Z. Such compounds have been shown to modulate the expression of cathepsin Z.  
       BACKGROUND OF THE INVENTION  
       [0002] Cysteine proteases belonging to the papain family comprise a major component of the lysosomal proteolytic system by playing an essential role in protein degradation. These enzymes can also be found extracellularly, in which case they have been implicated in the development of various diseases including cancer, bone remodeling, rheumatoid arthritis, osteoarthritis, Alzheimer&#39;s disease, multiple sclerosis, and muscular dystrophy. To date, eleven cysteine proteases of the papain family have been identified to date and they are generally termed cathepsins (Turk et al.,  Biochim. Biophys. Acta,  2000, 1477, 98-111).  
       [0003] The gene encoding human cathepsin Z (also called CTSZ, cathepsin X, CTSX, and cathepsin Y) was first cloned in 1998 from a human brain cDNA library and maps to chromosome 20q13, a unique location for any human cysteine protease mapped to date (Santamaria et al.,  J. Biol. Chem.,  1998, 273, 16816-16823). Chromosomal region 20q13 has a complex pattern of imprinted and non-imprinted transcription. The cathepsin z gene is oriented tail-to-tail with the TH1 gene, both of which do not appear to be imprinted and lie immediately downstream of GNAS1, a highly imprinted gene that encodes several G proteins (Bonthron et al.,  Hum. Genet.,  2000, 107, 165-175).  
       [0004] Claimed and disclosed in U.S. Pat. No. 5,783,434 is a nucleic acid sequence encoding cathepsin Z, a nucleic acid sequence fully complementary to that sequence, and an oligonucleotide comprising about 10-50 contiguous nucleotides of either sequence which is capable of selectively hybridizing to either sequence, but not to cathepsin genes other than cathepsin Y (Tung et al., 1998).  
       [0005] Cathepsin Z, like the other cathepsins, contains a signal sequence, a prodomain, and a catalytic domain responsible for proteolytic activity. Cathepsin Z contains several unique peptide insertions that may confer distinct substrate specificity or catalytic properties to cathepsin Z (Santamaria et al.,  J. Biol. Chem.,  1998, 273, 16816-16823). These distinctions between cathepsin Z and other cathepsin proteins may mean that cathepsin z represents a new subfamily of cathepsin-Z-like protease genes. Homologs of human cathepsin Z have been found in mouse, rat (cathepsin Y), O. volvulus larvae (LOVCP),  B. taurus, U. caupo, C. elegans , and  T. canis  (Tc-cpz-1) and phylogenic analysis suggests that the cathepsin Z subfamily represents a very old lineage (Deussing et al.,  Biochim. Biophys. Acta,  2000, 1491, 93-106.; Falcone et al.,  Exp. Parasitol.,  2000, 94, 201-207).  
       [0006] Cathepsin Z is widely expressed in human tissues and also ubiquitously distributed in cancer cell lines and in primary tumors, suggesting that cathepsin Z may participate in tumor progression as reported for other cathepsins (Santamaria et al.,  J. Biol. Chem.,  1998, 273, 16816-16823).  
       [0007] Sindbis virus proteins containing a single amino acid mutation can convert clinically silent encephalitis into uniformly lethal disease, however little is known about the host gene response during avirulent and virulent CNS infections. In a search for host genes that are important for modulating the viral pathogenesis, Cathepsin Z was identified as one gene whose mRNA was found to be uniquely upregulated in mice infected with a neurovirulent strain of the Sindbis virus (Johnston et al.,  J. Virol.,  2001, 75, 10431-10445).  
       [0008] Currently, there are no known therapeutic agents which effectively inhibit the synthesis of cathepsin Z. Consequently, there remains a long felt need for agents capable of effectively inhibiting cathepsin Z function.  
       [0009] 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 cathepsin Z expression.  
       [0010] The present invention provides compositions and methods for modulating cathepsin Z expression.  
       SUMMARY OF THE INVENTION  
       [0011] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding cathepsin Z, and which modulate the expression of cathepsin Z. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of cathepsin Z 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 cathepsin Z 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  
       [0012] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding cathepsin Z, ultimately modulating the amount of cathepsin Z produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding cathepsin Z. As used herein, the terms “target nucleic acid” and “nucleic acid encoding cathepsin Z” encompass DNA encoding cathepsin Z, 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 cathepsin Z. 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.  
       [0013] 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 cathepsin Z. 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 cathepsin Z, regardless of the sequence(s) of such codons.  
       [0014] 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.  
       [0015] 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.  
       [0016] 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.  
       [0017] 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.  
       [0018] 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.  
       [0019] 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.  
       [0020] 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.  
       [0021] 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.  
       [0022] 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).  
       [0023] 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.  
       [0024] 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.  
       [0025] 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.  
       [0026] 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.  
       [0027] 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.  
       [0028] 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.  
       [0029] 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.  
       [0030] 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).  
       [0031] 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.  
       [0032] 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.  
       [0033] 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.  
       [0034] 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.  
       [0035] 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.  
       [0036] 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.  
       [0037] 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.  
       [0038] 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.  
       [0039] 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.  
       [0040] 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.  
       [0041] 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.  
       [0042] 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.  
       [0043] 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.  
       [0044] 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.  
       [0045] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 21 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.  
       [0046] 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.  
       [0047] 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.  
       [0048] 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.  
       [0049] 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.  
       [0050] 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 triethyl-ammonium 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 U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.  
       [0051] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.  
       [0052] 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.  
       [0053] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.  
       [0054] 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.  
       [0055] 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.  
       [0056] 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.  
       [0057] 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.  
       [0058] 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.  
       [0059] 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.  
       [0060] 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.  
       [0061] 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 cathepsin Z 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.  
       [0062] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding cathepsin Z, 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 cathepsin Z 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 cathepsin Z in a sample may also be prepared.  
       [0063] 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.  
       [0064] 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.  
       [0065] 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. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.  
       [0066] 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.  
       [0067] 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.  
       [0068] 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.  
       [0069] 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.  
       [0070] 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.  
       [0071] Emulsions  
       [0072] 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.  
       [0073] 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).  
       [0074] 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).  
       [0075] 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.  
       [0076] 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).  
       [0077] 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.  
       [0078] 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.  
       [0079] 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.  
       [0080] 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).  
       [0081] 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.  
       [0082] 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 (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.  
       [0083] 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.  
       [0084] 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.  
       [0085] Liposomes  
       [0086] 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.  
       [0087] 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.  
       [0088] 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.  
       [0089] 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.  
       [0090] 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.  
       [0091] 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.  
       [0092] 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.  
       [0093] 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).  
       [0094] 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).  
       [0095] 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.  
       [0096] 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).  
       [0097] 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).  
       [0098] 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 G M1 , 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).  
       [0099] 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.).  
       [0100] 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.  
       [0101] 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.  
       [0102] 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.  
       [0103] 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).  
       [0104] 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.  
       [0105] 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.  
       [0106] 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.  
       [0107] 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.  
       [0108] 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).  
       [0109] Penetration Enhancers  
       [0110] 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.  
       [0111] 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.  
       [0112] 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).  
       [0113] 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).  
       [0114] 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).  
       [0115] 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).  
       [0116] 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).  
       [0117] 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.  
       [0118] 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.  
       [0119] Carriers  
       [0120] 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).  
       [0121] Excipients  
       [0122] 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.).  
       [0123] 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.  
       [0124] 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.  
       [0125] 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.  
       [0126] Other Components  
       [0127] 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.  
       [0128] 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.  
       [0129] 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.  
       [0130] 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.  
       [0131] 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.  
       [0132] 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  
     [0133] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy Amidites  
     [0134] 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.  
     [0135] 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).  
     [0136] 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:  
     [0137] Preparation of 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC Amidite  
     [0138] 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.  
     [0139] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine Intermediate for 5-methyl-dC Amidite  
     [0140] 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).  
     [0141] TLC indicated a complete reaction (product Rf 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.  
     [0142] 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.  
     [0143] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine Penultimate Intermediate for 5-methyl dC Amidite  
     [0144] 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.  
     [0145] 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.  
     [0146] [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N 4 -benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC Amidite)  
     [0147] 5′-(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%).  
     [0148] 2′-Fluoro Amidites  
     [0149] 2′-Fluorodeoxyadenosine Amidites  
     [0150] 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 S N 2-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.  
     [0151] 2′-Fluorodeoxyguanosine  
     [0152] 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.  
     [0153] 2′-Fluorouridine  
     [0154] 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.  
     [0155] 2′-Fluorodeoxycytidine  
     [0156] 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.  
     [0157] 2′-O-(2-Methoxyethyl) modified amidites  
     [0158] 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).  
     [0159] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate  
     [0160] 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.  
     [0161] 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.).  
     [0162] 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.  
     [0163] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine Penultimate Intermediate  
     [0164] 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.  
     [0165] 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.  
     [0166] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T Amidite)  
     [0167] 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%).  
     [0168] Preparation of 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate  
     [0169] 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, R f  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, R f  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  
     [0170] 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.  
     [0171] Preparation of 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N-4-benzoyl-5-methyl-cytidine Penultimate Intermediate:  
     [0172] 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%.  
     [0173] 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)  
     [0174] 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%).  
     [0175] 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)  
     [0176] 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%).  
     [0177] 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)  
     [0178] 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%).  
     [0179] 2′-O-(Aminooxyethyl) Nucleoside Amidites and 2′-O-(dimethylaminooxyethyl) Nucleoside Amidites  
     [0180] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites  
     [0181] 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.  
     [0182] 5′-O-tert-Butyldiphenylsilyl-O 2 -2′-anhydro-5-methyluridine  
     [0183] 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, 1.1 eq, 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.  
     [0184] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine  
     [0185] 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.  
     [0186] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine  
     [0187] 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.  
     [0188] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine  
     [0189] 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.  
     [0190] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine  
     [0191] 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 SO 4 , 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.  
     [0192] 2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0193] 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.  
     [0194] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0195] 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.  
     [0196] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0197] 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,N′-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.  
     [0198] 2′-(Aminooxyethoxy) Nucleoside Amidites  
     [0199] 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.  
     [0200] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0201] 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].  
     [0202] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites  
     [0203] 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.  
     [0204] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine  
     [0205] 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.  
     [0206] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyl Uridine  
     [0207] 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 CH 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.  
     [0208] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite  
     [0209] 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  
     [0210] Oligonucleotide Synthesis  
     [0211] 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.  
     [0212] 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.  
     [0213] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.  
     [0214] 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.  
     [0215] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.  
     [0216] 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.  
     [0217] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.  
     [0218] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.  
     [0219] 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  
     [0220] Oligonucleoside Synthesis  
     [0221] 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 oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.  
     [0222] 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.  
     [0223] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.  
     Example 4  
     [0224] PNA Synthesis  
     [0225] 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  
     [0226] Synthesis of Chimeric Oligonucleotides  
     [0227] 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”.  
     [0228] [2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides  
     [0229] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-β-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.  
     [0230] [2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides  
     [0231] [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.  
     [0232] [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides  
     [0233] [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.  
     [0234] 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  
     [0235] Oligonucleotide Isolation  
     [0236] 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  
     [0237] Oligonucleotide Synthesis—96 Well Plate Format  
     [0238] 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.  
     [0239] 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  
     [0240] Oligonucleotide Analysis—96-Well Plate Format  
     [0241] 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  
     [0242] Cell Culture and Oligonucleotide Treatment  
     [0243] 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.  
     [0244] T-24 Cells:  
     [0245] 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.  
     [0246] 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.  
     [0247] A549 Cells:  
     [0248] 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.  
     [0249] NHDF Cells:  
     [0250] 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.  
     [0251] HEK Cells:  
     [0252] 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.  
     [0253] Treatment with Antisense Compounds:  
     [0254] 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.  
     [0255] 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  
     [0256] Analysis of Oligonucleotide Inhibition of Cathepsin Z Expression  
     [0257] Antisense modulation of cathepsin Z expression can be assayed in a variety of ways known in the art. For example, cathepsin Z 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.  
     [0258] Protein levels of cathepsin Z 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 cathepsin Z 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).  
     [0259] 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  
     [0260] Poly(A)+mRNA Isolation  
     [0261] 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.  
     [0262] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.  
     Example 12  
     [0263] Total RNA Isolation  
     [0264] 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 QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 170 μL water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.  
     [0265] 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  
     [0266] Real-time Quantitative PCR Analysis of Cathepsin Z mRNA Levels  
     [0267] Quantitation of cathepsin Z mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 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, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISMT™ 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.  
     [0268] 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.  
     [0269] 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).  
     [0270] 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 RiboGreenTM (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 RiboGreenTM RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreenTM are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).  
     [0271] In this assay, 170 μL of RiboGreenTM 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.  
     [0272] Probes and primers to human cathepsin Z were designed to hybridize to a human cathepsin Z sequence, using published sequence information (GenBank accession number NM — 001336.1, incorporated herein as SEQ ID NO:4). For human cathepsin Z the PCR primers were: forward primer: AACGATGGGACCTCAGTCTTCTT (SEQ ID NO: 5) reverse primer: GCCATATTATTGCCCACAGTTG (SEQ ID NO: 6) and the PCR probe was: FAM-AGCAGAGGACTTGATATTTTGTATTTG-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.  
     Example 14  
     [0273] Northern Blot Analysis of Cathepsin Z mRNA Levels  
     [0274] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer&#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, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer&#39;s recommendations for stringent conditions.  
     [0275] To detect human cathepsin Z, a human cathepsin Z specific probe was prepared by PCR using the forward primer AACGATGGGACCTCAGTCTTCTT (SEQ ID NO: 5) and the reverse primer GCCATATTATTGCCCACAGTTG (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.).  
     [0276] 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  
     [0277] Antisense Inhibition of Human Cathepsin Z Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap  
     [0278] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human cathepsin Z RNA, using published sequences (GenBank accession number NM — 001336.1, incorporated herein as SEQ ID NO: 4, the complement of residues 22622000-22636000 of GenBank accession number NT — 011362.5, incorporated herein as SEQ ID NO: 11, GenBank accession number AF136273.1, incorporated herein as SEQ ID NO: 12, and GenBank accession number AI739190.1, the complement of which is incorporated herein as SEQ ID NO: 13). 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 cathepsin Z 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 antisense 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 cathepsin Z mRNA levels by chimeric           phosphorothioate oligonucleotides having 2′-MOE wings and a       deoxy gap                                                         TARGET                   CONTROL                   SEQ ID   TARGET       %   SEQ   SEQ ID       ISIS #   REGION   NO   SITE   SEQUENCE   INHIB   ID NO   NO                                                         206854   5′UTR   4   160   ggctcccgctctggatcccg   81   14   1                   206856   5′UTR   4   184   gctccgagtcccagatcccg   0   15   1               206858   5′UTR   4   189   atcccgctccgagtcccaga   89   16   1               206860   5′UTR   4   205   ctgggtcccgctccggatcc   86   17   1               206863   Coding   4   306   ggcggaagtagaggccgccc   52   18   1               206864   Coding   4   311   tccccggcggaagtagaggc   77   19   1               206867   Coding   4   323   gtagcaggtctgtccccggc   92   20   1               206868   Coding   4   331   agaggccggtagcaggtctg   82   21   1               206871   Coding   4   429   tgcgccagtcccagctcttg   86   22   1               206873   Coding   4   442   acaccatccacattgcgcca   89   23   1               206875   Coding   4   539   gttgatccgatccgccatag   67   24   1               206876   Coding   4   641   cacggacaggtcattacccc   90   25   1               206879   Coding   4   662   gtgctggtgggcgtagtccc   69   26   1               206881   Coding   4   775   tagttccggatggcgtggca   89   27   1               206883   Coding   4   846   ttgcgtagatttctgccatc   91   28   1               206884   Coding   4   852   gaccatttgcgtagatttct   88   29   1               206886   Coding   4   887   cagtctttctgttgccatta   91   30   1               206888   Coding   4   896   gtagttagccagtctttctg   86   31   1               206890   Coding   4   917   ttcggcatagatgcctccgg   85   32   1               206892   Coding   4   952   gaaacgacatggtttatata   79   33   1               206894   Coding   4   961   ccagccacggaaacgacatg   69   34   1               206897   Coding   4   993   tccagtactcagtcccatca   88   35   1               206898   Coding   4   1031   tctctcgccccatggttcac   57   36   1               206901   Coding   4   1075   cccttcccatccttataggt   0   37   1               206903   Coding   4   1106   gtgctcctcgatggcaaggt   89   38   1               206904   Coding   4   1120   tccccaaatgtacagtgctc   95   39   1               206905   Stop   4   1141   acatggccttaaacgatggg   66   40   1           Codon               206906   3′UTR   4   1163   ttcttaaactgcgcttctag   93   41   1               206907   3′UTR   4   1171   catgccttttcttaaactgc   93   42   1               206908   3′UTR   4   1183   gtcatgggtcaccatgcctt   93   43   1               206909   3′UTR   4   1196   aggatcccctctggtcatgg   73   44   1               206910   3′UTR   4   1206   acataaccataggatcccct   92   45   1               206911   3′UTR   4   1215   gcctggcacacataaccata   72   46   1               206912   3′UTR   4   1220   agccagcctggcacacataa   84   47   1               206913   3′UTR   4   1229   gttcctgccagccagcctgg   90   48   1               206914   3′UTR   4   1244   ttgatagccaccccagttcc   91   49   1               206915   3′UTR   4   1252   atccaatattgatagccacc   93   50   1               206916   3′UTR   4   1258   ctcgccatccaatattgata   91   51   1               206917   3′UTR   4   1264   gctgtcctcgccatccaata   86   52   1               206918   3′UTR   4   1274   ccagtaccacgctgtcctcg   80   53   1               206919   3′UTR   4   1281   ctcgcagccagtaccacgct   93   54   1               206920   3′UTR   4   1286   gaacactcgcagccagtacc   81   55   1               206921   3′UTR   4   1294   actctcaggaacactcgcag   93   56   1               206922   3′UTR   4   1301   actttcaactctcaggaaca   85   57   1               206923   3′UTR   4   1306   atcccactttcaactctcag   90   58   1               206924   3′UTR   4   1313   ataagtcatcccactttcaa   84   59   1               206925   3′UTR   4   1322   gcaagtgtcataagtcatcc   92   60   1               206926   3′UTR   4   1328   tgctgtgcaagtgtcataag   91   61   1               206927   3′UTR   4   1347   atcattgtgaggcagagcat   87   62   1               206928   3′UTR   4   1357   ggctgactgcatcattgtga   78   63   1               206929   3′UTR   4   1368   cttcaccaggtggctgactg   90   64   1               206930   3′UTR   4   1373   cacttcttcaccaggtggct   90   65   1               206931   3′UTR   4   1378   caggtcacttcttcaccagg   82   66   1               206932   3′UTR   4   1392   cgtttcctgtgttgcaggtc   91   67   1               206933   3′UTR   4   1400   ggtcccatcgtttcctgtgt   77   68   1               206934   3′UTR   4   1406   gactgaggtcccatcgtttc   85   69   1               206935   3′UTR   4   1412   gaagaagactgaggtcccat   0   70   1               206936   3′UTR   4   1424   aagtcctctgctgaagaaga   84   71   1               206937   3′UTR   4   1432   aaaatatcaagtcctctgct   89   72   1               206938   3′UTR   4   1456   ttattgcccacagttgccaa   94   73   1               206939   3′UTR   4   1470   tcttaaatgccatattattg   75   74   1               206940   3′UTR   4   1479   tctttcacctcttaaatgcc   88   75   1               206941   3′UTR   4   1512   taaagtgacataagaatggt   81   76   1               206942   Intron   11   1746   ggccacaatcaccagaggtg   61   77   1               206943   Intron   11   3180   tccacatcaaagggaccttt   81   78   1               206944   Intron   11   5792   cctccatctcttgtcacctt   72   79   1               206945   Exon:   11   6892   agcagcctaccctggtcctt   70   80   1                   Intron           Junction               206946   Intron   11   8999   ggtttctgatgagctccatc   70   81   1               206947   Intron:   11   11555   tattccacagctgagagcaa   79   82   1           Exon           Junction               206948   Exon:   11   11718   aacatcttacccatggttca   74   83   1           Intron           Junction               206949   Intron:   11   12597   ctctctcgccctgtgagaag   86   84   1           Exon           Junction               206950   3′UTR   12   1444   gatttgaaactgccaactcc   88   85   1               206951   3′UTR   12   1452   agttgggcgatttgaaactg   82   86   1               206952   3′UTR   12   1463   ctttatttatcagttgggcg   11   87   1               206953   3′UTR   12   1469   cagatactttatttatcagt   36   88   1               206954   5′UTR   13   3   gctcaaaaccagggagcccc   26   89   1               206955   5′UTR   13   92   cggctgctgagaaggcccag   6   90   1               206956   5′UTR   13   119   atcagcgactagcccccaac   20   91   1                  
 
     [0279] As shown in Table 1, SEQ ID NOs 14, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84, 85 and 86 demonstrated at least 65% inhibition of human cathepsin Z 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 2. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number of the corresponding target nucleic acid. Also shown in Table 2 is the species in which each of the preferred target regions was found.  
                   TABLE 2                          Sequence and position of preferred target regions identified           in cathepsin Z.                                                 TARGET                               SITE   SEQ ID   TARGET       REV COMP       SEQ ID       ID   NO   SITE   SEQUENCE   OF SEQ ID   ACTIVE IN   NO                                                     124515   4   160   cgggatccagagcgggagcc   14     H. sapiens     92                   124517   4   189   tctgggactcggagcgggat   16     H. sapiens     93               124518   4   205   ggatccggagcgggacccag   17     H. sapiens     94               124520   4   311   gcctctacttccgccgggga   19     H. sapiens     95               124521   4   323   gccggggacagacctgctac   20     H. sapiens     96               124522   4   331   cagacctgctaccggcctct   21     H. sapiens     97               124523   4   429   caagagctgggactggcgca   22     H. sapiens     98               124524   4   442   tggcgcaatgtggatggtgt   23     H. sapiens     99               124525   4   539   ctatggcggatcggatcaac   24     H. sapiens     100               124526   4   641   ggggtaatgacctgtccgtg   25     H. sapiens     101               124527   4   662   gggactacgcccaccagcac   26     H. sapiens     102               124528   4   775   tgccacgccatccggaacta   27     H. sapiens     103               124529   4   846   gatggcagaaatctacgcaa   28     H. sapiens     104               124530   4   852   agaaatctacgcaaatggtc   29     H. sapiens     105               124531   4   887   taatggcaacagaaagactg   30     H. sapiens     106               124532   4   896   cagaaagactggctaactac   31     H. sapiens     107               124533   4   917   ccggaggcatctatgccgaa   32     H. sapiens     108               124534   4   952   tatataaaccatgtcgtttc   33     H. sapiens     109               124535   4   961   catgtcgtttccgtggctgg   34     H. sapiens     110               124536   4   993   tgatgggactgagtactgga   35     H. sapiens     111               124539   4   1106   accttgccatcgaggagcac   38     H. sapiens     112               124540   4   1120   gagcactgtacatttgggga   39     H. sapiens     113               124541   4   1141   cccatcgtttaaggccatgt   40     H. sapiens     114               124542   4   1163   ctagaagcgcagtttaagaa   41     H. sapiens     115               124543   4   1171   gcagtttaagaaaaggcatg   42     H. sapiens     116               124544   4   1183   aaggcatggtgacccatgac   43     H. sapiens     117               124545   4   1196   ccatgaccagaggggatcct   44     H. sapiens     118               124546   4   1206   aggggatcctatggttatgt   45     H. sapiens     119               124547   4   1215   tatggttatgtgtgccaggc   46     H. sapiens     120               124548   4   1220   ttatgtgtgccaggctggct   47     H. sapiens     121               124549   4   1229   ccaggctggctggcaggaac   48     H. sapiens     122               124550   4   1244   ggaactggggtggctatcaa   49     H. sapiens     123               124551   4   1252   ggtggctatcaatattggat   50     H. sapiens     124               124552   4   1258   tatcaatattggatggcgag   51     H. sapiens     125               124553   4   1264   tattggatggcgaggacagc   52     H. sapiens     126               124554   4   1274   cgaggacagcgtggtactgg   53     H. sapiens     127               124555   4   1281   agcgtggtactggctgcgag   54     H. sapiens     128               124556   4   1286   ggtactggctgcgagtgttc   55     H. sapiens     129               124557   4   1294   ctgcgagtgttcctgagagt   56     H. sapiens     130               124558   4   1301   tgttcctgagagttgaaagt   57     H. sapiens     131               124559   4   1306   ctgagagttgaaagtgggat   58     H. sapiens     132               124560   4   1313   ttgaaagtgggatgacttat   59     H. sapiens     133               124561   4   1322   ggatgacttatgacacttgc   60     H. sapiens     134               124562   4   1328   cttatgacacttgcacagca   61     H. sapiens     135               124563   4   1347   atgctctgcctcacaatgat   62     H. sapiens     136               124564   4   1357   tcacaatgatgcagtcagcc   63     H. sapiens     137               124565   4   1368   cagtcagccacctggtgaag   64     H. sapiens     138               124566   4   1373   agccacctggtgaagaagtg   65     H. sapiens     139               124567   4   1378   cctggtgaagaagtgacctg   66     H. sapiens     140               124568   4   1392   gacctgcaacacaggaaacg   67     H. sapiens     141               124569   4   1400   acacaggaaacgatgggacc   68     H. sapiens     142               124570   4   1406   gaaacgatgggacctcagtc   69     H. sapiens     143               124572   4   1424   tcttcttcagcagaggactt   71     H. sapiens     144               124573   4   1432   agcagaggacttgatatttt   72     H. sapiens     145               124574   4   1456   ttggcaactgtgggcaataa   73     H. sapiens     146               124575   4   1470   caataatatggcatttaaga   74     H. sapiens     147               124576   4   1479   ggcatttaagaggtgaaaga   75     H. sapiens     148               124577   4   1512   accattcttatgtcacttta   76     H. sapiens     149               124579   11   3180   aaaggtccctttgatgtgga   78     H. sapiens     150               124580   11   5792   aaggtgacaagagatggagg   79     H. sapiens     151               124581   11   6892   aaggaccagggtaggctgct   80     H. sapiens     152               124582   11   8999   gatggagctcatcagaaacc   81     H. sapiens     153               124583   11   11555   ttgctctcagctgtggaata   82     H. sapiens     154               124584   11   11718   tgaaccatgggtaagatgtt   83     H. sapiens     155               124585   11   12597   cttctcacagggcgagagag   84     H. sapiens     156               124586   12   1444   ggagttggcagtttcaaatc   85     H. sapiens     157               124587   12   1452   cagtttcaaatcgcccaact   86     H. sapiens     158                  
 
     [0280] 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 cathepsin Z.  
     Example 16  
     [0281] Western Blot Analysis of Cathepsin Z Protein Levels  
     [0282] 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 cathepsin Z 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.).  
    
     
       
         1 
         
           
             158  
           
           
             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  
             1541  
             DNA  
             H. sapiens  
             
 
             
               CDS  
               (241)...(1152)  
             
           
            4 

aaatggtgaa agggggggag gggagaaaag aaaaaaaaaa aaagaaaggg gaaagggggg     60 

aaaaataaga aaaagcgaga cagaggcgct gccgcgtccg ctcgcgggga aggctggggt    120 

cggccgggtg ctaggccggg gccgaggccg aggccggggc gggatccaga gcgggagccg    180 

gcgcgggatc tgggactcgg agcgggatcc ggagcgggac ccaggagccg gcgcggggcc    240 

atg gcg agg cgc ggg cca ggg tgg cgg ccg ctt ctg ctg ctc gtg ctg     288 
Met Ala Arg Arg Gly Pro Gly Trp Arg Pro Leu Leu Leu Leu Val Leu 
   1               5                  10                  15 

ctg gcg ggc gcg gcg cag ggc ggc ctc tac ttc cgc cgg gga cag acc      336 
Leu Ala Gly Ala Ala Gln Gly Gly Leu Tyr Phe Arg Arg Gly Gln Thr 
             20                  25                  30 

tgc tac cgg cct ctg cgg ggg gac ggg ctg gct ccg ctg ggg cgc acg      384 
Cys Tyr Arg Pro Leu Arg Gly Asp Gly Leu Ala Pro Leu Gly Arg Thr 
         35                  40                  45 

aca tac ccc cgg cct cat gag tac ctg tcc cca gcg gat ctg ccc aag      432 
Thr Tyr Pro Arg Pro His Glu Tyr Leu Ser Pro Ala Asp Leu Pro Lys 
     50                  55                  60 

agc tgg gac tgg cgc aat gtg gat ggt gtc aac tat gcc agc atc acc      480 
Ser Trp Asp Trp Arg Asn Val Asp Gly Val Asn Tyr Ala Ser Ile Thr 
 65                  70                  75                  80 

cgg aac cag cac atc ccc caa tac tgc ggc tcc tgc tgg gcc cac gcc      528 
Arg Asn Gln His Ile Pro Gln Tyr Cys Gly Ser Cys Trp Ala His Ala 
                 85                  90                  95 

agc acc agc gct atg gcg gat cgg atc aac atc aag agg aag gga gcg      576 
Ser Thr Ser Ala Met Ala Asp Arg Ile Asn Ile Lys Arg Lys Gly Ala 
            100                 105                 110 

tgg ccc tcc acc ctc ctg tcc gtg cag aac gtc atc gac tgc ggt aac      624 
Trp Pro Ser Thr Leu Leu Ser Val Gln Asn Val Ile Asp Cys Gly Asn 
        115                 120                 125 

gct ggc tcc tgt gaa ggg ggt aat gac ctg tcc gtg tgg gac tac gcc      672 
Ala Gly Ser Cys Glu Gly Gly Asn Asp Leu Ser Val Trp Asp Tyr Ala 
    130                 135                 140 

cac cag cac ggc atc cct gac gag acc tgc aac aac tac cag gcc aag      720 
His Gln His Gly Ile Pro Asp Glu Thr Cys Asn Asn Tyr Gln Ala Lys 
145                 150                 155                 160 

gac cag gag tgt gac aag ttt aac caa tgt ggg aca tgc aat gaa ttc      768 
Asp Gln Glu Cys Asp Lys Phe Asn Gln Cys Gly Thr Cys Asn Glu Phe 
                165                 170                 175 

aaa gag tgc cac gcc atc cgg aac tac acc ctc tgg agg gtg gga gac      816 
Lys Glu Cys His Ala Ile Arg Asn Tyr Thr Leu Trp Arg Val Gly Asp 
            180                 185                 190 

tac ggc tcc ctc tct ggg agg gag aag atg atg gca gaa atc tac gca      864 
Tyr Gly Ser Leu Ser Gly Arg Glu Lys Met Met Ala Glu Ile Tyr Ala 
        195                 200                 205 

aat ggt ccc atc agc tgt gga ata atg gca aca gaa aga ctg gct aac      912 
Asn Gly Pro Ile Ser Cys Gly Ile Met Ala Thr Glu Arg Leu Ala Asn 
    210                 215                 220 

tac acc gga ggc atc tat gcc gaa tac cag gac acc aca tat ata aac      960 
Tyr Thr Gly Gly Ile Tyr Ala Glu Tyr Gln Asp Thr Thr Tyr Ile Asn 
225                 230                 235                 240 

cat gtc gtt tcc gtg gct ggg tgg ggc atc agt gat ggg act gag tac     1008 
His Val Val Ser Val Ala Gly Trp Gly Ile Ser Asp Gly Thr Glu Tyr 
                245                 250                 255 

tgg att gtc cgg aat tca tgg ggt gaa cca tgg ggc gag aga ggc tgg     1056 
Trp Ile Val Arg Asn Ser Trp Gly Glu Pro Trp Gly Glu Arg Gly Trp 
            260                 265                 270 

ctg agg atc gtg acc agc acc tat aag gat ggg aag ggc gcc aga tac     1104 
Leu Arg Ile Val Thr Ser Thr Tyr Lys Asp Gly Lys Gly Ala Arg Tyr 
        275                 280                 285 

aac ctt gcc atc gag gag cac tgt aca ttt ggg gac ccc atc gtt taa     1152 
Asn Leu Ala Ile Glu Glu His Cys Thr Phe Gly Asp Pro Ile Val 
    290                 295                 300 

ggccatgtca ctagaagcgc agtttaagaa aaggcatggt gacccatgac cagaggggat   1212 

cctatggtta tgtgtgccag gctggctggc aggaactggg gtggctatca atattggatg   1272 

gcgaggacag cgtggtactg gctgcgagtg ttcctgagag ttgaaagtgg gatgacttat   1332 

gacacttgca cagcatgctc tgcctcacaa tgatgcagtc agccacctgg tgaagaagtg   1392 

acctgcaaca caggaaacga tgggacctca gtcttcttca gcagaggact tgatattttg   1452 

tatttggcaa ctgtgggcaa taatatggca tttaagaggt gaaagagttc agacttatca   1512 

ccattcttat gtcactttag aatcaaggg                                     1541 

 
           
             5  
             23  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            5 

aacgatggga cctcagtctt ctt                                           23 

 
           
             6  
             22  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            6 

gccatattat tgcccacagt tg                                            22 

 
           
             7  
             27  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            7 

agcagaggac ttgatatttt gtatttg                                       27 

 
           
             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  
             14001  
             DNA  
             Homo sapiens  
             
 
           
            11 

ataagagaca catcagtagg tagagccccg gggccacact ttgcctcagt gaccactttt     60 

tggggaacaa ggactgaaac ttctgggctg acgaagcagc tctccagcct tgctctccac    120 

tcggacagtc atgcggggat tccatggcca cctcagcgct tccgggaatg gtcatggaag    180 

cttctggaag tcaggaggca gccactgtga cttccccttg cccacgtggc acgcttggaa    240 

tgtggtgagt gccactgagt atggagagag tcaggcaagc tcatctgtgg gccctgtgcc    300 

aagggccccc agcaggggcc tgtcaggtcg cagcccagaa tgccgggccc tgttcttacc    360 

agagaagaag gccatggtgt gggcccaagg gccatgacaa acagaggggc cgcagggagc    420 

gagaagccct cccccagtta caaaaccacg tcctgggggg ccacttctgc ttttggttcc    480 

tcatttgact aagaagagtt tcgttagcag aaaacctttt caaggcgtct ttggaagtca    540 

cattggataa ctcctgatgc ctgcgccaag tggaatcttc ctttggggca ttttctagaa    600 

ggatccctcc ttccttctct gcaaaaggga accgtccccc cgaaagggcc tgggcctttg    660 

ggaaaagggg tttagaaagc caggcgcggg gacccttctt tgggggcggt ggccgcatcc    720 

caggatccct tcctagggga ggcctgggcc cagatgtgag gctgggggcc tctccccagg    780 

gctcagggcc ccaggaatgc agggccaagg gggcccatga gcctgcggcc ccccaccccg    840 

aagccaggaa ccccggcaaa tgagtccagc cggacagtcc cgcgctcagg gcggcggccg    900 

gcggcggggt caggctgggg tctgcccggc gacctcgcgc ctggcccgcg agttcaggtg    960 

ccccggagcc agggagggag cggggcgcgg agccgggcgg ggagggcagg ggtcaccggg   1020 

cggggcagga gcagcgatgg gacagccccg ccccggggga gtgtcccggc ctggccgccg   1080 

cagccttaag ggccgggcgg ggcaggggcc caaagtgcgg ggtcggccgg gtgctgggcc   1140 

gaggccgagg ccggggcggg atccagagcg ggagccggcg cgggatctgg gactcggagc   1200 

gggatccgga gcgggaccca ggagccggcg cggggccatg gcgaggcgcg ggccagggtg   1260 

gcggccgctt ctgctgctcg tgctgctggc gggcgcggcg cagggcggcc tctacttccg   1320 

ccggggacag acctgctacc ggcctctgcg gggggacggg ctggctccgc tggggcgcag   1380 

gtgggcaccg gcggggaggc agccctgggg ggacgggagg cccatcgccc gggccaggcg   1440 

cgcgctctgc ctcctggggg ccctgcgtcg ccaacaggcc ttggaaggtc agatccagcc   1500 

gcttcgtttt gggggtcgct ccttctcttc cacatccagg gtgagccctg atgtttgtgg   1560 

ggttggacaa gccccacctg gccagaaagc caggccagcc ccacctcctc ggcctctggg   1620 

ggtggccctt acggcgcggc atggctgggg gagcaacccg gttccctcct ttcgggggtg   1680 

ccctgcctcc ttcacattct gagagcgctg gagcctccta gtgtgaggtg ggcccacacc   1740 

tggctcacct ctggtgattg tggccccact cccatgccct caggtgttgg gaaaggcggg   1800 

agggccctac cgagggcttc ctgggcccac ggcccccggg agccccagca ccgatctggg   1860 

tggggatgat ctcttctcag cacatacccc cggcctcatg agtacctgtc cccagcggat   1920 

ctgcccaaga gctgggactg gcgcaatgtg gatggtgtca actatgccag catcacccgg   1980 

aaccagcaca tcccccaata ctgcggctcc tgctgggccc acgccagcac cagcgctatg   2040 

gcgggtgagt ggccgccctt tgactgtggg ccaggaaagc ccatcccggc tgcctgttta   2100 

taggcccagg cagctcagcc cgcggttcca ggggcctgtg attcaaggcc aggtcagcct   2160 

gggctggggt gctaaggccg aggagcgcgg gcctttgccc tctgctgagg tgacccaagc   2220 

tgggtcattc tggggctttt gggtgacagg caggactagg actgaatccc agtgacaagt   2280 

ggctgccagg tgccctctct cccccaaagc ttgtgacctc agccattggg atagaaacag   2340 

aaatgcaaaa tcactgagtc cccaggctgt atttgaggat acagaagtaa actgcttctc   2400 

agcacagatg aaagcctgtc ctgggcttgg aatattgggt gaggctagtt ggctacagcc   2460 

catggcctaa cctggtcact acctccagcc tggtgggttt ctatcttagg cttttggcag   2520 

gagtagggta gggaggctct tagcaaagag gaaggggaaa ctaaggccct ccctatcaac   2580 

gaggctgctg caccctgctt tgggagagaa ggcgggagaa gggacagctg caaaccagcc   2640 

ccaggcccac cccacagcat ttggaaggga ctctgctatc ccctctccag tcacagcaca   2700 

gggtccagga gcctggattt cccctggaga tgggacttgc gtggcctggg agtttggccc   2760 

atgagcactg tggtgctgtt gcctcttccc tggccttggt cctcatgggt caggcctgct   2820 

gtacacccat gtgaaaagga tagactacag tgtggctgcc gctgtgggcc aaggctgggg   2880 

gacatgcgtt tgcgtgtgct ccatctcact gacgcctcct caatgccatg attgcttcca   2940 

gtttgcagat gaggaagcag gctttttgca gagagatggg gaggcttgag catagtgtga   3000 

gtgacagctg agatttgagc ccggaagcct gggctttgtg ccaggatgtc tgatgggctc   3060 

agtcttgtga atgaggggtt cttgctctgg gggtagggtc tggaccccag agccagccag   3120 

ctgaagctgt ggcatccgtg gcgtctcccc cgggggctgg aacacacttc tgatcgttca   3180 

aaggtccctt tgatgtggat ggagctggaa ggtctgggag accttgggcg ggctgcaggg   3240 

tgtgggtggg gctagaggca gaggactggg cagagtctag gggaagtgtc caggcctggc   3300 

acccgctgag gaagtgaaag agcctttgag ggtggggtga gggtgagggg agaatagaaa   3360 

ggtcaacagg tgcctgtggg aagccttgaa ctctttgaga gggagtcagg tgacaagatg   3420 

tgcgtctgtg aaggactgcg ggtagaaagg agatggcttt cagatggatg tgggaccctg   3480 

gccaggggtc agccctgccg catttcccca ggggactctc tctgggcgga tgactgcttc   3540 

accccacttt ctcctgaaac ctccttctca gcgcacccag gcccaggcac tcgcttcctc   3600 

tttcactgag gaatagaagc caccagaagg ggacttccac ggggccctgg ccatttgcca   3660 

gccaaggcag caggtcctcc atttgtcttc cctcctctcc cccaggatca actgtcttgt   3720 

cccctcactg agcccgtccc ttcccactca gggaagctct cccacctgga aaaaaggaac   3780 

acaaagcccc cggccccacc ccgccccaat tctgggctcc ctagggaagt atctggagtc   3840 

cctccacttc ctcttctcta cccttgaacc cactccagcc tcagtcccac ctctgccact   3900 

ccaccggcac tgttctttcc actgccacct ggggcccacc gtgaagcatt ctcctgatca   3960 

cgtctcccca ggatgcttcc ttctcctgca ccatactgcc cgcctccacc tccctggcca   4020 

ctccttcccc tctccccagc tgtctgctcc tcagagttct gagtgtcagc ttccccagga   4080 

ccaacccttc acctgaactc agcttgacat ccacctgccc actcgcccgc tgtcccggtc   4140 

acctgacacc tccagcctca gaccttcagg tctggatggc tccttccact actgtcacct   4200 

gacagcccat ttcttcccca gtcctccctg gctgtccttc cggctatgca ggcaaaagcc   4260 

tggactggta cccgactcct ctgttttctg tccctcatat aatccaccca caagtcttgt   4320 

cggttctacc ttcgaagcag atgcagaaat cacgcaccgc ccaccgcctc ctccaggtct   4380 

aagccacacc gccccactgc cgactgccct cactggtctc ctggcctctg cccctgaggc   4440 

cacagtgagc ctgtgacaac ccatcagaac atgttctttg gcttgaaacc ctctcaggat   4500 

tccactcaga taaaagacag aaccctctgg ccttcaagag ccttcaccat cgggctccct   4560 

cccagtccgc ccttgctccc ctgctcctgc ggagagtgga gcagggaggt agcttctccc   4620 

accaggcacc tcctctctcc ctgaggtccc atggctccct ccctccctcc tatggatgtc   4680 

acctcagcgg ggtcttcctc agccacccta tgcatatgac accccacttc ctgaattatc   4740 

tcccggattt atttttcttc atggtgctgt agactttgca cagttacaga tgctccttga   4800 

cttaactgtg gggtcatgtc ccgacaaccc cctcgtaagt tgaaaacacc atgagtggaa   4860 

aatgtgtttg acacacccag cctcctgcac accctggcct ggctttgtct gcattaaacg   4920 

tattcaggac acttgtgtta gcccacagtg tgagtaataa gcccgttccc agatggggct   4980 

ggtgggatga aggattgagt ttggccgtgg gagaagcttg tgcatacctc agtgagctgg   5040 

ttaggccggg tccagatgct tcaggcagga gtagacggag aggccagaac cgaaactgct   5100 

catccatccg agctctgcct ctgggccagg actgctgtcg ccgggttggg ttgggcatag   5160 

aaggataggg ctcgggggca ggccatggga cgccctgcac tgccaagtgg aactccagaa   5220 

gcccaggcag gaggtgcttc cagaaggatg gagcgaccgc tgtgctgact gctgccggag   5280 

agtggtcagc tcggatgaca gtggagaagt gacctttagc aagagagaaa gggctgaaga   5340 

ggagcggggc agtaggggtc ggggcactgt ctcaaaccat tgagtgaagc agaaaaccgt   5400 

gatagtgagt gggaggggcc gggggcttcc tctgttgttg tgagaaaggc aagtttgggg   5460 

tgtgcagatc tgcttagagg gggcatagct gacaagggag cggggagaag gaaagctgga   5520 

gggagccaag tccttgggag gaaacaggat ggaacctagg gtgggtcggt caggtgggag   5580 

gggacaaacc agccaccggc tgctccagga agagggtttg gggtgcagag catgggcagg   5640 

ctgggtggcg gcaggatgga ggtctgcttt gggacttttc agtgatgcgt gaagtcacca   5700 

gagcagggac agttccagaa ggcccctggc tgggcgaccg ggaggaggcc aggtggagga   5760 

gcggactgtg gggcagtgtg ggagagtctg gaaggtgaca agagatggag gggatgtgca   5820 

ggtccagagg gggctgccaa tgggcagagg gactccctgc tcattgtgcc ctgggggagg   5880 

gacaggtgag gctgcaggaa gagttccgac ttgaacgtgg ggtcaggaga tgaggcctga   5940 

ggcaggtgca ggggtgtgtg gctgtgaaat cattgtcctg ggagggcaga gtgagatgac   6000 

cctgaacatg ggcccggatt tccttctagg atctgtttta ttgcatttgt gcatgggaag   6060 

ggaagcaggt ccctcggtga ccaccctgag ctgtccctgg ctgcaccttg ggatttagag   6120 

cccagccacc tgggcagtga gggcatggtc agccagccgc tgggagccct ctgcatcccc   6180 

cagtgccttc ggggctgggt ccctgctggc cttccccagc cctcaggacc gcatctggcc   6240 

cctcactggg gctcatgcgg ctccctgggc tatactcgtt cacagagctg acaccttggc   6300 

cccctgtcca cgagggcagg gacggtctct cctggctggc tttggggtgt ggcccacttg   6360 

ccatgggtgg ctgagtctct gtccattcag cccttcattc agcaggcact tctcaggtgt   6420 

ctgcagggcc agatgctggt gaccgcctgg ccctggccct gctctcatgg catttgtagc   6480 

gggaggtgtg gcagacagga cagtgcccaa agaaggccag gagaggcctc tctgcagagc   6540 

gacgtctgag cggagacctc catgaagagg tcagcaggga gaaggagaca caggcagctg   6600 

ggcagagctg aaggcaggcc aggctgggca gcgcctgcat cccgcctgag gcgtccaggt   6660 

ccgttcgggg aagtgggtgg agagtgggtg agtgctgact ggcaggtgct gactgttgca   6720 

gatcggatca acatcaagag gaagggagcg tggccctcca ccctcctgtc cgtgcagaac   6780 

gtcatcgact gcggtaacgc tggctcctgt gaagggggta atgacctgtc cgtgtgggac   6840 

tacgcccacc agcacggcat ccctgacgag acctgcaaca actaccaggc caaggaccag   6900 

ggtaggctgc tgcccgtgcc cccactccac cctcctgccc tccactccct cttccacagg   6960 

ccttcgtggc tgaggaccca gcctgacccc gtgctcctca cattggctgg tggctgggga   7020 

gctgcgggag ggcagaggtt gcagccccca ggggcgctgg tgctcttgac catgaggggt   7080 

agtgttccca gtgccagctc cagcctctcg ctgcagaagc cccctctgct atcttgcacc   7140 

cgcttgggct ggggatcaga aaactggacc ccgtagtccc tgttgagaag cccaacgaaa   7200 

aagcctgctg caggagcggt ctgaagagca tccttggcca tgcgcgcggt ggctcatgcc   7260 

tgtaatctca gcactttggg aggccgagac aggcagatca ctggaggtca ggagttcaag   7320 

accagcctgg ccaacacggt gaaatcccgt ccctaccaaa agcacaaaaa ttagctgggt   7380 

gtggtggtgc acacctgtaa tcccaggtac tcgggcagct gaggcaggag cattgcttga   7440 

cccgggaggc agaggttgca gtgagccaag atcgcgccac tgcactctag cctgggtgac   7500 

agagcaagac tacggctcaa aaaaaaaaaa aaaaaaatac cggccctgtc cccatgtctc   7560 

cagcctcgtg gaagagtgtc ttgctcctgt gcccctttgg ctgggcctga atgtggtgtt   7620 

tgagttctga catgctggca ttttggtggc tttttgggcc tttgtagggg agaggtgtag   7680 

ggggccttgt ggagggagag ttcattttcc aggagctttg gtggagagca ggtggggagg   7740 

tgctgcccca aactggaagg agccattggc ttggttcctg ttctggtgac ccagggccca   7800 

ggtcccccac attcaggtgc ggctacggcc aagggggtgc tgtgtggatg agcagaccca   7860 

agctagcctg gaatccctag agctggcaca ttcctttcag gctgctccca ttagaaacag   7920 

aaggaaagaa aagtccagac cctaatccca taattggtag gttttgtttg tttgtttgtt   7980 

tgtttttgag agggagtttt gctcttgttg cccaggctgg agtgcaatgg cacgatctct   8040 

gctcactgca acctccgcct cccgggtttg agtgattctc ctgcctcagc ctcctgagta   8100 

gctgggatta caggcatgcg ccaccacgcc cagctacttt tgtatttttt agtagagacg   8160 

gggtttctcc atgttgttca ggctggtctt aaactcccaa cctcaagtga tccacctgcc   8220 

ttggcctccc aaaatggtgg gattacaggc atgagctgct gcgcccggcc ataactggta   8280 

gtttgttttt tttttttgag acggagtctc gctctttcgc ccaggcagga ctgcagtggc   8340 

gcgatctcgg ctcactgcaa gctccgcctc ccgggttcac gccactctcc tgcctcagcc   8400 

tccggagtag ctgggactac aggcgcccgc caccgcgccc ggctaatttt ttgtattttt   8460 

agtagaggcg ggatttcact gtgttaacca ggatggtctt gatctcctga cctcgtgata   8520 

tgcctgcctc ggcctcccaa agtgctggga ttacaggtgt gagccaccgt gcccggccat   8580 

aactggtagt tttaagccac ctgactcaga tctccctcac agccagagca gagagctggg   8640 

aaagcccagc agctgcttgc tttgctgcct ccacccctgc cccaggctat ataggctggc   8700 

cccagcgccc tgtgcactct gaagtggcag aaggtcctct gggcaccacc ctctgccaac   8760 

ggggctccct tgccttgcct ctcaggcctt attgttcagc cagaaattct cctggactgg   8820 

ctgagtgagt ccttcgagaa gggggacaga tggaaggtgc agagggaagg gctatcagat   8880 

ggctctggaa gcaggaagca ggtatcaggt ggtctccagg gccacgccca ctccacgcca   8940 

aagtgctctc gcccagctga ggccaggccc ctccaaccca cccagcctga caggagcaga   9000 

tggagctcat cagaaacctg attgtgtgac tggtgaccgc ggtggctcac acctgtaatc   9060 

ccagcacttt gggaggctga ggcgggcgga tcacttgaga ccaggagttc gagaccagac   9120 

tggccaacat ggtaaaaccc tgtccctact aaaaatacaa aaaatagccg agcgcggtgg   9180 

catgcgcctg taatcccagc tactcaggag gctaaggcag gagaatcact tgaacccagg   9240 

aggcagaagt tgcagtgagc caggatcata ccactgaact ccagcctggg tgacagagtg   9300 

agactctgtc tccaaaaaaa aaagaaaaga aaagaataaa aaaacctgat tgcgataaat   9360 

agtcatcttt gtagtgttag caaaagtgtg gccacacatc caccattttc ccgtagtact   9420 

gggcactggg cacttggtgt gcaaggaaca aattgaacca gtgtctgagc ctcaaggtgc   9480 

tcatgttcct gggggaggaa tcccgacaga tgtgtaacta tgacagacag gtcgcgccaa   9540 

agcagcgaat ggcaggccga gggccgccgt gtggatggag tgaaaagtgg gaacacagtg   9600 

actctgagcc ttgcaaaacc gagagctgta ctttttcttt tttttaaatt aatttataaa   9660 

attttttttc tgtagagatg aggtctcgct ctgttgtcca ggctggtctc agactcctgg   9720 

tatcaagcca tcttcctgcc tcggcctccc gaagtgctgg gattacaggt gtgagccacc   9780 

acgcctgtcc tttctttctt tctttctttc tttttttttg agacggagtc tcgctcttgt   9840 

cgctcaggct ggagtgcaat ggcgcgatcc tggctcactg caacctctgc ctcccgggtt   9900 

caagcgattc tcctgcctca gcctccactg agtagctggg attacaggca tccaccacca   9960 

cacccagcta atttttgtac tttattagta gagacagggg tttcaccatg ttggccaggc  10020 

tggtctcaaa ctcctgacct caggtgatcc acccacctcg gcctcccaaa gagctgggat  10080 

tacaggcgcc tggccctctt tattttattt tttaagccta attcttgata acctcttaag  10140 

tcgaaataac cagaattttt ttttttggtc ccctcagcgt gggcacagaa gaaaatgctt  10200 

ttttgtggtt gttgttaccc aaagtattgg cctatttatt caccagccag caatagagac  10260 

attttattaa gtgctaatca tttgattatg ctccattttc agctagaaag tattgtggaa  10320 

aggtccttct ccagccatta gaggaaaaag gcgaactcct gggtgcctac tatgtgctaa  10380 

ttaatgtctg tctcttttct tctctgccaa ggcctgggga agggcttttc ataactccct  10440 

ggcagctgaa agaagagcgt cattaagcag cacctaatag caagatgatt tccgttctta  10500 

gctgctgagt ggtaggctgc atgatgagag tggagagtgg gcttggcggg cttctttgtc  10560 

agtgggttga tgaggagacg gcctcatgct gacttttctt gctctccccc agagtgtgac  10620 

aagtttaacc aatgtgggac atgcaatgaa ttcaaagagt gccacgccat ccggaactac  10680 

accctctgga gggtgggaga ctacggctcc ctctctggga gggagaagat gatggcagaa  10740 

atctatgcaa atggtcccat caggtgagga gaggtcccgc gggcaaagag aggtttgcgg  10800 

gtgaggaaag gtcccgcggg tgaggaaagg tcccgcgggt gaggagagat ccgcggtgac  10860 

agcgccagtg tgggctcatg tcggtgctca tgtaggcatc tccgtccttc gccactttcc  10920 

ttctggccgg ttgtactcag taggctcgag acagccacat catttcccct ctaagaactt  10980 

cagttttctc agagttaaca ctcagctgat agcaagtctg cccctatgga tggttgtgcg  11040 

gaatgcggat gagacgctcc aagctgtacc gagggggcag tttcagcagg gaagacacta  11100 

tgtgaataac aacagaagtt tatccagaac cgtcagatat acactacctg agtcgggttt  11160 

acactttgtt gtcataccag attttttttt tttttttttt ttttgaaaca cagtctcact  11220 

ctgtcaccca ggctggagtg cagtggtatg atcttcgctc actgcagcct tgaccgcccg  11280 

ggctcaagct atcctcccgc ctcagccccc tccaccgagt agctgggact actggcgcac  11340 

accaccacac ccagctaatt tttgtatttt ctgtagaggt ggggtttcgc catcttgccc  11400 

aggctggtct tgaattcctg gactcaagcg atccacctgc ctcggcctcc caaagtgctg  11460 

gaattacagg cgtgagccac ggcgcccagc catatcagat tatttctaat gacggtaaat  11520 

tctctcaatt taaaagtggt aattctttaa ctgcttgctc tcagctgtgg aataatggca  11580 

acagaaagac tggctaacta caccggaggc atctatgccg aataccagga caccacatat  11640 

ataaaccatg tcgtttctgt ggctgggtgg ggcatcagtg atgggactga gtactggatt  11700 

gtccggaatt catggggtga accatgggta agatgttttc attttcttaa ggtcattcct  11760 

agaaaaaaac gtcctggtac tcctctcgct tgaatggttc tgtttacgtt tagctaaaaa  11820 

ctggggcctt gacagagggt tccttgctcc agctgtctca gccaccgcgc gagtggcttt  11880 

tggggccaga cccttctctg acatgcgcgg ctgccgccat cctgtgcact ttgggtgttc  11940 

agcagcatcc ctggcccctc cacccactag gtgactcccc gcaatcccag tgtgaccaaa  12000 

accgtctcca gatggtgcct ggtgccctgg ggaagtccct gtgagcactg ctggagaggg  12060 

gctctgccac cctcttcctt tacaaccagc tcattctttt tcccatcatg gtttttgggc  12120 

tcccctaaaa tactgtgtcg gtttttcaaa aatactcgag ttgggccaaa aatccaggag  12180 

agagaaatta actgccagac ctagtcttag ccattaataa tttaagacac agaaattcat  12240 

gattaagaaa tggggggaaa gcggttcatg tgcaggtgat gaaatgtcgt cattttttac  12300 

taggtgacta atgggggtag tagtagctgg aggaggcacc agtctagagt caacatgttt  12360 

tgtgagatga attcacttct ggcatagagg gccctcatgt taaacattaa ccaagtgaaa  12420 

tgtttcacaa aaggtatttc agaacagggg tcatgggaaa gctgaggggc agctgagtgg  12480 

gctgtctgga tcccccaagg ctggcctgag agatggaggc agaaagcagg gggcaggggg  12540 

ctgacggcag caaggggagc gggagacggc tgcagatggg ctgacgcgcg atcccacttc  12600 

tcacagggcg agagaggctg gctgaggatc gtgaccagca cctataagga tgggaagggc  12660 

gccagataca accttgccat cgaggagcac tgtacatttg gggaccccat cgtttaaggc  12720 

catgtcacta gaagcgcagt ttaagaaaag gcatggtgac ccatgaccag aggggatcct  12780 

atggttatgt gtgccaggct ggctggcagg aactggggtg gctatcaata ttggatggcg  12840 

aggacagcgt ggcactggct gcgagtgttc ctgagagttg aaagtgggat gacttatgac  12900 

acttgcacag catggctctg cctcacaatg atgcagtcag ccacctggtg aagaagtgac  12960 

ctgcgacaca ggaaacgatg ggacctcagt cttcttcagc agaggacttg atattttgta  13020 

tttggcaact gtgggcaata atatggcatt taagaggtga aagagttcag acttatcacc  13080 

attcttatgt cactttagaa tcaagggtgg gggagggagg gagggagttg gcagtttcaa  13140 

atcgcccaag tgatgaataa agtatctggc tctgcacgag actgaggctg cccgtgcctt  13200 

attcgcacac aaaaacaatg tcacatcagg acttaatcca acccagcaga aaggcttttt  13260 

aaaaatttac ttattacttg ttcttagcaa attaagacaa ttacaataaa acatcagcta  13320 

actgggttct tgtgagaaaa ctgaggtcag cttggaaagg agttccccga gtggagttcc  13380 

cagcggcccg cggctgacgg ccagatctgt cctgaggggt cgtgggagcc cagcgcctgc  13440 

cttgagggaa atgaacactg aaaacaggat ttgggagcag tattggattg acagcagaga  13500 

agggactgtt tgtaagggca gtttctcact gaagctgcta ccattttcct ttgtaaagaa  13560 

gtcatccacc tcctcccagc ggtgcccatt ttcaagacgc tgcccgagcc tcttaaaaca  13620 

gcttcttgaa agggtttttc cacaacgggt tctggaatgt tctgcttcag ctctggagga  13680 

tgctctaaat tagttcacca tgatgaagtt agatttgcag tgagctttaa cgaggaaaac  13740 

agtgactgtg ttagaaacag gaagtagaac atgacatttt ctttcttttt tttttttttt  13800 

tttgagatgc agttttgctc ttgttgccca ggatagagtg caatggcgcg atcttggctc  13860 

actgcaacct ctgcctccca ggttaagcga ttctcctgcc tcagcctccc gagtagctag  13920 

gattacaggc acgtgccacc atgcccagct aatgctgtat ttttagtaga gatggggttt  13980 

ctccatgttg gtcgggctgg t                                            14001 

 
           
             12  
             1500  
             DNA  
             Homo sapiens  
             
 
           
            12 

ggggtcggcc gggtgctagg ccggggccga ggccgaggcc ggggcgggat ccagagcggg     60 

agccggcgcg ggatctggga ctcggagcgg gatccggagc gggacccagg agccggcgcg    120 

gggccatggc gaggcgcggg ccagggtggc ggccgcttct gctgctcgtg ctgctggcgg    180 

gcgcggcgca gggcggcctc tacttccgcc ggggacagac ctgctaccgg cctctgcggg    240 

gggacgggct ggctccgctg gggcgcagca catacccccg gcctcatgag tacctgtccc    300 

cagcggatct gcccaagagc tgggactggc gcaatgtgga tggtgtcaac tatgccagca    360 

tcacccggaa ccagcacatc ccccaatact gcggctcctg ctgggcccac gccagcacca    420 

gcgctatggc ggatcggatc aacatcaaga ggaagggagc gtggccctcc accctcctgt    480 

ccgtgcagaa cgtcatcgac tgcggtaacg ctggctcctg tgaagggggt aatgacctgt    540 

ccgtgtggga ctacgcccac cagcacggca tccctgacga gacctgcaac aactaccagg    600 

ccaaggacca ggagtgtgac aagtttaacc aatgtgggac atgcaatgaa ttcaaagagt    660 

gccacgccat ccggaactac accctctgga gagtgggaga ctacggctcc ctctctggga    720 

gggagaagat gatggcagaa atctacgcaa atggtcccat cagctgtgga ataatggcaa    780 

cagaaagact ggctaactac accggaggca tctatgccga ataccaggac accacatata    840 

taaaccatgt cgtttccgtg gctgggtggg gcatcagtga tgggactgag tactggattg    900 

tccggaattc atggggtgaa ccatggggcg agagaggctg gctgaggatc gtgaccagca    960 

cctataagga tgggaagggc gccagataca accttgccat cgaggagcac tgtacatttg   1020 

gggaccccat cgtttaaggc catgtcacta gaagcgcagt ttaagaaaag gcatggtgac   1080 

ccatgaccag aggggatcct atggttatgt gtgccaggct ggctggcagg aactggggtg   1140 

gctatcaata ttggatggcg aggacagcgt ggtactggct gcgagtgttc ctgagagttg   1200 

aaagtgggat gacttatgac acttgcacag catggctctg cctcacaatg atgcagtcag   1260 

ccacctggtg aagaagtgac ctgcaacaca ggaaacgatg ggacctcagt cttcttcagc   1320 

agaggacttg atattttgta tttggcaact gtgggcaata atatggcatt taagaggtga   1380 

aagagttcag acttatcacc attcttatgt cactttagaa tcaagggtgg gggagggagg   1440 

gagggagttg gcagtttcaa atcgcccaac tgataaataa agtatctggc tctgcacgag   1500 

 
           
             13  
             300  
             DNA  
             H. sapiens  
             
               unsure  
               27  
               unknown  
             
           
            13 

ggggggctcc ctggttttga gcataanntn nnncnnnntc cctccctttc nnnccnnctt     60 

tcccttgtac ttgttttgca cagaacgtgg gctgggcctt ctcagcagcc gccttctagt    120 

tgggggctag tcgctgatct gccggctccc gcccagcctg tgtaaaacaa gtaaacaaag    180 

tgcggggtcg gccgggtgct aggccggggc cgaggccgag gccggggcgg gatccagagc    240 

gggagccggc gcgggatctg ggactcggag cgggatccgg agcgggaccc aggagccggc    300 

 
           
             14  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            14 

ggctcccgct ctggatcccg                                                 20 

 
           
             15  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            15 

gctccgagtc ccagatcccg                                                 20 

 
           
             16  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            16 

atcccgctcc gagtcccaga                                                 20 

 
           
             17  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            17 

ctgggtcccg ctccggatcc                                                 20 

 
           
             18  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            18 

ggcggaagta gaggccgccc                                                 20 

 
           
             19  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            19 

tccccggcgg aagtagaggc                                                 20 

 
           
             20  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            20 

gtagcaggtc tgtccccggc                                                 20 

 
           
             21  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            21 

agaggccggt agcaggtctg                                                 20 

 
           
             22  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            22 

tgcgccagtc ccagctcttg                                                 20 

 
           
             23  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            23 

acaccatcca cattgcgcca                                                 20 

 
           
             24  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            24 

gttgatccga tccgccatag                                                 20 

 
           
             25  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            25 

cacggacagg tcattacccc                                                 20 

 
           
             26  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            26 

gtgctggtgg gcgtagtccc                                                 20 

 
           
             27  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            27 

tagttccgga tggcgtggca                                                 20 

 
           
             28  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            28 

ttgcgtagat ttctgccatc                                                 20 

 
           
             29  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            29 

gaccatttgc gtagatttct                                                 20 

 
           
             30  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            30 

cagtctttct gttgccatta                                                 20 

 
           
             31  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            31 

gtagttagcc agtctttctg                                                 20 

 
           
             32  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            32 

ttcggcatag atgcctccgg                                                 20 

 
           
             33  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            33 

gaaacgacat ggtttatata                                                 20 

 
           
             34  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            34 

ccagccacgg aaacgacatg                                                 20 

 
           
             35  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            35 

tccagtactc agtcccatca                                                 20 

 
           
             36  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            36 

tctctcgccc catggttcac                                                 20 

 
           
             37  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            37 

cccttcccat ccttataggt                                                 20 

 
           
             38  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            38 

gtgctcctcg atggcaaggt                                                 20 

 
           
             39  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            39 

tccccaaatg tacagtgctc                                                 20 

 
           
             40  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            40 

acatggcctt aaacgatggg                                                 20 

 
           
             41  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            41 

ttcttaaact gcgcttctag                                                 20 

 
           
             42  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            42 

catgcctttt cttaaactgc                                                 20 

 
           
             43  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            43 

gtcatgggtc accatgcctt                                                 20 

 
           
             44  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            44 

aggatcccct ctggtcatgg                                                 20 

 
           
             45  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            45 

acataaccat aggatcccct                                                 20 

 
           
             46  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            46 

gcctggcaca cataaccata                                                 20 

 
           
             47  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            47 

agccagcctg gcacacataa                                                 20 

 
           
             48  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            48 

gttcctgcca gccagcctgg                                                 20 

 
           
             49  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            49 

ttgatagcca ccccagttcc                                                 20 

 
           
             50  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            50 

atccaatatt gatagccacc                                                 20 

 
           
             51  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            51 

ctcgccatcc aatattgata                                                 20 

 
           
             52  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            52 

gctgtcctcg ccatccaata                                                 20 

 
           
             53  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            53 

ccagtaccac gctgtcctcg                                                 20 

 
           
             54  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            54 

ctcgcagcca gtaccacgct                                                 20 

 
           
             55  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            55 

gaacactcgc agccagtacc                                                 20 

 
           
             56  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            56 

actctcagga acactcgcag                                                 20 

 
           
             57  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            57 

actttcaact ctcaggaaca                                                 20 

 
           
             58  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            58 

atcccacttt caactctcag                                                 20 

 
           
             59  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            59 

ataagtcatc ccactttcaa                                                 20 

 
           
             60  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            60 

gcaagtgtca taagtcatcc                                                 20 

 
           
             61  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            61 

tgctgtgcaa gtgtcataag                                                 20 

 
           
             62  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            62 

atcattgtga ggcagagcat                                                 20 

 
           
             63  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            63 

ggctgactgc atcattgtga                                                 20 

 
           
             64  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            64 

cttcaccagg tggctgactg                                                 20 

 
           
             65  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            65 

cacttcttca ccaggtggct                                                 20 

 
           
             66  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            66 

caggtcactt cttcaccagg                                                 20 

 
           
             67  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            67 

cgtttcctgt gttgcaggtc                                                 20 

 
           
             68  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            68 

ggtcccatcg tttcctgtgt                                                 20 

 
           
             69  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            69 

gactgaggtc ccatcgtttc                                                 20 

 
           
             70  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            70 

gaagaagact gaggtcccat                                                 20 

 
           
             71  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            71 

aagtcctctg ctgaagaaga                                                 20 

 
           
             72  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            72 

aaaatatcaa gtcctctgct                                                 20 

 
           
             73  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            73 

ttattgccca cagttgccaa                                                 20 

 
           
             74  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            74 

tcttaaatgc catattattg                                                 20 

 
           
             75  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            75 

tctttcacct cttaaatgcc                                                 20 

 
           
             76  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            76 

taaagtgaca taagaatggt                                                 20 

 
           
             77  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            77 

ggccacaatc accagaggtg                                                 20 

 
           
             78  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            78 

tccacatcaa agggaccttt                                                 20 

 
           
             79  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            79 

cctccatctc ttgtcacctt                                                 20 

 
           
             80  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            80 

agcagcctac cctggtcctt                                                 20 

 
           
             81  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            81 

ggtttctgat gagctccatc                                                 20 

 
           
             82  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            82 

tattccacag ctgagagcaa                                                 20 

 
           
             83  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            83 

aacatcttac ccatggttca                                                 20 

 
           
             84  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            84 

ctctctcgcc ctgtgagaag                                                 20 

 
           
             85  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            85 

gatttgaaac tgccaactcc                                                 20 

 
           
             86  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            86 

agttgggcga tttgaaactg                                                 20 

 
           
             87  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            87 

ctttatttat cagttgggcg                                                 20 

 
           
             88  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            88 

cagatacttt atttatcagt                                                 20 

 
           
             89  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            89 

gctcaaaacc agggagcccc                                                 20 

 
           
             90  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            90 

cggctgctga gaaggcccag                                                 20 

 
           
             91  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            91 

atcagcgact agcccccaac                                                 20 

 
           
             92  
             20  
             DNA  
             H. sapiens  
             
 
           
            92 

cgggatccag agcgggagcc                                                 20 

 
           
             93  
             20  
             DNA  
             H. sapiens  
             
 
           
            93 

tctgggactc ggagcgggat                                                 20 

 
           
             94  
             20  
             DNA  
             H. sapiens  
             
 
           
            94 

ggatccggag cgggacccag                                                 20 

 
           
             95  
             20  
             DNA  
             H. sapiens  
             
 
           
            95 

gcctctactt ccgccgggga                                                 20 

 
           
             96  
             20  
             DNA  
             H. sapiens  
             
 
           
            96 

gccggggaca gacctgctac                                                 20 

 
           
             97  
             20  
             DNA  
             H. sapiens  
             
 
           
            97 

cagacctgct accggcctct                                                 20 

 
           
             98  
             20  
             DNA  
             H. sapiens  
             
 
           
            98 

caagagctgg gactggcgca                                                 20 

 
           
             99  
             20  
             DNA  
             H. sapiens  
             
 
           
            99 

tggcgcaatg tggatggtgt                                                 20 

 
           
             100  
             20  
             DNA  
             H. sapiens  
             
 
           
            100 

ctatggcgga tcggatcaac                                                 20 

 
           
             101  
             20  
             DNA  
             H. sapiens  
             
 
           
            101 

ggggtaatga cctgtccgtg                                                 20 

 
           
             102  
             20  
             DNA  
             H. sapiens  
             
 
           
            102 

gggactacgc ccaccagcac                                                 20 

 
           
             103  
             20  
             DNA  
             H. sapiens  
             
 
           
            103 

tgccacgcca tccggaacta                                                 20 

 
           
             104  
             20  
             DNA  
             H. sapiens  
             
 
           
            104 

gatggcagaa atctacgcaa                                                 20 

 
           
             105  
             20  
             DNA  
             H. sapiens  
             
 
           
            105 

agaaatctac gcaaatggtc                                                 20 

 
           
             106  
             20  
             DNA  
             H. sapiens  
             
 
           
            106 

taatggcaac agaaagactg                                                 20 

 
           
             107  
             20  
             DNA  
             H. sapiens  
             
 
           
            107 

cagaaagact ggctaactac                                                 20 

 
           
             108  
             20  
             DNA  
             H. sapiens  
             
 
           
            108 

ccggaggcat ctatgccgaa                                                 20 

 
           
             109  
             20  
             DNA  
             H. sapiens  
             
 
           
            109 

tatataaacc atgtcgtttc                                                 20 

 
           
             110  
             20  
             DNA  
             H. sapiens  
             
 
           
            110 

catgtcgttt ccgtggctgg                                                 20 

 
           
             111  
             20  
             DNA  
             H. sapiens  
               
           
            111 

tgatgggact gagtactgga                                                 20 

 
           
             112  
             20  
             DNA  
             H. sapiens  
             
 
           
            112 

accttgccat cgaggagcac                                                 20 

 
           
             113  
             20  
             DNA  
             H. sapiens  
             
 
           
            113 

gagcactgta catttgggga                                                 20 

 
           
             114  
             20  
             DNA  
             H. sapiens  
             
 
           
            114 

cccatcgttt aaggccatgt                                                 20 

 
           
             115  
             20  
             DNA  
             H. sapiens  
             
 
           
            115 

ctagaagcgc agtttaagaa                                                 20 

 
           
             116  
             20  
             DNA  
             H. sapiens  
             
 
           
            116 

gcagtttaag aaaaggcatg                                                 20 

 
           
             117  
             20  
             DNA  
             H. sapiens  
             
 
           
            117 

aaggcatggt gacccatgac                                                 20 

 
           
             118  
             20  
             DNA  
             H. sapiens  
             
 
           
            118 

ccatgaccag aggggatcct                                                 20 

 
           
             119  
             20  
             DNA  
             H. sapiens  
             
 
           
            119 

aggggatcct atggttatgt                                                 20 

 
           
             120  
             20  
             DNA  
             H. sapiens  
             
 
           
            120 

tatggttatg tgtgccaggc                                                 20 

 
           
             121  
             20  
             DNA  
             H. sapiens  
             
 
           
            121 

ttatgtgtgc caggctggct                                                 20 

 
           
             122  
             20  
             DNA  
             H. sapiens  
             
 
           
            122 

ccaggctggc tggcaggaac                                                 20 

 
           
             123  
             20  
             DNA  
             H. sapiens  
             
 
           
            123 

ggaactgggg tggctatcaa                                                 20 

 
           
             124  
             20  
             DNA  
             H. sapiens  
             
 
           
            124 

ggtggctatc aatattggat                                                 20 

 
           
             125  
             20  
             DNA  
             H. sapiens  
             
 
           
            125 

tatcaatatt ggatggcgag                                                 20 

 
           
             126  
             20  
             DNA  
             H. sapiens  
             
 
           
            126 

tattggatgg cgaggacagc                                                 20 

 
           
             127  
             20  
             DNA  
             H. sapiens  
             
 
           
            127 

cgaggacagc gtggtactgg                                                 20 

 
           
             128  
             20  
             DNA  
             H. sapiens  
             
 
           
            128 

agcgtggtac tggctgcgag                                                 20 

 
           
             129  
             20  
             DNA  
             H. sapiens  
             
 
           
            129 

ggtactggct gcgagtgttc                                                 20 

 
           
             130  
             20  
             DNA  
             H. sapiens  
             
 
           
            130 

ctgcgagtgt tcctgagagt                                                 20 

 
           
             131  
             20  
             DNA  
             H. sapiens  
             
 
           
            131 

tgttcctgag agttgaaagt                                                 20 

 
           
             132  
             20  
             DNA  
             H. sapiens  
             
 
           
            132 

ctgagagttg aaagtgggat                                                 20 

 
           
             133  
             20  
             DNA  
             H. sapiens  
             
 
           
            133 

ttgaaagtgg gatgacttat                                                 20 

 
           
             134  
             20  
             DNA  
             H. sapiens  
             
 
           
            134 

ggatgactta tgacacttgc                                                 20 

 
           
             135  
             20  
             DNA  
             H. sapiens  
             
 
           
            135 

cttatgacac ttgcacagca                                                 20 

 
           
             136  
             20  
             DNA  
             H. sapiens  
             
 
           
            136 

atgctctgcc tcacaatgat                                                 20 

 
           
             137  
             20  
             DNA  
             H. sapiens  
             
 
           
            137 

tcacaatgat gcagtcagcc                                                 20 

 
           
             138  
             20  
             DNA  
             H. sapiens  
             
 
           
            138 

cagtcagcca cctggtgaag                                                 20 

 
           
             139  
             20  
             DNA  
             H. sapiens  
             
 
           
            139 

agccacctgg tgaagaagtg                                                 20 

 
           
             140  
             20  
             DNA  
             H. sapiens  
             
 
           
            140 

cctggtgaag aagtgacctg                                                 20 

 
           
             141  
             20  
             DNA  
             H. sapiens  
             
 
           
            141 

gacctgcaac acaggaaacg                                                 20 

 
           
             142  
             20  
             DNA  
             H. sapiens  
             
 
           
            142 

acacaggaaa cgatgggacc                                                 20 

 
           
             143  
             20  
             DNA  
             H. sapiens  
             
 
           
            143 

gaaacgatgg gacctcagtc                                                 20 

 
           
             144  
             20  
             DNA  
             H. sapiens  
             
 
           
            144 

tcttcttcag cagaggactt                                                 20 

 
           
             145  
             20  
             DNA  
             H. sapiens  
             
 
           
            145 

agcagaggac ttgatatttt                                                 20 

 
           
             146  
             20  
             DNA  
             H. sapiens  
             
 
           
            146 

ttggcaactg tgggcaataa                                                 20 

 
           
             147  
             20  
             DNA  
             H. sapiens  
             
 
           
            147 

caataatatg gcatttaaga                                                 20 

 
           
             148  
             20  
             DNA  
             H. sapiens  
             
 
           
            148 

ggcatttaag aggtgaaaga                                                 20 

 
           
             149  
             20  
             DNA  
             H. sapiens  
             
 
           
            149 

accattctta tgtcacttta                                                 20 

 
           
             150  
             20  
             DNA  
             H. sapiens  
             
 
           
            150 

aaaggtccct ttgatgtgga                                                 20 

 
           
             151  
             20  
             DNA  
             H. sapiens  
             
 
           
            151 

aaggtgacaa gagatggagg                                                 20 

 
           
             152  
             20  
             DNA  
             H. sapiens  
             
 
           
            152 

aaggaccagg gtaggctgct                                                 20 

 
           
             153  
             20  
             DNA  
             H. sapiens  
             
 
           
            153 

gatggagctc atcagaaacc                                                 20 

 
           
             154  
             20  
             DNA  
             H. sapiens  
             
 
           
            154 

ttgctctcag ctgtggaata                                                 20 

 
           
             155  
             20  
             DNA  
             H. sapiens  
               
           
            155 

tgaaccatgg gtaagatgtt                                                 20 

 
           
             156  
             20  
             DNA  
             H. sapiens  
             
 
           
            156 

cttctcacag ggcgagagag                                                 20 

 
           
             157  
             20  
             DNA  
             H. sapiens  
             
 
           
            157 

ggagttggca gtttcaaatc                                                 20 

 
           
             158  
             20  
             DNA  
             H. sapiens  
             
 
           
            158 

cagtttcaaa tcgcccaact                                                 20