Patent Publication Number: US-2003224516-A1

Title: Antisense modulation of prox-1 expression

Description:
FIELD OF THE INVENTION  
       [0001] The present invention provides compositions and methods for modulating the expression of prox-1. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding prox-1. Such compounds have been shown to modulate the expression of prox-1.  
       BACKGROUND OF THE INVENTION  
       [0002] Homeoproteins are a large class of transcription factors containing the very common DNA binding domain called the homeodomain. The homeodomain is a 60 amino acid sequence which contains 3 helices, with the C-terminal helix binding to DNA in the major groove. It is well known that proteins containing a homeodomain play an essential role in the determination of cell fate and the establishment of body plan. Even in evolutionarily distant organisms, homologous homeobox genes are often involved in the development of analogous organs (Prochiantz,  Ann. N. Y. Acad. Sci ., 1999, 886, 172-179). Only a few homeobox genes are known to be expressed in the eye and the identification of such genes may help to identify the molecular basis for some human eye pathologies (Zinovieva et al.,  Genomics , 1996, 35, 517-522).  
       [0003] One such gene encoding prox-1 (also called PROX-1, prospero-related homeobox 1, and homeodomain protein) was cloned in 1996 and maps to chromosome 1q32.2-q32.3, which is a region close to the location of Usher syndrome type II, a syndrome associated with hearing loss and retinitis pigmentosa (Zinovieva et al.,  Genomics , 1996, 35, 517-522). The homologous mouse gene maps to position 106.3 cM from the centromere of chromosome 1, which is very close to the retinal degeneration mutation, rd3 (Tomarev et al.,  Biochem. Biophys. Res. Commun ., 1998, 248, 684-689). Thus, prox-1 has been considered as a candidate for these conditions.  
       [0004] Prox-1 is expressed in several human tissues including lens, heart, brain, lung, kidney, and liver, with the highest expression found in lens. In embryonic lens tissue, two cDNAs of different lengths were detected, indicating that the prox-1 gene may be alternatively spliced in the lens (Zinovieva et al.,  Genomics , 1996, 35, 517-522). In human and rat lenses the subcellular distribution of prox-1 changes during development, with prox-1 predominantly in the cytoplasm until differentiation at which point prox-1 protein redistributes to the nucleus (Duncan et al.,  Mech. Dev ., 2002, 112, 195-198).  
       [0005] The biological function of prox-1 has been studied by generating prox-1 null mice. From these studies it was determined that prox-1 is required for hepatocyte migration during liver development, development of the lens and the lymphatic system, but not the vascular system (Sosa-Pineda et al.,  Nat. Genet ., 2000, 25, 254-255; Wigle et al.,  Nat. Genet ., 1999, 21, 318-322.; Wigle and Oliver,  Cell , 1999, 98, 769-778.). Prox-1 function is also required for the expression of the cell-cycle inhibitors Cdkn1b and Cdkn1c (Wigle et al.,  Nat. Genet ., 1999, 21, 318-322.).  
       [0006] Several other functions for prox-1 as a transcription factor have been described. Prox-1 activates the SIX3 promoter, a human transcription factor essential for eye development (Lengler and Graw,  Biochem. Biophys. Res. Commun ., 2001, 287, 372-376). The homeodomain of prox-1 can bind to Pax6, a transcription factor that controls the development of the eyes and central nervous system (Mikkola et al.,  J. Biol. Chem ., 2001, 276, 4109-4118.). Prox-1 regulates differentiation of neurons and glia in neural progenitors (Yamamoto et al.,  J. Neurosci .; 2001, 21, 9814-9823.) and prox-1 also stimulates the Crygf promoter, a gene which has been reported to have mutations that result in a variety of lens opacities (Lengler et al.,  Nucleic Acids Res ., 2001, 29, 515-526).  
       [0007] Currently, there are no known therapeutic agents which effectively inhibit the synthesis of prox-1. Consequently, there remains a long felt need for agents capable of effectively inhibiting prox-1 function.  
       [0008] 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 prox-1 expression.  
       [0009] The present invention provides compositions and methods for modulating prox-1 expression.  
       SUMMARY OF THE INVENTION  
       [0010] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding prox-1, and which modulate the expression of prox-1. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of prox-1 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 prox-1 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  
       [0011] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding prox-1, ultimately modulating the amount of prox-1 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding prox-1. As used herein, the terms “target nucleic acid” and “nucleic acid encoding prox-1” encompass DNA encoding prox-1, 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 prox-1. 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.  
       [0012] 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 prox-1. 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 prox-1, regardless of the sequence(s) of such codons.  
       [0013] 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.  
       [0014] 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.  
       [0015] 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.  
       [0016] 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.  
       [0017] 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.  
       [0018] 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.  
       [0019] 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.  
       [0020] 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.  
       [0021] 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).  
       [0022] 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.  
       [0023] 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.  
       [0024] 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.  
       [0025] 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.  
       [0026] 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.  
       [0027] 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.  
       [0028] 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.  
       [0029] 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).  
       [0030] 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.  
       [0031] 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.  
       [0032] 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.  
       [0033] 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.  
       [0034] 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. 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.  
       [0035] 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.  
       [0036] 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.  
       [0037] 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.  
       [0038] 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.  
       [0039] 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.  
       [0040] 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.  
       [0041] 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.  
       [0042] 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.  
       [0043] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1  to C 10  alkyl or C 2  to C 10  alkenyl and alkynyl. Particularly preferred are O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 ] 2 , where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C 1  to C 10  lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al.,  Helv. Chim. Acta , 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2  group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH 2 —O—CH 2 —N(CH 3 ) 2 , also described in examples hereinbelow.  
       [0044] 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.  
       [0045] 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.  
       [0046] 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 O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (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.  
       [0047] 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.  
       [0048] 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.  
       [0049] 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.  
       [0050] 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.  
       [0051] 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.  
       [0052] 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.  
       [0053] 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.  
       [0054] 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.  
       [0055] 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.  
       [0056] 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.  
       [0057] 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.  
       [0058] 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.  
       [0059] 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 prox-1 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.  
       [0060] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding prox-1, 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 prox-1 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 prox-1 in a sample may also be prepared.  
       [0061] 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.  
       [0062] 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.  
       [0063] 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. applications Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673 (filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624 (filed May 21, 1998) and 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.  
       [0064] 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.  
       [0065] 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.  
       [0066] 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.  
       [0067] 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.  
       [0068] 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.  
       [0069] Emulsions  
       [0070] 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.  
       [0071] 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).  
       [0072] 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).  
       [0073] 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.  
       [0074] 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).  
       [0075] 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.  
       [0076] 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.  
       [0077] 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.  
       [0078] 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).  
       [0079] 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.  
       [0080] 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 (SO750), decaglycerol decaoleate (DAO750), 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.  
       [0081] 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.  
       [0082] 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.  
       [0083] Liposomes  
       [0084] 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.  
       [0085] 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.  
       [0086] 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.  
       [0087] 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.  
       [0088] 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.  
       [0089] 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.  
       [0090] 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.  
       [0091] 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).  
       [0092] 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).  
       [0093] 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.  
       [0094] 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).  
       [0095] 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) .  
       [0096] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G Ml , 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).  
       [0097] 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 Ml , 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 Ml  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.).  
       [0098] 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. No, 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.  
       [0099] 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.  
       [0100] 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.  
       [0101] 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).  
       [0102] 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.  
       [0103] 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.  
       [0104] 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.  
       [0105] 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.  
       [0106] 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).  
       [0107] Penetration Enhancers  
       [0108] 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.  
       [0109] 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.  
       [0110] 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).  
       [0111] 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).  
       [0112] 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).  
       [0113] 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).  
       [0114] 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).  
       [0115] 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.  
       [0116] 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.  
       [0117] Carriers  
       [0118] 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).  
       [0119] Excipients  
       [0120] 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.).  
       [0121] 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.  
       [0122] 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.  
       [0123] 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.  
       [0124] Other Components  
       [0125] 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.  
       [0126] 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.  
       [0127] 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.  
       [0128] 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.  
       [0129] 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.  
       [0130] 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  
     [0131] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy Amidites  
     [0132] 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.  
     [0133] 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).  
     [0134] 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:  
     [0135] Preparation of 5′-O-Dimethoxytrityl-thymidine Intermediate for 5-methyl dC Amidite  
     [0136] 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 (R f  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.  
     [0137] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine Intermediate for 5-methyl-dC Amidite  
     [0138] 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).  
     [0139] TLC indicated a complete reaction (product R f  0.35 in EtOAc-MeOH 4:1). The reaction solution was concentrated on a rotary evaporator to a dense foam. Each foam was slowly redissolved in warm EtOAc (4 L; 50° C.), combined in a 50 L glass reactor vessel, and extracted with water (2×4L) to remove the triazole by-product. The water was back-extracted with EtOAc (2 L). The organic layers were combined and concentrated to about 8 kg total weight, cooled to 0° C. and seeded with crystalline product. After 24 hours, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc (3×3L) until a white powder was left and then washed with ethyl ether (2×3L). The solid was put in pans (1″ deep) and allowed to air dry overnight. The filtrate was concentrated to an oil, then redissolved in EtOAc (2 L), cooled and seeded as before. The second crop was collected and washed as before (with proportional solvents) and the filtrate was first extracted with water (2×1L) and then concentrated to an oil. The residue was dissolved in EtOAc (1 L) and yielded a third crop which was treated as above except that more washing was required to remove a yellow oily layer.  
     [0140] 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.  
     [0141] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine Penultimate Intermediate for 5-methyl dC Amidite  
     [0142] 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.  
     [0143] 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 re-equilibrated 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.  
     [0144] [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N 4 -benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC Amidite)  
     [0145] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N 4 -benzoyl-5-methylcytidine (998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (300 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (15 ml) was added and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2.5 L) and water (600 ml), and extracted with hexane (3×3 L). The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (7.5 L) and hexane (6 L). The two layers were separated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L) and water (3×2 L), and the phases were separated. The organic layer was dried (Na 2 SO 4 ), filtered and rotary evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried to a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 g an off-white foam solid (96%).  
     [0146] 2′-Fluoro Amidites  
     [0147] 2′-Fluorodeoxyadenosine Amidites  
     [0148] 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.  
     [0149] 2′-Fluorodeoxyguanosine  
     [0150] 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.  
     [0151] 2′-Fluorouridine  
     [0152] 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.  
     [0153] 2′-Fluorodeoxycytidine  
     [0154] 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.  
     [0155] 2′-O-(2-Methoxyethyl) modified amidites  
     [0156] 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).  
     [0157] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate  
     [0158] 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.  
     [0159] 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.).  
     [0160] 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.  
     [0161] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine Penultimate Intermediate  
     [0162] 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.  
     [0163] 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.  
     [0164] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T Amidite)  
     [0165] 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%).  
     [0166] Preparation of 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate  
     [0167] 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  
     [0168] 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.  
     [0169] Preparation of 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidine Penultimate Intermediate:  
     [0170] 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%.  
     [0171] 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)  
     [0172] 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%).  
     [0173] 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)  
     [0174] 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%).  
     [0175] 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)  
     [0176] 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%).  
     [0177] 2′-O-(Aminooxyethyl) Nucleoside Amidites and 2′-O-(dimethylaminooxyethyl) Nucleoside Amidites  
     [0178] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites  
     [0179] 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.  
     [0180] 5′-O-tert-Butyldiphenylsilyl-O 2 -2′-anhydro-5-methyluridine  
     [0181] 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 (R f  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 (3×2 00 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.  
     [0182] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine  
     [0183] 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, R f  0.67 for desired product and R f  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.  
     [0184] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine  
     [0185] 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.  
     [0186] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine  
     [0187] 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.  
     [0188] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine  
     [0189] 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.  
     [0190] 2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0191] 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.  
     [0192] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0193] 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.  
     [0194] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0195] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried over P 2 O 5  under high vacuum overnight at 40° C., This was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N 1 ,N 1 ′-tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 h under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, then the residue was dissolved in EtOAc (70 mL) and washed with 5% aqueous NaHCO 3  (40 mL). The EtOAc layer was dried over anhydrous Na 2 SO 4 , filtered, and concentrated. The residue obtained was purified by column chromatography (EtOAc as eluent) to afford 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%) upon rotary evaporation.  
     [0196] 2′-(Aminooxyethoxy) Nucleoside Amidites  
     [0197] 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.  
     [0198] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0199] 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 a minor 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].  
     [0200] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites  
     [0201] 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.  
     [0202] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine  
     [0203] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) was slowly added to a solution of borane in tetrahydrofuran (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.  
     [0204] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyl uridine  
     [0205] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. The reaction mixture was poured into water (200 mL) and extracted with CH 2 Cl 2  (2×200 mL). The combined CH 2 Cl 2  layers were washed with saturated NaHCO 3  solution, followed by saturated NaCl solution, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography (eluted with 5:100:1 MeOH/CH 2 Cl 2 /TEA) to afford the product.  
     [0206] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite  
     [0207] 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  
     [0208] Oligonucleotide Synthesis  
     [0209] 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.  
     [0210] 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.  
     [0211] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.  
     [0212] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporated by reference.  
     [0213] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. Nos., 5,256,775 or 5,366,878, herein incorporated by reference.  
     [0214] 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.  
     [0215] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.  
     [0216] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.  
     [0217] 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  
     [0218] Oligonucleoside Synthesis  
     [0219] 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.  
     [0220] 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.  
     [0221] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.  
     Example 4  
     [0222] PNA Synthesis  
     [0223] 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,  Dioorganic  &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  
     [0224] Synthesis of Chimeric Oligonucleotides  
     [0225] 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”.  
     [0226] [2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides  
     [0227] 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-phosphor-amidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH 4 OH) for 12-16 hr at 55° C., The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.  
     [0228] [2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides  
     [0229] [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.  
     [0230] [2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxy Phosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides  
     [0231] [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.  
     [0232] 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  
     [0233] Oligonucleotide Isolation  
     [0234] 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  
     [0235] Oligonucleotide Synthesis—96 Well Plate Format  
     [0236] 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.  
     [0237] 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  
     [0238] Oligonucleotide Analysis—96-Well Plate Format  
     [0239] 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  
     [0240] Cell culture and Oligonucleotide Treatment  
     [0241] 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.  
     [0242] T-24 Cells:  
     [0243] 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.  
     [0244] 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.  
     [0245] A549 Cells:  
     [0246] 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.  
     [0247] NHDF Cells:  
     [0248] 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.  
     [0249] HEK Cells:  
     [0250] 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.  
     [0251] HepG2 Cells:  
     [0252] The human hepatoblastoma cell line HepG2 was obtained from the American Type Culture Collection (Manassas, Va.). HepG2 cells were routinely cultured in Eagle&#39;s MEM supplemented with 10% fetal calf serum, non-essential amino acids, and 1 mM sodium pyruvate (Gibco/Life Technologies, Gaithersburg, Md.). 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.  
     [0253] For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.  
     [0254] Treatment with Antisense Compounds:  
     [0255] 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.  
     [0256] 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  
     [0257] Analysis of Oligonucleotide Inhibition of Prox-1 Expression  
     [0258] Antisense modulation of prox-1 expression can be assayed in a variety of ways known in the art. For example, prox-1 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.  
     [0259] Protein levels of prox-1 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 prox-1 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).  
     [0260] 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  
     [0261] Poly(A)+ mRNA Isolation  
     [0262] 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.  
     [0263] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.  
     Example 12  
     [0264] Total RNA Isolation  
     [0265] 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.  
     [0266] 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  
     [0267] Real-Time Quantitative PCR Analysis of Prox-1 mRNA Levels  
     [0268] Quantitation of prox-1 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 PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.  
     [0269] 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.  
     [0270] 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).  
     [0271] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPbH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).  
     [0272] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.  
     [0273] Probes and primers to human prox-1 were designed to hybridize to a human prox-1 sequence, using published sequence information (GenBank accession number NM — 002763.1, incorporated herein as SEQ ID NO:4). For human prox-1 the PCR primers were: 
     [0274] forward primer: TGCCATGATGCCTTTTCCA (SEQ ID NO: 5)  
     [0275] reverse primer: TGCCACCATTTTTGTTCATGTT (SEQ ID NO: 6) and the  
     [0276] PCR probe was: FAM-CAACCATAATTTCCCAGCTGTTGAAA-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were:  
     [0277] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8)  
     [0278] 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  
     [0279] Northern Blot Analysis of Prox-1 mRNA Levels  
     [0280] 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.  
     [0281] To detect human pro-1, a human prox-1 specific probe was prepared by PCR using the forward primer TGCCATGATGCCTTTTCCA (SEQ ID NO: 5) and the reverse primer TGCCACCATTTTTGTTCATGTT (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.).  
     [0282] 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  
     [0283] Antisense Inhibition of Human Prox-1 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap  
     [0284] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human prox-1 RNA, using published sequences (GenBank accession number NM — 002763.1, incorporated herein as SEQ ID NO: 4, and the complement of residues 1195126-1243521 of GenBank accession number NT — 004612.7, incorporated herein as SEQ ID NO: 11). 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 prox-1 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which HepG2 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 prox-1 mRNA levels by chimeric           phosphorothioate oligonucleotides having 2′-MOE wings and a       deoxy gap                                                         TARGET                                       SEQ ID   TARGET       %   SEQ ID   CONTROL       ISIS #   REGION   NO   SITE   SEQUENCE   INHIB   NO   SEQ ID NO                                                         232058   5′UTR   4   362   ggacggtgagtctgtgcgac   48   12   1                   232059   5′UTR   4   540   agctcaagaatcccgggacc   86   13   1               232060   5′UTR   4   550   agctgggcacagctcaagaa   69   14   1               232061   5′UTR   4   560   aaagctcgtcagctgggcac   61   15   1               232062   5′UTR   4   570   gccatcttcaaaagctcgtc   67   16   1               232063   Start   4   599   atggtcaggcatcactggac   62   17   1           Codon               232064   Start   4   604   ctgtcatggtcaggcatcac   71   18   1           Codon               232065   Start   4   609   ctgtgctgtcatggtcaggc   71   19   1           Codon               232066   Coding   4   614   gagggctgtgctgtcatggt   43   20   1               232067   Coding   4   619   cttaagagggctgtgctgtc   75   21   1               232068   Coding   4   624   gccggcttaagagggctgtg   59   22   1               232069   Coding   4   629   ggtttgccggcttaagaggg   39   23   1               232070   Coding   4   634   ctcttggtttgccggcttaa   66   24   1               232071   Coding   4   661   cttttcactccaatgtcaac   53   25   1               232072   Coding   4   666   ccgtccttttcactccaatg   74   26   1               232073   Coding   4   672   tccctaccgtccttttcact   49   27   1               232074   Coding   4   677   tgctgtccctaccgtccttt   61   28   1               232075   Coding   4   682   gcagatgctgtccctaccgt   63   29   1               232076   Coding   4   687   aaaatgcagatgctgtccct   53   30   1               232077   Coding   4   817   gccctcttcagcagcttgcg   75   31   1               232078   Coding   4   822   agttcgccctcttcagcagc   65   32   1               232079   Coding   4   827   atacgagttcgccctcttca   78   33   1               232080   Coding   4   832   tcttcatacgagttcgccct   50   34   1               232081   Coding   4   837   tggcatcttcatacgagttc   64   35   1               232082   Coding   4   842   catcatggcatcttcatacg   37   36   1               232083   Coding   4   847   aaaggcatcatggcatcttc   80   37   1               232084   Coding   4   852   ctggaaaaggcatcatggca   92   38   1               232085   Coding   4   857   tgctcctggaaaaggcatca   94   39   1               232086   Coding   4   880   ttcaacagctgggaaattat   75   40   1               232087   Coding   4   885   tatttttcaacagctgggaa   87   41   1               232088   Coding   4   928   ctggcttggaaactgggctc   72   42   1               232089   Coding   4   961   tgatgtacttcggagcctgt 74   43   1               232090   Coding   4   971   tatatcctcctgatgtactt   54   44   1               232091   Coding   4   994   ctgtctcttgaagagttgct   42   45   1               232092   Coding   4   1032   tagtaggcctgccaaaaggg   71   46   1               232093   Coding   4   1042   aactggctcatagtaggcct   57   47   1               232094   Coding   4   1067   ctcatcacataagcgatcca   84   48   1               232095   Coding   4   1077   ctctcaggtgctcatcacat   55   49   1               232096   Coding   4   1082   ctttgctctcaggtgctcat   46   50   1               232097   Coding   4   1093   acccgggcccgctttgctct   75   51   1               232098   Coding   4   1123   gaatggctcataccccgaat   48   52   1               232099   Coding   4   1144   ccccttaatgccacactggg   61   53   1               232100   Coding   4   1154   attttcattgccccttaatg   27   54   1               232101   Coding   4   1170   gggccatctctctttcattt   80   55   1               232102   Coding   4   1205   ttctctgtaactttctcggg   63   56   1               232103   Coding   4   1247   actctgttgctgctgctggg   78   57   1               232104   Coding   4   1252   tggaaactctgttgctgctg   88   58   1               232105   Coding   4   1262   aaccagctgctggaaactct   68   59   1               232106   Coding   4   1272   ttcgggctgaaaccagctgc   87   60   1               232107   Coding   4   1311   gctgtttcagctgtcggcgc   81   61   1               232108   Coding   4   1424   catgctgtcttcagacaggt   75   62   1               232109   Coding   4   1434   tctccgagcgcatgctgtct   65   63   1               232110   Coding   4   1444   gcatccaggatctccgagcg   41   64   1               232111   Coding   4   1735   aagacctgaggaacctggcg   70   65   1               232112   Coding   4   1740   gtgggaagacctgaggaacc   47   66   1               232113   Coding   4   1795   tggaaattgtggttttcccc   68   67   1               232114   Coding   4   2093   ggagccggagggagcaccta   61   68   1               232115   Coding   4   2170   gacatcttggtcctcagact   41   69   1               232116   Coding   4   2314   tgcattgcacttcccgaata   78   70   1               232117   Coding   4   2344   tttttcaagtgattgggtga   47   71   1               232118   Coding   4   2407   gagaagtaggtcttcagcat   22   72   1               232119   Coding   4   2427   atctgttgaactttacgtcg   58   73   1               232120   Coding   4   2628   ggaatctctctggaacctca   51   74   1               232121   Coding   4   2668   gcattgaaaaactcccgtaa   51   75   1               232122   Coding   4   2698   gaaggatcaacatctttgcc   42   76   1               232123   Coding   4   2703   tccaggaaggatcaacatct   41   77   1               232124   Coding   4   2734   agcttgcagatgaccttgta   47   78   1               232125   Coding   4   2744   ttcactatccagcttgcaga   53   79   1               232126   Stop   4   2806   tgaaatttctactcatgaag   54   80   1           Codon               232127   3′UTR   4   2858   acttggacatccaaagagga   26   81   1               232128   exon:   11   732   agatacttacgcggtgcagg   30   82   1           intron           junction               232129   exon:   11   10504   gatattgcacttcccgaata   56   83   1           intron           junction               232130   intron:   11   17408   tgcatgtaggatgcaattgg   30   84   1           exon           junction               232131   exon:   11   23964   tagttcctacctcaaagtca   26   85   1           intron           junction               232132   intron   11   29523   ttatgaagcaggaggagaaa   41   86   1               232133   intron   11   36453   caagacaggtaaatagattg   0   87   1               232134   intron   11   39446   agcaaacccagggaaaggct   33   88   1               232135   intron   11   43588   tgttttgataaaaaggcatc   37   89   1                  
 
     [0285] As shown in Table 1, SEQ ID NOs 13, 14, 15, 16, 17, 18, 22, 24, 26, 28, 29, 31, 32, 33, 35, 37, 38, 39, 40, 41, 42, 43, 46, 47, 48, 49, 51, 53, 55, 56, 57, 58, 59, 60, 61, 62, 63, 65, 67, 68, 70, 73 and 83 demonstrated at least 55% inhibition of human prox-1 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 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 correponding 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 prox-1.                                                 TARGET                               SITE   SEQ ID   TARGET       REV COMP       SEQ ID       ID   NO   SITE   SEQUENCE   OF SEQ ID   ACTIVE IN   NO                                                     148614   4   540   ggtcccgggattcttgagct   13     H. sapiens     90                   148615   4   550   ttcttgagctgtgcccagct   14     H. sapiens     91               148616   4   560   gtgcccagctgacgagcttt   15     H. sapiens     92               148617   4   570   gacgagcttttgaagatggc   16     H. sapiens     93               148618   4   599   gtccagtgatgcctgaccat   17     H. sapiens     94               148619   4   604   gtgatgcctgaccatgacag   18     H. sapiens     95               148620   4   609   gcctgaccatgacagcacag   19     H. sapiens     96               148622   4   619   gacagcacagccctcttaag   21     H. sapiens     97               148623   4   624   cacagccctcttaagccggc   22     H. sapiens     98               148625   4   634   ttaagccggcaaaccaagag   24     H. sapiens     99               148627   4   666   cattggagtgaaaaggacgg   26     H. sapiens     100               148629   4   677   aaaggacggtagggacagca   28     H. sapiens     101               148630   4   682   acggtagggacagcatctgc   29     H. sapiens     102               148632   4   817   cgcaagctgctgaagagggc   31     H. sapiens     103               148633   4   822   gctgctgaagagggcgaact   32     H. sapiens     104               148634   4   827   tgaagagggcgaactcgtat   33     H. sapiens     105               148636   4   837   gaactcgtatgaagatgcca   35     H. sapiens     106               148638   4   847   gaagatgccatgatgccttt   37     H. sapiens     107               148639   4   852   tgccatgatgccttttccag   38     H. sapiens     108               148640   4   857   tgatgccttttccaggagca   39     H. sapiens     109               148641   4   880   ataatttcccagctgttgaa   40     H. sapiens     110               148642   4   885   ttcccagctgttgaaaaata   41     H. sapiens     111               148643   4   928   gagcccagtttccaagccag   42     H. sapiens     112               148644   4   961   acaggctccgaagtacatca   43     H. sapiens     113               148647   4   1032   cccttttggcaggcctacta   46     H. sapiens     114               148648   4   1042   aggcctactatgagccagtt   47     H. sapiens     115               148649   4   1067   tggatcgcttatgtgatgag   48     H. sapiens     116               148650   4   1077   atgtgatgagcacctgagag   49     H. sapiens     117               148652   4   1093   agagcaaagcgggcccgggt   51     H. sapiens     118               148654   4   1144   cccagtgtggcattaagggg   53     H. sapiens     119               148656   4   1170   aaatgaaagagagatggccc   55     H. sapiens     120               148657   4   1205   cccgagaaagttacagagaa   56     H. sapiens     121               148658   4   1247   cccagcagcagcaacagagt   57     H. sapiens     122               148659   4   1252   cagcagcaacagagtttcca   58     H. sapiens     123               148660   4   1262   agagtttccagcagctggtt   59     H. sapiens     124               148661   4   1272   gcagctggtttcagcccgaa   60     H. sapiens     125               148662   4   1311   gcgccgacagctgaaacagc   61     H. sapiens     126               148663   4   1424   acctgtctgaagacagcatg   62     H. sapiens     127               148664   4   1434   agacagcatgcgctcggaga   63     H. sapiens     128               148666   4   1735   cgccaggttcctcaggtctt   65     H. sapiens     129               148668   4   1795   ggggaaaaccacaatttcca   67     H. sapiens     130               148669   4   2093   taggtgctccctccggctcc   68     H. sapiens     131               148671   4   2314   tattcgggaagtgcaatgca   70     H. sapiens     132               148674   4   2427   cgacgtaaagttcaacagat   73     H. sapiens     133               148684   11   10504   tattcgggaagtgcaatatc   83     H. sapiens     134                  
 
     [0286] 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 prox-1.  
     Example 16  
     [0287] Western Blot Analysis of Prox-1 Protein Levels  
     [0288] 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 prox-1 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 
         
           
             134  
           
           
             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  
             2924  
             DNA  
             H. sapiens  
             
 
             
               CDS  
               (607)...(2817)  
             
           
            4 

aagtaaatct tgttgtggag cggagccctc agctgagggt gcgctctgaa ataatacacc     60 

attgcagccg gggaaagcag agcgcgcaaa agagctctcg ccgggtccgc ctgctccctc    120 

tccgcttcgc tcctcttctc ttctttaccc ttctcctctc tcctcctctg ctgctctctc    180 

ctctcctccg ctcttctctc tcctcctctc ctgctctctc ctcttccctt agctcctctt    240 

cttttcttct cctcttcttc cctctcctcg cctctcccct gctcctcttc tctcgtctcc    300 

cctcccctcc cgcctctctc tcccctctcc ctctcccact cgccccgctc gctcgctcgt    360 

cgtcgcacag actcaccgtc ccttgtccaa ttatcatatt catcacccgc aagatatcac    420 

cgtgtgtgca ctcgcgtgtt ttcctctctc tgccggggga aaaaaaagag agagagaggg    480 

atagagagag agagagagag agagagagag aggctcggtc ccactgctcc ctgcaccgcg    540 

gtcccgggat tcttgagctg tgcccagctg acgagctttt gaagatggca caataaccgt    600 

ccagtg atg cct gac cat gac agc aca gcc ctc tta agc cgg caa acc       648 
       Met Pro Asp His Asp Ser Thr Ala Leu Leu Ser Arg Gln Thr 
         1               5                  10 

aag agg aga aga gtt gac att gga gtg aaa agg acg gta ggg aca gca      696 
Lys Arg Arg Arg Val Asp Ile Gly Val Lys Arg Thr Val Gly Thr Ala 
 15                  20                  25                  30 

tct gca ttt ttt gct aag gca aga gca acg ttt ttt agt gcc atg aat      744 
Ser Ala Phe Phe Ala Lys Ala Arg Ala Thr Phe Phe Ser Ala Met Asn 
                 35                  40                  45 

ccc caa ggt tct gag cag gat gtt gag tat tca gtg gtg cag cat gca      792 
Pro Gln Gly Ser Glu Gln Asp Val Glu Tyr Ser Val Val Gln His Ala 
             50                  55                  60 

gat ggg gaa aag tca aat gta cta cgc aag ctg ctg aag agg gcg aac      840 
Asp Gly Glu Lys Ser Asn Val Leu Arg Lys Leu Leu Lys Arg Ala Asn 
         65                  70                  75 

tcg tat gaa gat gcc atg atg cct ttt cca gga gca acc ata att tcc      888 
Ser Tyr Glu Asp Ala Met Met Pro Phe Pro Gly Ala Thr Ile Ile Ser 
     80                  85                  90 

cag ctg ttg aaa aat aac atg aac aaa aat ggt ggc acg gag ccc agt      936 
Gln Leu Leu Lys Asn Asn Met Asn Lys Asn Gly Gly Thr Glu Pro Ser 
 95                 100                 105                 110 

ttc caa gcc agc ggt ctc tct agt aca ggc tcc gaa gta cat cag gag      984 
Phe Gln Ala Ser Gly Leu Ser Ser Thr Gly Ser Glu Val His Gln Glu 
                115                 120                 125 

gat ata tgc agc aac tct tca aga gac agc ccc cca gag tgt ctt tcc     1032 
Asp Ile Cys Ser Asn Ser Ser Arg Asp Ser Pro Pro Glu Cys Leu Ser 
            130                 135                 140 

cct ttt ggc agg cct act atg agc cag ttt gat atg gat cgc tta tgt     1080 
Pro Phe Gly Arg Pro Thr Met Ser Gln Phe Asp Met Asp Arg Leu Cys 
        145                 150                 155 

gat gag cac ctg aga gca aag cgg gcc cgg gtt gag aat ata att cgg     1128 
Asp Glu His Leu Arg Ala Lys Arg Ala Arg Val Glu Asn Ile Ile Arg 
    160                 165                 170 

ggt atg agc cat tcc ccc agt gtg gca tta agg ggc aat gaa aat gaa     1176 
Gly Met Ser His Ser Pro Ser Val Ala Leu Arg Gly Asn Glu Asn Glu 
175                 180                 185                 190 

aga gag atg gcc ccg cag tct gtg agt ccc cga gaa agt tac aga gaa     1224 
Arg Glu Met Ala Pro Gln Ser Val Ser Pro Arg Glu Ser Tyr Arg Glu 
                195                 200                 205 

aac aaa cgc aag caa aag ctt ccc cag cag cag caa cag agt ttc cag     1272 
Asn Lys Arg Lys Gln Lys Leu Pro Gln Gln Gln Gln Gln Ser Phe Gln 
            210                 215                 220 

cag ctg gtt tca gcc cga aaa gaa cag aag cga gag gag cgc cga cag     1320 
Gln Leu Val Ser Ala Arg Lys Glu Gln Lys Arg Glu Glu Arg Arg Gln 
        225                 230                 235 

ctg aaa cag cag ctg gag gac atg cag aaa cag ctg ctc cac gtg cag     1368 
Leu Lys Gln Gln Leu Glu Asp Met Gln Lys Gln Leu Leu His Val Gln 
    240                 245                 250 

gaa aag ttc tac caa atc tat gac agc act gat tcg gaa aat gat gaa     1416 
Glu Lys Phe Tyr Gln Ile Tyr Asp Ser Thr Asp Ser Glu Asn Asp Glu 
255                 260                 265                 270 

gat ggt aac ctg tct gaa gac agc atg cgc tcg gag atc ctg gat gcc     1464 
Asp Gly Asn Leu Ser Glu Asp Ser Met Arg Ser Glu Ile Leu Asp Ala 
                275                 280                 285 

agg gcc cag gac tct gtc gga agg tca gat aat gag atg tgc gag cta     1512 
Arg Ala Gln Asp Ser Val Gly Arg Ser Asp Asn Glu Met Cys Glu Leu 
            290                 295                 300 

gac cca gga cag ttt att gac cga gct cga gcc ctg atc aga gag cag     1560 
Asp Pro Gly Gln Phe Ile Asp Arg Ala Arg Ala Leu Ile Arg Glu Gln 
        305                 310                 315 

gaa atg gct gaa aac aag ccg aag cga gaa ggc aac aac aaa gaa aga     1608 
Glu Met Ala Glu Asn Lys Pro Lys Arg Glu Gly Asn Asn Lys Glu Arg 
    320                 325                 330 

gac cat ggg cca aac tcc tta caa ccg gaa ggc aaa cat ttg gct gag     1656 
Asp His Gly Pro Asn Ser Leu Gln Pro Glu Gly Lys His Leu Ala Glu 
335                 340                 345                 350 

acc ttg aaa cag gaa ctg aac act gcc atg tcg caa gtt gtg gac act     1704 
Thr Leu Lys Gln Glu Leu Asn Thr Ala Met Ser Gln Val Val Asp Thr 
                355                 360                 365 

gtg gtc aaa gtc ttt tcg gcc aag ccc tcc cgc cag gtt cct cag gtc     1752 
Val Val Lys Val Phe Ser Ala Lys Pro Ser Arg Gln Val Pro Gln Val 
            370                 375                 380 

ttc cca cct ctc cag atc ccc cag gcc aga ttt gca gtc aat ggg gaa     1800 
Phe Pro Pro Leu Gln Ile Pro Gln Ala Arg Phe Ala Val Asn Gly Glu 
        385                 390                 395 

aac cac aat ttc cac acc gcc aac cag cgc ctg cag tgc ttt ggc gac     1848 
Asn His Asn Phe His Thr Ala Asn Gln Arg Leu Gln Cys Phe Gly Asp 
    400                 405                 410 

gtc atc att ccg aac ccc ctg gac acc ttt ggc aat gtg cag atg gcc     1896 
Val Ile Ile Pro Asn Pro Leu Asp Thr Phe Gly Asn Val Gln Met Ala 
415                 420                 425                 430 

agt tcc act gac cag aca gaa gca ctg ccc ctg gtt gtc cgc aaa aac     1944 
Ser Ser Thr Asp Gln Thr Glu Ala Leu Pro Leu Val Val Arg Lys Asn 
                435                 440                 445 

tcc tct gac cag tct gcc tcc ggc ctg gtg ggc ggc cac cac cag ccc     1992 
Ser Ser Asp Gln Ser Ala Ser Gly Leu Val Gly Gly His His Gln Pro 
            450                 455                 460 

ctg cac cag tcg cct ctc tct gcc acc acg ggc ttc acc acg tcc acc     2040 
Leu His Gln Ser Pro Leu Ser Ala Thr Thr Gly Phe Thr Thr Ser Thr 
        465                 470                 475 

ttc cgc cac ccc ttc ccc ctt ccc ttg atg gcc tat cca ttt cag agc     2088 
Phe Arg His Pro Phe Pro Leu Pro Leu Met Ala Tyr Pro Phe Gln Ser 
    480                 485                 490 

cca tta ggt gct ccc tcc ggc tcc ttc tct gga aaa gac aga gcc tct     2136 
Pro Leu Gly Ala Pro Ser Gly Ser Phe Ser Gly Lys Asp Arg Ala Ser 
495                 500                 505                 510 

cct gaa tcc tta gac tta act agg gat acc acg agt ctg agg acc aag     2184 
Pro Glu Ser Leu Asp Leu Thr Arg Asp Thr Thr Ser Leu Arg Thr Lys 
                515                 520                 525 

atg tca tct cac cac ctg agc cac cac cct tgt tca cca gca cac ccg     2232 
Met Ser Ser His His Leu Ser His His Pro Cys Ser Pro Ala His Pro 
            530                 535                 540 

ccc agc acc gcc gaa ggg ctc tcc ttg tcg ctc ata aag tcc gag tgc     2280 
Pro Ser Thr Ala Glu Gly Leu Ser Leu Ser Leu Ile Lys Ser Glu Cys 
        545                 550                 555 

ggc gat ctt caa gat atg tct gaa ata tca cct tat tcg gga agt gca     2328 
Gly Asp Leu Gln Asp Met Ser Glu Ile Ser Pro Tyr Ser Gly Ser Ala 
    560                 565                 570 

atg cag gaa gga ttg tca ccc aat cac ttg aaa aaa gca aag ctc atg     2376 
Met Gln Glu Gly Leu Ser Pro Asn His Leu Lys Lys Ala Lys Leu Met 
575                 580                 585                 590 

ttt ttt tat acc cgt tat ccc agc tcc aat atg ctg aag acc tac ttc     2424 
Phe Phe Tyr Thr Arg Tyr Pro Ser Ser Asn Met Leu Lys Thr Tyr Phe 
                595                 600                 605 

tcc gac gta aag ttc aac aga tgc att acc tct cag ctc atc aag tgg     2472 
Ser Asp Val Lys Phe Asn Arg Cys Ile Thr Ser Gln Leu Ile Lys Trp 
            610                 615                 620 

ttt agc aat ttc cgt gag ttt tac tac att cag atg gag aag tac gca     2520 
Phe Ser Asn Phe Arg Glu Phe Tyr Tyr Ile Gln Met Glu Lys Tyr Ala 
        625                 630                 635 

cgt caa gcc atc aac gat ggg gtc acc agt act gaa gag ctg tct ata     2568 
Arg Gln Ala Ile Asn Asp Gly Val Thr Ser Thr Glu Glu Leu Ser Ile 
    640                 645                 650 

acc aga gac tgt gag ctg tac agg gct ctg aac atg cac tac aat aaa     2616 
Thr Arg Asp Cys Glu Leu Tyr Arg Ala Leu Asn Met His Tyr Asn Lys 
655                 660                 665                 670 

gca aat gac ttt gag gtt cca gag aga ttc ctg gaa gtg gct cag atc     2664 
Ala Asn Asp Phe Glu Val Pro Glu Arg Phe Leu Glu Val Ala Gln Ile 
                675                 680                 685 

aca tta cgg gag ttt ttc aat gcc att atc gca ggc aaa gat gtt gat     2712 
Thr Leu Arg Glu Phe Phe Asn Ala Ile Ile Ala Gly Lys Asp Val Asp 
            690                 695                 700 

cct tcc tgg aag aag gcc ata tac aag gtc atc tgc aag ctg gat agt     2760 
Pro Ser Trp Lys Lys Ala Ile Tyr Lys Val Ile Cys Lys Leu Asp Ser 
        705                 710                 715 

gaa gtc cct gag ttt ttc aaa tcc ccg aac tgc cta caa gag ctg ctt     2808 
Glu Val Pro Glu Phe Phe Lys Ser Pro Asn Cys Leu Gln Glu Leu Leu 
    720                 725                 730 

cat gag tag aaatttcaac aactcttttt gaatgtatga agagtagcag tcctctttgg  2867 
His Glu 
735 

atgtccaagt tatatgtgtc tagattttga tttcatatat atgtgtatgg gaggcgg      2924 

 
           
             5  
             19  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            5 

tgccatgatg ccttttcca                                                  19 

 
           
             6  
             22  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            6 

tgccaccatt tttgttcatg tt                                              22 

 
           
             7  
             26  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            7 

caaccataat ttcccagctg ttgaaa                                          26 

 
           
             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  
             48396  
             DNA  
             Homo sapiens  
             
 
           
            11 

agggggagcg cagggcccct ctcccctcct ctcttcccag cccctcaccc ccaccccttt     60 

tatatttttt tttcctccca agttctcttg ccttgctatc cccccttgaa tccgaaggcg    120 

cctcgcgatt gggtgctggg gccgggtacg tcagatagac tgtgacgtgc agtcttcctg    180 

tttccttcag ctgtgtctta aagtaaatct tgttgtggag cggagccctc agctgaggga    240 

gcgctctgaa ataatacacc attgcagccg gggaaagcag agcggcgcaa aagagctctc    300 

gccgggtccg cctgctccct ctccgcttcg ctcctcttct cttctttacc cttctcctct    360 

ctcctcctct gctgctctct cctctcctcc cgctcttctc tctcctcctc tcctgctctc    420 

tcctcttccc ttagctcctc ttcttttctt ctcctcttct tccctctcct cgcctctccc    480 

ctgctcctct tctctcgtct cccctcccct cccgcctctc tctcccctct ccctctccca    540 

ctcgccccgc tcgctcgctc gctgtcgcac agactcaccg tcccttgtcc aattatcata    600 

ttcatcaccc gcaagatatc accgtgtgtg cactcgcgtg ttttcctctc tctgccgggg    660 

gaaaaaaaag agagagagag agatagagag agagagagag agagagagag agaggctcgg    720 

tcccactgct ccctgcaccg cgtaagtatc ttcttcttcc cctcgtgagt ccctcccctt    780 

ttccagaatc acttgcactg tcttgttctt gaatgagaaa ggaagaaaag agcctcccat    840 

tactcagacc cgtgtaaaca ttattccccc caggagaaaa tggtgttatt caaatgaatc    900 

ataataaaat agcctctaaa cagtttctaa gcgggagcct ccgtggaact cagcgctccg    960 

ctcctcccag ttcctaagag taagtgatcc tcttggcttt tatttctttc tctttcctgc   1020 

tggtggctgg gggtggcggt ggcgatgggg gggaggctga tgttgctgga cttgtcgctg   1080 

atcttgtcac cttttgtgta ctgtttctgg ggtgtgagga ggcgtttgct ccctttcctt   1140 

ctttctcctg ctctctcttc tcaggagaga ggaccgcgag agggaccggg tcgctttttt   1200 

gttcgtggag atccccgctt tccgccaaac cccatccttc cgatctcccc aggctaaaac   1260 

tccggggccg gtccccttgt cctttctctt tgtcttgttt attatagctg cctttcttcc   1320 

cggctcttcc aatttgcttg tcatttgcat acctttcact tctccttttt taaccccagc   1380 

agaggaccgg gaactgggag gaggagagag ggaggtgggg gggcgctctg ttactttcgt   1440 

ctcaaaacgc tgtcgaagcc gaattgtgga aatccggctt ggaggggagc ggtgatgggt   1500 

cccgggaaac gcgcgcggcg cccctcttcc gagctcctgg acccagggct gggtcaagtt   1560 

gagtagggta aggcggcacc gggaggctcg gggggtcgcg tggcggtggg attgggacac   1620 

cagcacgagg aggaccggag gatcgcgggc cgggtaagag tagggggttc ttgggcagca   1680 

gaaatgggag gcgatgaatc tcccagccat cgctggcaga ctatggtgtt gggcagcttc   1740 

ggtctggtct cgtctgggtg gtacctaccg ttttgcccca gttaggagga ctggggaggg   1800 

aggacaggag aggtgagagt aattgttact gggaagacta gtgaggaggg cgggaagagg   1860 

gagggaagag ctgctatctt gcctgagcag atcaggaggg ggacgcagtg ggcgggggga   1920 

gacatcaccc aaagtccagt ttagcaagtt gttgattctt ctggtgtgcc agcccgttac   1980 

tccccctgct gaagctgaag gttggtggag tgatggagcg tggggatggt aaaggaggag   2040 

taagtagctt tccacagact cccaggtctc tggccccttc ccagcttctt gggaaattga   2100 

gagccctcca ggcagacaga gaacagaact agaaggaggg gtggtgctta gtcttaaata   2160 

gctcaaggag gcaggttgga gtgtgaaact gctgttcttg gcaacccaga aggctactct   2220 

gcctggggga aggctggaaa ctcacctgct tgtttttatt tttccgagaa gatctgtgct   2280 

gtctccttga gcttataaaa acagaggaag cacagggtgg cctcctcgca aagtcaaggc   2340 

tagaagactc ccttctcctg ttctcttttc cactcatgcc ctcccttatt taaaaaaaaa   2400 

aaaaaaaaga aagaaaagaa aaaaaaaaga actcatttcc tttcctaacc taggtaggca   2460 

gaaatctatt agcagagtgc gcatgggcag ggcctgacag gtgtgttgtg tcaagaaaga   2520 

caggtgcaaa tttcctctgt gtctgtgtgt gtctgtacag ctctagacca caatgcttgc   2580 

tcgagggttg gagaggttta tgaatttatg gttgtcctgg ttaataggat tgtctgggct   2640 

aatgggaatt gggctgttgt tcttttgagc cctgccatgt gagttcttgg ggtggggggt   2700 

gggggcaagt tggtatgtgt ttgtttattt ttcttaagga tattggcagt ctactgctga   2760 

ggctgtgtcc caggcttctg tctgccagtc agcccaaagc acccccactt taggcagcag   2820 

gtggagggag actgactttt cctttgcttc ctaccagttt atgcctatct cccaggtctg   2880 

tgcttggcag agagagagag agagagagag aactgtcgtg tgtgtgtgtg tgtgtgtgtg   2940 

tgtgtgtgtg tgtgtgtgtt tgtgtgtgtg tggtgtatgc tttggatagc aatgagtggt   3000 

gtgtaactgc caagaattcc aaagtcagtt tgaaagtgtt actgttgtta aagcttatct   3060 

ttttaagcat gctttctcct tgcccagaaa gaataggtat gtacataaac tctttcaagt   3120 

catatgttaa ataatctcat aaagtagaat gagcctgtca ttgtcccaga catgtgccaa   3180 

atgtcctaga tatgaatttg atggagaaag aaaatctcaa gtacatgaga aggtaactgt   3240 

gcttttctat tctgatgcaa gatgtgagaa gtcagttcta cagggaattt cttgcaagaa   3300 

cttctgagta tttccaaaat gaaatttttt gtgtgtgttg agggaggaaa acgagagtat   3360 

tcacattaac ttgtccatgg gttaaaacat ggacatgtat atgtaatagt aaaataggtg   3420 

aagctaagga ctgtggcttg atgtgtgagg aaagttgttg ggaattcaat gtaagcacta   3480 

tatctggctt cttaaaactt gaccttttaa aattatcttt aaacagacta cttctgtaga   3540 

ctgagttgca caggaatagg ttggttggca aatggttttt gctcattggc tttgtgtttg   3600 

ggtagttatt gtttccatga aaatgagatc gtatgtgtca tttattctgt agacttcaac   3660 

attaacgtcc ccccacctcc caaacacaca cacacacacc caatactttc cttggatgct   3720 

tttgaagttc tttggtaatt aaaatgtcat ctatgcctat gttcatttgc tttattttta   3780 

ataggggtta tctgtgcttg gcacttattg atattttatg tgtccattat gcagaattct   3840 

atttagttta atcaccacct tgtgggaaaa aaaagtcatg catacataac atgcatcttt   3900 

gttctcactt tattcatttc ctagcatcat tcctctataa gcagcacatg ctatcttaaa   3960 

acctaagctg gcttattctg taagttgcca gacttcctct ttatttgttt aaaactcaaa   4020 

caggcctctt ttcatgaatg tcttatatca ttttagggat tgtcttgaat ttgcagtgtt   4080 

aatataagaa gttttaggtt tcagattaac aaaagaaatt ataaaatgtg actgatgtta   4140 

taatatgaaa atagattgtg catgatgtat cattatagga ttttaattaa gtacctgtgt   4200 

aacttggaaa ggaaccatat acataaggaa tttctcagac ttattgcctg tgcattctca   4260 

aaggacattt agagagttca attttctgca aaaagaaaaa agtgtatttt cttaagatta   4320 

tttcacactc tgtcttattt acctatctga taagttgtta ctttttaaac aagtagaaat   4380 

taatatttta ggcatgtctc agaaaatgtt ctgtgttcat tttgcaggtg aaaagtgtgt   4440 

ggaatttttg atgggatggg agaatcttaa atgaaatctt aaatgatttg agaagtatat   4500 

tatgacagga aatttaaaaa cctgataacg caatcttagt taatttaggt attaacttat   4560 

gtcaagtgag ttcttcaaaa taaatatcaa aggttttctt aacctgatag ggagcagaaa   4620 

tatctccaat atctctgaag aaaaagttgc taattagcag aaacaaattc ttgaatgtag   4680 

tgaaggggac aatttaatga ttcaggggct acttaaatca gaccatctga tttttcccct   4740 

ttgaatcact aatttccaga ttgatttgaa atattctttg ttaatgatat cctatttgaa   4800 

atttcataac caggttgacc caagtagatt agaggcccat acaaagatga ttttctaaaa   4860 

gaagtcaagt gtaggcttgc acaatttctt caaataattt tatcaacaaa gacagatcat   4920 

ctaaataatc caagcaggaa accatgccaa ccttacactc tccctgcctc ataaaagatt   4980 

tgtctgaact atctggataa ttaccgtaat gaaacacttc tttgtccaga atctggactc   5040 

cagatagatg cagtaaaagt tgaatcctcc tccccgaaat aacttcttta ttaaagtaga   5100 

gcacttaacc actttatact tcacgctgca gtgttccttt gaaattcttt actgaaaatt   5160 

ctttcctcct aaccttaagt catcagtttc cttagaattt tgcatgttaa agagaatgtc   5220 

agataattca gatattaaag gagactcttt tggagtagtt aaaacctgtt ttgattatac   5280 

ctggatgttt attcttctaa tatctttttc tgggaggaat ctgctatgtt aagatatgca   5340 

ttgtataaga attactaaag catttgtgta ggttatatac gaagtgatgc aacaaaatat   5400 

ttaatgatga aaaactctat atagactttc acattaatta aagaggggtt tacaggaata   5460 

gagtaagtgt atccgatcaa taatacattt gggttcaaat tctcatcagt atttttctgc   5520 

atccttgctg atttggacat ccaccagtgt tgatcaaaag cttcatattg cctagtgaaa   5580 

ctgaaaatta atgttaaaat gcaaatatga tatgcatcaa taataattgc aggtgaaaca   5640 

tgatagctta atacatatct tgagaaataa aggagtttaa aaaatatcaa tgataaagtc   5700 

attccatggc ttcctttaaa ttctgaactg gaatatcatg gaagcacttg ggaaatgttt   5760 

ttaagagatt taatttatat tatggtaacg taacagtaca ttttcttatg tggtaaatat   5820 

attcatatag atatcttgtt tatgaaatgt gatgctaata aagtgctgtg tcaaccggtt   5880 

attattattt aatcatgcct atagcttcca tgggttatgg ttccagtgtg tgctaccact   5940 

atacttttat ttctaaatta aatctaagct atatggagag atatatttat ttgtgcctat   6000 

taatataatg ccttgtcctg gattatataa tttatcttat ttttcccatt tgttttgtct   6060 

tatttgttat gttccagctg gacattttac aacaagacct aaaagtattt aaattctttt   6120 

agcccaagac agatacaaat cgttatttaa tctaaaaatg ttgactgaaa tagaattaca   6180 

aaattagttt agtttggtga atatcaaggg agttatatct tgttcttaac agactccaca   6240 

agcatttctt tccaccttag gaagagcaca gccctcctct tggctccagc atggggcagg   6300 

gatgcagctg ttgataccta ggctagatga gaggaagtgc agttgacgca gaggtaaatg   6360 

gcagttggaa aaggaaggat gcctggggat gaccttgtgc tcatcagcga caccagtctg   6420 

tcctttccaa gcctctgtgg cagagctgct cttcccacag caaggatggc aggaggaaag   6480 

tccagtttgg gtgttagggt gaacagggag agaaaaaata ctgcaaaaag tttgtttgac   6540 

attttgattg gagatccatg tgctttgcag gtgatagtca agagaaaagg atttgcatac   6600 

aaatagaaaa gatgtaaaat ttaaaaataa gggcaataag ctctattttg gggaaggtga   6660 

tatacacaca gaaaaaagtc ttccttgtaa ccgcccccca tgcaagtgtt tctttgatta   6720 

acagagcttt gaaatgattc atcctttttc ttgtctcagc ctctccttgt tctttctgtc   6780 

atctgacagc taacctgatt tatcagatct aatgtgtttg tgtagtattt gtcactgcat   6840 

ttttgtattc ctgaaaccaa ttttattatt agtgtttgaa agggtctcaa tcattctgaa   6900 

ttcaattttg aacccaatgt tgtagttctt gagaactcca tctccattct aagttcagga   6960 

aattttatcc tgaagcatgc aaaaagtatt tcattctcaa gcatgcaaat atatatatat   7020 

atatatatat atatatatat atatatatat atatatatat aaagaggtat cattttgctt   7080 

tcatgatacc ctaaagcagg ctcttttaaa atgttttatc tttctataga aaccaggagc   7140 

aaagatttca tgaggaaatc actgtcactt aaaaaaatat acatattgtt gccatctaag   7200 

cattgagcat tttcttgatt tttacaggtt atttcatgct gaaattatgc ctatttgcat   7260 

ggatagtcat tctttaaagc tagccacaga tgcagtccta gggagcacgt agatgttttt   7320 

acaggtgaac cgaaagagat gggagccgtt ccagacactc tgcatgctgc ctttggcaat   7380 

ggaccctgtt attgtgaaga tgtgctctgt taagcaaacg tgaagtttaa tattagataa   7440 

acccaacgtg aaaaaaattt tcattttctt cataaaatgt taattataaa caaaaagatg   7500 

tgacatctta tatgtctaca aaatttggga ttagcatcac tagttaataa gttacacaat   7560 

gtcaagtgcc ttttatgaaa ttcaaagaag gatgttctct ttttatactg tgtttccaag   7620 

aaacaatgga agttcatata caaagaaata tttccctttc tcacacattt gatggacatt   7680 

attttctttc ttctttatat atcttctttc agttttttct gttttttttt ttcctttaat   7740 

ttggcacagg aaataaggtt cacaaatcct gtatgttaaa gagtttcttt gggcattgga   7800 

catattattt tggcagattt aaacagaagg aaactagtcc tgaagatata tttatcttta   7860 

tctcggtcaa taacttatta ttcctcatat tgatttctaa aatgtggtaa catccttgtt   7920 

ttgcagtgaa tccaactttg taataatttg tcattaaaag gacattatga aaatgtataa   7980 

atattcttat agttacatta agatatatca acagatatca tcttcaccta tgattttaca   8040 

agtaaaaaat gcatagctaa gctaaataag cagacttata aaatgactat tgtgcattta   8100 

tttcaatgct aaactgacca tttatgtttg aaagatgctg ctgctaaggg tgttctcctt   8160 

cccattttac atatgacaaa aatattgtaa aattcaagaa taaaagctct ctattatata   8220 

tttgcattta ttttagagtc cttttccttt aatagcgtta aaaccacact aattgtaatg   8280 

cagaaatgca atttttcatg tgaatttctc atagtctcaa aatttaacct tatttcttaa   8340 

gtatagagca gtttcatctt ccttataata tgaatctcaa tgcccaaaat ttaatcaatt   8400 

ggttgtcaga ggctgtgttc ttataatcta ctgtttcttc tgaagataaa cagtatcatt   8460 

ttaggcattt gtgagagaga atcatattac tggtgcttaa gcagtttttg cttaattttt   8520 

ttttaatctt aatccatctt aaaccagtgg agcagaaata tttaaaaatg tttcatttca   8580 

agcagagtgc ataataaatt gcaataattg taatgtgcca taaatcccag agcctatgca   8640 

ttttgcattt gattcaggat tgaggtcagg aaatttggag aaatttaaag aaaatgattc   8700 

atcagtcctt ttgttctgtt ggccagggtc ccgggattct tgagctgtgc ccagctgacg   8760 

agcttttgaa gatggcacaa taaccgtcca gtgatgcctg accatgacag cacagccctc   8820 

ttaagccggc aaaccaagag gagaagagtt gacattggag tgaaaaggac ggtagggaca   8880 

gcatctgcat tttttgctaa ggcaagagca acgtttttta gtgccatgaa tccccaaggt   8940 

tctgagcagg atgttgagta ttcagtggtg cagcatgcag atggggaaaa gtcaaatgta   9000 

ctccgcaagc tgctgaagag ggcgaactcg tatgaagatg ccatgatgcc ttttccagga   9060 

gcaaccataa tttcccagct gttgaaaaat aacatgaaca aaaatggtgg cacggagccc   9120 

agtttccaag ccagcggtct ctctagtaca ggctccgaag tacatcagga ggatatatgc   9180 

agcaactctt caagagacag ccccccagag tgtctttccc cttttggcag gcctactatg   9240 

agccagtttg atatggatcg cttatgtgat gagcacctga gagcaaagcg cgcccgggtt   9300 

gagaatataa ttcggggtat gagccattcc cccagtgtgg cattaagggg caatgaaaat   9360 

gaaagagaga tggccccgca gtctgtgagt ccccgagaaa gttacagaga aaacaaacgc   9420 

aagcaaaagc ttccccagca gcagcaacag agtttccagc agctggtttc agcccgaaaa   9480 

gaacagaagc gagaggagcg ccgacagctg aaacagcagc tggaggacat gcagaaacag   9540 

ctgcgccagc tgcaggaaaa gttctaccaa atctatgaca gcactgattc ggaaaatgat   9600 

gaagatggta acctgtctga agacagcatg cgctcggaga tcctggatgc cagggcccag   9660 

gactctgtcg gaaggtcaga taatgagatg tgcgagctag acccaggaca gtttattgac   9720 

cgagctcgag ccctgatcag agagcaggaa atggctgaaa acaagccgaa gcgagaaggc   9780 

aacaacaaag aaagagacca tgggccaaac tccttacaac cggaaggcaa acatttggct   9840 

gagaccttga aacaggaact gaacactgcc atgtcgcaag ttgtggacac tgtggtcaaa   9900 

gtcttttcgg ccaagccctc ccgccaggtt cctcaggtct tcccacctct ccagatcccc   9960 

caggccagat ttgcagtcaa tggggaaaac cacaatttcc acaccgccaa ccagcgcctg  10020 

cagtgctttg gcgacgtcat cattccgaac cccctggaca cctttggcaa tgtgcagatg  10080 

gccagttcca ctgaccagac agaagcactg cccctggttg tccgcaaaaa ctcctctgac  10140 

cagtctgcct ccggccctgc cgctggcggc caccaccagc ccctgcacca gtcgcctctc  10200 

tctgccacca cgggcttcac cacgtccacc ttccgccacc ccttccccct tcccttgatg  10260 

gcctatccat ttcagagccc attaggtgct ccctccggct ccttctctgg aaaagacaga  10320 

gcctctcctg aatccttaga cttaactagg gataccacga gtctgaggac caagatgtca  10380 

tctcaccacc tgagccacca cccttgttca ccagcacacc cgcccagcac cgccgaaggg  10440 

ctctccttgt cgctcataaa gtccgagtgc ggcgatcttc aagatatgtc tgaaatatca  10500 

ccttattcgg gaagtgcaat atccttttat tttcccctcg aggaaaaaac aaaccaaaaa  10560 

aggtttccca aaaggttggg tttacacaat atctagagta atgtagatta gtatcttctt  10620 

aagaaggcaa cctttcccat tattcaaagg aataggcttt tatcagcatg cgtgtgccat  10680 

tcctgattgc agaaaagctt aaaactaagc caacatcttt gcagcttcca caagttgttc  10740 

actgccttga ggagctccta tttaatatgt gctttctcag cagtgttttt tttctgctgt  10800 

tcttcctgca ttatcttctt atccctatct cttaaaaaaa ataaagaagt agatttagag  10860 

atgagaaaac agtctcattg taaatactga ttgaattctc tcagatattt tttaaagatg  10920 

gtaagtttaa tagaataagg agaaaagtca gttttcagat ccctaagatc ccataagaag  10980 

aattctcagt gtaaaccatc tgcaaggctt ctggtccgtt taaagacagc ccgatgaaat  11040 

cttaggaaga gcgctttaca agtgggaggt tgaggaggaa gaaaaatgga tgtgggtggg  11100 

gagttagtct ctctttcatc tttaagtgag actttttttt ttaaggaaat atacaggtac  11160 

tgatttattc agacagcatc ggtctctctc ccgttcaccc aaggtctgtt ctttgggtct  11220 

ggtgcagctg cctctatgca tgattaacct ctgttcagcc atacacagaa atcttttgtc  11280 

ccaacataca caaagcaaat tattttggaa agcgagagag cacaattaaa tataaaactc  11340 

agctgtattc gacttaaaaa tggctctttt tatgattctt ttaaattctg aaactgacgt  11400 

ttatgtagag ataacagtta tattttttta ttaggcctat cccgaactcc agctattttt  11460 

aactgaagat ttttttttct ctctgtatat cggttctttc tgtaaatttt ttaaaaatct  11520 

tgtggtcgtt ggtcttttgg gagtagtaaa atagtagcat ttgggggcag gtggaggcat  11580 

gtttcttata taataaacag atggatataa aatttagcaa ttaagttggc tgtgactaaa  11640 

tttaggattt tgagcaattg tcttgatgac tagagattga cattttcata tctaagccca  11700 

ctccagaggc tgccacgtaa gtgcaaagtc ccagctattg gtggaaatat gttttcctgg  11760 

ttagtggagg tcgtacttca agccacctct caggataata gtgtagattt ctgatagggt  11820 

gaactactag ggccctaatc atgagtcctg cttgggcagt taaacatgga gtctctctta  11880 

tactgagcaa gagaagaaca ttgtaacaga aagggaagag aaagatgtgg gagatttcta  11940 

catatacgta gaaatggagt tttagcttgg ttgttgattt cacttggacc ttttgaagat  12000 

ctaaaattca atccaccagc catgaatcaa agctgcacca agcaccatgc cttacatatt  12060 

ataagcaggc agtaaatatt gatcaaatga ttggaatatc gctgttggtg atgagaaagg  12120 

caaagtaaga agacacaatg gcttgaatgg tttttgtgcc ctttgcaaaa agagcatctt  12180 

cagaggttca tgtaaggcta atgtctaggg ctaagacccc attgcacccc agagatctct  12240 

taacttcatt ttgaaccagg tagttgtgat agtgggttct ttctgtctct ctctctctct  12300 

tacacacaca cacacacaca cacagacaca cacacacaga gtaaagtgac atgcgtgcca  12360 

attttggtga atatttaaag atttaatgcc aggtttcaaa actcctgtaa gtccacacta  12420 

agctctttag ttcaagatgc cagtttatgg tttttcttta aattagactt ttcattataa  12480 

ccagatcatt ataattatgg ctgtgctttt tgtttttagt cttctaggaa aaaaatcttt  12540 

tagattgctt taagtgttgg ctatgttcat tgtctcaacc tctccaaatc cccggaggaa  12600 

ttttgaggat ttgaattgaa ataagttcct tttattttga tacatatcaa aggctttaaa  12660 

gaaaatatag ttgcttcttc ttcagaggca tgacttctcc tttcttctat caacataact  12720 

ttctgtcgag cggtgattct gttgggaaac acccgtgttc atgtgaaatg ttagttgctc  12780 

acactcagaa ttgtttcttt catatagcta aataatgtcg gcctctcgtg gcaattagtg  12840 

attacatttt ccaccttttg gccttctatg ctcctattct tttcccccct ctactattaa  12900 

tacattgcac ttttaaccat ttatctcatt ggtatattat ttctcaggaa gagtaagata  12960 

ggcaaacaac cttttctata gttcccacaa ttctgaaacc agtgaggatc tgttggtttg  13020 

tagagagatt gggcccactt ttctcctgtc tctacctctg tatggcagtg tgttcttccc  13080 

ttgatttaac tgttagtgtg taggcaaaat tctcaagctt ttactttgaa gaaatatctg  13140 

ggaatcacag tgagtgatgt cttacttcaa ttttagggat acggggccat atatgatccg  13200 

gttgtacagt tattcctcga aaagatcaat agaaatgggc agaaatgtaa tgaaatggta  13260 

caactgtgat tgctattatt atgttttaat ttttcgttca tggctttcca aactgttata  13320 

tataatttaa tttttcagga aaaattatct cccactccaa aaggtaccat ctgttttttg  13380 

aacaaagtag ctaagataag aactattaag aacaccagct tatcaggtca acccattcta  13440 

cattcaccac attaaacata tatgttctgt aggatagaac acactacctc attatcccat  13500 

ctagtagaag ggaaatagtg aatgtgtatg caagttaaac tgaatttcag tgcacctgct  13560 

ccaagggctc atgtcttgga ttttaaaaat atgttcagta tctttgcaaa tgaatctgtt  13620 

taatcaaata ttaagtttta ttcaaattcc aaaagaaaca gtcagccaat tgcttttctt  13680 

catgatgttc cttgtcattc atcctctttg catctcaaga aaaatagcct agtttaggcc  13740 

ccaaacattt gcatgcaccc agttaaagca caagaggagt agtataagcc gttaagacgt  13800 

gcaggtgaag aaattgagcc tgttctctga aacagccggc tttttctact caacttttag  13860 

ggagaatgtt agaaagactt gaagtttaga aaggaaaatg gtttagtaat ttgaaattaa  13920 

aatccaacca ggaaccatag attagaaatg aatttctgaa atttgaaacc atccacagaa  13980 

attgatctta tacattttta gaagtcttgt ggaggctata gtacttatat tagctagagc  14040 

aaaacatgta gattaaagac taaaagactt tgggctccta cactaccccc ctcccctgaa  14100 

aaaaattata aagtaagtaa attaaaaaaa aaaaatccct acactacaca gccctccgat  14160 

tatggtgaac ttcctagtgg gagttacgac ttgctctatc actgtcatta tgtgagagag  14220 

tttagatctt ttctccccat tttagtttct agggggaaaa cctcttagaa acttagcaaa  14280 

ttagggaata aggcagaact aaaattcttt aggtttcaaa tgttttggaa aatgtaagta  14340 

gtctcaaccc atttgctggg aactgcagca cgtacaatct ctagctacaa tccagagttt  14400 

agctggaaaa aaagaatttt cttcctccgc tttcacagct tattattctc ccatttgcct  14460 

ttttgctgcc tccgctgctc ctcccgtggc tgctgtttag gtaaggttat attgtacttg  14520 

gtaaacagac aacacttagg ttctcaggtt gtttgaacac tgctttacgt tcagctgcag  14580 

taccctgctt ctctgatctt ttatattccc gagcagatgt ctttcattaa tttatggatt  14640 

tatcatcttt tctttttttt ttcttttttc tttttttttt ttttttacac ctggcagctg  14700 

tctcaagttt caacagttat tgtctatttt gcattacaca tagaattgaa tgtcatctgt  14760 

cttcacaaag ctatggctaa gagaattgag gcacagccac atgagctgct gggacagatc  14820 

ttgtttgcgt tccatccccc ctcaccccac tcccctttac ctccttaata tttatttgtg  14880 

ctcattttct ttcctggcct tgaatggagc ttagctcgtg ttcagtacag ctgtatgttt  14940 

actgaatcta ttccatcatg agtcattgtg cgtgtgtaag tatcctggaa acagctagtg  15000 

ctttcttgga agaacagttg cttttcagca caagcactta aaagggaaat taaccaattg  15060 

gtcagttcag atttattttg aggagaaaaa aaggattatc taactgttgc cttttaaatg  15120 

tttcattagt tatttttaat agtttattag aaacatatat tttatgggaa ttttatctta  15180 

attacacaat aagcaagaga taaagattaa ttctgtgttc catttcaact gatcagttcc  15240 

aagtattacc aacaggaaac attttaaagc aaaaatgaac ttgagaaatc caaatcagaa  15300 

taattttttg ttagataaaa agcctctaaa tactgatcaa aataaaatgg atattttact  15360 

ttttttagat aaaaagaaca aaaacatctt agcataaatt agatgtatta aaagcttcag  15420 

gaagttttgg tagctcagtg cccatctaag aaacacagaa aaacactttg tattttgtat  15480 

gacaccaaat tttaaaagat ttgtgacttc caattaaatg catgacgttg tcttaatgta  15540 

gccatctgaa agaaaagatt agaacccaga tctgagagtg tctgtcaaag tttggacttg  15600 

cctaaaactc ttatcacaag gcagtcgcag acagcttgca actattattt cacttatcca  15660 

tttggacaga tggtcctgaa gtgtgctggg ctcctttagt cttctgtatc agtctaatgg  15720 

aggttactgg agggcctttc agccctctcc ttggcacaag aagtatgtca gtcataaatt  15780 

atcgtctttg taatcattaa ggatctcaaa caaaaacaca agttcagtta agctgctttg  15840 

gcttacagat ataaaatcaa aatttctttc tttagtgttt attttcagtt taacaaaaaa  15900 

taaaaaaata aaaaacctgc actacttaac ttttctattt acagaccaag gtgatctttt  15960 

taaaattgca tgggatatta aagggaatgt taattgaaca aattctcagc agaatatttg  16020 

gttaaacacc ctgttataag tagtcaagag cttatccata ttaatttgat tatgcttctc  16080 

tagtaacttt ctggtttccc tccattctta agattagtca cgctagactt gatgaaggtc  16140 

atttggaaaa ttttaccttt cctaaatatc tgtgtttatt tgacatttct gcctaagggg  16200 

tgaaattttt gttgggtagt tgtgtgagtg tgtttgtgtg tgtgtttgca cacacaagca  16260 

cactttcttt tctttttttt cttatttttc ttagacactc ttctaaaaga aaatccttag  16320 

agaagcttct aggaagggcc cttaattgac cttgtggggg accacattga ttttctccac  16380 

gtgcatcttc atttctgata aattataaag ccattaattt gctgaggaaa tggcagggcc  16440 

aggctgcggc acagatgtga ccagagccat cccagctctg agtctgctga ggagtgccaa  16500 

gaatctgggg gagaatcagg aagcctggat tgttatggtt agcctcacat tctcttggga  16560 

actgttttag ttgctgctgt ttacagatct aaaaggtaat gatgtttcca gataaatagg  16620 

ccttcttatt ttgggtaagt ggccatttat tgatctgcta acccacatgt attgatttgt  16680 

tagccccaac tactgcgtca ctctcaaagg agttaactat aaatccaaga caggcaaatt  16740 

gtatttggtt ttggaccatt gctttcacaa aagcaacagc cccctccctg tcctctccat  16800 

gccaaaacta ctcttcccaa gttttagcta ttatttaaaa ggaaaaacaa ttaaaaggat  16860 

ataataagat aaaaagcaag tgagtcaaga tgctccatta gattaacact aaaaggtaaa  16920 

atgtgaaact tgcatagcag tgttcaaaat aatgcatttt atattttcat gtacattagt  16980 

agaataattt gctttaaact gcagagtgtg gagagaagaa caaacagaac tgtaattgca  17040 

aggaagaaaa aaaaacctct tatgacaaga gttgtgtagt acatgttggg tgcatttgtc  17100 

tccttagcaa caagtgaatg tatagatagc ctaccgacct aaagcaagga aaatattttg  17160 

ccatcctcac cctaaagtag ccaagattct gcaactcaat tgtgcatcct caccattgca  17220 

tgtggcaacc tctgacaggc gacggtcact gagcaaatgg cagcaagtta gcaatggatg  17280 

ccatagccag tgtcatatac cttccagcac tcccaccgca gcttgatgga cccccagact  17340 

ctatggaggt ggggactgga gggagggagg tgggagtcct tgtgcttaca gaattgcttt  17400 

tccttaacca attgcatcct acatgcagga aggattgtca cccaatcact tgaaaaaagc  17460 

aaagctcatg tttttttata cccgttatcc cagctccaat atgctgaaga cctacttctc  17520 

cgacgtaaag gtagggactt tttttattct taattttttc attttctatg catgtggcag  17580 

taatttgaac tcccggaagt taatggagat gaatgtggaa ttggtttatt cctacacctg  17640 

tgttataatt gatttaatgc acttgtcttt ttgtctaaag gtgtgttaag caaagatgcc  17700 

acttgtgtat taagattgga agactggtgt taataagttg catgggtttc caatgtagtc  17760 

tgaaaaactt agcctctgtc tttatatgtt tgagtagctt ctttgaagaa atttcagctg  17820 

gtaatggatg ggtgtgcttt agagaatgtt ttttccctcc cctcagcaac agtaaactgt  17880 

ttctgttttt gtttctgttg gtttccccat atttgtgctt atgaaagcaa actctagcac  17940 

ctctttttcc ccctgtcgaa aaggagcgta cattgaaatt ctctatgcag tagctgctta  18000 

aaaacaaaag tgatgattgt ctcttattta caacttaatt tgttgttgat gtagagtaca  18060 

ctgagcataa ggagaatgaa taaagtgaca gattcaggac acattattca aatgaggata  18120 

tgaaagctgt cggcctacag ctgcagcctc cctcattcta cagaatattg ggacctcctg  18180 

gttctctctg tgtgtgtatg cgtgtgtgtg tgtgtgtgtg tatgtgtctg tgtctgtgtg  18240 

tgggttttaa gtaattgttt gcatcaactt gatgttgtgt taatcatctg taacttttta  18300 

aaacatagat tgggttttga tgatgataat gacacacatg gtatcattat cccaggaact  18360 

tgataaacac tacattagct gagattagtt tattaggggt gggtgttttt tccccactcc  18420 

tcccctgccc acccccatat gtacaagttc ttctttctgc catggagaac tcacaagctg  18480 

ccaaaacaca ctcgctcttc cactgctccc cgcacgcagc ttgttttgtg cttgatgccc  18540 

aagtggcttc attggcccca ttttgcaggc caactcattt cagtttcctt cactggtgtt  18600 

ttatttggcc ttataagaaa agttctgttt tccctcctgt ttgcttttga attgtgtatc  18660 

aacttcagcc ttttatcttt ctccttccct ggctgtgctc cttaagtgga aggcttgttt  18720 

tctccttgtt cagcaccagc aaactgggca agatggggag gcagggaaag tccatcacgt  18780 

aaatgtctgg ataagactaa gtgagcacaa acaaggctga gtgacacaga ggccaggaaa  18840 

agggtttggg ctttgtagag gacaatctag aatacacaaa ttgaaggcaa tttgtcacct  18900 

ggttgaggac tgaccagctt ctagagtcta gtagaacctg gtaaagtttg tcttccaggg  18960 

aatcctccca acattttagt tctaggaggg gacatggagg acagggagaa aagggttatt  19020 

gtgtgcacat atgtgtgtgt gtgtgtctgt gtgtgcagat gtccatgtta ctcattcctt  19080 

ttagggcaat gatcttcagt gttgtgaaat aataatgaca ataacttata ttctttgcat  19140 

agcaattttc acccagaagt aggccaaaga gctttaccaa ctgcacacat aggtgtcact  19200 

cacccaccac ggaaacacag ccacctggag ggtgggaaac agcagccatt ctgagccaac  19260 

actacccaac agtagacgtc aatattagaa acaatcattt tttgtgagag ttcaagcatg  19320 

cgtgcatgtg tgtggtgtgt ggtggcaagt ggggaagatt attgatctgt agctttataa  19380 

ataccatgca atacaaacca acaagaaact gttcccattc ctctagaatg cccctagcaa  19440 

ttcagctttg caaataacca ctgactctgt gtagataaca atggaatacc tgggtgaata  19500 

ttttattttc aaaagcacta atattcagat tgttgattct atccatacct tacccatact  19560 

ggaagagaag gctgttaaag tatatgtgag tctggttact accaattatc cactgtaatg  19620 

gaggggaaac agtagaacat atcaggcaaa gcagaaaatc actgaaggtc acttctcttt  19680 

tatttttgga aggaattata catttttaac tttcctaatt atgttttttc tttggttagt  19740 

aataaatgaa tttgtatttc ttgagcttac actgatgaga gtagaaagcc atgcaaagaa  19800 

agggaaaggt agtccaggca atgtggtcca gagactttcc agaaaacaat ggcagagcat  19860 

tctgggattt cttcaatatt aaggataatc acagatgtga atattgacaa tgtatacaca  19920 

cacatatgtg catgtgcatg ggttcacaat acacatatac atatatacac atatctatag  19980 

cttgacattg acatacagat agacaagtgt gtctatttat ttgcaaggct gaaagaaata  20040 

gatatttctt tatatatgaa tatacaatcc aaacttttat tttggccagg attcaagaaa  20100 

tcactagaga aattggggaa gagaacttag ggtcttctca gaaatgaaac ctgcatcatt  20160 

tatctggaac aagatatatg catgtatcta tggaccatgt aatgcttgtt ataatgacat  20220 

gaggctctac ttggtcatgg ccacattcat ctaggagaaa attcctaact ttagtaaaat  20280 

gtactctttc aaataataaa gttattttat tcaatttttt ttttttgaga cggaatttca  20340 

ctcttgtcac ccaggctgga gtgcaatggt gcaatctcag ctcactgcaa cctccacctc  20400 

ctgggttcaa gagattctcc tgcctcagcc tcccaagaag ctgggattac aggaatgtgc  20460 

caccacgcct ggctaatttt tgtatttttt ttagtagaga cggggtttca ccatgttggc  20520 

gaagcttgtc ttgaactcct gacctcaaat gatctgcctg ccttggcgtc ccaaagtgct  20580 

gggattacag gcatgagcca ccgcgctcag ccctcatatt ttatttagtg atcataagtt  20640 

cattttgcaa gcaaaaacaa aaaacaaaca acaacaacaa caacaaaaaa aaccaggaga  20700 

aaaaaatgtg agcagaaaat atcttgtttc ctgaatatgg tataacgtaa tggtccatca  20760 

aagccacact tggaggatag agctagatgg ggtaaatcct ctgacttgct ctagaaggtg  20820 

agtcatgcca aagtggtgcc cactcctttg tatttctcct taggaatgga cacagtgctt  20880 

aactctccac aaatgacttc cacctgggta agaggtaaat gcttttcaat taccttggaa  20940 

cgaaagaggt agagggaaat catacaattc agagatgttg gcatggcgag agttcttctt  21000 

ctacaggggt gatgtatatg aaggatgaaa ccagggccga cctagtttaa ctcctagagc  21060 

aagaatctaa acaaagttct atgttctcac agagagccaa cttaattccc tcataatgac  21120 

atttagccaa acaaaaagct cagctcatcg gggctacaaa tcctttgaga aggacaagtg  21180 

gacaaatgtg agagagctgc cagggatcga tgggccgcac cagctccctg ttcactactg  21240 

ggtgctgatt ttaatgtaca aactaataac tcttagacca ctaagtacag cagattcagt  21300 

gtcattttag ctttgaagaa cagacgctca cagcttttca agccggcagt gttaaatgat  21360 

gtatctcatt ccctccaccc cttgagtcaa ctgctgccta gccagattaa ggtgtcagat  21420 

tgatttgttt tatacatctt ttgaccatgc tcattgaata tttaggaagt ttcttcagcc  21480 

catattgagg ctgagatgtc ccgtgggaag cattaatcaa agtcacagag actcgtacac  21540 

tgtggaaaca cagcctcttt attgtagcga ttagtttttg cagtaacaca ttaacacact  21600 

acagagcttt cctttataga acaattgatc cttttcttgt aagccactac agaatgaggg  21660 

aaattaactc tttaaagttt aatacttttt ctcccccagt gtgaatatct agaaaagcgg  21720 

gggcttgctt ttgcttttag ccggcgacta aaactgaaca aattttagtt cacttctcct  21780 

ggagggaaac cctgttcctt aggctgttgg gctggtcatt tcgcttgcct catgtttggg  21840 

gagtctgttg tttttgtcca ttctttctct ctggtatttc cattctccaa caataagctt  21900 

taaatctccc tttatgtccc attcgtaaat aatggcaagt gcacttactt ttttgtcctc  21960 

cccattaggt cattcgtgac cattctagaa aaaaaatacc cttctatttt tttcctctac  22020 

agtactcttg tccatatgag acaatgtctt gtaacaatgc agaagcctaa tctccatgtc  22080 

aaagcaattt tcattcccca gtgcacagcc tgctatcatt ttgtaatgtt ttgtttctta  22140 

ttctaaaaga attaaaaagg aacagtaagc cgtcacgggg gcctgtagtc cttatctcag  22200 

tgtctggaaa tttggacagt gtattttact gctgagataa aatggaaaga actccaagtt  22260 

cagcaaatcg taatgggttt aagttctatt gaaatcggca accagaagat cagataatgg  22320 

gggtccttca gttgtctttt taatcgggtt ccccgcgagg ctgaatagag acagagcaga  22380 

cacacagagt gaaaatataa ttcttggata ggttaagtac atgtttgaac tcttgcaagc  22440 

agaagcgatt tgctgatgac ttaatcattt tctggtcaat tatctgtaag ggcccttgca  22500 

actccatggc aattatgatg caagttggcc ttttgggaga aacaccagtc tctctgcttc  22560 

tgtttccttg tgacttccat tctctgccat aaattttcat tcatttatta tctttgctag  22620 

tatagaaaca actttctgtg tagtaattag agccccaata cacactttag ctgtcatctt  22680 

gttggagtct ggatgttctc atggcctgtg tttgataagt gctctttgtt gatttttgat  22740 

gaatgtacat ctttttctgg gggcccaggg aaggggatgc ctgtgatgac aaaaggcagg  22800 

gggttgtctg tcagcccgcc tgatatagag ctatggattt attggttttg acttggcaag  22860 

ttgagactca tctgtccttt acgtgagcag aggactgtca ataaggatgg tatcatttgc  22920 

agtgcatcca gaaagacatc ttcatttcaa aggtcatcag gaaaccttgg taaacaaagt  22980 

tttaaggcct aaccatgtta tagtaacttg gcatttaaaa aaatgtaata aagctcctgt  23040 

ctatgccatc tgtgtactgt gtcctaacca tgcctcccaa atggcagaga taccaaggga  23100 

gggggacatg ggtcttatcc aatgctggct tcaggaagca ggtgaacagg caccaggagc  23160 

tgaccagacc tcaccagaca tgaatgccgt gggcaaacat taagtggaat cacagttgga  23220 

tggacatggg aatcactcat tgccaaaaaa ataagcaaat gccaactcct cccattttgt  23280 

gggaaggcca tttgtctgca ttgaaggggg ctgtaatgcg gtgatacaaa tcctcactta  23340 

aaaaaaaaaa gtatatcaaa ctagtggtag agtcatgtgg cacatcacct ctggtacatg  23400 

ggagtaacaa cacttccagg attctatggc ttcaatgaat gtccataaga agtatataaa  23460 

tgcaagttgt tctactgaaa gatgaagaac aatggttaaa aataaagatg ttcggcttaa  23520 

ggaaagtctg atttagaatg tgacttttcc acttgaaagg tagagggttg tgatatgatt  23580 

tccattactg acaggttttt ataatttctt gtaagtatat tcttcctctt gcctctcttg  23640 

ccaccatttt ggtggagtta aatacgtatc tttccaagta aagaagggac gggaacatta  23700 

aaaatgcttc agacacttaa aaaaataaat gaagaaaatg gcaatgttct tatccttttc  23760 

aacatttaaa tttaacagtt caacagatgc attacctctc agctcatcaa gtggtttagc  23820 

aatttccgtg agttttacta cattcagatg gagaagtacg cacgtcaagc catcaacgat  23880 

ggggtcacca gtactgaaga gctgtctata accagagact gtgagctgta cagggctctg  23940 

aacatgcact acaataaagc aaatgacttt gaggtaggaa ctaatcttta ttttttggtc  24000 

atctcccttt tcctttttta aaaaatttat tttctttaga aatgtaccca aatctgtttt  24060 

tgtgttggtt tcgcatacaa gcatccccca atagagtaac aggtagagct gtgatgagga  24120 

gcttccatag tccccattgg aatcatgagg ctctgaccca ctgccatttt ttccccattc  24180 

cctggctttt cagcttgtgt ggaagactca tttggccaca gaaaagggaa ctgtagaatc  24240 

caaagaaaaa tggcagcaag cagcaaagac agagtgattc attttccaag gaagaggtcc  24300 

ctactccaat agaccttttt catatttagg ttctgagagg tcaatgagct gatacatgct  24360 

atgtgcaatg gtagctacca atgttatttt cttaaaaagt ctagaaacgt tgatggggga  24420 

gtgatcatgg tttctgactt tgacatttag tccctttgtg gaggaaatgg tatgataatt  24480 

tactaagtac atagcataag agatccattg acatcttttt ttgggatttt gtttctgttt  24540 

ttgttctttt tggaggagag actcgtgtgt tttgcctaag tgtaccttca caagcatgct  24600 

gctctttgta caaacactct catacacact tatatatatc tgtgacgtgt atattctaga  24660 

tccacacaaa gcagcataga gaattcccag aaagcaatat ccatgcaaca atgaaagatg  24720 

tgtggctatg agtaaggcat ttctttatgg gctaatgtgg tgcctcagca aacagttttc  24780 

atcacaacgt gatgactctc tgtgagacaa cactagcaaa tctcccagta ctcacaaagg  24840 

cattttgctg agccctgctg gctgaggcaa cagtagttgg aggtgggaac atggcaagaa  24900 

ttctgcaggc tgaactccct gatgatgaga tcagacaggc tgtggcttga caaagttggt  24960 

ccatttcttg tattatcttg gctagatgct gtgccatctt gagggtagga attttttctc  25020 

caacgtctgt gtgcacttgg accttatgtt aatattcttg ctttcttctt gtagataggt  25080 

atccaggaat acccaggaag ttccaaattt caaaggaaag aggacacctt ggcctcgctc  25140 

tgtcaattaa ggggtctgac ccctagtact cttcctgctt gcccccctcc tttttttcgg  25200 

ctcttgtccc tacagttctt ggcaatgcag accagttata gtggcttata aagaattgaa  25260 

tatggaagct cagcaatggg gaagtcatag tttttctttg aaagtttgag tagttatagt  25320 

gtaagctacc tatttgtctt tgctctctaa gactaatata ttttttgcca aatgtgtgat  25380 

aaatgaagtt tgggtggtgt gtgtgtgtgt gtgtgtgtgt gtgtgtttgc taaatacatt  25440 

aaaagtgaga attcttcgtg tactgctcca ctattttaaa atctgttttt aaagtctcag  25500 

ttgtaataga gcactggctc actataatga cagagcacta gcaggcttct tctaaagctg  25560 

aagaatatga ttatggctaa ccattttaaa gaaatctcat taagagcatc ttttctcccc  25620 

tgcctttctg ctaagcctgt tgccctaaac cttaagctaa gagacttctg tgtgctagtg  25680 

aattatttac attacatgat gacataagta tctgtttggc agcatacatc aagcttcatg  25740 

aaagaattgc ccaagattca tgagatgact tctgcatttt tgctatataa aatacccaag  25800 

aggacaagtc cttaaagtgc gcacgagggt tttcgggttg cttaaacctt acctggttgg  25860 

aatttaatcc gctacccaca ggccaggggc caaaatgaca caaacagggg atggctggca  25920 

tcaggaggta cccgacaagc tgctccattt agcatcatct aaatcctctt taatatgatt  25980 

aacatctaat atttctctct ttgtgaatca tatccacttc cagccaggcc acctctcctt  26040 

tatctgcagt gtctatttta agactgcttc actgcaagga gtatggggcc cgggcaggaa  26100 

ttttgtcact tctcatgtga cttcggacag ttattggact attctggatc tgattcctcc  26160 

ttcagtgaaa agaagggaag aaagcaggac catgcagtgt gtcctgcccc ctctactcac  26220 

acacttacac atccatatgc acacacgcgt accgaccacc acacataatc ctaatatcac  26280 

gaaatcgttt ttcttttagc ctctcggtct ggctcattta ctgacaaaag tttcagataa  26340 

ggtgagccct tcttttccgt gcctttgtgc atggaggtca ctgcttaagt gagatgctta  26400 

aaaagccacc gttcttatcg tggtagcttt gctagtgtgg gccgtggctg agagccaaaa  26460 

gtagatccgg caccttcagc tgaatacctc cactgatact gtgtgcacgg ctttactttt  26520 

gtatttaagt ttctcctctt aaggtcaagt aaaatgaacc tatagtttaa gtattagcaa  26580 

gtgaagagga tggcaaaatg gagaactgtg ctacaaacag agctaaacca tggtagaggg  26640 

actttgaagc tacgtctaca cggtgcccca agatccagtc gattccaagg aatcgtgtca  26700 

cccagcttag taggagctgg tcaaacaata aaatgtctta ttgattgtat tcccagactt  26760 

ctcaatcaat tgttgggaac aataataaaa tagctaacat ttattgactg tttactaatg  26820 

acctaggcac tcttctaagt gttttaccaa aatagggctt atttaatgtg ggtaataata  26880 

atgacagtga taccaatata ataacaagaa aaacttcagt ttgcccaaag ctttactatt  26940 

cttcaagtta ttctaactgg gcagaggcag atcgagccag ggagagagaa ggaggtttga  27000 

cgtctcttca ctactacttt attccttctt tctctcctct accccttgtc ttctctcagc  27060 

cttctactcc catctctgcc tctgtcagaa gcttgctagt ggcacctttg tcactgctta  27120 

gcaccacctc cgtccagccc ctgctgctga tggctctcaa ggctggagag gctgctgacc  27180 

cctggcctac aggaaaataa agcagatggg gaaagtttat cagcagcgaa gagggagtgg  27240 

cttgcctgct ctcctctcct agaccctgca tttcctggcc tttatgagta caggaccttc  27300 

taagtggcag tagagcttgt tctgcctttt gtatcagttt acacaattgc cagaattctt  27360 

ggcacggtgt gcagacttag ggtggtgagc gtttgagaag acccaaggga tgtggaagaa  27420 

gacacccaag gggaaaaata cgaaatacac ttttagtttg tgctaaaggg cagaagcttg  27480 

gccatatcac accgggtggg gtgtcttgct tctgtgcgtg agtgtgtgag gcacgcagga  27540 

gaggggtgtg taattatgtg ctgtatcctt catttctgct cctcacattt aatgagattg  27600 

gcaacaataa atttgtcttt ccaggtgtga tggtatatat ttctatgctt cattctcact  27660 

tcactttgaa gggcttccaa aaaaaatttt atgggcagaa agagcaagtt tgggattcct  27720 

tcccagtttt taaatcatac tgatacttgt gactttaggg gcgtatgagt tggattttat  27780 

cgcttttgtt gttttcctca caactgtggc aggaaaagaa gatgacgatc tctgtcagtt  27840 

tctgaggctg gtttacctgt tttgcaaaga gctccaccga gacaactaac ttgtgtaact  27900 

cacaaaggtt aattgcacaa cgtaaggagc caaaagacat agcagctata tgtgcagctg  27960 

cgaaaggcag aatcatccaa aggttggagg gtttgttacc gcctgagtgt aggttgagaa  28020 

aagaatgtgc cagattcctt catccagtca cattgagctc tctttctcat tccagggtac  28080 

cgggaggtag tgtttcccac gccatggtaa gccacacatc cctcctgggc ccctcagtgg  28140 

ctagtcattc acctgtaggc agggtctaag tttccagtaa gaatgacaga tctcccctat  28200 

cctcgctaaa ggcccaggtt tggggatgga aggcttcaaa ataaattgaa tagggaactt  28260 

gattcactca ttagtggcct tatgaatgcc attttctaag gtactaatac ctcactgggc  28320 

agatgctcca tcttagagac tgtgggtttg acatttttct gggtgacaca tgacagggaa  28380 

gaagggtact tccgcacacc tttgaatgtg ttttcttact ttcctcttgg aaatagaaaa  28440 

taaaaaacaa caccccaccc cacccccaac acacacacac actaatacat acacacttgc  28500 

tgaatatgtt ctctacccca tacctaccct tttcttaacc tactcccact ttcaatagaa  28560 

cccacatttc agaagattta atatatttgg aagactttta ttcgcattgt catctcttta  28620 

aagaaaaatg aggacaggtg gatttaggaa gcgcttccct ctgctccaaa tagatcctta  28680 

aatatgagtg atcgtttaga aaactggcac atgagtgaga gcctttcact gctgttgcag  28740 

tcttttggcc tcaaagctgc tgagccgttt aaataatcgc ataacacact cttggtgggt  28800 

ggcgaggagg aaaagaaacc cttaccattt cttcccttgc cagtcccacc gttgacaagc  28860 

caaattgatc ttttaagaga tcaaatgaat gttctctaaa tatatgtaca cacatggctg  28920 

cctggaaacg tattccttcc acagaatgat tgcctgaaat ttgaaggaga gcgcagtaaa  28980 

gacaccaggt tggaagtggg gttgaagggc tagggggtgg agtggaggta gaattctatg  29040 

cgtgcatgag gcttcacttt tgtacactgt ccttttggga ttcaaggtgt tcatcagtat  29100 

aatgaagcgg gcccattgat ttatcatcta tttggtaatg tcattgcatt tttagctccc  29160 

tgtgtctttt ttgtcattgg gttacattca agcacagtaa gatcaacttt aaaacctcct  29220 

tactcaacag ctttattagt tatagcattc catgaccttt ctcaacattc ttaaagaaaa  29280 

agatacagtg taatgtcgct ttactttgct tattgtcctt tgttggggtg aacaaagcat  29340 

tttctacagt ggctatatca cataattata cagctttcaa tagcagtgtc ttggcacata  29400 

tcaaagttca gaggagcctt tagaaaaaaa aaaagatgtt ttgtggcagc ctagggaggg  29460 

tctcatcttt ccttcagaaa atagttcaag gctcttctgt caagcttccc tacttagagc  29520 

tttttctcct cctgcttcat aaagtttaaa ggggattcag tggagttcta tgatctattt  29580 

cctttgaaag attgttcctc ggcacagaga ggccctttga cttcaagagt tcacagattc  29640 

atgtctttag gtatcatatg tctgacctta tcagttactc catttaatgt aggagaaaaa  29700 

gtctcaactc tttgtgtttg tctgttttgc ctctgtgaaa tgatttggtg aaaagaccat  29760 

cctttttaac acaccactga gaggccgttt ctgactgtaa cctaccctgt ggcttttctc  29820 

tctttaaaaa aaaaaaaaat cgtccttgtg ttttgtgtat ggatgagttc acagtgagaa  29880 

tagaattata caagggcagg cgcacacaca aaaaaatctt tgctttcctc cctcacctcc  29940 

cgcacccccc cacaaatgat ctattggctc tctcggcggc tgtaccccaa caggcgaagc  30000 

catttagcaa acacagaggt agcggctgtg gtgctgggac agtggtgggt tttcccttgc  30060 

ttcgacctac ccctaaggcc ttcataatta attgtccttc agcgatgagg aaagttcaga  30120 

aacagtgtgt ggagtgatgc ctattgtctg atattcagtt ctccttgcct tggttctttt  30180 

tcttcatccc acaaagggtt atcaatggga gaaagagagc aagttctctt ctgagagctg  30240 

ctggtggtgg ctgtagcttt cagtgggatg ttatcattgt gttcagccca tcctggatta  30300 

aatgtctgaa gaagttctaa caaccttttg aaagacagcc tgtttatttc gcctagatga  30360 

aacaaattca tttagcaaac caaagcttgt tcgaagttgg ccaccccttt tcacatggca  30420 

gataacatta tagatcaaat ttcttcattt ttccccccgc aggatgttat ttaacttgaa  30480 

ctgtttggtt ctttgtcagt cacagggcag aaattttaat gactattcac tcactgctct  30540 

taaatacatc aatattaatt tacaataata cagtttttgc taacatcctt tttgatgaag  30600 

cgtagacgtt taatacttga aagcagataa ttagtttaaa aatattgttt ctccttcaat  30660 

gactgccttc agccaatctt caattctatc ttgtaagatg atgtgaaaca aacgcatttt  30720 

gtcttcctgc accccccaat ttttggctga gatacaaaat aaagatgcag tgtggagaga  30780 

gctatttgag aagggtagga aaaagagaac cgtctattaa tgatcattat actactgttc  30840 

ctgttaaata gggtgaagcc aagaaaaaca aatataatcg ttcttccgag gagagcagtt  30900 

gaactagtaa atcacagagg tttaaaataa ctacattgta gtgttcatga caacttcaag  30960 

gctgaaggga accatattta aaggcaatct ctgtgtctct tatagcagtt tcttttggag  31020 

gaagagaccg acaggatggc cagaatcaat tctgccccct ttgctctttg aaaacaattt  31080 

cacaacagac cttttggtat ttaaagagaa cctgtatatg gaagttgaca caactaatat  31140 

agtcatacca aaaagggggt cataaaaaat taaagttctt cttatgaatc tttcatgaga  31200 

agcaatgaaa agggacacta gtgtagccaa gttctttgtg ctacaagctc ttcttccggg  31260 

ctctgagcta ttgttctttc agctcctcaa acagactttc actttcaaac tgacaaaagt  31320 

cacttaaaag ccagacagct gtactaacac acccacctta ctgagcaaga gccactggca  31380 

ggtgacaagg cctgctgaga gaccttgttg aaaatgagca ggggtgactt tctcgtgcct  31440 

taacgttgct tttgcactca ctttgagatg gcccattgac tgctcttttt gcccccccac  31500 

cccaaaacag gctccccaaa atatgttgtg cattttcttt gcagtgtgca acattgacat  31560 

ccgtgatcat atttctgcct tacacctgtg tggctaggca cgggttctgg gaaatttgtg  31620 

cccttctagc agaagacagg gagtttgact cacaaaactc ctgctgcctc ttttcctttt  31680 

gcccctccat tcagttcaaa tctcacttaa ggttttcaga tttctgttgc ctcactaggg  31740 

ttggatagaa aacacccacc aaagatgggt gcaaacctca ccttcggatt taagatctag  31800 

gcagagatcg ttaggtgggt agtcctgcct gcatcccgac cctcagggca gcagccgtcg  31860 

tgggccatgg gaggcctccc tgtgtgcgca ttacaggcct cccctcccct gtcaccttgt  31920 

gtacagtctg gtctgtgaca ctgatggtga ttatgtcatt attttgctct gggggccctg  31980 

gcacatctgc agagcccaag cacatcttct ttgttgcgtt ggcaaatgtc ccacgccgca  32040 

aatgcttcat tagccctgct gccggcctcc ttgccagacg cctgtgccca aatcccggct  32100 

tctttttgct ccgttctttt gtgtagctga tgatcatgta ttcatcttcc tggttcttcc  32160 

ccattttcct cgacttctga actccagatg tcccagtttt cttgcccaaa tcactccgaa  32220 

gtctacaatg cgaaatgaag tgactcttta cccttgaatc cttccccact cctgaccacc  32280 

tttcctactt tttttccccc aaatgaatag tgactttgaa tagctcgcca ccatgaagac  32340 

taacgttttc aaacttgcaa tctgaaaaga caccaagtga ttgcttccag tttatgatga  32400 

gagacagggt tagaatgagt ttggcattat tagatattgc ttattatctg tgtgccttcc  32460 

tcctccgtcc ccactctgcc cccctcacta tttccttgga tcctttattt gcacctgtgc  32520 

attgccacat tttaccaatt ttctgaaagc actttgaaat gtgagtacag aaaatactct  32580 

tcatgcctcg ctgtgcacgt tacagtcttc tgaaggttcc tttctctaag tgaatcttca  32640 

tctccactct accctctccc aaaaccactg ccccctcctt ctgccccagc cctcaacaat  32700 

gacctactat tagatactta cagtgattaa cacttggctg ttttggaaac agctaaaaca  32760 

tttctctctc taaagtttta ttctatatat ctaacagagc cacagctttt gtgaaggtgt  32820 

actggtttct acattagctg cagtaaattt tagagcttaa tatcttgggc tgtgatggat  32880 

actacataat tggtatgttt aattttccct taaatttgaa ttaattgatc tgtgttagca  32940 

tattatgagc agcttttcca atagagttta actagttttt aaattctcta actactgcaa  33000 

cataaaatga tttaaatgtc tccatctttg agcaaaccat aagattttag ttttcaggtg  33060 

tagttaaagg agttaagtgt atattttatg gaaatcatgg ttagatcact gccatgaatt  33120 

gtaatttgaa attcaagaca aagactctgt taagggttaa agaaaacttc ctcagaggaa  33180 

tgagttgcca cattgtaccg ggttgctgag attttcaaat acctatcaaa gaggggcaca  33240 

agaatatgca tgttgcaaat attaggacca atgtagccaa caaggtgaga agagaggtgg  33300 

tcagatcagg cgggtgggct ccccaaccca ttgtcagccc tgtgcaggga gcatattggg  33360 

agaggctggt acctgtcatt gaatcatttt tcaaaaggct cgagatatat ccaaaatatt  33420 

cctaacctcc cagttgccca ccattatggt tttatcaccc atgagtttta cttaaacctt  33480 

ttttaaactt aatctcattg tcagaatata ccactcctta agataataat tctctaagtg  33540 

tattacctgc tgggaaaata ctatcttctt tttacggctc taaacgtgat tcccctagaa  33600 

ctccacaggg atagcccttg ttataatatc ctgggattgt gaagagggtt gtgtccatat  33660 

tctccatttc ctttctgatt ttacagactt tgatcattac tccctcttaa tcttcatctc  33720 

tccagattaa ggagctctaa tcctttttaa aagcctaatc tcatacagta agtgggctgc  33780 

cctggatcat tttagctgcc ctgctgtaat gcgcttccag cctgactgtg tttttctgag  33840 

ggacagttac agttactaac tcacacagca gaactccagg tgtgggcagt catgccacgg  33900 

tttggtgatg gtgccttgtg cacacccaat gggacttttt tgattacccc aaaagtttat  33960 

cctcagaagc tggaattcga gttggatctc agtagtgctt attggttaaa atgatcctat  34020 

gagaccagct gatcagactc ttggcaaata ctctggcaaa tatgattgtg tctataggac  34080 

atacccagcc aaatagaaaa taggcagatc caccctgccc tccagatgtt ttcagtgttc  34140 

ttgtagatca agcactgggg tatttgacat catgaggaga tagccttagt cttgaacttg  34200 

agtctataat aatgacagct ctgggggaaa gctccagttt ctgctttatt tgatgttatt  34260 

ctcaggcagg caatgaaatg ttcacctgca agtagtcaat attttatata aaacatcccc  34320 

ttgaaatctt acaaagaaaa tgctttgggg agtctttcca ctgtcagtgg tcctggatca  34380 

ataccgttgt aggacttaca gcatggactc tccagccagg ccctgggatc aaatcccagc  34440 

tctgctgctt tctagcagtg aaaccctggc aagtgtctta ccctgcctgt acttcagttt  34500 

ccttatctgt aaaatagggg atgtaatagt gactacttca cagagtgttg tgagaattaa  34560 

atgaatctac acaattgtat tagcacaaag taagtgctgt ataagcattc acatttattc  34620 

atttgcagag ccaagtaaat gttaccttgt tgctgtgaca tctgtggtcc aattattgca  34680 

ccatttcctg ctgaccctaa ataggaaagt aaacaaacgg gcaatgaggg agctctcatc  34740 

agaattggaa catatattca acgtaaaact ggttttcaca agagcaagtg ttcctgctct  34800 

gaatgtggct gaaaaggcga cactagcctg gaacagctcc aggactctgg ggtcatccgt  34860 

tccagatgag aaggacacga tgagatgctg ggggtggtgg aaggagcact ggcctggagg  34920 

gtctggctct ggccatacct gcctcattgt ggtctactgt gctcaccttt tggaaagtga  34980 

taagattaaa ttcaagagtt tcattctagc tctgaaattt tgtgactcta gagtagaggg  35040 

gcagtttcat tctagctctg aaattttgtg actctagaat agaggggtat tctgcattct  35100 

ctaaataaag tctcttttga gtcttggtca tgttgcaaag ctttaagcag tgagtataga  35160 

ggccctggga atccagatgg cttccatgtg aggccccttc taccctggtg actctgctgc  35220 

agcttaatta tctcagtcaa aatctccagg gtgcccattt tcgttttctc ccaaggccct  35280 

atttgcagat ctgaatctca acagtgccct tggagacatg gcaattccct tactgggatt  35340 

atagagacta atttttcaaa ttcatacaca atttattgac tgaattggca ctatcattag  35400 

acttgctgct cactttattt gttgccttgg ccagggtggc caaacaatga ggaaatttgt  35460 

cagtgaagcc ctcatgccat tgggttttct cacacattcc atgcaggcct caacacagac  35520 

tatcagcatt tataatatgc attaacttct atataatgta cgtctcctct ctttcagagc  35580 

agaattggct atgttttttt ttttattctt ttattttttt atttttttga gacacagagt  35640 

gttgcactgt tgcctaagct ggagtacagt ggcatgattt cagctcacca caacctccac  35700 

ctctcgggct ccagcgattc tcctgcctca gcctcccaag tagctgggat tacaggtgtg  35760 

catcactatg cccagccaga attggcagtt ttagatgata taactacctt ccctactaag  35820 

cctacttggt agtgtttgca aaagcaacac cacccttttc tttaaatatt ccccaaatga  35880 

tagtaatata gatcatgaaa gtcttttccc ttgagattgt tttgtatgtg tgagagtttg  35940 

tggttgggag gtattgagtc ctcatacaag ccatttggat atgtattctt catatttctt  36000 

atggctattg cacctaagtt ctgttttctt aaggctacat taacatttta aattagaata  36060 

tggtgctaaa agtgactttc agtaaaaggt aatgtattcc ctgagaacaa gtaaatactt  36120 

gggcagggag ggatggtttg agtagaggtg aaaacagaga aatgatggga agctgaccat  36180 

atgtagaaga agctgaaagg tcatggtttc aaggccactg tgtttccttt catttagagc  36240 

atccactttt aaagatttat cattttcagt gacctgaagg cgtacaagat aatctgtgta  36300 

gatacctgaa actgcctttc aacaaggcca gtcctaggta ttgacagcat cctaggttgt  36360 

cccaccctaa acattacctc aagtcccatt gggtaggagt ctagtggact tccaaaagcc  36420 

cccgagttca ttctgcaatc tgcctgtctt tgcaatctat ttacctgtct tgaaaaaggg  36480 

attccaaagc ccttcacaag ctcttaagta gcatttgaaa tacagcccat ccttagtttt  36540 

gcaaagggtg attgcagaga aagacaaata gaattccctg gaaatacaga atagaatttc  36600 

tctgacagaa caaagatctt gcagtcaaaa ccaagggatg ggattgaggc caataatccc  36660 

catcctttcc taaagcaact cggatattat ttggggtgtc ataagctatt gccagcagag  36720 

tgccagcatc ccccatgaac ttgtgttctc tgaagctctg tctgatttcc taccatctgt  36780 

atcacaagcg ctttctttgg tgtttactat gagcaatccc tttctcatca caacctgcct  36840 

gaaccccact tcctaacagc ttctccctag gctccttact cacattgctc catcaatagc  36900 

aatacagggc acacagacta gttttaatat tagcctaggc aaagcttaat tatgaaggta  36960 

aagctgtggc agaaaacaat cacgtaatac attctcgaac gaaacaggag taactgtgga  37020 

ttatctgtgc cccagcttcc cttcatgcaa tattggagtg tttgtgctat gttgtttttg  37080 

gataatgtcc catccaagaa tggcaccaag cttggccctg cttcttttac cacctcaccc  37140 

agtaattgta gcaaaagtta aacttcaagg gctgtcagct tgtcttgaac tcagacacca  37200 

atggcaccaa atttacgggg ctgacttaaa ggggaatttg ttaacactac aaagtgactg  37260 

gtatatgatt gcagggctta tttttccacc taagtattga gctgatttgt cagatgtgtc  37320 

atgaagcagg gatacattcc tctgtttagc acatttaaat atgtactggc aggaaagctc  37380 

ccaattaaac gttcctaatc agagcagggt aagactgaag tcttcctggt ccttgaccac  37440 

cacgtgtgtg gtttattaac tctgttcccg tagacatagg cagccttaac tccatcgggg  37500 

gaatggtctg gccttacagg tcgaattcaa gtgaatcaat cgaactatcc tccaagatag  37560 

agcagaatga aagacccagg atcagtgcag aatgaaagac cattaggcct ctagaaaagc  37620 

tgttagccct caagtttggc taaaagcagg ggctggcaaa gtatggccta tgggcagagc  37680 

tgcccctcaa tctgttttta tggcttcaag ctaagaatga cttaaatttt taaacagttg  37740 

taaaaaataa ggagaatatc caacctagac caaatatggc ccacagagcc tatgtattta  37800 

ttacctggcc ctttactggc aaattttgct gaccaccggc tgaaggtttt ttctcttctg  37860 

tgggacatga actctctgag attccttcta gttctgaagt tccaaaattc tgtgattcct  37920 

tttttttttt ttttttgaga tggagtctca ctctgtcacc caggctggag tgcagtggca  37980 

tgatctcagc tcactgcaac ctccgcctct tgggttcaag caattctctg cctcagcctc  38040 

ctgaatagct gggattgcgg gcgccagcca ccacgcccgg ctaatttttt tgtatttcta  38100 

gtagagacgg ggtttcacca tcttggccag gttggtattg aactcctgac ctcatgattc  38160 

acccgcctca gcctcccaaa gtgctgggat tacaggcgtg agccaccgca cccggccaat  38220 

tccatgagtc tttgatggaa tagtcttggt ccagctctta cctgaacagc ctaccagatg  38280 

agcaatttct gcacagtgct tccagttgtt tttaagatct taacagtatc tgtgtagtat  38340 

ctcaggggga gagaatgagg tattaggttt tagtttttga tgctttttcc ttgattttgc  38400 

ttgcatattt gtttgtttgt ttaaacttgg aatcactttt taagacctat gcagagtttg  38460 

ggagagaagg aaaatttgct tcatcgcgac caataatgtg acaattatgt ttcctaacac  38520 

gtataatacc aagacctcca tgtgtgagca aataaactag ccacttaaag cacgttcact  38580 

gaccaaattt cagccccacg aaataatttt gacagtctct catagacatt tgtcattctg  38640 

ctcctagcaa gctagtacta tcttctactg gggctatgga agagatggtt ttacttacct  38700 

tgatctctac atgcagaatt gccaatggaa tacttacata atttaaaatg tatgcacaat  38760 

ttattaaacg tagaatagaa gatgttaaga catccttttc tattacctga aagtcacaat  38820 

tattcgaaat gctcaaatct agaacattgt tgataattat ataatatttt aacaacacat  38880 

atgttatcaa catcataatg ctgtagaaat tttattgtga attttgtatt ttctaaatac  38940 

tcttaaaaga caaagactca aattcaggta gaaaaacaaa gaagatactc agggtgtatc  39000 

tctgcccttc attcattgct gtggtcagag aagtctgtgt gaggggtttg gccggtagca  39060 

gccccccaga tccgtacact gcagaccaaa attcagctcc tgtgatgctt ttccatggag  39120 

tttccctgtc aattcaaggt agatcctcaa cctccctcct tggcagtttg catgtgactg  39180 

ttcattcttt ttattacatt tcctccaggg ggccattttc accatgtcat atctgtttgc  39240 

tatcagcatt tataagggct ggtgtggcat tggaggatgt caagtggtct gacttggaag  39300 

tgtactgcca caaactccat gtaggtgaca ggaggagaga cctgctttcc cgttgccact  39360 

ttttggatta tccctgcaac tctttccgtc tggctgacaa aaaccttggg gctattgggt  39420 

ggctcatcac ttctgctcct tctctagcct ttccctgggt ttgcttcccc caacccccac  39480 

accccctcgc acattaacat gacattgcct ggtgagcaca gaagagagca gcttccacca  39540 

gctgaaacct ctgatctcaa actcactaga gagtttggct tcgggatttt ggcaagaagg  39600 

ccgattgccc atcaggtcag catgaataaa gatttctttc ttcccttctt ttttaaagtc  39660 

aagcatcaac cgaaactgct cccaaagctc tgtctctcaa gacaatttaa cccctttcac  39720 

ctaagtacat tttctatttt gaatgcatgg tactttgttt tattcttttc ctgtgagatg  39780 

accaagaaat ctactatatg taaaatttga aagccaagtc aattctaaac caggcttatc  39840 

atttttaaag tatgtttatc cagctttgta gtaggaacaa gcagactgtt tgaaggccac  39900 

atacttttca aaccctggtt gcaacacgtc tgccccgttt tgaaactgtc tttatctagc  39960 

cgagaaaacg aaaatctatt tgacaaagtg gcactctggc cagtttatct tgcaatatgg  40020 

ctttagctca ctgagtctat tgatttcctt aaattaatgt ttacagaatg ctactgaatt  40080 

ttgctcaaca gaacattgtt ctttcgaagc tttatatata tatatatata aaacagatac  40140 

agactgttat tgccatgtgt tcctttgttt agaccaagga aacatagttt ttaggttttt  40200 

ttttttctta agacagcctt gaactatagc cacttcctac aagcatttac ttttcacata  40260 

tttaaacagc aaaacatgta actagaaagt gggcccaaac tgcatgggta ttagacgaat  40320 

ctaatcctca gtgttcctga aagctgaatg ccacctggag catcagaggg agaaagcctt  40380 

tagtcctaag cccagatgtt gctggagaac cttcctctgc ctcatttggg gtaactcggc  40440 

aggcacccga aagcaacttc acagccagtg ctcctggatc ctgctagttt ttccaaacac  40500 

aagcatccta ataaaattca aacaccattt agctgtttgg gaactctaaa tataacatct  40560 

tgccctttga ccacggtgct cagtgttcaa tacacaaaac ctaatctcta aagatgattt  40620 

taaaactgac cttcccagag aagtacacgt atccattcag ctacgaacag tgcagaaaac  40680 

aggattttga ctcataatta tgaaatggcc aaaataaaac ttagggaaca caaagcaact  40740 

tttctcaacc ggttgactca gccaacaaac tcacccaagc gaacctcctc agagcacctc  40800 

tcaaaacgat gctttgcaga catttattaa tcacagtgaa tgcttcccag gaattagggc  40860 

tcctctttaa aatctcaaac ttgtaaacca ccttatattt ggatgatatt ttatgcttcc  40920 

caaagtgcat tcatgttttc ttttccattt gatcctcccc tggaatgaga gggcactgga  40980 

atagaatctc aggattcact gtgtatagca tcctgcacca ttccttctct tctggagggc  41040 

ctgttagtcc ccggctgtac acacaggata aatgcatgca tgactgcaaa gggagaccct  41100 

tagtaaccac atcttgtgac catattttac agctccatga ttcctctttt cagcctctgg  41160 

caggagagtt tagtgtgagt gagacagtga agaggagcag caataacgta tctgttcttg  41220 

gcttttcatc tgataatctc tatgaggagt tactaaagca tctgagttta tccatttaag  41280 

tccactctgt ctgcagtgta agtccccagc ttgtgccact gctgtcagga gatgagtctc  41340 

tccttgatcg atatttactt aacaaacagc agggatggga gagtttgttt agaggaatca  41400 

tgtgcactct agggtgaatg aatgctcggg aaagtacttc aactatttgt ctccttccct  41460 

aagatttttg tgtacgtgtg tgtgcacaca cgtgtgcaga tgcccattct ctttttaact  41520 

tctccaaaga cacttcgaag tcatctagaa aaatacctcg ctatgtatga ttggtacatc  41580 

attataccgt taaggagcta atgatgcaga tgcagttttt ctaacccagc aaagtttggt  41640 

tcttcttttg tgctcttata tagagcacaa aagagactct taggataaac taaatgcaca  41700 

agcatctacc tttgacccct ttcagatgag tggaagggaa gaaaatacgg atggaaacaa  41760 

taaaagcagt ttgacaaggc agctcttcac tatgtatttt tgatggcatt acctatatat  41820 

ttttaaaggc ccacagggac aaaaagtaac tttctccaat ttttcagagc tgcttcagca  41880 

ttagatatat ttaactctac tactgtatat gaattccacg gtgtgaaaat tgagagagca  41940 

ctgttctttc gagttccctg aaacaattgc ttgaaggctc aagtcagcct cttgaatgca  42000 

gttgacttgg aggcatctgg ggctagatcg aggggttttg tttctgggtg tggggagagg  42060 

ctggggggtg gctggggagt tatttattta tttgattttg tgaatcggag ttgtaaaagc  42120 

catctgaaat attcatgcag aatagtctga gaagcccgtt tctgttttat ttaccgcaca  42180 

gtagaacagc cacagcggat tagttctaca atacccgtaa caaaagccca acagctgatg  42240 

catgtgatgt taggaggtga caaaacagtt aaagtatgct gctggctaca ggcaagcagt  42300 

cagcagatgc agacaaaagg gtttgtgaca agaataactc tctctccaag gcgagcagtg  42360 

aagagtatcc aaaataccag tacccttttc tccttgacat tgtcttctta cagtcagcat  42420 

tttattgccc ttttatagta taaaaaaaaa tggaggagga agaagaagga aaacccacac  42480 

acaaactaat tcaccaaaat actaggcagg attgtacttt cccattcgct agccatgcct  42540 

gccagtacac gtgtcctttt ccatttctcc atcgaagcaa gtttgaaaaa aaaaattagc  42600 

ttaaaagatc agctataaag atgatttccc ttgaaaagtt tgtaatctat tgataggctt  42660 

gataggccat tggagccttt ggttacgggt tggggggtgg gtggccaggg aaagaagtcg  42720 

atgcctggtt tgttttctgt ccatttcagt gaagatcatt tcagtgatga aatgaggcca  42780 

gagggccaat ttttaaaggg gattgaggag ggaggagtgt ccatggagaa ctgagcaagg  42840 

ggcaaggttt aggtcccccg caagaggctg atgaatgagc ttacggacgg ttcagaggtg  42900 

tgaaaaatga gcttctctgt ctccagaaaa taggagaggc tgtcttcttt ttaacctttg  42960 

taattcccct tctattctct gtgacattca ttcagctgcc aagagcgttt ggcaaggttt  43020 

gggccagcga gcacacttcc agtgaccgct aaccttggta tgtcctgaca cttatgatga  43080 

gtatctgcag gacacagaag gcaggcagcc tgctatgtca ggcttttatt atgtactgca  43140 

gaggctaggg acagtcagtt taataaaaca aatcatcctt gaaggtaaag caactgggaa  43200 

gaggaggaag acaggagaaa aatgtgtctt tgccactcat tccgatggaa aaaaaaaaga  43260 

acagcaaaac aaccacccac ccaacacacc gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt  43320 

gtgcgcgcgc gcgcattcgc gcacgctaca cacacgcgca acccagctgt ggactgggca  43380 

gacttgaaaa cctcctctca ttttctgcat ttcatggaag cccagaaggc tcttgtttgc  43440 

tctgaggaga ctcaagtctg tgatgaaatt ggtagaagct gatagccaac ccccttcaaa  43500 

tttatgcata tcttcaagta cctcattact ttatattctt ctccaaatat caaggcaaga  43560 

ccatctgggg tgacgttcct atattgggat gcctttttat caaaacaaag tttccactct  43620 

cctctcctga ggaacgctgg gcaaagcagc tcccacaata gcctcagagt tccagccaaa  43680 

gactttggaa gccttttgtt ttttccctgt ggcatgtcca aaggcagggc cttctcccct  43740 

cctccgcccg ccctccccag ccgcctgcat tgtcttgcat tccagtgact tgattgactg  43800 

ttaccacctg atgctgagga gatactctag ggttcattct gcagattgtt gggttctatt  43860 

aaaagaaacc tagataaggg attacttgtc actaagggat tttctgcaga tgtttattgg  43920 

tgatgggaaa gccattaggt gtgaagaggt gcagaaaaat atggacaaca tcattctgat  43980 

aagactggtt tctaagatgc tcccacaaaa catcagaaag taccccctat tattctgtta  44040 

aatggagctg ggtgttttca agcagaggta aaggtctctt tttccatggg tgatgtttct  44100 

atgtgtggat gaaattcact ggaaccctct cagaagatca gttgctaccc aaaagtgtac  44160 

ctctgggagc caccaaacac atgagttgct ccagtagttc agtatctcat tacaactttc  44220 

ttttgtccag tccagtccat tgcatgagta tcacctcaaa gtaagcacta tattaactaa  44280 

tcattttatt tgttcacaaa gaattcattt cttcccaaat ataaaccaat aaccaaagtc  44340 

tcctccaggg catcttttat accatttcca tttattttga agttactaga ttctctgtgg  44400 

tttttcaaga ttacagaggc acagcttttc aaggttttgg tgcctcatat aaatagtaga  44460 

aattgctgaa aaagcattaa aagggagcca gcatcgttta atgcaaagac accttacctc  44520 

acagtaatct cttcatctca tcatttcttc atctcataca atctcatgct ttcttcatct  44580 

ataaagtgat gatttctgag atctattcga actctttgaa ttctacctta ctttaccatt  44640 

attttaaact tctttttttt tttttatttt tgagatgggg tctcactgtc acccaggctg  44700 

tagtgcaatg gtgcaacctc agctcactgc aacctccgcc acctgggctc aagccatcct  44760 

ctcacctcca cctcccagta gctgggacca caggcatgtg ccaccacacc cagctaattt  44820 

ttttgcattt ttggtagaga cggggtttca tcatgttgct caggctgaag cttcccttta  44880 

ttaagtattg ttaaagtatt aagtaactgc cactctagag caatatggag taaagcagaa  44940 

ggcaagatct cactatgagc tatttaccaa ataactttgc aaaagatact ctgctgaggc  45000 

tccttatcta gagacacctt atgatgaggt aattgaaagt acataaaagt agataaaaag  45060 

ttaaacagca tcaagacaca aatgcaaaag gtgataaagg ataacctatg attgccacca  45120 

caagaaagga atatttaaaa cagattaaaa cccactaaaa accattaaca agcatgacga  45180 

actataaaaa tgatgaagag gagactgcat acaaccccca aagaagttgc cttgttctca  45240 

tgcaaatcct acaactacac ttccctccct cccctgctgc tgatgttcta gatgtacctc  45300 

ttctctctcc tctgacagtc ttgaacaatg cctgcccttc ccctgtccct ggttccccag  45360 

acctcctgtg cagttcttgg tgtgggcagg gcttccggcc ttctctggct tctctggggc  45420 

agctgcccac accttcaccc ctcaaagctc tctgccatgt catgctgcat ccctgagtgc  45480 

tcaaggaaca tagaatttca ctgaggctgt attgccgttg gctgatgaaa ccacccttct  45540 

tgaaacgttt attttaataa atgcctataa ttggccaggt gcagtggctc acacctgtaa  45600 

tctcagcact ttgggaggcc aagacgggca gatcacctga ggttgggagt tggagaccag  45660 

cctggccaac atggtgaaac cccatctcta ctgaaaatac aaaagttagc caggcgtgat  45720 

ggcacttgcc tgtaatctca gctactcagg aggctaaggc aggagagtct catgaaccca  45780 

ggaggcagag gttgcagtga gccaggatca tgccactgca ctccagcctg ggtgacagag  45840 

caaaactcca tctcaaataa ataaattaat aaatgcctat gattatgttt ctgtagcatt  45900 

tggctaacag ctcccaatcc aaggagtgag agtgggcagt tgctccgctt cactgttctc  45960 

cagccacatt ccctccctca gtgatgctca tttgatagaa tgtggaggat tatctttggg  46020 

ggtggaggtg actgtgctag aaaagattgc ttcacgaatt tttatttgta taatgtgagt  46080 

gggagggcta agctctcctc caacaaatac tcatgtatac aagacatttg ggaggaaatc  46140 

acccaaaggc ctgtagaaaa tccacatgaa ttctcagcag agaatggccc ttgaggtgta  46200 

tgggtttgca cattcatggc ggacaaggcg gcactttgaa ggattttcca ggcaacactg  46260 

ggaattatgt cctaagaaat gggccagtgt gaaagtcttt aggagggtct gataaaaatg  46320 

taagcttaag actgattggc cccaaaagga gtccctttca tttttttctg cagagttatt  46380 

acatttcttt ataaacaaca attaacttgc catagggaac aatgaacttc tttgtccaat  46440 

tttaaacgtg aaaaacagtg atgtcgggtg atgattctgg ttttctttac cagttactac  46500 

tattgttaaa aagtacattg cacccaaggt gggaagaaag agatgaaaca tgttcaacat  46560 

tacactactt cctttttact ttggtacgtg gcatgtctga acttagatga aatgtctttc  46620 

atctcttgta tatgcgtaga taaatatggc tacatgtaca cctatgatac gtttatgtcc  46680 

tcatacgtct gcacttaatg taaaaatgaa actttactgg tgtataagta ccccactaaa  46740 

agaaatctac taagtgtcaa tgtgtacttg gaaaatcatg agttcatgga ttattctgtg  46800 

attccattat gttggtgtgg ggatagatag accatgctgt actataagta acttccaaag  46860 

aacactaaat aagtacatca gtagctactg ctttccttag tcaagagatc agattaataa  46920 

gtaattaaga gaacacacac acacacacaa cacacataca tattaattgc tgtggaagaa  46980 

aagccttaag aaattggggt tctaaaatga atatttgggg aatgtttatt ttggatgata  47040 

aggaccttga ggaatttcct taccctctct gagcctcagt tttctattgt gtaactggga  47100 

taataacacc ccttagagag attgggagaa ctgaatgaca taattcacat tcagtacata  47160 

aaacatagcc tggcaagtag taaatactcg aaaaaagtta gtttgtatta ttattattat  47220 

cagctgaata aatcactctc ttatggagca attctaatct caaggttaag tagtttctga  47280 

tgtaatattt taggatcagt tttgtgactt catgttaata ttattatttt actcctttat  47340 

gtatatagaa tactttatat tgcagattaa tatacaactt agcatctgag tcaacaatcc  47400 

tctgagacaa acagataact gagattttag aagattttct tcatttaaag cttgggttta  47460 

atttataaag aagcccaact atttgttatt ctattttgag aacgtatttt gttttcatca  47520 

tggcaatcaa aaagaaatag gattcaaatt ctgaaaaaat aattggagac tttcttctgg  47580 

atagcactta tttaataaag tgaggaatcc caaaagtcac atcccatatt cctatcctaa  47640 

tatccacaat gaaatcccag tttttcaata ggtctgcgtt ggatctttca tacactcttc  47700 

ttaaaacaaa gctgtcaacc ccacatcaca atgcttctat atataatgac tttacattaa  47760 

aagaatagaa gccagctatt tttagaaaat gcaggtgcca tgtaagcccc tttctgcaag  47820 

aatgatctta gctcagtttc cttggaataa ctgtagactt gaaactgaaa actttattaa  47880 

tgccattgtc tccttgtatc agcaggttcc agagagattc ctggaagttg ctcagatcac  47940 

attacgggag tttttcaatg ccattatcgc aggcaaagat gttgatcctt cctggaagaa  48000 

ggccatatac aaggtcatct gcaagctgga tagtgaagtc cctgagattt tcaaatcccc  48060 

gaactgccta caagagctgc ttcatgagta gaaatttcaa caactctttt tgaatgtatg  48120 

aagagtagca gtcccctttg gatgtccaag ttatatgtgt ctagattttg atttcatata  48180 

tatgtgtatg ggaggcatgg atatgttatg aaatcagctg gtaattcctc ctcatcacgt  48240 

ttctctcatt ttcttttgtt ttccattgca aggggatggt tgttttcttt ctgcctttag  48300 

tttgcttttg cccaaggccc ttaacatttg gacacttaaa atagggttaa ttttcaggga  48360 

aaaagaatgt tggcgtgtgt aaagtctcta ttagca                            48396 

 
           
             12  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            12 

ggacggtgag tctgtgcgac                                                 20 

 
           
             13  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            13 

agctcaagaa tcccgggacc                                                 20 

 
           
             14  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            14 

agctgggcac agctcaagaa                                                 20 

 
           
             15  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            15 

aaagctcgtc agctgggcac                                                 20 

 
           
             16  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            16 

gccatcttca aaagctcgtc                                                 20 

 
           
             17  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            17 

atggtcaggc atcactggac                                                 20 

 
           
             18  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            18 

ctgtcatggt caggcatcac                                                 20 

 
           
             19  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            19 

ctgtgctgtc atggtcaggc                                                 20 

 
           
             20  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            20 

gagggctgtg ctgtcatggt                                                 20 

 
           
             21  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            21 

cttaagaggg ctgtgctgtc                                                 20 

 
           
             22  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            22 

gccggcttaa gagggctgtg                                                 20 

 
           
             23  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            23 

ggtttgccgg cttaagaggg                                                 20 

 
           
             24  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            24 

ctcttggttt gccggcttaa                                                 20 

 
           
             25  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            25 

cttttcactc caatgtcaac                                                 20 

 
           
             26  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            26 

ccgtcctttt cactccaatg                                                 20 

 
           
             27  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            27 

tccctaccgt ccttttcact                                                 20 

 
           
             28  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            28 

tgctgtccct accgtccttt                                                 20 

 
           
             29  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            29 

gcagatgctg tccctaccgt                                                 20 

 
           
             30  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            30 

aaaatgcaga tgctgtccct                                                 20 

 
           
             31  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            31 

gccctcttca gcagcttgcg                                                 20 

 
           
             32  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            32 

agttcgccct cttcagcagc                                                 20 

 
           
             33  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            33 

atacgagttc gccctcttca                                                 20 

 
           
             34  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            34 

tcttcatacg agttcgccct                                                 20 

 
           
             35  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            35 

tggcatcttc atacgagttc                                                 20 

 
           
             36  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            36 

catcatggca tcttcatacg                                                 20 

 
           
             37  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            37 

aaaggcatca tggcatcttc                                                 20 

 
           
             38  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            38 

ctggaaaagg catcatggca                                                 20 

 
           
             39  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            39 

tgctcctgga aaaggcatca                                                 20 

 
           
             40  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            40 

ttcaacagct gggaaattat                                                 20 

 
           
             41  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            41 

tatttttcaa cagctgggaa                                                 20 

 
           
             42  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            42 

ctggcttgga aactgggctc                                                 20 

 
           
             43  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            43 

tgatgtactt cggagcctgt                                                 20 

 
           
             44  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            44 

tatatcctcc tgatgtactt                                                 20 

 
           
             45  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            45 

ctgtctcttg aagagttgct                                                 20 

 
           
             46  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            46 

tagtaggcct gccaaaaggg                                                 20 

 
           
             47  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            47 

aactggctca tagtaggcct                                                 20 

 
           
             48  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            48 

ctcatcacat aagcgatcca                                                 20 

 
           
             49  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            49 

ctctcaggtg ctcatcacat                                                 20 

 
           
             50  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            50 

ctttgctctc aggtgctcat                                                 20 

 
           
             51  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            51 

acccgggccc gctttgctct                                                 20 

 
           
             52  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            52 

gaatggctca taccccgaat                                                 20 

 
           
             53  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            53 

ccccttaatg ccacactggg                                                 20 

 
           
             54  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            54 

attttcattg ccccttaatg                                                 20 

 
           
             55  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            55 

gggccatctc tctttcattt                                                 20 

 
           
             56  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            56 

ttctctgtaa ctttctcggg                                                 20 

 
           
             57  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            57 

actctgttgc tgctgctggg                                                 20 

 
           
             58  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            58 

tggaaactct gttgctgctg                                                 20 

 
           
             59  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            59 

aaccagctgc tggaaactct                                                 20 

 
           
             60  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            60 

ttcgggctga aaccagctgc                                                 20 

 
           
             61  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            61 

gctgtttcag ctgtcggcgc                                                 20 

 
           
             62  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            62 

catgctgtct tcagacaggt                                                 20 

 
           
             63  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            63 

tctccgagcg catgctgtct                                                 20 

 
           
             64  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            64 

gcatccagga tctccgagcg                                                 20 

 
           
             65  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            65 

aagacctgag gaacctggcg                                                 20 

 
           
             66  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            66 

gtgggaagac ctgaggaacc                                                 20 

 
           
             67  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            67 

tggaaattgt ggttttcccc                                                 20 

 
           
             68  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            68 

ggagccggag ggagcaccta                                                 20 

 
           
             69  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            69 

gacatcttgg tcctcagact                                                 20 

 
           
             70  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            70 

tgcattgcac ttcccgaata                                                 20 

 
           
             71  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            71 

tttttcaagt gattgggtga                                                 20 

 
           
             72  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            72 

gagaagtagg tcttcagcat                                                 20 

 
           
             73  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            73 

atctgttgaa ctttacgtcg                                                 20 

 
           
             74  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            74 

ggaatctctc tggaacctca                                                 20 

 
           
             75  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            75 

gcattgaaaa actcccgtaa                                                 20 

 
           
             76  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            76 

gaaggatcaa catctttgcc                                                 20 

 
           
             77  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            77 

tccaggaagg atcaacatct                                                 20 

 
           
             78  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            78 

agcttgcaga tgaccttgta                                                 20 

 
           
             79  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            79 

ttcactatcc agcttgcaga                                                 20 

 
           
             80  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            80 

tgaaatttct actcatgaag                                                 20 

 
           
             81  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            81 

acttggacat ccaaagagga                                                 20 

 
           
             82  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            82 

agatacttac gcggtgcagg                                                 20 

 
           
             83  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            83 

gatattgcac ttcccgaata                                                 20 

 
           
             84  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            84 

tgcatgtagg atgcaattgg                                                 20 

 
           
             85  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            85 

tagttcctac ctcaaagtca                                                 20 

 
           
             86  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            86 

ttatgaagca ggaggagaaa                                                 20 

 
           
             87  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            87 

caagacaggt aaatagattg                                                 20 

 
           
             88  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            88 

agcaaaccca gggaaaggct                                                 20 

 
           
             89  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            89 

tgttttgata aaaaggcatc                                                 20 

 
           
             90  
             20  
             DNA  
             H. sapiens  
             
 
           
            90 

ggtcccggga ttcttgagct                                                 20 

 
           
             91  
             20  
             DNA  
             H. sapiens  
             
 
           
            91 

ttcttgagct gtgcccagct                                                 20 

 
           
             92  
             20  
             DNA  
             H. sapiens  
             
 
           
            92 

gtgcccagct gacgagcttt                                                 20 

 
           
             93  
             20  
             DNA  
             H. sapiens  
             
 
           
            93 

gacgagcttt tgaagatggc                                                 20 

 
           
             94  
             20  
             DNA  
             H. sapiens  
             
 
           
            94 

gtccagtgat gcctgaccat                                                 20 

 
           
             95  
             20  
             DNA  
             H. sapiens  
             
 
           
            95 

gtgatgcctg accatgacag                                                 20 

 
           
             96  
             20  
             DNA  
             H. sapiens  
             
 
           
            96 

gcctgaccat gacagcacag                                                 20 

 
           
             97  
             20  
             DNA  
             H. sapiens  
             
 
           
            97 

gacagcacag ccctcttaag                                                 20 

 
           
             98  
             20  
             DNA  
             H. sapiens  
             
 
           
            98 

cacagccctc ttaagccggc                                                 20 

 
           
             99  
             20  
             DNA  
             H. sapiens  
             
 
           
            99 

ttaagccggc aaaccaagag                                                 20 

 
           
             100  
             20  
             DNA  
             H. sapiens  
             
 
           
            100 

cattggagtg aaaaggacgg                                                 20 

 
           
             101  
             20  
             DNA  
             H. sapiens  
             
 
           
            101 

aaaggacggt agggacagca                                                 20 

 
           
             102  
             20  
             DNA  
             H. sapiens  
               
           
            102 

acggtaggga cagcatctgc                                                 20 

 
           
             103  
             20  
             DNA  
             H. sapiens  
             
 
           
            103 

cgcaagctgc tgaagagggc                                                 20 

 
           
             104  
             20  
             DNA  
             H. sapiens  
             
 
           
            104 

gctgctgaag agggcgaact                                                 20 

 
           
             105  
             20  
             DNA  
             H. sapiens  
             
 
           
            105 

tgaagagggc gaactcgtat                                                 20 

 
           
             106  
             20  
             DNA  
             H. sapiens  
             
 
           
            106 

gaactcgtat gaagatgcca                                                 20 

 
           
             107  
             20  
             DNA  
             H. sapiens  
             
 
           
            107 

gaagatgcca tgatgccttt                                                 20 

 
           
             108  
             20  
             DNA  
             H. sapiens  
             
 
           
            108 

tgccatgatg ccttttccag                                                 20 

 
           
             109  
             20  
             DNA  
             H. sapiens  
             
 
           
            109 

tgatgccttt tccaggagca                                                 20 

 
           
             110  
             20  
             DNA  
             H. sapiens  
             
 
           
            110 

ataatttccc agctgttgaa                                                 20 

 
           
             111  
             20  
             DNA  
             H. sapiens  
             
 
           
            111 

ttcccagctg ttgaaaaata                                                 20 

 
           
             112  
             20  
             DNA  
             H. sapiens  
             
 
           
            112 

gagcccagtt tccaagccag                                                 20 

 
           
             113  
             20  
             DNA  
             H. sapiens  
             
 
           
            113 

acaggctccg aagtacatca                                                 20 

 
           
             114  
             20  
             DNA  
             H. sapiens  
             
 
           
            114 

cccttttggc aggcctacta                                                 20 

 
           
             115  
             20  
             DNA  
             H. sapiens  
             
 
           
            115 

aggcctacta tgagccagtt                                                 20 

 
           
             116  
             20  
             DNA  
             H. sapiens  
             
 
           
            116 

tggatcgctt atgtgatgag                                                 20 

 
           
             117  
             20  
             DNA  
             H. sapiens  
             
 
           
            117 

atgtgatgag cacctgagag                                                 20 

 
           
             118  
             20  
             DNA  
             H. sapiens  
             
 
           
            118 

agagcaaagc gggcccgggt                                                 20 

 
           
             119  
             20  
             DNA  
             H. sapiens  
             
 
           
            119 

cccagtgtgg cattaagggg                                                 20 

 
           
             120  
             20  
             DNA  
             H. sapiens  
             
 
           
            120 

aaatgaaaga gagatggccc                                                 20 

 
           
             121  
             20  
             DNA  
             H. sapiens  
             
 
           
            121 

cccgagaaag ttacagagaa                                                 20 

 
           
             122  
             20  
             DNA  
             H. sapiens  
             
 
           
            122 

cccagcagca gcaacagagt                                                 20 

 
           
             123  
             20  
             DNA  
             H. sapiens  
             
 
           
            123 

cagcagcaac agagtttcca                                                 20 

 
           
             124  
             20  
             DNA  
             H. sapiens  
             
 
           
            124 

agagtttcca gcagctggtt                                                 20 

 
           
             125  
             20  
             DNA  
             H. sapiens  
             
 
           
            125 

gcagctggtt tcagcccgaa                                                 20 

 
           
             126  
             20  
             DNA  
             H. sapiens  
             
 
           
            126 

gcgccgacag ctgaaacagc                                                 20 

 
           
             127  
             20  
             DNA  
             H. sapiens  
             
 
           
            127 

acctgtctga agacagcatg                                                 20 

 
           
             128  
             20  
             DNA  
             H. sapiens  
             
 
           
            128 

agacagcatg cgctcggaga                                                 20 

 
           
             129  
             20  
             DNA  
             H. sapiens  
             
 
           
            129 

cgccaggttc ctcaggtctt                                                 20 

 
           
             130  
             20  
             DNA  
             H. sapiens  
             
 
           
            130 

ggggaaaacc acaatttcca                                                 20 

 
           
             131  
             20  
             DNA  
             H. sapiens  
             
 
           
            131 

taggtgctcc ctccggctcc                                                 20 

 
           
             132  
             20  
             DNA  
             H. sapiens  
             
 
           
            132 

tattcgggaa gtgcaatgca                                                 20 

 
           
             133  
             20  
             DNA  
             H. sapiens  
             
 
           
            133 

cgacgtaaag ttcaacagat                                                 20 

 
           
             134  
             20  
             DNA  
             H. sapiens  
             
 
           
            134 

tattcgggaa gtgcaatatc                                                 20