Patent Publication Number: US-2004043956-A1

Title: Antisense modulation of complement component C3 expression

Description:
INTRODUCTION  
     [0001] This application is a continuation of U.S. Ser. No. 10/001,076 filed Oct. 23, 2001, which is herein incorporated by reference in its entirety. 
    
    
     
       FIELD OF THE INVENTION  
       [0002] The present invention provides compositions and methods for modulating the expression of complement component C3. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding complement component C3. Such compounds have been shown to modulate the expression of complement component C3.  
       BACKGROUND OF THE INVENTION  
       [0003] The complement system provides a rapid and efficient means of protecting a host from invasive microorganisms. The complement system consists of about 30 proteins, acting within a cascade-like reaction sequence, that serve as control proteins or as cellular receptors. The complement system can be activated by any of three pathways, either the antibody-dependent classical pathway, the alternative pathway, or the mannan-binding lectin (MBL)/MBL-associated serine protease pathway. Following activation, the complement components form membrane attack complexes which elicit a number of biological effects such as chemotaxis of leukocytes, degranulation of phagocytic cells, mast cells and basophils, smooth muscle contraction, and the increase of vascular permeability (Kirschfink,  Immunopharmacology , 1997, 38, 51-62).  
       [0004] Due to its diverse biological activities, complement is a key mediator of inflammation, a natural response to the host tissue response to any injury. There is also increasing evidence that complement significantly contributes to the regulation of the immune response. Inappropriate or excessive activation of the complement system can lead to harmful, potentially life-threatening consequences due to severe inflammatory tissue destruction. These consequences are clinically manifested in various disorders including septic shock, multiple organ failure, hyperacute organ failure, autoimmune disorders, and CNS inflammation (Kirschfink,  Immunopharmacology , 1997, 38, 51-62).  
       [0005] The role of complement component C3 (also known as C3) is indispensable because it functions in all three pathways in complement activation. The physiological activities of complement component C3 include opsonization and cellular activation via ligation of complement receptors CR1, CR2, and CR3; anaphylatoxic activities mediated by C3a; and binding to Factor B to form the alternative pathway C3bBb C3 convertase enzyme and participation in the classical and alternative pathway with C4 convertase enzymes, C4b2a3b and C3bBbC3b. These different activities of C3 are mediated by different regions of the polypeptide and by attached carbohydrate residues (Fong et al.,  Genomics , 1990, 7, 579-586).  
       [0006] Complement component C3 was isolated and cloned from human liver (de Bruijn and Fey,  Proc. Natl. Acad. Sci. U.S.A ., 1985, 82, 708-712) and mapped to chromosome 19p13.3-p13.2 (Whitehead et al.,  Proc. Natl. Acad. Sci. U.S.A ., 1982, 79, 5021-5025).  
       [0007] Nucleic acid sequences encoding human complement component C3 are disclosed in PCT publication WO 97/32981 (Farries and Harrison, 1997). Disclosed and claimed in U.S. Pat. No. 6,221,657 is a DNA sequence encoding a modified human complement component C3 and a vector comprising said sequence (Harrison and Farries, 2001).  
       [0008] The complete gene is 41 kb and consists of 41 exons. The protein is produced as a single polypeptide of approximately 200 kDa, which is then proteolytically processed to yield the mature protein. The mature protein consists of two disulfide-linked subunits, α and β, of 100 and 75 kDa, respectively (Fong et al.,  Genomics , 1990, 7, 579-586). While the primary site of complement component C3 synthesis is the liver, extra-hepatic synthesis is common and a number of cell types such as macrophages, keratinocytes, kidney tubular epithelial cells, and endothelial cells (Carroll,  Annu. Rev. Immunol ., 1998, 16, 545-568). Adipocytes are also an important source for complement component C3 (Yudkin,  Eur. Heart J ., 2000, 21, 1036-1039).  
       [0009] Muscari et al. demonstrated strong associations between serum levels of complement component C3 and a history of myocardial infarction and stroke. They also report multivariate associations between serum complement component C3 concentrations and those of insulin, triglyceride, and high-density lipoprotein cholesterol (inversely), as well as high blood pressure and obesity (Muscari et al.,  Eur. Heart J ., 2000, 21, 1081-1090).  
       [0010] Nataf et al. used complement component C3 knockout mice to study the mechanisms leading to CNS inflammation and mylein destruction in multiple sclerosis in its animal model, experimental allergic encephalomyelitis (EAE). Although induction of EAE led to inflammatory changes in the meninges and perivascular spaces both wild-type and the knockout animals, there was little infiltration of the parenchyma by macrophages and T-cells. In addition, the knockout mice were protected from demyelination. These results suggest that complement component C3 might be a target for the therapeutic treatment of inflammatory demyelinating diseases of the CNS (Nataf et al.,  J. Immunol ., 2000, 165, 5867-5873).  
       [0011] Mabbott et al. showed that a temporary depletion of complement component C3 significantly delays the onset of scrapie in mice. Transmissable spongiform encephalopathies, like scrapie, require host prion proteins for replication. Depletion of complement component C3 reduces the early accumulation of detergent insoluble, proteinase-resistant prion proteins on the follicular dendritic cells (Mabbott et al.,  Nat. Med ., 2001, 7, 485-487).  
       [0012] The pharmacological modulation of complement component C3 expression is therefore believed to be an appropriate point of therapeutic intervention in pathological conditions.  
       [0013] Currently, there are no known therapeutic agents that effectively inhibit the synthesis of complement component C3.  
       [0014] Anti-complement component C3 antibodies have been used to block the complement cascade (Kirschfink,  Immunopharmacology , 1997, 38, 51-62).  
       [0015] To date, investigative strategies aimed at modulating C3 function have involved the use of antibodies and gene knockouts in mice.  
       [0016] Consequently, there remains a long felt need for additional agents capable of inhibiting complement component C3 function.  
       [0017] 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 complement component C3 expression.  
       [0018] The present in invention provides compositions and methods for modulation complement component C3 expression.  
       SUMMARY OF THE INVENTION  
       [0019] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding complement component C3, and which modulate the expression of complement component C3. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of complement component C3 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 complement component C3 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  
       [0020] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding complement component C3, ultimately modulating the amount of complement component C3 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding complement component C3. As used herein, the terms “target nucleic acid” and “nucleic acid encoding complement component C3” encompass DNA encoding complement component C3, 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, 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 complement component C3. 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.  
       [0021] 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 complement component C3. 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 complement component C3, regardless of the sequence(s) of such codons.  
       [0022] 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.  
       [0023] 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.  
       [0024] 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. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.  
       [0025] 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.  
       [0026] 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. 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 utility, 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.  
       [0027] Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target are identified through experimentation, and the sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The target sites to which these preferred sequences are complementary are hereinbelow referred to as “active sites” and are therefore preferred sites for targeting. Therefore another embodiment of the invention encompasses compounds which hybridize to these active sites.  
       [0028] Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.  
       [0029] For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in combination with other antisense compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.  
       [0030] Expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.  
       [0031] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo,  FEBS Lett ., 2000, 480, 17-24; Celis, et al.,  FEBS Lett ., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al.,  Drug Discov. Today , 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman,  Methods Enzymol ., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al.,  Proc. Natl. Acad. Sci. U.S.A ., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al.,  FEBS Lett ., 2000, 480, 2-16; Jungblut, et al.,  Electrophoresis , 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al.,  FEBS Lett ., 2000, 480, 2-16; Larsson, et al.,  J. Biotechnol ., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al.,  Anal. Biochem ., 2000, 286, 91-98; Larson, et al.,  Cytometry , 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont,  Curr. Opin. Microbiol ., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al.,  J. Cell Biochem. Suppl ., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson,  Eur. J. Cancer , 1999, 35, 1895-904) and mass spectrometry methods (reviewed in (To,  Comb. Chem. High Throughput Screen , 2000, 3, 235-41).  
       [0032] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.  
       [0033] In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.  
       [0034] While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 50 nucleobases (i.e. from about 8 to about 50 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.  
       [0035] 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. 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, aminoalkyl-phosphotriesters, 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 3 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-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH 2 O—CH 2 —N(CH 2 ) 2 , also described in examples hereinbelow.  
       [0044] A further prefered 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 —) 2  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.  
       [0045] Other preferred modifications include 2′-methoxy (2′-O—CH 2 ), 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.  
       [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-aminopropyl-adenine, 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′-0-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 inter-calators, 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 conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al.,  Proc. Natl. Acad. Sci. USA , 1989, 86, 6553-6556), cholic acid (Manoharan et al.,  Bioorg. Med. Chem. Let ., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,  Ann. N.Y. Acad. Sci ., 1992, 660, 306-309; Manoharan et al.,  Bioorg. Med. Chem. Let ., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,  Nucl. Acids Res ., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,  EMBO J ., 1991, 10, 1111-1118; Kabanov et al.,  FEBS Lett ., 1990, 259, 327-330; Svinarchuk et al.,  Biochimie , 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,  Tetrahedron Lett ., 1995, 36, 3651-3654; Shea et al.,  Nucl. Acids Res ., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al.,  Nucleosides  &amp;  Nucleotides , 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al.,  Tetrahedron Lett ., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,  Biochim. Biophys. Acta , 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al.,  J. Pharmacol. Exp. Ther ., 1996, 277, 923-937. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in 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, 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. 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 antisense compounds of the invention are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules.  
       [0054] 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.  
       [0055] 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.  
       [0056] 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.  
       [0057] 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.  
       [0058] 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.  
       [0059] 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.  
       [0060] 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 complement component C3 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.  
       [0061] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding complement component C3, 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 complement component C3 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 complement component C3 in a sample may also be prepared.  
       [0062] 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.  
       [0063] 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.  
       [0064] 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, 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. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999) each of which is incorporated herein by reference in their entirety.  
       [0065] 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.  
       [0066] 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.  
       [0067] 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.  
       [0068] 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.  
       [0069] 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.  
       [0070] Emulsions  
       [0071] 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 of two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be either water-in-oil (w/o) or of 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 provides an o/w/o emulsion.  
       [0072] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).  
       [0073] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).  
       [0074] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.  
       [0075] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).  
       [0076] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.  
       [0077] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.  
       [0078] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailability standpoint. (Rosoff, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.  
       [0079] In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in:  Controlled Release of Drugs: Polymers and Aggregate Systems , Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in  Remington&#39;s Pharmaceutical Sciences , Mack Publishing Co., Easton, Pa., 1985, p. 271).  
       [0080] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.  
       [0081] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic 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.  
       [0082] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al.,  Pharmaceutical Research , 1994, 11, 1385-1390; Ritschel,  Meth. Find. Exp. Clin. Pharmacol ., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al.,  Pharmaceutical Research , 1994, 11, 1385; Ho et al.,  J. Pharm. Sci ., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.  
       [0083] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems , 1991, p. 92). Each of these classes has been discussed above.  
       [0084] Liposomes  
       [0085] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.  
       [0086] Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.  
       [0087] In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.  
       [0088] Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.  
       [0089] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes. As the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.  
       [0090] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.  
       [0091] Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.  
       [0092] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al.,  Biochem. Biophys. Res. Commun ., 1987, 147, 980-985).  
       [0093] Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al.,  Journal of Controlled Release , 1992, 19, 269-274).  
       [0094] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.  
       [0095] Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al.,  Journal of Drug Targeting , 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al.,  Antiviral Research , 1992, 18, 259-265).  
       [0096] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al.  S.T.P.Pharma. Sci ., 1994, 4, 6, 466).  
       [0097] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G M1 , or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al.,  FEBS Letters , 1987, 223, 42; Wu et al.,  Cancer Research , 1993, 53, 3765). Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. ( Ann. N.Y. Acad. Sci ., 1987, 507, 64) reported the ability of monosialoganglioside G M1 , galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. ( Proc. Natl. Acad. Sci. U.S.A ., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G M1  or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).  
       [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 2 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-dihydrofusidate (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, nonchelating 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 2 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′-0-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, the standard cycle for unmodified oligonucleotides was utilized, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds.  
     [0133] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C) 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.).  
     [0134] 2′-Fluoro Amidites  
     [0135] 2′-Fluorodeoxyadenosine Amidites  
     [0136] 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. Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2′-alpha-fluoro atom is introduced by a S N 2-displacement of a 2′-beta-trityl 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 and standard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.  
     [0137] 2′-Fluorodeoxyguanosine  
     [0138] 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 diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give diisobutyryl 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.  
     [0139] 2′-Fluorouridine  
     [0140] 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′-DMT3′phosphoramidites.  
     [0141] 2′-Fluorodeoxycytidine  
     [0142] 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.  
     [0143] 2′-O-(2-Methoxyethyl) Modified Amidites  
     [0144] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared as follows, or alternatively, as per the methods of Martin, P.,  Helvetica Chimica Acta , 1995, 78, 486-504.  
     [0145] 2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine] 
     [0146] 5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid that was crushed to a light tan powder (57 g, 85% crude yield). The NMR spectrum was consistent with the structure, contaminated with phenol as its sodium salt (ca. 5%). The material was used as is for further reactions (or it can be purified further by column chromatography using a gradient of methanol in ethyl acetate (10-25%) to give a white solid, mp 222-4° C.).  
     [0147] 2′-O-Methoxyethyl-5-methyluridine  
     [0148] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160° C. After heating for 48 hours at 155-160° C., the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH 3 CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH 2 Cl 2 /acetone/MeOH (20:5:3) containing 0.5% Et 3 NH. The residue was dissolved in CH 2 Cl 2  (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product. Additional material was obtained by reworking impure fractions.  
     [0149] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine  
     [0150] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH 3 CN (200 mL). The residue was dissolved in CHCl 3  (1.5 L) and extracted with 2×500 mL of saturated NaHCO 3  and 2×500 mL of saturated NaCl. The organic phase was dried over Na 2 SO 4 , filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et 3 NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).  
     [0151] 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine  
     [0152] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by TLC by first quenching the TLC sample with the addition of MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl 3  (800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl 3 . The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%). An additional 1.5 g was recovered from later fractions.  
     [0153] 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine  
     [0154] A first solution was prepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH 3 CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH 3 CN (1 L), cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl 3  was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10° C., and the resulting mixture stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to the latter solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1×300 mL of NaHCO 3  and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.  
     [0155] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine  
     [0156] A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH 4 OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH 3  gas was added and the vessel heated to 100° C. for 2 hours (TLC showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.  
     [0157] N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine  
     [0158] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, TLC showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL). The residue was dissolved in CHCl 3  (700 mL) and extracted with saturated NaHCO 3  (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO 4  and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et 3 NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.  
     [0159] N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite  
     [0160] N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH 2 Cl 2  (1 L). Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (TLC showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO 3  (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH 2 Cl 2  (300 mL), and the extracts were combined, dried over MgSO 4  and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound.  
     [0161] 2′-O-(Aminooxyethyl) Nucleoside Amidites and 2′-O-(dimethylaminooxyethyl) Nucleoside Amidites  
     [0162] 2′-(Dimethylaminooxyethoxy) nucleoside amidites 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.  
     [0163] 5′-O-tert-Butyldiphenylsilyl-O 2 -2′-anhydro-5-methyluridine  
     [0164] O 2 -2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) and the solution was cooled to −10° C. The resulting crystalline product was collected by filtration, washed with ethyl ether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMR were consistent with pure product.  
     [0165] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine  
     [0166] In a 2 L stainless steel, unstirred pressure reactor was added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and with manual stirring, ethylene glycol (350 mL, excess) was added cautiously at first until the evolution of hydrogen gas subsided. 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 and opened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T side product, ethyl acetate) indicated about 70% conversion to the product. In order to avoid additional side product formation, the reaction was stopped, 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 low boiling solvent is gone, the remaining solution can be partitioned between ethyl acetate and water. The product will be in the organic phase.] The residue was purified by column chromatography (2 kg silica gel, ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, stripped and dried to product as a white crisp foam (84 g, 50%), contaminated starting material (17.4 g) and pure reusable starting material 20 g. The yield based on starting material less pure recovered starting material was 58%. TLC and NMR were consistent with 99% pure product.  
     [0167] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine  
     [0168] 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). It was then dried over P 2 O 5  under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture. The rate of addition is maintained such that resulting deep red coloration is just discharged before adding the next drop. After the addition was complete, the reaction was stirred for 4 hrs. By that time TLC showed the completion of the reaction (ethylacetate:hexane, 60:40). The solvent was evaporated in vacuum. Residue obtained was placed on a flash column and eluted with ethyl acetate:hexane (60:40), to get 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%).  
     [0169] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine  
     [0170] 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 was washed with ice cold CH 2 Cl 2  and the combined organic phase was washed with water, brine and dried over anhydrous Na 2 SO 4 . The solution was concentrated to get 2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was strirred for 1 h. Solvent was removed under vacuum; residue chromatographed to get 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%).  
     [0171] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine  
     [0172] 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). Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at 10° C. under inert atmosphere. The reaction mixture was stirred for 10 minutes at 10° C. After that the reaction vessel was removed from the ice bath and stirred at room temperature for 2 h, the reaction monitored by TLC (5% MeOH in CH 2 Cl 2 ). Aqueous NaHCO 3  solution (5%, 10 mL) was added and extracted with ethyl acetate (2×20 mL). Ethyl acetate phase was dried over anhydrous Na 2 SO 4 , evaporated to dryness. Residue was dissolved in a solution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and the reaction mixture was stirred at room temperature for 10 minutes. Reaction mixture cooled to 10° C. in an ice bath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction mixture stirred at 10° C. for 10 minutes. After 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature for 2 hrs. To the reaction mixture 5% NaHCO 3  (25 mL) solution was added and extracted with ethyl acetate (2×25 mL). Ethyl acetate layer was dried over anhydrous Na 2 SO 4  and evaporated to dryness . The residue obtained was purified by flash column chromatography and eluted with 5% MeOH in CH 2 Cl 2  to get 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%).  
     [0173] 2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0174] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). This mixture of triethylamine-2HF was then added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reaction was monitored by TLC (5% MeOH in CH 2 Cl 2 ). Solvent was removed under vacuum and the residue placed on a flash column and eluted with 10% MeOH in CH 2 Cl 2  to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).  
     [0175] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0176] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P 2 O 2  under high vacuum overnight at 40° C. It was then co-evaporated with anhydrous pyridine (20 mL). The residue obtained was dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the mixture and the reaction mixture was stirred at room temperature until all of the starting material disappeared. Pyridine was removed under vacuum and the residue chromatographed and eluted with 10% MeOH in CH 2 Cl 2  (containing a few drops of pyridine) to get 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).  
     [0177] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0178] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and dried over P 2 O 2  under high vacuum overnight at 40° C. Then the reaction mixture 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 hrs under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated, then the residue was dissolved in ethyl acetate (70 mL) and washed with 5% aqueous NaHCO 3  (40 mL). Ethyl acetate layer was dried over anhydrous Na 2 SO 2  and concentrated. Residue obtained was chromatographed (ethyl acetate as eluent) to get 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%).  
     [0179] 2′-(Aminooxyethoxy) nucleoside amidites  
     [0180] 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.  
     [0181] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0182] 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 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].  
     [0183] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites  
     [0184] 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.  
     [0185] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine  
     [0186] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the solid dissolves. O 2 -,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oil bath and heated to 155° C. for 26 hours. The bomb is cooled to room temperature and opened. The crude solution is concentrated and the residue partitioned between water (200 mL) and hexanes (200 mL). The excess phenol is extracted into the hexane layer. The aqueous layer is extracted with ethyl acetate (3×200 mL) and the combined organic layers are washed once with water, dried over anhydrous sodium sulfate and concentrated. The residue is columned on silica gel using methanol/methylene chloride 1:20 (which has 2% triethylamine) as the eluent. As the column fractions are concentrated a colorless solid forms which is collected to give the title compound as a white solid.  
     [0187] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine  
     [0188] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reaction mixture is poured into water (200 mL) and extracted with CH 2 Cl 2  (2×200 mL). The combined CH 2 Cl 2  layers are washed with saturated NaHCO 3  solution, followed by saturated NaCl solution and dried over anhydrous sodium sulfate. Evaporation of the solvent followed by silica gel chromatography using MeOH:CH 2 Cl 2 :Et 3 N (20:1, v/v, with 1% triethylamine) gives the title compound.  
     [0189] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite  
     [0190] Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are 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 is stirred overnight and the solvent evaporated. The resulting residue is purified by silica gel flash column chromatography with ethyl acetate as the eluent to give the title compound.  
     Example 2  
     [0191] Oligonucleotide Synthesis  
     [0192] Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine.  
     [0193] Phosphorothioates (P═S) are synthesized as for the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation wait step was increased to 68 sec and was followed by the capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (18 h), the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.  
     [0194] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.  
     [0195] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 5,610,289 or U.S. Pat. No. 5,625,050, herein incorporated by reference.  
     [0196] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.  
     [0197] 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.  
     [0198] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.  
     [0199] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.  
     [0200] 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  
     [0201] Oligonucleoside Synthesis  
     [0202] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.  
     [0203] 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.  
     [0204] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.  
     Example 4  
     [0205] PNA Synthesis  
     [0206] Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications,  Bioorganic  &amp;  Medicinal Chemistry , 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.  
     Example 5  
     [0207] Synthesis of Chimeric Oligonucleotides  
     [0208] 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”.  
     [0209] [2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides  
     [0210] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 380B, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for RNA and twice for 2′-O-methyl. The fully protected oligonucleotide is cleaved from the support and the phosphate group is deprotected in 3:1 ammonia/ethanol at room temperature overnight then lyophilized to dryness. Treatment in methanolic ammonia for 24 hrs at room temperature is then done to deprotect all bases and sample was again lyophilized to dryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at room temperature to deprotect the 2′ positions. The reaction is then quenched with 1M TEAA and the sample is then reduced to ½ volume by rotovac before being desalted on a G25 size exclusion column. The oligo recovered is then analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.  
     [0211] [2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides  
     [0212] [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.  
     [0213] [2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxy Phosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides  
     [0214] [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, oxidization 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.  
     [0215] 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  
     [0216] Oligonucleotide Isolation  
     [0217] After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C for 18 hours, the oligonucleotides or oligonucleosides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by  31 p nuclear magnetic resonance spectroscopy, and 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  
     [0218] Oligonucleotide Synthesis—96 Well Plate Format  
     [0219] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a standard 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-cyanoethyldiisopropyl 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 known literature or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites. 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  
     [0220] Oligonucleotide Analysis—96 Well Plate Format  
     [0221] 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  
     [0222] Cell Culture and Oligonucleotide Treatment  
     [0223] 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 6 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.  
     [0224] T-24 Cells:  
     [0225] 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 (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (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.  
     [0226] 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.  
     [0227] A549 Cells:  
     [0228] 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 (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.  
     [0229] NHDF Cells:  
     [0230] 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.  
     [0231] HEK Cells:  
     [0232] 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.  
     [0233] HepG2 Cells:  
     [0234] 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.  
     [0235] 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.  
     [0236] HEPA 1-6 Cells:  
     [0237] The mouse hepatoma cell line HEPA 1-6 is a derivative of the BW7756 mouse hepatoma that arose in a C57/L mouse and is supplied by the American Type Culture Collection (Manassas, Va.). The cells are propagated in Dulbecco&#39;s minimal essential medium with 10% fetal bovine serum. Cells are subcultured by removing the medium, adding fresh 0.25% trypsin, 0.03% EDTA solution and letting the culture sit at room temperature for 3 minutes. Trypsin is then removed and the culture allowed to sit an additional 5 minutes until the cells begin to detach, at which point, fresh medium is added.  
     [0238] Treatment with Antisense Compounds:  
     [0239] When cells reached 80% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTINTM (Gibco BRL) 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.  
     [0240] 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 ISIS 13920, TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to human H-ras. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, 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.  
     Example 10  
     [0241] Analysis of oligonucleotide inhibition of complement component C3 expression  
     [0242] Antisense modulation of complement component C3 expression can be assayed in a variety of ways known in the art. For example, complement component C3 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. 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.  
     [0243] Protein levels of complement component C3 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 complement component C3 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.  
     [0244] 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  
     [0245] Poly(A)+mRNA Isolation  
     [0246] 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.  
     [0247] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.  
     Example 12  
     [0248] Total RNA Isolation  
     [0249] 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. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 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 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 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 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water.  
     [0250] 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  
     [0251] Real-Time Quantitative PCR Analysis of Complement Component C3 mRNA Levels  
     [0252] Quantitation of complement component C3 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., JOE, FAM, or VIC, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) 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.  
     [0253] 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.  
     [0254] PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1× TAQMAN™ buffer A, 5.5 mM MgCl 2 , 300 μM each of DATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 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 AMPLITAQ GOLD™, 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).  
     [0255] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, OR). 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.  
     [0256] In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 25 uL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.  
     [0257] Probes and primers to human complement component C3 were designed to hybridize to a human complement component C3 sequence, using published sequence information (GenBank accession number K02765, incorporated herein as SEQ ID NO:3). For human complement component C3 the PCR primers were:  
     [0258] forward primer: CGTGATACACCAAGAAATGATTGG (SEQ ID NO: 4)  
     [0259] reverse primer: CTGCAGCGAGATGAGAACAAAG (SEQ ID NO: 5) and the PCR probe was: FAM-ACAACGAGAAAGACATGGCCCTCACG-TAMRA (SEQ ID NO: 6) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied  
     [0260] Biosystems, Foster City, Calif.) is the quencher dye. For human GAPDH the PCR primers were:  
     [0261] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:7)  
     [0262] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:8) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 9) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.  
     [0263] Probes and primers to mouse complement component C3 were designed to hybridize to a mouse complement component C3 sequence, using published sequence information (GenBank accession number J00367, incorporated herein as SEQ ID NO:10). For mouse complement component C3 the PCR primers were:  
     [0264] forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO:11)  
     [0265] reverse primer: CGACTCCGGGCTCACAAG (SEQ ID NO: 12) and the PCR probe was: FAM-TAGCCGGACATTCAGGTTGATCTTCTCCT-TAMRA (SEQ ID NO: 13) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. For mouse GAPDH the PCR primers were:  
     [0266] forward primer: GGCAAATTCAACGGCACAGT(SEQ ID NO:14)  
     [0267] reverse primer: GGGTCTCGCTCCTGGAAGAT(SEQ ID NO:15) and the PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 16) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMPA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.  
     Example 14  
     [0268] Northern Blot Analysis of Complement Component C3 mRNA Levels  
     [0269] 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 robed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer&#39;s recommendations for stringent conditions.  
     [0270] To detect human complement component C3, a human complement component C3 specific probe was prepared by PCR using the forward primer CGTGATACACCAAGAAATGATTGG (SEQ ID NO: 4) and the reverse primer CTGCAGCGAGATGAGAACAAAG (SEQ ID NO: 5). 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.).  
     [0271] To detect mouse complement component C3, a mouse complement component C3 specific probe was prepared by PCR using the forward primer AAGCTGTGCCACAGTGAAATGT (SEQ ID NO: 11) and the reverse primer CGACTCCGGGCTCACAAG (SEQ ID NO: 12). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).  
     [0272] 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  
     [0273] Antisense Inhibition of Human Complement Component C3 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap  
     [0274] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human complement component C3 RNA, using published sequences (GenBank accession number K02765, incorporated herein as SEQ ID NO: 3 and GenBank accession number M55658, incorporated herein as SEQ ID NO: 17). 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 complement component C3 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, “N.D.” indicates “no data”.  
                   TABLE 1                          Inhibition of human complement component C3 mRNA levels by           chimeric phosphorothioate oligonucleotides having 2′-MOE       wings and a deoxy gap                                                     TARGET   TARGET           SEQ ID           ISIS #   REGION   SEQ ID NO   SITE   SEQUENCE   % INHIB   NO                                                     139964   Start   3   45   ccatggtgctgggacagtgc   44   19               Codon                                   139965   Start   3   54   aggtgggtcccatggtgctg   26   20           Codon                                   139966   Coding   3   84   ttagtagcaggagcagcagg   25   21               139967   5′UTR   17   179   ctgttggacccactttgtag   11   22               139968   Coding   3   185   gtcgtgggcctccagcacca   76   23               139969   Coding   3   214   gtaacagtgactggaacatc   58   24               139970   5′UTR   17   231   ctgccctggactctcccagg   9   25               139971   Coding   3   254   actggacagcactagttttt   68   26               139972   5′UTR   17   257   cccagcagcatgaatgcagc   2   27               139973   5′UTR   17   271   ctgagggcatgttccccagc   16   28               139974   Coding   3   330   tgaactccctgttggctggg   79   29               139975   Coding   3   445   tctgtctggatgaagaggta   61   30               139976   Coding   3   450   tcttgtctgtctggatgaag   69   31               139977   Coding   3   455   gatggtcttgtctgtctgga   56   32               139978   Coding   3   460   gtgtagatggtcttgtctgt   20   33               139979   Coding   3   505   ttgtggttgacggtgaagat   54   34               139980   Coding   3   725   cacgtactccttcacctcaa   6   35               139981   Coding   3   766   aatttctctgtaggctccac   1   36               139982   Coding   3   822   agaggaacctggcggtgatg   32   37               139983   Coding   3   877   tcgccatcctggatcccgaa   40   38               139984   Coding   3   922   tcaatcggaatgcgcttgag   26   39               139985   Coding   3   1009   gacttccccaccaggtcttc   7   40               139986   Coding   3   1064   tgcctgcaccatgtcactgc   47   41               139987   Coding   3   1117   ttggtgaagtggatctggta   60   42               139988   Coding   3   1123   ggtgtcttggtgaagtggat   75   43               139989   Coding   3   1163   caccatgaggtcaaagggca   63   44               139990   Coding   3   1170   tcacgaacaccatgaggtca   31   45               139991   Coding   3   1193   ggctggagagccatcagggt   41   46               139992   Coding   3   1344   ctgcctccgagagctcctgc   68   47               139993   Coding   3   1402   ttgttggagttgcccacggt   21   48               139994   Coding   3   1430   tgtacgtagcactgagagat   25   49               139995   Coding   3   1583   caggtcctggccgggctctc   66   50               139996   Coding   3   1652   cagcgtgtagtacgccacca   49   51               139997   Coding   3   1860   tattcagcacgaacacgccc   43   52               139998   Coding   3   2246   ccgcagctctgtgatgtagt   29   53               139999   Coding   3   2279   caggcccaggtggctggccc   26   54               140000   Coding   3   2406   tcgtagagattccatttttc   45   55               140001   Coding   3   2448   cccacgtggtgatggagtct   56   56               140002   Coding   3   2483   ccctttcttgtccgacatgc   66   57               140003   Coding   3   2492   cacacagatccctttcttgt   18   58               140004   Coding   3   2499   ggtctgccacacagatccct   49   59               140005   Coding   3   2768   ttcctgcaggccggtcttta   53   60               140006   Coding   3   2788   acggcagccttgacttccac   61   61               140007   Coding   3   2953   acttggtcactgaggtctgc   57   62               140008   Coding   3   2987   caggagaattctggtctcag   15   63               140009   Coding   3   2992   ccttgcaggagaattctggt   48   64               140010   Coding   3   2997   gggtcccttgcaggagaatt   45   65               140011   Coding   3   3127   tccaggtaatgcacagcgat   48   66               140012   Coding   3   3214   gccagctgctgggtgtaccc   74   67               140013   Coding   3   3219   tgaaggccagctgctgggtg   50   68               140014   Coding   3   3298   aagaccttgaccacgtaggc   42   69               140015   Coding   3   3304   agagagaagaccttgaccac   34   70               140016   Coding   3   3330   agtcgatggcgatgaggttg   66   71               140017   Coding   3   3383   gggcttctgcttctccagga   59   72               140018   Coding   3   3395   gaagaccccgtcgggcttct   37   73               140019   Coding   3   3425   ttcttggtgtatcacgggcg   60   74               140020   Coding   3   3436   ccaccaatcatttcttggtg   44   75               140021   Coding   3   3517   tcgcaaatatctttagcctc   0   76               140022   Coding   3   3533   gctgttgacctgctcctcgc   0   77               140023   Coding   3   3606   cagtgtaggatctctgtagg   52   78               140024   Coding   3   3692   cttatctttggctgtggtca   42   79               140025   Coding   3   3714   taccagggtcctcccagcgg   45   80               140026   Coding   3   3748   gcataggatgtggcctccac   27   81               140027   Coding   3   3871   tggaacaccatgaaggtggc   3   82               140028   Coding   3   3887   gtattgagccaaggcttgga   46   83               140029   Coding   3   3965   ggtgatcttggagctgcggc   47   84               140030   Coding   3   4187   gttcttggcatcctgaggcc   55   85               140031   Coding   3   4276   gcaaagccagtcatcatgga   61   86               140032   Coding   3   4281   ctggagcaaagccagtcatc   14   87               140033   Coding   3   4286   tgtgtctggagcaaagccag   24   88               140034   Coding   3   4355   gaaggctttgtccagctcat   79   89               140035   Coding   3   4383   ggtagatgatgagggtgttc   60   90               140036   Coding   3   4406   ctcagagtgtgagaccttgt   75   91               140037   Coding   3   4533   ccttttccggatggtagaac   48   92               140038   Coding   3   4724   gtactcgtcaaagtcattgg   59   93               140039   Coding   3   4856   ccacatgaggtagtgtttct   35   94               140040   Coding   3   4870   tcggaggagagaccccacat   28   95               140041   Coding   3   4939   tcaggccagtgctccaccca   66   96                  
 
     [0275] As shown in Table 1, SEQ ID NOs 19, 20, 21, 23, 24, 30, 31, 32, 34, 37, 38, 39, 41, 42, 43, 44, 45, 46, 50, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 64, 67, 68, 69, 70, 71, 72, 73, 74, 75, 78, 79, 80, 81, 85, 86, 89, 90, 91, 92, 93, 94, 95, 96, 100 and 107 demondsrated at least 25% inhibition of human complement component C3 expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention.  
     Example 16  
     [0276] Antisense Inhibition of Mouse Complement Component C3 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap.  
     [0277] In accordance with the present invention, a second series of oligonucleotides were designed to target different regions of the mouse complement component C3 RNA, using published sequences (GenBank accession number J00367, incorporated herein as SEQ ID NO: 10, GenBank accession number K02782, incorporated herein as SEQ ID NO: 111, and GenBank accession number Z37998, incorporated herein as SEQ ID NO: 112). The oligonucleotides are shown in Table 2. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on mouse complement component C3 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, “N.D.” indicates “no data”.  
                   TABLE 2                          Inhibition of mouse complement component C3 mRNA levels by           chimeric phosphorothioate oligonucleotides having 2′-MOE       wings and a deoxy gap                                                     TARGET   TARGET           SEQ ID           ISIS #   REGION   SEQ ID NO   SITE   SEQUENCE   % INHIB   NO                                                     139975   Exon   10   1973   tctgtctggatgaagaggta   59   30                   139976   Exon   10   1978   tcttgtctgtctggatgaag   79   31               139977   Exon   10   1983   gatggtcttgtctgtctgga   76   32               139978   Exon   10   1988   gtgtagatggtcttgtctgt   52   33               139987   Exon   10   2645   ttggtgaagtggatctggta   61   42               139988   Exon   10   2651   ggtgtcttggtgaagtggat   69   43               140000   Exon   10   3931   tcgtagagattccatttttc   65   55               140012   Exon   10   4739   gccagctgctgggtgtaccc   31   67               140016   Exon   11   2377   agtcgatggcgatgaggttg   65   71               140020   Exon   10   4961   ccaccaatcatttcttggtg   71   75               140035   Exon   10   5908   ggtagatgatgagggtgttc   0   90               140044   Start   11   148   gctggtcccatagtgaagga   56   113           Codon                                   140045   Coding   11   179   cagcagtagcactagtagct   79   114               140046   Coding   11   195   gggagctggccaacagcagc   68   115               140047   Coding   11   1276   gctcctgtcaacactgtctt   71   116               140048   Coding   11   1320   tactggctggaatcttgatg   74   117               140049   Coding   11   1380   ccccgaagtttgccaccact   87   118               140050   Coding   11   1535   gatgacgactgtcttgccca   4   119               140051   Coding   11   1730   gggcagcacgtattccttca   62   120               140052   Coding   11   1895   ggccagagaaatcttcttat   57   121               140053   Coding   11   11079   ggatcccactgcgctctgcc   15   122               140054   Coding   11   11128   aatttgggtgtcttggtgaa   57   123               140055   Coding   11   11389   ttgtgcatagtgctgtaggg   63   124               140056   Coding   11   11470   cgcaggtggaagttgacatt   77   125               140057   Coding   11   11534   cttccccttgttcataacca   54   126               140058   Coding   11   11645   ggtgtagtaagccaccaggc   68   127               140059   Coding   11   11834   cttgtccacagccactagcc   32   128               140060   Coding   11   11937   agttcttcccactgcctggg   80   129               140061   Coding   11   12094   gtgtactgaccagctttgtc   74   130               140062   Coding   11   12191   ctcgccctgggtgatgaggc   43   131               140063   Coding   11   12286   tcactcctggccaggcccag   80   132               140064   Coding   11   12341   tgggaagtggcttctagaga   47   133               140065   Coding   11   12373   ttcaactcttctatggtcca   10   134               140066   Coding   11   12432   tggaatctttgagaaagatg   62   135               140067   Coding   11   12442   caggtggtgatggaatcttt   62   136               140068   Coding   11   12465   acaagctcactgccagaatc   0   137               140069   Coding   11   12595   ttgaagagcacagctctgat   55   138               140070   Coding   11   12665   ggccatgctgcagaaggctg   37   139               140071   Coding   11   12842   tccttctggcacgaccttca   75   140               140072   Coding   11   12961   tctgtgtctggcacttggtc   62   141               140073   Coding   11   13043   gtgtttcagccgctccccgt   0   142               140074   Coding   11   13053   tcacgatcaggtgtttcagc   82   143               140075   Coding   11   13095   gtgtcatgccaatcatgttc   79   144               140076   Coding   11   13193   ccctttcttgatgagctcca   82   145               140077   Coding   11   13247   gttgaaggcagcataggcag   52   146               140078   Coding   11   13276   gctgtcagccaggtgctggg   62   147               140079   Coding   11   13376   cttctgtttctccagaatca   0   148               140080   Coding   11   13399   tcctcctgaaagacaccatc   84   149               140081   Coding   11   13496   ttcctgcagtgcgatgagga   59   150               140082   Coding   11   13525   ttgacctgcccctcacagat   75   151               140083   Coding   11   13555   cctgccttgttgatgctccc   82   152               140084   Coding   11   13598   gtatggtctctgcaggttca   58   153               140085   Coding   11   13624   agggcatacccagcaatggc   71   154               140086   Coding   11   13644   ccagtttgttcatcagggcc   0   155               140087   Coding   11   13655   gtaaggttcctccagtttgt   75   156               140088   Coding   111   3702   tcctcccagcggttccgatc   75   157               140089   Coding   111   3768   ttcagcagcagcagggccag   67   158               140090   Coding   111   3786   ggcacagagtcaaagtcttt   80   159               140091   Coding   111   3865   gaataccatgaaggtagcct   47   160               140092   Coding   111   3875   ccaaggcttggaataccatg   81   161               140093   Coding   111   3925   cacatccatgttcaagtcct   39   162               140094   Coding   111   4133   tgaccctgaggtcaaacttc   55   163               140095   Coding   111   4167   ggcttcttggctgtctcagg   72   164               140096   Coding   111   4252   gatgtccaggatggacatag   27   165               140097   Coding   111   4270   aaagccagtcatcatggaga   86   166               140098   Coding   111   4361   tcttgttggagaaggctttg   21   167               140099   Coding   111   4389   atcttttctaggtagatgat   2   168               140100   Coding   111   4479   gagtagaccttgaccgaccc   84   169               140101   Coding   111   4519   atgatagaaccgggtgcatg   32   170               140102   Coding   111   4653   ggctcacaagccttgtctag   29   171               140103   Coding   111   4753   gcctgacttgatgacctgct   21   172               140104   Coding   111   4773   cctgcctgcacctcatctga   13   173               140105   Coding   111   4858   gaggccccacatgaggtact   78   174               140106   Coding   111   4885   gggcttttctccccagaggt   62   175               140107   Coding   111   4909   cttcccaatgatgtagctgg   88   176               140108   Coding   111   4929   cagtgctccacccacgtgtc   85   177               140109   Stop   111   5037   ggctgtagtcagttgggaca   50   178           Codon                                   140110   3′UTR   111   5067   aaatacaactgaagctttat   77   179                  
 
     [0278] As shown in Table 2, SEQ ID NOs 30, 31, 32, 33, 42, 43, 55, 71, 75, 113, 114, 115, 116, 117, 118, 120, 121, 123,124, 125, 126, 127, 129, 130, 131, 132, 133, 135, 136, 138, 140, 141, 143, 144, 145, 146, 147, 149, 150, 151, 152, 153, 154, 156, 157, 158, 159, 160, 161, 163, 164, 166, 169, 174, 175, 176, 177, 178 and 179 demonstrated at least 43% inhibition of mouse complement component C3 expression in this periment and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention.  
     Example 17  
     [0279] Western Blot Analysis of Complement Component C3 Protein Levels  
     [0280] 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 complement component C3 is used, with a radiolabelled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).  
    
     
       
         1 
         
           
             179  
           
           
             1  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            1 

tccgtcatcg ctcctcaggg                                                 20 

 
           
             2  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            2 

atgcattctg cccccaagga                                                 20 

 
           
             3  
             5067  
             DNA  
             Homo sapiens  
             
               CDS  
               (61)...(5052)  
             
           
            3 

ctcctcccca tcctctccct ctgtccctct gtccctctga ccctgcactg tcccagcacc     60 

atg gga ccc acc tca ggt ccc agc ctg ctg ctc ctg cta cta acc cac      108 
Met Gly Pro Thr Ser Gly Pro Ser Leu Leu Leu Leu Leu Leu Thr His 
  1               5                  10                  15 

ctc ccc ctg gct ctg ggg agt ccc atg tac tct atc atc acc ccc aac      156 
Leu Pro Leu Ala Leu Gly Ser Pro Met Tyr Ser Ile Ile Thr Pro Asn 
             20                  25                  30 

atc ttg cgg ctg gag agc gag gag acc atg gtg ctg gag gcc cac gac      204 
Ile Leu Arg Leu Glu Ser Glu Glu Thr Met Val Leu Glu Ala His Asp 
         35                  40                  45 

gcg caa ggg gat gtt cca gtc act gtt act gtc cac gac ttc cca ggc      252 
Ala Gln Gly Asp Val Pro Val Thr Val Thr Val His Asp Phe Pro Gly 
     50                  55                  60 

aaa aaa cta gtg ctg tcc agt gag aag act gtg ctg acc cct gcc acc      300 
Lys Lys Leu Val Leu Ser Ser Glu Lys Thr Val Leu Thr Pro Ala Thr 
 65                  70                  75                  80 

aac cac atg ggc aac gtc acc ttc acg atc cca gcc aac agg gag ttc      348 
Asn His Met Gly Asn Val Thr Phe Thr Ile Pro Ala Asn Arg Glu Phe 
                 85                  90                  95 

aag tca gaa aag ggg cgc aac aag ttc gtg acc gtg cag gcc acc ttc      396 
Lys Ser Glu Lys Gly Arg Asn Lys Phe Val Thr Val Gln Ala Thr Phe 
            100                 105                 110 

ggg acc caa gtg gtg gag aag gtg gtg ctg gtc agc ctg cag agc ggg      444 
Gly Thr Gln Val Val Glu Lys Val Val Leu Val Ser Leu Gln Ser Gly 
        115                 120                 125 

tac ctc ttc atc cag aca gac aag acc atc tac acc cct ggc tcc aca      492 
Tyr Leu Phe Ile Gln Thr Asp Lys Thr Ile Tyr Thr Pro Gly Ser Thr 
    130                 135                 140 

gtt ctc tat cgg atc ttc acc gtc aac cac aag ctg cta ccc gtg ggc      540 
Val Leu Tyr Arg Ile Phe Thr Val Asn His Lys Leu Leu Pro Val Gly 
145                 150                 155                 160 

cgg acg gtc atg gtc aac att gag aac ccg gaa ggc atc ccg gtc aag      588 
Arg Thr Val Met Val Asn Ile Glu Asn Pro Glu Gly Ile Pro Val Lys 
                165                 170                 175 

cag gac tcc ttg tct tct cag aac cag ctt ggc gtc ttg ccc ttg tct      636 
Gln Asp Ser Leu Ser Ser Gln Asn Gln Leu Gly Val Leu Pro Leu Ser 
            180                 185                 190 

tgg gac att ccg gaa ctc gtc aac atg ggc cag tgg aag atc cga gcc      684 
Trp Asp Ile Pro Glu Leu Val Asn Met Gly Gln Trp Lys Ile Arg Ala 
        195                 200                 205 

tac tat gaa aac tca cca cag cag gtc ttc tcc act gag ttt gag gtg      732 
Tyr Tyr Glu Asn Ser Pro Gln Gln Val Phe Ser Thr Glu Phe Glu Val 
    210                 215                 220 

aag gag tac gtg ctg ccc agt ttc gag gtc ata gtg gag cct aca gag      780 
Lys Glu Tyr Val Leu Pro Ser Phe Glu Val Ile Val Glu Pro Thr Glu 
225                 230                 235                 240 

aaa ttc tac tac atc tat aac gag aag ggc ctg gag gtc acc atc acc      828 
Lys Phe Tyr Tyr Ile Tyr Asn Glu Lys Gly Leu Glu Val Thr Ile Thr 
                245                 250                 255 

gcc agg ttc ctc tac ggg aag aaa gtg gag gga act gcc ttt gtc atc      876 
Ala Arg Phe Leu Tyr Gly Lys Lys Val Glu Gly Thr Ala Phe Val Ile 
            260                 265                 270 

ttc ggg atc cag gat ggc gaa cag agg att tcc ctg cct gaa tcc ctc      924 
Phe Gly Ile Gln Asp Gly Glu Gln Arg Ile Ser Leu Pro Glu Ser Leu 
        275                 280                 285 

aag cgc att ccg att gag gat ggc tcg ggg gag gtt gtg ctg agc cgg      972 
Lys Arg Ile Pro Ile Glu Asp Gly Ser Gly Glu Val Val Leu Ser Arg 
    290                 295                 300 

aag gta ctg ctg gac ggg gtg cag aac ctc cga gca gaa gac ctg gtg     1020 
Lys Val Leu Leu Asp Gly Val Gln Asn Leu Arg Ala Glu Asp Leu Val 
305                 310                 315                 320 

ggg aag tct ttg tac gtg tct gcc acc gtc atc ttg cac tca ggc agt     1068 
Gly Lys Ser Leu Tyr Val Ser Ala Thr Val Ile Leu His Ser Gly Ser 
                325                 330                 335 

gac atg gtg cag gca gag cgc agc ggg atc ccc atc gtg acc tct ccc     1116 
Asp Met Val Gln Ala Glu Arg Ser Gly Ile Pro Ile Val Thr Ser Pro 
            340                 345                 350 

tac cag atc cac ttc acc aag aca ccc aag tac ttc aaa cca gga atg     1164 
Tyr Gln Ile His Phe Thr Lys Thr Pro Lys Tyr Phe Lys Pro Gly Met 
        355                 360                 365 

ccc ttt gac ctc atg gtg ttc gtg acg aac cct gat ggc tct cca gcc     1212 
Pro Phe Asp Leu Met Val Phe Val Thr Asn Pro Asp Gly Ser Pro Ala 
    370                 375                 380 

tac cga gtc ccc gtg gca gtc cag ggc gag gac act gtg cag tct cta     1260 
Tyr Arg Val Pro Val Ala Val Gln Gly Glu Asp Thr Val Gln Ser Leu 
385                 390                 395                 400 

acc cag gga gat ggc gtg gcc aaa ctc agc atc aac aca cac ccc agc     1308 
Thr Gln Gly Asp Gly Val Ala Lys Leu Ser Ile Asn Thr His Pro Ser 
                405                 410                 415 

cag aag ccc ttg agc atc acg gtg cgc acg aag aag cag gag ctc tcg     1356 
Gln Lys Pro Leu Ser Ile Thr Val Arg Thr Lys Lys Gln Glu Leu Ser 
            420                 425                 430 

gag gca gag cag gct acc agg acc atg cag gct ctg ccc tac agc acc     1404 
Glu Ala Glu Gln Ala Thr Arg Thr Met Gln Ala Leu Pro Tyr Ser Thr 
        435                 440                 445 

gtg ggc aac tcc aac aat tac ctg cat ctc tca gtg cta cgt aca gag     1452 
Val Gly Asn Ser Asn Asn Tyr Leu His Leu Ser Val Leu Arg Thr Glu 
    450                 455                 460 

ctc aga ccc ggg gag acc ctc aac gtc aac ttc ctc ctg cga atg gac     1500 
Leu Arg Pro Gly Glu Thr Leu Asn Val Asn Phe Leu Leu Arg Met Asp 
465                 470                 475                 480 

cgc gcc cac gag gcc aag atc cgc tac tac acc tac ctg atc atg aac     1548 
Arg Ala His Glu Ala Lys Ile Arg Tyr Tyr Thr Tyr Leu Ile Met Asn 
                485                 490                 495 

aag ggc agg ctg ttg aag gcg gga cgc cag gtg cga gag ccc ggc cag     1596 
Lys Gly Arg Leu Leu Lys Ala Gly Arg Gln Val Arg Glu Pro Gly Gln 
            500                 505                 510 

gac ctg gtg gtg ctg ccc ctg tcc atc acc acc gac ttc atc cct tcc     1644 
Asp Leu Val Val Leu Pro Leu Ser Ile Thr Thr Asp Phe Ile Pro Ser 
        515                 520                 525 

ttc cgc ctg gtg gcg tac tac acg ctg atc ggt gcc agc ggc cag agg     1692 
Phe Arg Leu Val Ala Tyr Tyr Thr Leu Ile Gly Ala Ser Gly Gln Arg 
    530                 535                 540 

gag gtg gtg gcc gac tcc gtg tgg gtg gac gtc aag gac tcc tgc gtg     1740 
Glu Val Val Ala Asp Ser Val Trp Val Asp Val Lys Asp Ser Cys Val 
545                 550                 555                 560 

ggc tcg ctg gtg gta aaa agc ggc cag tca gaa gac cgg cag cct gta     1788 
Gly Ser Leu Val Val Lys Ser Gly Gln Ser Glu Asp Arg Gln Pro Val 
                565                 570                 575 

cct ggg cag cag atg acc ctg aag ata gag ggt gac cac ggg gcc cgg     1836 
Pro Gly Gln Gln Met Thr Leu Lys Ile Glu Gly Asp His Gly Ala Arg 
            580                 585                 590 

gtg gta ctg gtg gcc gtg gac aag ggc gtg ttc gtg ctg aat aag aag     1884 
Val Val Leu Val Ala Val Asp Lys Gly Val Phe Val Leu Asn Lys Lys 
        595                 600                 605 

aac aaa ctg acg cag agt aag atc tgg gac gtg gtg gag aag gca gac     1932 
Asn Lys Leu Thr Gln Ser Lys Ile Trp Asp Val Val Glu Lys Ala Asp 
    610                 615                 620 

atc ggc tgc acc ccg ggc agt ggg aag gat tac gcc ggt gtc ttc tcc     1980 
Ile Gly Cys Thr Pro Gly Ser Gly Lys Asp Tyr Ala Gly Val Phe Ser 
625                 630                 635                 640 

gac gca ggg ctg acc ttc acg agc agc agt ggc cag cag acc gcc cag     2028 
Asp Ala Gly Leu Thr Phe Thr Ser Ser Ser Gly Gln Gln Thr Ala Gln 
                645                 650                 655 

agg gca gaa ctt cag tgc ccg cag cca gcc gcc cgc cga cgc cgt tcc     2076 
Arg Ala Glu Leu Gln Cys Pro Gln Pro Ala Ala Arg Arg Arg Arg Ser 
            660                 665                 670 

gtg cag ctc acg gag aag cga atg gac aaa gtc ggc aag tac ccc aag     2124 
Val Gln Leu Thr Glu Lys Arg Met Asp Lys Val Gly Lys Tyr Pro Lys 
        675                 680                 685 

gag ctg cgc aag tgc tgc gag gac ggc atg cgg gag aac ccc atg agg     2172 
Glu Leu Arg Lys Cys Cys Glu Asp Gly Met Arg Glu Asn Pro Met Arg 
    690                 695                 700 

ttc tcg tgc cag cgc cgg acc cgt ttc atc tcc ctg ggc gag gcg tgc     2220 
Phe Ser Cys Gln Arg Arg Thr Arg Phe Ile Ser Leu Gly Glu Ala Cys 
705                 710                 715                 720 

aag aag gtc ttc ctg gac tgc tgc aac tac atc aca gag ctg cgg cgg     2268 
Lys Lys Val Phe Leu Asp Cys Cys Asn Tyr Ile Thr Glu Leu Arg Arg 
                725                 730                 735 

cag cac gcg cgg gcc agc cac ctg ggc ctg gcc agg agt aac ctg gat     2316 
Gln His Ala Arg Ala Ser His Leu Gly Leu Ala Arg Ser Asn Leu Asp 
            740                 745                 750 

gag gac atc att gca gaa gag aac atc gtt tcc cga agt gag ttc cca     2364 
Glu Asp Ile Ile Ala Glu Glu Asn Ile Val Ser Arg Ser Glu Phe Pro 
        755                 760                 765 

gag agc tgg ctg tgg aac gtt gag gac ttg aaa gag cca ccg aaa aat     2412 
Glu Ser Trp Leu Trp Asn Val Glu Asp Leu Lys Glu Pro Pro Lys Asn 
    770                 775                 780 

gga atc tct acg aag ctc atg aat ata ttt ttg aaa gac tcc atc acc     2460 
Gly Ile Ser Thr Lys Leu Met Asn Ile Phe Leu Lys Asp Ser Ile Thr 
785                 790                 795                 800 

acg tgg gag att ctg gct gtc agc atg tcg gac aag aaa ggg atc tgt     2508 
Thr Trp Glu Ile Leu Ala Val Ser Met Ser Asp Lys Lys Gly Ile Cys 
                805                 810                 815 

gtg gca gac ccc ttc gag gtc aca gta atg cag gac ttc ttc atc gac     2556 
Val Ala Asp Pro Phe Glu Val Thr Val Met Gln Asp Phe Phe Ile Asp 
            820                 825                 830 

ctg cgg cta ccc tac tct gtt gtt cga aac gag cag gtg gaa atc cga     2604 
Leu Arg Leu Pro Tyr Ser Val Val Arg Asn Glu Gln Val Glu Ile Arg 
        835                 840                 845 

gcc gtt ctc tac aat tac cgg cag aac caa gag ctc aag gtg agg gtg     2652 
Ala Val Leu Tyr Asn Tyr Arg Gln Asn Gln Glu Leu Lys Val Arg Val 
    850                 855                 860 

gaa cta ctc cac aat cca gcc ttc tgc agc ctg gcc acc acc aag agg     2700 
Glu Leu Leu His Asn Pro Ala Phe Cys Ser Leu Ala Thr Thr Lys Arg 
865                 870                 875                 880 

cgt cac cag cag acc gta acc atc ccc ccc aag tcc tcg ttg tcc gtt     2748 
Arg His Gln Gln Thr Val Thr Ile Pro Pro Lys Ser Ser Leu Ser Val 
                885                 890                 895 

cca tat gtc atc gtg ccg cta aag acc ggc ctg cag gaa gtg gaa gtc     2796 
Pro Tyr Val Ile Val Pro Leu Lys Thr Gly Leu Gln Glu Val Glu Val 
            900                 905                 910 

aag gct gcc gtc tac cat cat ttc atc agt gac ggt gtc agg aag tcc     2844 
Lys Ala Ala Val Tyr His His Phe Ile Ser Asp Gly Val Arg Lys Ser 
        915                 920                 925 

ctg aag gtc gtg ccg gaa gga atc aga atg aac aaa act gtg gct gtt     2892 
Leu Lys Val Val Pro Glu Gly Ile Arg Met Asn Lys Thr Val Ala Val 
    930                 935                 940 

cgc acc ctg gat cca gaa cgc ctg ggc cgt gaa gga gtg cag aaa gag     2940 
Arg Thr Leu Asp Pro Glu Arg Leu Gly Arg Glu Gly Val Gln Lys Glu 
945                 950                 955                 960 

gac atc cca cct gca gac ctc agt gac caa gtc ccg gac acc gag tct     2988 
Asp Ile Pro Pro Ala Asp Leu Ser Asp Gln Val Pro Asp Thr Glu Ser 
                965                 970                 975 

gag acc aga att ctc ctg caa ggg acc cca gtg gcc cag atg aca gag     3036 
Glu Thr Arg Ile Leu Leu Gln Gly Thr Pro Val Ala Gln Met Thr Glu 
            980                 985                 990 

gat gcc gtc gac gcg gaa cgg ctg aag cac ctc att gtg acc ccc tcg     3084 
Asp Ala Val Asp Ala Glu Arg Leu Lys His Leu Ile Val Thr Pro Ser 
        995                 1000                1005 

ggc tgc ggg gaa cag aac atg atc ggc atg acg ccc acg gtc atc gct     3132 
Gly Cys Gly Glu Gln Asn Met Ile Gly Met Thr Pro Thr Val Ile Ala 
    1010                1015                1020 

gtg cat tac ctg gat gaa acg gag cag tgg gag aag ttc ggc cta gag     3180 
Val His Tyr Leu Asp Glu Thr Glu Gln Trp Glu Lys Phe Gly Leu Glu 
1025                1030                1035                1040 

aag cgg cag ggg gcc ttg gag ctc atc aag aag ggg tac acc cag cag     3228 
Lys Arg Gln Gly Ala Leu Glu Leu Ile Lys Lys Gly Tyr Thr Gln Gln 
                1045                1050                1055 

ctg gcc ttc aga caa ccc agc tct gcc ttt gcg gcc ttc gtg aaa cgg     3276 
Leu Ala Phe Arg Gln Pro Ser Ser Ala Phe Ala Ala Phe Val Lys Arg 
            1060                1065                1070 

gca ccc agc acc tgg ctg acc gcc tac gtg gtc aag gtc ttc tct ctg     3324 
Ala Pro Ser Thr Trp Leu Thr Ala Tyr Val Val Lys Val Phe Ser Leu 
        1075                1080                1085 

gct gtc aac ctc atc gcc atc gac tcc caa gtc ctc tgc ggg gct gtt     3372 
Ala Val Asn Leu Ile Ala Ile Asp Ser Gln Val Leu Cys Gly Ala Val 
    1090                1095                1100 

aaa tgg ctg atc ctg gag aag cag aag ccc gac ggg gtc ttc cag gag     3420 
Lys Trp Leu Ile Leu Glu Lys Gln Lys Pro Asp Gly Val Phe Gln Glu 
1105                1110                1115                1120 

gat gcg ccc gtg ata cac caa gaa atg att ggt gga tta cgg aac aac     3468 
Asp Ala Pro Val Ile His Gln Glu Met Ile Gly Gly Leu Arg Asn Asn 
                1125                1130                1135 

aac gag aaa gac atg gcc ctc acg gcc ttt gtt ctc atc tcg ctg cag     3516 
Asn Glu Lys Asp Met Ala Leu Thr Ala Phe Val Leu Ile Ser Leu Gln 
            1140                1145                1150 

gag gct aaa gat att tgc gag gag cag gtc aac agc ctg cca ggc agc     3564 
Glu Ala Lys Asp Ile Cys Glu Glu Gln Val Asn Ser Leu Pro Gly Ser 
        1155                1160                1165 

atc act aaa gca gga gac ttc ctt gaa gcc aac tac atg aac cta cag     3612 
Ile Thr Lys Ala Gly Asp Phe Leu Glu Ala Asn Tyr Met Asn Leu Gln 
    1170                1175                1180 

aga tcc tac act gtg gcc att gct ggc tat gct ctg gcc cag atg ggc     3660 
Arg Ser Tyr Thr Val Ala Ile Ala Gly Tyr Ala Leu Ala Gln Met Gly 
1185                1190                1195                1200 

agg ctg aag ggg cct ctt ctt aac aaa ttt ctg acc aca gcc aaa gat     3708 
Arg Leu Lys Gly Pro Leu Leu Asn Lys Phe Leu Thr Thr Ala Lys Asp 
                1205                1210                1215 

aag aac cgc tgg gag gac cct ggt aag cag ctc tac aac gtg gag gcc     3756 
Lys Asn Arg Trp Glu Asp Pro Gly Lys Gln Leu Tyr Asn Val Glu Ala 
            1220                1225                1230 

aca tcc tat gcc ctc ttg gcc cta ctg cag cta aaa gac ttt gac ttt     3804 
Thr Ser Tyr Ala Leu Leu Ala Leu Leu Gln Leu Lys Asp Phe Asp Phe 
        1235                1240                1245 

gtg cct ccc gtc gtg cgt tgg ctc aat gaa cag aga tac tac ggt ggt     3852 
Val Pro Pro Val Val Arg Trp Leu Asn Glu Gln Arg Tyr Tyr Gly Gly 
    1250                1255                1260 

ggc tat ggc tct acc cag gcc acc ttc atg gtg ttc caa gcc ttg gct     3900 
Gly Tyr Gly Ser Thr Gln Ala Thr Phe Met Val Phe Gln Ala Leu Ala 
1265                1270                1275                1280 

caa tac caa aag gac gcc cct gac cac cag gaa ctg aac ctt gat gtg     3948 
Gln Tyr Gln Lys Asp Ala Pro Asp His Gln Glu Leu Asn Leu Asp Val 
                1285                1290                1295 

tcc ctc caa ctg ccc agc cgc agc tcc aag atc acc cac cgt atc cac     3996 
Ser Leu Gln Leu Pro Ser Arg Ser Ser Lys Ile Thr His Arg Ile His 
            1300                1305                1310 

tgg gaa tct gcc agc ctc ctg cga tca gaa gag acc aag gaa aat gag     4044 
Trp Glu Ser Ala Ser Leu Leu Arg Ser Glu Glu Thr Lys Glu Asn Glu 
        1315                1320                1325 

ggt ttc aca gtc aca gct gaa gga aaa ggc caa ggc acc ttg tcg gtg     4092 
Gly Phe Thr Val Thr Ala Glu Gly Lys Gly Gln Gly Thr Leu Ser Val 
    1330                1335                1340 

gtg aca atg tac cat gct aag gcc aaa gat caa ctc acc tgt aat aaa     4140 
Val Thr Met Tyr His Ala Lys Ala Lys Asp Gln Leu Thr Cys Asn Lys 
1345                1350                1355                1360 

ttc gac ctc aag gtc acc ata aaa cca gca ccg gaa aca gaa aag agg     4188 
Phe Asp Leu Lys Val Thr Ile Lys Pro Ala Pro Glu Thr Glu Lys Arg 
                1365                1370                1375 

cct cag gat gcc aag aac act atg atc ctt gag atc tgt acc agg tac     4236 
Pro Gln Asp Ala Lys Asn Thr Met Ile Leu Glu Ile Cys Thr Arg Tyr 
            1380                1385                1390 

cgg gga gac cag gat gcc act atg tct ata ttg gac ata tcc atg atg     4284 
Arg Gly Asp Gln Asp Ala Thr Met Ser Ile Leu Asp Ile Ser Met Met 
        1395                1400                1405 

act ggc ttt gct cca gac aca gat gac ctg aag cag ctg gcc aat ggt     4332 
Thr Gly Phe Ala Pro Asp Thr Asp Asp Leu Lys Gln Leu Ala Asn Gly 
    1410                1415                1420 

gtt gac aga tac atc tcc aag tat gag ctg gac aaa gcc ttc tcc gat     4380 
Val Asp Arg Tyr Ile Ser Lys Tyr Glu Leu Asp Lys Ala Phe Ser Asp 
1425                1430                1435                1440 

agg aac acc ctc atc atc tac ctg gac aag gtc tca cac tct gag gat     4428 
Arg Asn Thr Leu Ile Ile Tyr Leu Asp Lys Val Ser His Ser Glu Asp 
                1445                1450                1455 

gac tgt cta gct ttc aaa gtt cac caa tac ttt aat gta gag ctt atc     4476 
Asp Cys Leu Ala Phe Lys Val His Gln Tyr Phe Asn Val Glu Leu Ile 
            1460                1465                1470 

cag cct gga gca gtc aag gtc tac gcc tat tac aac ctg gag gaa agc     4524 
Gln Pro Gly Ala Val Lys Val Tyr Ala Tyr Tyr Asn Leu Glu Glu Ser 
        1475                1480                1485 

tgt acc cgg ttc tac cat ccg gaa aag gag gat gga aag ctg aac aag     4572 
Cys Thr Arg Phe Tyr His Pro Glu Lys Glu Asp Gly Lys Leu Asn Lys 
    1490                1495                1500 

ctc tgc cgt gat gaa ctg tgc cgc tgt gct gag gag aat tgc ttc ata     4620 
Leu Cys Arg Asp Glu Leu Cys Arg Cys Ala Glu Glu Asn Cys Phe Ile 
1505                1510                1515                1520 

caa aag tcg gat gac aag gtc acc ctg gaa gaa cgg ctg gac aag gcc     4668 
Gln Lys Ser Asp Asp Lys Val Thr Leu Glu Glu Arg Leu Asp Lys Ala 
                1525                1530                1535 

tgt gag cca gga gtg gac tat gtg tac aag acc cga ctg gtc aag gtt     4716 
Cys Glu Pro Gly Val Asp Tyr Val Tyr Lys Thr Arg Leu Val Lys Val 
            1540                1545                1550 

cag ctg tcc aat gac ttt gac gag tac atc atg gcc att gag cag acc     4764 
Gln Leu Ser Asn Asp Phe Asp Glu Tyr Ile Met Ala Ile Glu Gln Thr 
        1555                1560                1565 

atc aag tca ggc tcg gat gag gtg cag gtt gga cag cag cgc acg ttc     4812 
Ile Lys Ser Gly Ser Asp Glu Val Gln Val Gly Gln Gln Arg Thr Phe 
    1570                1575                1580 

atc agc ccc atc aag tgc aga gaa gcc ctg aag ctg gag gag aag aaa     4860 
Ile Ser Pro Ile Lys Cys Arg Glu Ala Leu Lys Leu Glu Glu Lys Lys 
1585                1590                1595                1600 

cac tac ctc atg tgg ggt ctc tcc tcc gat ttc tgg gga gag aag ccc     4908 
His Tyr Leu Met Trp Gly Leu Ser Ser Asp Phe Trp Gly Glu Lys Pro 
                1605                1610                1615 

aac ctc agc tac atc atc ggg aag gac act tgg gtg gag cac tgg cct     4956 
Asn Leu Ser Tyr Ile Ile Gly Lys Asp Thr Trp Val Glu His Trp Pro 
            1620                1625                1630 

gag gag gac gaa tgc caa gac gaa gag aac cag aaa caa tgc cag gac     5004 
Glu Glu Asp Glu Cys Gln Asp Glu Glu Asn Gln Lys Gln Cys Gln Asp 
        1635                1640                1645 

ctc ggc gcc ttc acc gag agc atg gtt gtc ttt ggg tgc ccc aac tga     5052 
Leu Gly Ala Phe Thr Glu Ser Met Val Val Phe Gly Cys Pro Asn 
    1650                1655                1660 

ccacaccccc attcc                                                    5067 

 
           
             4  
             24  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            4 

cgtgatacac caagaaatga ttgg                                            24 

 
           
             5  
             22  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            5 

ctgcagcgag atgagaacaa ag                                              22 

 
           
             6  
             26  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            6 

acaacgagaa agacatggcc ctcacg                                          26 

 
           
             7  
             19  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            7 

gaaggtgaag gtcggagtc                                                  19 

 
           
             8  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            8 

gaagatggtg atgggatttc                                                 20 

 
           
             9  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            9 

caagcttccc gttctcagcc                                                 20 

 
           
             10  
             6435  
             DNA  
             Mus musculus  
             
 
           
            10 

aagcttagga aactatgttg cgaaattttg ggcagtccct ggtgcaggaa cagggaggga     60 

ccagagagga gagccatata aagagccagc ggctacagcc ccagctcgcc tctgcccacc    120 

cctgcccctt accccttcat tccttccacc tttttccttc actatgggac cagcttcagg    180 

gtcccagcta ctagtgctac tgctgctgtt ggccagctcc ccattagctc tggggatccc    240 

catgtaagta gttacctttt gggggtgcag tttcttatac aatttaggag tcacctaggt    300 

gagtcaccta ggagtcaccc acttgggggg agacagggat gttaagaatt tgtgctgggg    360 

gctggaggat ggctcagtgg gtatgaagtc ttgctgcatg gacataagga ccttaactca    420 

aactcccagc acccacagaa aagccaggag tggcctccag agcctgtaac cccacactgt    480 

ggggctggag accagatact ggggcttgct gactgccagc ccagtgccag gttcagagag    540 

agatgttgac tcaaggtgtg gggagggagg ggcatagaac aggacaccgg acatcttcct    600 

gtgacgtgac atacatacat acatacatac atacatacat acatacatac aaacagagag    660 

aaagagagag aatgtgagtg tttgggttgt cttatgttca tagaactcag gtattgtagc    720 

attggtgtgc ctacttatga agaagaaggt ctatagattt atggtgtctg tgatgtcttg    780 

tggtttaggc aatggtaagt tttaagatgt gaggactgga gggttgttgg ggtctatagc    840 

ccttgtgtgg actgtcaaaa gccaagctca gcattgggcc tgattacatg gacctgtcat    900 

cctgtccact ctggaagccc aggcaggaag tgtacacatt ccagccaagc ctgggctaca    960 

gagtgagttc agggctagcc tgaacaactt agtgagatgc tgtttccaaa cagaaaagtg   1020 

aggagaggct gaggtatagc ttagtggtag agagagcact ttctcagtac aagcaaagtc   1080 

cttatggctg gtacccagtg ctgaagccag gaaaagacag cttctagtgg ctaagggaaa   1140 

gtgggctgtg gtttggtacc ttcttgtgtg gggaggacgt ggatccaggc atctgctcca   1200 

gcactgagaa ctagaacacg ctagaactag ggactgggcc agccagagtg agggctgaag   1260 

tctcaggcct tgaggtgaca tctgctccca caccccacgg tcaggtattc catcattact   1320 

cccaatgtcc tacggctgga gagcgaagag accatcgtac tggaggccca cgatgctcag   1380 

ggtgacatcc cagtcacagt cactgtgcaa gacttcctaa agaggcaagt gctgaccagt   1440 

gagaagacag tgttgacagg agccagtgga catctgagaa gcgtctccat caaggtgggc   1500 

aaggaactgg aactacagtc agccgtagcc ccttccggtc cggcctgggt ccctcaggct   1560 

gcctcctata gcctcctcgg agctcctccc ttctgagtcc ctcccctctg acgcccctgc   1620 

cgtctgggtc cctctcgttt gatctcctcc cctctgagtc ccctaccctt tgagaccctc   1680 

tgctctgagc cccttccctc tgagattcct cctctcagtc cctctcctct tgagtctctt   1740 

cctccttgag cccctcctgt ctgaggggag atgacagaga ggaggcccag ggggatctag   1800 

gggatgcttt ctgggcacca ctccctgaca cagactcctg acatcccacg catagattcc   1860 

agccagtaag gaattcaact cagataagga ggggcacaag tacgtgacag tggtggcaaa   1920 

cttcggggaa acggtggtgg agaaagcagt gatggtaagc ttccagagtg ggtacctctt   1980 

catccagaca gacaagacca tctacacccc tggctccact gtcttatatc ggatcttcac   2040 

tgtggacaac aacctactgc ccgtgggcaa gacagtcgtc atcctcattg agacccccga   2100 

tggcattcct gtcaagagag acattctgtc ttccaacaac caacacggca tcttgccttt   2160 

gtcttggaac attcctgaac tggtcaacat ggggcagtgg aagatccgag ccttttacga   2220 

acatgcgccg aagcagatct tctccgcaga gtttgaggtg aaggaatacg tgctgcccag   2280 

ttttgaggtc cgggtggagc ccacagagac attttattac atcgatgacc caaatggcct   2340 

ggaagtttcc atcatagcca agttcctgta cgggaaaaac gtggacggga cagccttcgt   2400 

gatttttggg gtccaggatg gcgataagaa gatttctctg gcccactccc tcacgcgcgt   2460 

agtgattgag gatggtgtgg gggatgcagt gctgacccgg aaggtgctga tggagggggt   2520 

acggccttcc aacgccgacg ccctggtggg gaagtccctg tatgtctccg tcactgtcat   2580 

cctgcactca ggtagtgaca tggtagaggc agagcgcagt gggatcccga ttgtcacttc   2640 

cccgtaccag atccacttca ccaagacacc caaattcttc aagccagcca tgccctttga   2700 

cctcatggtg ttcgtgacca accccgatgg ctctccggcc agcaaagtgc tggtggtcac   2760 

tcagggatct aatgcaaagg ctctcaccca agatgatggc gtggccaagc taagcatcaa   2820 

cacacccaac agccgccaac ccctgaccat cacagtccgc accaagaagg acactctccc   2880 

agaatcacgg caggccacca agacaatgga ggcccatccc tacagcacta tgcacaactc   2940 

caacaactac ctacacttgt cagtgtcacg aatggagctc aagccggggg acaacctcaa   3000 

tgtcaacttc cacctgcgca cagacccagg ccatgaggcc aagatccgat actacaccta   3060 

cctggttatg aacaagggga agctcctgaa ggcaggccgc caggttcggg agcctggcca   3120 

ggacctggtg gtcttgtccc tgcccatcac tccagagttt attccttcat ttcgcctggt   3180 

ggcttactac accctgattg gagctagtgg ccagagggag gtggtggctg actctgtgtg   3240 

ggtggatgtg aaggattcct gtattggcac gctggtggtg aagggtgacc caagagataa   3300 

ccatctcgca cctgggcaac aaacgacact caggattgaa ggaaaccagg gggcccgagt   3360 

ggggctagtg gctgtggaca agggagtgtt tgtgctgaac aagaagaaca aactcacaca   3420 

gagcaagatc tgggatgtgg tagagaaggc agacattggc tgcaccccag gcagtgggaa   3480 

gaactatgct ggtgtcttca tggatgcagg cctggccttc aagacaagcc aaggactgca   3540 

gactgaacag agagcagatc ttgagtgcac caagccagca gcccgccgcc gtcgctcagt   3600 

acagttgatg gaaagaagga tggacaaagc tggtcagtac actgacaagg gtcttcggaa   3660 

gtgttgtgag gatggtatgc gggatatccc tatgagatac agctgccagc gccgggcacg   3720 

cctcatcacc cagggcgaga actgcataaa ggccttcata gactgctgca accacatcac   3780 

caagctgcgt gaacaacaca gaagagacca cgtgctgggc ctggccagga gtgaattgga   3840 

ggaagacata attccagaag aagatattat ctctagaagc cacttcccac agagctggtt   3900 

gtggaccata gaagagttga aagaaccaga gaaaaatgga atctctacga aggtcatgaa   3960 

catctttctc aaagattcca tcaccacctg ggagattctg gcagtgagct tgtcagacaa   4020 

gaaagggatc tgtgtggcag acccctatga gatcagagtg atgcaggact tcttcattga   4080 

cctgcggctg ccctactctg tagtgcgcaa cgaacaggtg gagatcagag ctgtgctctt   4140 

caactaccgt gaacaggagg aacttaaggt gagggtggaa ctgttgcata atccagcctt   4200 

ctgcagcatg gccaccgcca agaatcgcta cttccagacc atcaaaatcc ctcccaagtc   4260 

ctcggtggct gtaccgtatg tcattgtccc cttgaagatc ggccaacaag aggtggaggt   4320 

caaggctgct gtcttcaatc acttcatcag tgatggtgtc aagaagacac tgaaggtcgt   4380 

gccagaagga atgagaatca acaaaactgt ggccatccat acactggacc cagagaagct   4440 

cggtcaaggg ggagtgcaga aggtggatgt gcctgccgca gaccttagcg accaagtgcc   4500 

agacacagac tctgagacca gaattatcct gcaagggagc ccggtggttc agatggctga   4560 

agatgctgtg gacggggagc ggctgaaaca cctgatcgtg acccccgcag gctgtgggga   4620 

acagaacatg attggcatga caccaacagt cattgcggta cactacctgg accagaccga   4680 

acagtgggag aagttcggca tagagaagag gcaagaggcc ctggagctca tcaagaaagg   4740 

gtacacccag cagctggcct tcaaacagcc cagctctgcc tatgctgcct tcaacaaccg   4800 

gccccccagc acctggctga cagcctacgt ggtcaaggtc ttctctctag ctgccaacct   4860 

catcgccatc gactctcacg tcctgtgtgg ggctgttaaa tggttgattc tggagaaaca   4920 

gaagccggat ggtgtctttc aggaggatgg gcccgtgatt caccaagaaa tgattggtgg   4980 

cttccggaac gccaaggagg cagatgtgtc actcacagcc ttcgtcctca tcgcactgca   5040 

ggaagccagg gacatctgtg aggggcaggt caatagcctt cctgggagca tcaacaaggc   5100 

aggggagtat attgaagcca gttacatgaa cctgcagaga ccatacacag tggccattgc   5160 

tgggtatgcc ctggccctga tgaacaaact ggaggaacct tacctcggca agtttctgaa   5220 

cacagccaaa gatcggaacc gctgggagga gcctgaccag cagctctaca acgtagaggc   5280 

cacatcctac gccctcctgg ccctgctgct gctgaaagac tttgactctg tgccccctgt   5340 

agtgcgctgg ctcaatgagc aaagatacta cggaggcggc tatggctcca cccaggctac   5400 

cttcatggta ttccaagcct tggcccaata tcaaacagat gtccctgacc ataaggactt   5460 

gaacatggat gtgtccttcc acctccccag ccgtagctct gcaaccacgt ttcgcctgct   5520 

ctgggaaaat ggcaacctcc tgcgatcgga agagaccaag caaaatgagg ccttctctct   5580 

aacagccaaa ggaaaaggcc gaggcacatt gtcggtggtg gcagtgtatc atgccaaact   5640 

caaaagcaaa gtcacctgca agaagtttga cctcagggtc agcataagac cagcccctga   5700 

gacagccaag aagcccgagg aagccaagaa taccatgttc cttgaaatct gcaccaagta   5760 

cttgggagat gtggacgcca ctatgtccat cctggacatc tccatgatga ctggctttgc   5820 

tccagacaca aaggacctgg aactgctggc ctctggagta gatagataca tctccaagta   5880 

cgagatgaac aaagccttct ccaacaagaa caccctcatc atctacctag aaaagatttc   5940 

acacaccgaa gaagactgcc tgaccttcaa agttcaccag tactttaatg tgggacttat   6000 

ccagcccggg tcggtcaagg tctactccta ttacaacctc gaggaatcat gcacccggtt   6060 

ctatcatcca gagaaggacg atgggatgct cagcaagctg tgccacagtg aaatgtgccg   6120 

gtgtgctgaa gagaactgct tcatgcaaca gtcacaggag aagatcaacc tgaatgtccg   6180 

gctagacaag gcttgtgagc ccggagtcga ctatgtgtac aagaccgagc taaccaacat   6240 

aaagctgttg gatgattttg atgagtacac catgaccatc cagcaggtca tcaagtcagg   6300 

ctcagatgag gtgcaggcag ggcagcaacg caagttcatc agccacatca agtgcagaaa   6360 

cgccctgaag ctgcagaaag ggaagaagta cctcatgtgg ggcctctcct ctgacctctg   6420 

gggagaaaag cccaa                                                    6435 

 
           
             11  
             22  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            11 

aagctgtgcc acagtgaaat gt                                              22 

 
           
             12  
             18  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            12 

cgactccggg ctcacaag                                                   18 

 
           
             13  
             29  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            13 

tagccggaca ttcaggttga tcttctcct                                       29 

 
           
             14  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            14 

ggcaaattca acggcacagt                                                 20 

 
           
             15  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            15 

gggtctcgct cctggaagat                                                 20 

 
           
             16  
             27  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            16 

aaggccgaga atgggaagct tgtcatc                                         27 

 
           
             17  
             607  
             DNA  
             Homo sapiens  
             
 
           
            17 

tttctcccat tctacttccc tcctcagcat tggaagctcg taagtgggct ctgactccca     60 

gcctacagag agattcctag gaagtggttc gactgataaa cgcatggcca aaagtgaact    120 

ggggatgagg tccaagacat ctgcggtggg gggttctcca gaccttagtg ttcttccact    180 

acaaagtggg tccaacagag aaaggtctgt gttcaccagg tggccctgac cctgggagag    240 

tccagggcag ggtgcagctg cattcatgct gctggggaac atgccctcag gttactcacc    300 

ccatggacat gttggcccca gggactgaaa agcttaggaa atggtattga gaaatctggg    360 

gcagccaaaa ggggagaggc catggggaga agggggggct gagtggggga aagcaggagc    420 

cagataaaaa gccagctcca gcaggcgctg ctcactcctc cccatcctct ccctctgtcc    480 

ctctgtccct ctgaccctgc actgtcccag caccatggga cccacctcag gtcccagcct    540 

gctgctcctg ctactaaccc acctccccct ggctctgggg agtcccatgt gagtggttat    600 

gactcta                                                              607 

 
           
             18  
               
               
               
             
 
           
            18 

000 

 
           
             19  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            19 

ccatggtgct gggacagtgc                                                 20 

 
           
             20  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            20 

aggtgggtcc catggtgctg                                                 20 

 
           
             21  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            21 

ttagtagcag gagcagcagg                                                 20 

 
           
             22  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            22 

ctgttggacc cactttgtag                                                 20 

 
           
             23  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            23 

gtcgtgggcc tccagcacca                                                 20 

 
           
             24  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            24 

gtaacagtga ctggaacatc                                                 20 

 
           
             25  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            25 

ctgccctgga ctctcccagg                                                 20 

 
           
             26  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            26 

actggacagc actagttttt                                                 20 

 
           
             27  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            27 

cccagcagca tgaatgcagc                                                 20 

 
           
             28  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            28 

ctgagggcat gttccccagc                                                 20 

 
           
             29  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            29 

tgaactccct gttggctggg                                                 20 

 
           
             30  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            30 

tctgtctgga tgaagaggta                                                 20 

 
           
             31  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            31 

tcttgtctgt ctggatgaag                                                 20 

 
           
             32  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            32 

gatggtcttg tctgtctgga                                                 20 

 
           
             33  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            33 

gtgtagatgg tcttgtctgt                                                 20 

 
           
             34  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            34 

ttgtggttga cggtgaagat                                                 20 

 
           
             35  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            35 

cacgtactcc ttcacctcaa                                                 20 

 
           
             36  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            36 

aatttctctg taggctccac                                                 20 

 
           
             37  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            37 

agaggaacct ggcggtgatg                                                 20 

 
           
             38  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            38 

tcgccatcct ggatcccgaa                                                 20 

 
           
             39  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            39 

tcaatcggaa tgcgcttgag                                                 20 

 
           
             40  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            40 

gacttcccca ccaggtcttc                                                 20 

 
           
             41  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            41 

tgcctgcacc atgtcactgc                                                 20 

 
           
             42  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            42 

ttggtgaagt ggatctggta                                                 20 

 
           
             43  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            43 

ggtgtcttgg tgaagtggat                                                 20 

 
           
             44  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            44 

caccatgagg tcaaagggca                                                 20 

 
           
             45  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            45 

tcacgaacac catgaggtca                                                 20 

 
           
             46  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            46 

ggctggagag ccatcagggt                                                 20 

 
           
             47  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            47 

ctgcctccga gagctcctgc                                                 20 

 
           
             48  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            48 

ttgttggagt tgcccacggt                                                 20 

 
           
             49  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            49 

tgtacgtagc actgagagat                                                 20 

 
           
             50  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            50 

caggtcctgg ccgggctctc                                                 20 

 
           
             51  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            51 

cagcgtgtag tacgccacca                                                 20 

 
           
             52  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            52 

tattcagcac gaacacgccc                                                 20 

 
           
             53  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            53 

ccgcagctct gtgatgtagt                                                 20 

 
           
             54  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            54 

caggcccagg tggctggccc                                                 20 

 
           
             55  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            55 

tcgtagagat tccatttttc                                                 20 

 
           
             56  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            56 

cccacgtggt gatggagtct                                                 20 

 
           
             57  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            57 

ccctttcttg tccgacatgc                                                 20 

 
           
             58  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            58 

cacacagatc cctttcttgt                                                 20 

 
           
             59  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            59 

ggtctgccac acagatccct                                                 20 

 
           
             60  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            60 

ttcctgcagg ccggtcttta                                                 20 

 
           
             61  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            61 

acggcagcct tgacttccac                                                 20 

 
           
             62  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            62 

acttggtcac tgaggtctgc                                                 20 

 
           
             63  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            63 

caggagaatt ctggtctcag                                                 20 

 
           
             64  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            64 

ccttgcagga gaattctggt                                                 20 

 
           
             65  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            65 

gggtcccttg caggagaatt                                                 20 

 
           
             66  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            66 

tccaggtaat gcacagcgat                                                 20 

 
           
             67  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            67 

gccagctgct gggtgtaccc                                                 20 

 
           
             68  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            68 

tgaaggccag ctgctgggtg                                                 20 

 
           
             69  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            69 

aagaccttga ccacgtaggc                                                 20 

 
           
             70  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            70 

agagagaaga ccttgaccac                                                 20 

 
           
             71  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            71 

agtcgatggc gatgaggttg                                                 20 

 
           
             72  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            72 

gggcttctgc ttctccagga                                                 20 

 
           
             73  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            73 

gaagaccccg tcgggcttct                                                 20 

 
           
             74  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            74 

ttcttggtgt atcacgggcg                                                 20 

 
           
             75  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            75 

ccaccaatca tttcttggtg                                                 20 

 
           
             76  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            76 

tcgcaaatat ctttagcctc                                                 20 

 
           
             77  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            77 

gctgttgacc tgctcctcgc                                                 20 

 
           
             78  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            78 

cagtgtagga tctctgtagg                                                 20 

 
           
             79  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            79 

cttatctttg gctgtggtca                                                 20 

 
           
             80  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            80 

taccagggtc ctcccagcgg                                                 20 

 
           
             81  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            81 

gcataggatg tggcctccac                                                 20 

 
           
             82  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            82 

tggaacacca tgaaggtggc                                                 20 

 
           
             83  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            83 

gtattgagcc aaggcttgga                                                 20 

 
           
             84  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            84 

ggtgatcttg gagctgcggc                                                 20 

 
           
             85  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            85 

gttcttggca tcctgaggcc                                                 20 

 
           
             86  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            86 

gcaaagccag tcatcatgga                                                 20 

 
           
             87  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            87 

ctggagcaaa gccagtcatc                                                 20 

 
           
             88  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            88 

tgtgtctgga gcaaagccag                                                 20 

 
           
             89  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            89 

gaaggctttg tccagctcat                                                 20 

 
           
             90  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            90 

ggtagatgat gagggtgttc                                                 20 

 
           
             91  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            91 

ctcagagtgt gagaccttgt                                                 20 

 
           
             92  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            92 

ccttttccgg atggtagaac                                                 20 

 
           
             93  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            93 

gtactcgtca aagtcattgg                                                 20 

 
           
             94  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            94 

ccacatgagg tagtgtttct                                                 20 

 
           
             95  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            95 

tcggaggaga gaccccacat                                                 20 

 
           
             96  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            96 

tcaggccagt gctccaccca                                                 20 

 
           
             97  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            97 

ggcatcacac acgaagccca                                                 20 

 
           
             98  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            98 

tgcggcagcc gctgcccatg                                                 20 

 
           
             99  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            99 

gtcatagctg tggcagcctg                                                 20 

 
           
             100  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            100 

ggttgagctc cacatctgcc                                                 20 

 
           
             101  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            101 

ttgaagaaat cctccttgtc                                                 20 

 
           
             102  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            102 

atgcattcga tgcggcagcc                                                 20 

 
           
             103  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            103 

caaacagctc tcgggagatg                                                 20 

 
           
             104  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            104 

tagctcatgc ctgtcttgca                                                 20 

 
           
             105  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            105 

ggagggacta cccacagcaa                                                 20 

 
           
             106  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            106 

actcgctgaa gaatatgcat                                                 20 

 
           
             107  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            107 

catcatactc ctgggccacc                                                 20 

 
           
             108  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            108 

aacttctgct tgctctcctc                                                 20 

 
           
             109  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            109 

tgggcaccaa cttctgcttg                                                 20 

 
           
             110  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            110 

tctgcttgct ctcctccatg                                                 20 

 
           
             111  
             5087  
             DNA  
             Mus musculus  
             
               CDS  
               (57)...(5048)  
             
           
            111 

gcctctgccc acccctgccc cttacccctt cattccttcc acctttttcc ttcact atg     59 
                                                              Met 
                                                                1 

gga cca gct tca ggg tcc cag cta cta gtg cta ctg ctg ctg ttg gcc      107 
Gly Pro Ala Ser Gly Ser Gln Leu Leu Val Leu Leu Leu Leu Leu Ala 
              5                  10                  15 

agc tcc cca tta gct ctg ggg atc ccc atg tat tcc atc att act ccc      155 
Ser Ser Pro Leu Ala Leu Gly Ile Pro Met Tyr Ser Ile Ile Thr Pro 
         20                  25                  30 

aat gtc cta cgg ctg gag agc gaa gag acc atc gta ctg gag gcc cac      203 
Asn Val Leu Arg Leu Glu Ser Glu Glu Thr Ile Val Leu Glu Ala His 
     35                  40                  45 

gat gct cag ggt gac atc cca gtc aca gtc act gtg caa gac ttc cta      251 
Asp Ala Gln Gly Asp Ile Pro Val Thr Val Thr Val Gln Asp Phe Leu 
 50                  55                  60                  65 

aag agg caa gtg ctg acc agt gag aag aca gtg ttg aca gga gcc agt      299 
Lys Arg Gln Val Leu Thr Ser Glu Lys Thr Val Leu Thr Gly Ala Ser 
                 70                  75                  80 

gga cat ctg aga agc gtc tcc atc aag att cca gcc agt aag gaa ttc      347 
Gly His Leu Arg Ser Val Ser Ile Lys Ile Pro Ala Ser Lys Glu Phe 
             85                  90                  95 

aac tca gat aag gag ggg cac aag tac gtg aca gtg gtg gca aac ttc      395 
Asn Ser Asp Lys Glu Gly His Lys Tyr Val Thr Val Val Ala Asn Phe 
        100                 105                 110 

ggg gaa acg gtg gtg gag aaa gca gtg atg gta agc ttc cag agt ggg      443 
Gly Glu Thr Val Val Glu Lys Ala Val Met Val Ser Phe Gln Ser Gly 
    115                 120                 125 

tac ctc ttc atc cag aca gac aag acc atc tac acc cct ggc tcc act      491 
Tyr Leu Phe Ile Gln Thr Asp Lys Thr Ile Tyr Thr Pro Gly Ser Thr 
130                 135                 140                 145 

gtc tta tat cgg atc ttc act gtg gac aac aac cta ctg ccc gtg ggc      539 
Val Leu Tyr Arg Ile Phe Thr Val Asp Asn Asn Leu Leu Pro Val Gly 
                150                 155                 160 

aag aca gtc gtc atc ctc att gag acc ccc gat ggc att cct gtc aag      587 
Lys Thr Val Val Ile Leu Ile Glu Thr Pro Asp Gly Ile Pro Val Lys 
            165                 170                 175 

aga gac att ctg tct tcc aac aac caa cac ggc atc ttg cct ttg tct      635 
Arg Asp Ile Leu Ser Ser Asn Asn Gln His Gly Ile Leu Pro Leu Ser 
        180                 185                 190 

tgg aac att cct gaa ctg gtc aac atg ggg cag tgg aag atc cga gcc      683 
Trp Asn Ile Pro Glu Leu Val Asn Met Gly Gln Trp Lys Ile Arg Ala 
    195                 200                 205 

ttt tac gaa cat gcg ccg aag cag atc ttc tcc gca gag ttt gag gtg      731 
Phe Tyr Glu His Ala Pro Lys Gln Ile Phe Ser Ala Glu Phe Glu Val 
210                 215                 220                 225 

aag gaa tac gtg ctg ccc agt ttt gag gtc cgg gtg gag ccc aca gag      779 
Lys Glu Tyr Val Leu Pro Ser Phe Glu Val Arg Val Glu Pro Thr Glu 
                230                 235                 240 

aca ttt tat tac atc gat gac cca aat ggc ctg gaa gtt tcc atc ata      827 
Thr Phe Tyr Tyr Ile Asp Asp Pro Asn Gly Leu Glu Val Ser Ile Ile 
            245                 250                 255 

gcc aag ttc ctg tac ggg aaa aac gtg gac ggg aca gcc ttc gtg att      875 
Ala Lys Phe Leu Tyr Gly Lys Asn Val Asp Gly Thr Ala Phe Val Ile 
        260                 265                 270 

ttt ggg gtc cag gat ggc gat aag aag att tct ctg gcc cac tcc ctc      923 
Phe Gly Val Gln Asp Gly Asp Lys Lys Ile Ser Leu Ala His Ser Leu 
    275                 280                 285 

acg cgc gta gtg att gag gat ggt gtg ggg gat gca gtg ctg acc cgg      971 
Thr Arg Val Val Ile Glu Asp Gly Val Gly Asp Ala Val Leu Thr Arg 
290                 295                 300                 305 

aag gtg ctg atg gag ggg gta cgg cct tcc aac gcc gac gcc ctg gtg     1019 
Lys Val Leu Met Glu Gly Val Arg Pro Ser Asn Ala Asp Ala Leu Val 
                310                 315                 320 

ggg aag tcc ctg tat gtc tcc gtc act gtc atc ctg cac tca ggt agt     1067 
Gly Lys Ser Leu Tyr Val Ser Val Thr Val Ile Leu His Ser Gly Ser 
            325                 330                 335 

gac atg gta gag gca gag cgc agt ggg atc ccg att gtc act tcc ccg     1115 
Asp Met Val Glu Ala Glu Arg Ser Gly Ile Pro Ile Val Thr Ser Pro 
        340                 345                 350 

tac cag atc cac ttc acc aag aca ccc aaa ttc ttc aag cca gcc atg     1163 
Tyr Gln Ile His Phe Thr Lys Thr Pro Lys Phe Phe Lys Pro Ala Met 
    355                 360                 365 

ccc ttt gac ctc atg gtg ttc gtg acc aac ccc gat ggc tct ccg gcc     1211 
Pro Phe Asp Leu Met Val Phe Val Thr Asn Pro Asp Gly Ser Pro Ala 
370                 375                 380                 385 

agc aaa gtg ctg gtg gtc act cag gga tct aat gca aag gct ctc acc     1259 
Ser Lys Val Leu Val Val Thr Gln Gly Ser Asn Ala Lys Ala Leu Thr 
                390                 395                 400 

caa gat gat ggc gtg gcc aag cta agc atc aac aca ccc aac agc cgc     1307 
Gln Asp Asp Gly Val Ala Lys Leu Ser Ile Asn Thr Pro Asn Ser Arg 
            405                 410                 415 

caa ccc ctg acc atc aca gtc cgc acc aag aag gac act ctc cca gaa     1355 
Gln Pro Leu Thr Ile Thr Val Arg Thr Lys Lys Asp Thr Leu Pro Glu 
        420                 425                 430 

tca cgg cag gcc acc aag aca atg gag gcc cat ccc tac agc act atg     1403 
Ser Arg Gln Ala Thr Lys Thr Met Glu Ala His Pro Tyr Ser Thr Met 
    435                 440                 445 

cac aac tcc aac aac tac cta cac ttg tca gtg tca cga atg gag ctc     1451 
His Asn Ser Asn Asn Tyr Leu His Leu Ser Val Ser Arg Met Glu Leu 
450                 455                 460                 465 

aag ccg ggg gac aac ctc aat gtc aac ttc cac ctg cgc aca gac cca     1499 
Lys Pro Gly Asp Asn Leu Asn Val Asn Phe His Leu Arg Thr Asp Pro 
                470                 475                 480 

ggc cat gag gcc aag atc cga tac tac acc tac ctg gtt atg aac aag     1547 
Gly His Glu Ala Lys Ile Arg Tyr Tyr Thr Tyr Leu Val Met Asn Lys 
            485                 490                 495 

ggg aag ctc ctg aag gca ggc cgc cag gtt cgg gag cct ggc cag gac     1595 
Gly Lys Leu Leu Lys Ala Gly Arg Gln Val Arg Glu Pro Gly Gln Asp 
        500                 505                 510 

ctg gtg gtc ttg tcc ctg ccc atc act cca gag ttt att cct tca ttt     1643 
Leu Val Val Leu Ser Leu Pro Ile Thr Pro Glu Phe Ile Pro Ser Phe 
    515                 520                 525 

cgc ctg gtg gct tac tac acc ctg att gga gct agt ggc cag agg gag     1691 
Arg Leu Val Ala Tyr Tyr Thr Leu Ile Gly Ala Ser Gly Gln Arg Glu 
530                 535                 540                 545 

gtg gtg gct gac tct gtg tgg gtg gat gtg aag gat tcc tgt att ggc     1739 
Val Val Ala Asp Ser Val Trp Val Asp Val Lys Asp Ser Cys Ile Gly 
                550                 555                 560 

acg ctg gtg gtg aag ggt gac cca aga gat aac cat ctc gca cct ggg     1787 
Thr Leu Val Val Lys Gly Asp Pro Arg Asp Asn His Leu Ala Pro Gly 
            565                 570                 575 

caa caa acg aca ctc agg att gaa gga aac cag ggg gcc cga gtg ggg     1835 
Gln Gln Thr Thr Leu Arg Ile Glu Gly Asn Gln Gly Ala Arg Val Gly 
        580                 585                 590 

cta gtg gct gtg gac aag gga gtg ttt gtg ctg aac aag aag aac aaa     1883 
Leu Val Ala Val Asp Lys Gly Val Phe Val Leu Asn Lys Lys Asn Lys 
    595                 600                 605 

ctc aca cag agc aag atc tgg gat gtg gta gag aag gca gac att ggc     1931 
Leu Thr Gln Ser Lys Ile Trp Asp Val Val Glu Lys Ala Asp Ile Gly 
610                 615                 620                 625 

tgc acc cca ggc agt ggg aag aac tat gct ggt gtc ttc atg gat gca     1979 
Cys Thr Pro Gly Ser Gly Lys Asn Tyr Ala Gly Val Phe Met Asp Ala 
                630                 635                 640 

ggc ctg gcc ttc aag aca agc caa gga ctg cag act gaa cag aga gca     2027 
Gly Leu Ala Phe Lys Thr Ser Gln Gly Leu Gln Thr Glu Gln Arg Ala 
            645                 650                 655 

gat ctt gag tgc acc aag cca gca gcc cgc cgc cgt cgc tca gta cag     2075 
Asp Leu Glu Cys Thr Lys Pro Ala Ala Arg Arg Arg Arg Ser Val Gln 
        660                 665                 670 

ttg atg gaa aga agg atg gac aaa gct ggt cag tac act gac aag ggt     2123 
Leu Met Glu Arg Arg Met Asp Lys Ala Gly Gln Tyr Thr Asp Lys Gly 
    675                 680                 685 

ctt cgg aag tgt tgt gag gat ggt atg cgg gat atc cct atg aga tac     2171 
Leu Arg Lys Cys Cys Glu Asp Gly Met Arg Asp Ile Pro Met Arg Tyr 
690                 695                 700                 705 

agc tgc cag cgc cgg gca cgc ctc atc acc cag ggc gag aac tgc ata     2219 
Ser Cys Gln Arg Arg Ala Arg Leu Ile Thr Gln Gly Glu Asn Cys Ile 
                710                 715                 720 

aag gcc ttc ata gac tgc tgc aac cac atc acc aag ctg cgt gaa caa     2267 
Lys Ala Phe Ile Asp Cys Cys Asn His Ile Thr Lys Leu Arg Glu Gln 
            725                 730                 735 

cac aga aga gac cac gtg ctg ggc ctg gcc agg agt gaa ttg gag gaa     2315 
His Arg Arg Asp His Val Leu Gly Leu Ala Arg Ser Glu Leu Glu Glu 
        740                 745                 750 

gac ata att cca gaa gaa gat att atc tct aga agc cac ttc cca cag     2363 
Asp Ile Ile Pro Glu Glu Asp Ile Ile Ser Arg Ser His Phe Pro Gln 
    755                 760                 765 

agc tgg ttg tgg acc ata gaa gag ttg aaa gaa cca gag aaa aat gga     2411 
Ser Trp Leu Trp Thr Ile Glu Glu Leu Lys Glu Pro Glu Lys Asn Gly 
770                 775                 780                 785 

atc tct acg aag gtc atg aac atc ttt ctc aaa gat tcc atc acc acc     2459 
Ile Ser Thr Lys Val Met Asn Ile Phe Leu Lys Asp Ser Ile Thr Thr 
                790                 795                 800 

tgg gag att ctg gca gtg agc ttg tca gac aag aaa ggg atc tgt gtg     2507 
Trp Glu Ile Leu Ala Val Ser Leu Ser Asp Lys Lys Gly Ile Cys Val 
            805                 810                 815 

gca gac ccc tat gag atc aga gtg atg cag gac ttc ttc att gac ctg     2555 
Ala Asp Pro Tyr Glu Ile Arg Val Met Gln Asp Phe Phe Ile Asp Leu 
        820                 825                 830 

cgg ctg ccc tac tct gta gtg cgc aac gaa cag gtg gag atc aga gct     2603 
Arg Leu Pro Tyr Ser Val Val Arg Asn Glu Gln Val Glu Ile Arg Ala 
    835                 840                 845 

gtg ctc ttc aac tac cgt gaa cag cag gaa ctt aag gtg agg gtg gaa     2651 
Val Leu Phe Asn Tyr Arg Glu Gln Gln Glu Leu Lys Val Arg Val Glu 
850                 855                 860                 865 

ctg ttg cat aat cca gcc ttc tgc agc atg gcc acc gcc aag aat cgc     2699 
Leu Leu His Asn Pro Ala Phe Cys Ser Met Ala Thr Ala Lys Asn Arg 
                870                 875                 880 

tac ttc cag acc atc aaa atc cct ccc aag tcc tcg gtg gct gta ccg     2747 
Tyr Phe Gln Thr Ile Lys Ile Pro Pro Lys Ser Ser Val Ala Val Pro 
            885                 890                 895 

tat gtc att gtc ccc ttg aag atc ggc caa caa gag gtg gag gtc aag     2795 
Tyr Val Ile Val Pro Leu Lys Ile Gly Gln Gln Glu Val Glu Val Lys 
        900                 905                 910 

gct gct gtc ttc aat cac ttc atc agt gat ggt gtc aag aag aca ctg     2843 
Ala Ala Val Phe Asn His Phe Ile Ser Asp Gly Val Lys Lys Thr Leu 
    915                 920                 925 

aag gtc gtg cca gaa gga atg aga atc aac aaa act gtg gcc atc cat     2891 
Lys Val Val Pro Glu Gly Met Arg Ile Asn Lys Thr Val Ala Ile His 
930                 935                 940                 945 

aca ctg gac cca gag aag ctc ggt caa ggg gga gtg cag aag gtg gat     2939 
Thr Leu Asp Pro Glu Lys Leu Gly Gln Gly Gly Val Gln Lys Val Asp 
                950                 955                 960 

gtg cct gcc gca gac ctt agc gac caa gtg cca gac aca gac tct gag     2987 
Val Pro Ala Ala Asp Leu Ser Asp Gln Val Pro Asp Thr Asp Ser Glu 
            965                 970                 975 

acc aga att atc ctg caa ggg agc ccg gtg gtt cag atg gct gaa gat     3035 
Thr Arg Ile Ile Leu Gln Gly Ser Pro Val Val Gln Met Ala Glu Asp 
        980                 985                 990 

gct gtg gac ggg gag cgg ctg aaa cac ctg atc gtg acc ccc gca ggc     3083 
Ala Val Asp Gly Glu Arg Leu Lys His Leu Ile Val Thr Pro Ala Gly 
    995                 1000                1005 

tgt ggg gaa cag aac atg att ggc atg aca cca aca gtc att gcg gta     3131 
Cys Gly Glu Gln Asn Met Ile Gly Met Thr Pro Thr Val Ile Ala Val 
1010                1015                1020                1025 

cac tac ctg gac cag acc gaa cag tgg gag aag ttc ggc ata gag aag     3179 
His Tyr Leu Asp Gln Thr Glu Gln Trp Glu Lys Phe Gly Ile Glu Lys 
                1030                1035                1040 

agg caa gag gcc ctg gag ctc atc aag aaa ggg tac acc cag cag ctg     3227 
Arg Gln Glu Ala Leu Glu Leu Ile Lys Lys Gly Tyr Thr Gln Gln Leu 
            1045                1050                1055 

gcc ttc aaa cag ccc agc tct gcc tat gct gcc ttc aac aac cgg ccc     3275 
Ala Phe Lys Gln Pro Ser Ser Ala Tyr Ala Ala Phe Asn Asn Arg Pro 
        1060                1065                1070 

ccc agc acc tgg ctg aca gcc tac gtg gtc aag gtc ttc tct cta gct     3323 
Pro Ser Thr Trp Leu Thr Ala Tyr Val Val Lys Val Phe Ser Leu Ala 
    1075                1080                1085 

gcc aac ctc atc gcc atc gac tct cac gtc ctg tgt ggg gct gtt aaa     3371 
Ala Asn Leu Ile Ala Ile Asp Ser His Val Leu Cys Gly Ala Val Lys 
1090                1095                1100                1105 

tgg ttg att ctg gag aaa cag aag ccg gat ggt gtc ttt cag gag gat     3419 
Trp Leu Ile Leu Glu Lys Gln Lys Pro Asp Gly Val Phe Gln Glu Asp 
                1110                1115                1120 

ggg ccc gtg att cac caa gaa atg att ggt ggc ttc cgg aac gcc aag     3467 
Gly Pro Val Ile His Gln Glu Met Ile Gly Gly Phe Arg Asn Ala Lys 
            1125                1130                1135 

gag gca gat gtg tca ctc aca gcc ttc gtc ctc atc gca ctg cag gaa     3515 
Glu Ala Asp Val Ser Leu Thr Ala Phe Val Leu Ile Ala Leu Gln Glu 
        1140                1145                1150 

gcc agg gac atc tgt gag ggg cag gtc aat agc ctt cct ggg agc atc     3563 
Ala Arg Asp Ile Cys Glu Gly Gln Val Asn Ser Leu Pro Gly Ser Ile 
    1155                1160                1165 

aac aag gca ggg gag tat att gaa gcc agt tac atg aac ctg cag aga     3611 
Asn Lys Ala Gly Glu Tyr Ile Glu Ala Ser Tyr Met Asn Leu Gln Arg 
1170                1175                1180                1185 

cca tac aca gtg gcc att gct ggg tat gcc ctg gcc ctg atg aac aaa     3659 
Pro Tyr Thr Val Ala Ile Ala Gly Tyr Ala Leu Ala Leu Met Asn Lys 
                1190                1195                1200 

ctg gag gaa cct tac ctc ggc aag ttt ctg aac aca gcc aaa gat cgg     3707 
Leu Glu Glu Pro Tyr Leu Gly Lys Phe Leu Asn Thr Ala Lys Asp Arg 
            1205                1210                1215 

aac cgc tgg gag gag cct gac cag cag ctc tac aac gta gag gcc aca     3755 
Asn Arg Trp Glu Glu Pro Asp Gln Gln Leu Tyr Asn Val Glu Ala Thr 
        1220                1225                1230 

tcc tac gcc ctc ctg gcc ctg ctg ctg ctg aaa gac ttt gac tct gtg     3803 
Ser Tyr Ala Leu Leu Ala Leu Leu Leu Leu Lys Asp Phe Asp Ser Val 
    1235                1240                1245 

ccc cct gta gtg cgc tgg ctc aat gag caa aga tac tac gga ggc ggc     3851 
Pro Pro Val Val Arg Trp Leu Asn Glu Gln Arg Tyr Tyr Gly Gly Gly 
1250                1255                1260                1265 

tat ggc tcc acc cag gct acc ttc atg gta ttc caa gcc ttg gcc caa     3899 
Tyr Gly Ser Thr Gln Ala Thr Phe Met Val Phe Gln Ala Leu Ala Gln 
                1270                1275                1280 

tat caa aca gat gtc cct gac cat aag gac ttg aac atg gat gtg tcc     3947 
Tyr Gln Thr Asp Val Pro Asp His Lys Asp Leu Asn Met Asp Val Ser 
            1285                1290                1295 

ttc cac ctc ccc agc cgt agc tct gca acc acg ttt cgc ctg ctc tgg     3995 
Phe His Leu Pro Ser Arg Ser Ser Ala Thr Thr Phe Arg Leu Leu Trp 
        1300                1305                1310 

gaa aat ggc aac ctc ctg cga tcg gaa gag acc aag caa aat gag gcc     4043 
Glu Asn Gly Asn Leu Leu Arg Ser Glu Glu Thr Lys Gln Asn Glu Ala 
    1315                1320                1325 

ttc tct cta aca gcc aaa gga aaa ggc cga ggc aca ttg tcg gtg gtg     4091 
Phe Ser Leu Thr Ala Lys Gly Lys Gly Arg Gly Thr Leu Ser Val Val 
1330                1335                1340                1345 

gca gtg tat cat gcc aaa ctc aaa agc aaa gtc acc tgc aag aag ttt     4139 
Ala Val Tyr His Ala Lys Leu Lys Ser Lys Val Thr Cys Lys Lys Phe 
                1350                1355                1360 

gac ctc agg gtc agc ata aga cca gcc cct gag aca gcc aag aag ccc     4187 
Asp Leu Arg Val Ser Ile Arg Pro Ala Pro Glu Thr Ala Lys Lys Pro 
            1365                1370                1375 

gag gaa gcc aag aat acc atg ttc ctt gaa atc tgc acc aag tac ttg     4235 
Glu Glu Ala Lys Asn Thr Met Phe Leu Glu Ile Cys Thr Lys Tyr Leu 
        1380                1385                1390 

gga gat gtg gac gcc act atg tcc atc ctg gac atc tcc atg atg act     4283 
Gly Asp Val Asp Ala Thr Met Ser Ile Leu Asp Ile Ser Met Met Thr 
    1395                1400                1405 

ggc ttt gct cca gac aca aag gac ctg gaa ctg ctg gcc tct gga gta     4331 
Gly Phe Ala Pro Asp Thr Lys Asp Leu Glu Leu Leu Ala Ser Gly Val 
1410                1415                1420                1425 

gat aga tac atc tcc aag tac gag atg aac aaa gcc ttc tcc aac aag     4379 
Asp Arg Tyr Ile Ser Lys Tyr Glu Met Asn Lys Ala Phe Ser Asn Lys 
                1430                1435                1440 

aac acc ctc atc atc tac cta gaa aag att tca cac acc gaa gaa gac     4427 
Asn Thr Leu Ile Ile Tyr Leu Glu Lys Ile Ser His Thr Glu Glu Asp 
            1445                1450                1455 

tgc ctg acc ttc aaa gtt cac cag tac ttt aat gtg gga ctt atc cag     4475 
Cys Leu Thr Phe Lys Val His Gln Tyr Phe Asn Val Gly Leu Ile Gln 
        1460                1465                1470 

ccc ggg tcg gtc aag gtc tac tcc tat tac aac ctc gag gaa tca tgc     4523 
Pro Gly Ser Val Lys Val Tyr Ser Tyr Tyr Asn Leu Glu Glu Ser Cys 
    1475                1480                1485 

acc cgg ttc tat cat cca gag aag gac gat ggg atg ctc agc aag ctg     4571 
Thr Arg Phe Tyr His Pro Glu Lys Asp Asp Gly Met Leu Ser Lys Leu 
1490                1495                1500                1505 

tgc cac agt gaa atg tgc cgg tgt gct gaa gag aac tgc ttc atg caa     4619 
Cys His Ser Glu Met Cys Arg Cys Ala Glu Glu Asn Cys Phe Met Gln 
                1510                1515                1520 

cag tca cag gag aag atc aac ctg aat gtc cgg cta gac aag gct tgt     4667 
Gln Ser Gln Glu Lys Ile Asn Leu Asn Val Arg Leu Asp Lys Ala Cys 
            1525                1530                1535 

gag ccc gga gtc gac tat gtg tac aag acc gag cta acc aac ata aag     4715 
Glu Pro Gly Val Asp Tyr Val Tyr Lys Thr Glu Leu Thr Asn Ile Lys 
        1540                1545                1550 

ctg ttg gat gat ttt gat gag tac acc atg acc atc cag cag gtc atc     4763 
Leu Leu Asp Asp Phe Asp Glu Tyr Thr Met Thr Ile Gln Gln Val Ile 
    1555                1560                1565 

aag tca ggc tca gat gag gtg cag gca ggg cag caa cgc aag ttc atc     4811 
Lys Ser Gly Ser Asp Glu Val Gln Ala Gly Gln Gln Arg Lys Phe Ile 
1570                1575                1580                1585 

agc cac atc aag tgc aga aac gcc ctg aag ctg cag aaa ggg aag aag     4859 
Ser His Ile Lys Cys Arg Asn Ala Leu Lys Leu Gln Lys Gly Lys Lys 
                1590                1595                1600 

tac ctc atg tgg ggc ctc tcc tct gac ctc tgg gga gaa aag ccc aac     4907 
Tyr Leu Met Trp Gly Leu Ser Ser Asp Leu Trp Gly Glu Lys Pro Asn 
            1605                1610                1615 

acc agc tac atc att ggg aag gac acg tgg gtg gag cac tgg cct gag     4955 
Thr Ser Tyr Ile Ile Gly Lys Asp Thr Trp Val Glu His Trp Pro Glu 
        1620                1625                1630 

gca gaa gaa tgc cag gat cag aag tac cag aaa cag tgc gaa gaa ctt     5003 
Ala Glu Glu Cys Gln Asp Gln Lys Tyr Gln Lys Gln Cys Glu Glu Leu 
    1635                1640                1645 

ggg gca ttc aca gaa tct atg gtg gtt tat ggt tgt ccc aac tga         5048 
Gly Ala Phe Thr Glu Ser Met Val Val Tyr Gly Cys Pro Asn 
1650                1655                1660 

ctacagccca gccctctaat aaagcttcag ttgtatttc                          5087 

 
           
             112  
             494  
             DNA  
             Mus musculus  
             
 
           
            112 

gctggcttca aacagcccag ctctgcctat gctgccttca acaaccggcc ccccagcacc     60 

tgggtagcgg gttgtcagct ctgtcccctc tgcctcaaca tccacgtgag caaagcctga    120 

ttccccacca gtggtggtct ggcctctctc tgtcaaggct gcagggactg aatgagcctt    180 

agagtccttt aagcaccagc tttatgcggc tttgaaatta aaaatccata actgagggct    240 

ctgcaccagg ccctctctgg tcattggtgg gtgaagatgt caatctatct actaaaacca    300 

atcgagtctc agctggtgtt cctataactc cgccccagct gacagcctac gtggtcaagg    360 

tcttctctct agctgccaac ctcatcgcca tcgactctca cgtcctgtgt ggggctgtta    420 

aatggttgat tctggagaaa cagaagccgg atggtgtctt tcaggaggat gggcccgtga    480 

ttcaccaaga aatg                                                      494 

 
           
             113  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            113 

gctggtccca tagtgaagga                                                 20 

 
           
             114  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            114 

cagcagtagc actagtagct                                                 20 

 
           
             115  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            115 

gggagctggc caacagcagc                                                 20 

 
           
             116  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            116 

gctcctgtca acactgtctt                                                 20 

 
           
             117  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            117 

tactggctgg aatcttgatg                                                 20 

 
           
             118  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            118 

ccccgaagtt tgccaccact                                                 20 

 
           
             119  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            119 

gatgacgact gtcttgccca                                                 20 

 
           
             120  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            120 

gggcagcacg tattccttca                                                 20 

 
           
             121  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            121 

ggccagagaa atcttcttat                                                 20 

 
           
             122  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            122 

ggatcccact gcgctctgcc                                                 20 

 
           
             123  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            123 

aatttgggtg tcttggtgaa                                                 20 

 
           
             124  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            124 

ttgtgcatag tgctgtaggg                                                 20 

 
           
             125  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            125 

cgcaggtgga agttgacatt                                                 20 

 
           
             126  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            126 

cttccccttg ttcataacca                                                 20 

 
           
             127  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            127 

ggtgtagtaa gccaccaggc                                                 20 

 
           
             128  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            128 

cttgtccaca gccactagcc                                                 20 

 
           
             129  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            129 

agttcttccc actgcctggg                                                 20 

 
           
             130  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            130 

gtgtactgac cagctttgtc                                                 20 

 
           
             131  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            131 

ctcgccctgg gtgatgaggc                                                 20 

 
           
             132  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            132 

tcactcctgg ccaggcccag                                                 20 

 
           
             133  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            133 

tgggaagtgg cttctagaga                                                 20 

 
           
             134  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            134 

ttcaactctt ctatggtcca                                                 20 

 
           
             135  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            135 

tggaatcttt gagaaagatg                                                 20 

 
           
             136  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            136 

caggtggtga tggaatcttt                                                 20 

 
           
             137  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            137 

acaagctcac tgccagaatc                                                 20 

 
           
             138  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            138 

ttgaagagca cagctctgat                                                 20 

 
           
             139  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            139 

ggccatgctg cagaaggctg                                                 20 

 
           
             140  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            140 

tccttctggc acgaccttca                                                 20 

 
           
             141  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            141 

tctgtgtctg gcacttggtc                                                 20 

 
           
             142  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            142 

gtgtttcagc cgctccccgt                                                 20 

 
           
             143  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            143 

tcacgatcag gtgtttcagc                                                 20 

 
           
             144  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            144 

gtgtcatgcc aatcatgttc                                                 20 

 
           
             145  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            145 

ccctttcttg atgagctcca                                                 20 

 
           
             146  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            146 

gttgaaggca gcataggcag                                                 20 

 
           
             147  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            147 

gctgtcagcc aggtgctggg                                                 20 

 
           
             148  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            148 

cttctgtttc tccagaatca                                                 20 

 
           
             149  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            149 

tcctcctgaa agacaccatc                                                 20 

 
           
             150  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            150 

ttcctgcagt gcgatgagga                                                 20 

 
           
             151  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            151 

ttgacctgcc cctcacagat                                                 20 

 
           
             152  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            152 

cctgccttgt tgatgctccc                                                 20 

 
           
             153  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            153 

gtatggtctc tgcaggttca                                                 20 

 
           
             154  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            154 

agggcatacc cagcaatggc                                                 20 

 
           
             155  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            155 

ccagtttgtt catcagggcc                                                 20 

 
           
             156  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            156 

gtaaggttcc tccagtttgt                                                 20 

 
           
             157  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            157 

tcctcccagc ggttccgatc                                                 20 

 
           
             158  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            158 

ttcagcagca gcagggccag                                                 20 

 
           
             159  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            159 

ggcacagagt caaagtcttt                                                 20 

 
           
             160  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            160 

gaataccatg aaggtagcct                                                 20 

 
           
             161  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            161 

ccaaggcttg gaataccatg                                                 20 

 
           
             162  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            162 

cacatccatg ttcaagtcct                                                 20 

 
           
             163  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            163 

tgaccctgag gtcaaacttc                                                 20 

 
           
             164  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            164 

ggcttcttgg ctgtctcagg                                                 20 

 
           
             165  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            165 

gatgtccagg atggacatag                                                 20 

 
           
             166  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            166 

aaagccagtc atcatggaga                                                 20 

 
           
             167  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            167 

tcttgttgga gaaggctttg                                                 20 

 
           
             168  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            168 

atcttttcta ggtagatgat                                                 20 

 
           
             169  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            169 

gagtagacct tgaccgaccc                                                 20 

 
           
             170  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            170 

atgatagaac cgggtgcatg                                                 20 

 
           
             171  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            171 

ggctcacaag ccttgtctag                                                 20 

 
           
             172  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            172 

gcctgacttg atgacctgct                                                 20 

 
           
             173  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            173 

cctgcctgca cctcatctga                                                 20 

 
           
             174  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            174 

gaggccccac atgaggtact                                                 20 

 
           
             175  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            175 

gggcttttct ccccagaggt                                                 20 

 
           
             176  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            176 

cttcccaatg atgtagctgg                                                 20 

 
           
             177  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            177 

cagtgctcca cccacgtgtc                                                 20 

 
           
             178  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            178 

ggctgtagtc agttgggaca                                                 20 

 
           
             179  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            179 

aaatacaact gaagctttat                                                 20