AU2011203319B2 - Antisense modulation of C-reactive protein expression - Google Patents

Abstract

-101 ANTISENSE MODULATION OF C-REACTIVE PROTEIN EXPRESSION Abstract Antisense compounds, compositions and methods are provided for modulating the expression of C-reactive protein. The compositions comprise antisense compounds, particularly 5 antisense oligonucleotides, targeted to nucleic acids encoding C-reactive protein. Methods of using these compounds for modulation of C-reactive protein expression and for treatment of diseases associated with expression of C-reactive protein are provided.

Description

EDITORIAL NOTE Number: 2011203319 The following Specification contains page numbers Description: 1 to 95 Claims: 96 to 98 Abstract: 101 Australian Patents Act 1990 - Regulation 3.2 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Antisense modulation of C-reactive protein expression The following statement is a full description of this invention, including the best method of performing it known to me: P/00/011 Cos I WO 03/010284 PCT/US02/22656 ANTISENSE MODULATION OF C-REACTIVE PROTEIN EXPRESSION FIELD OF THE INVENTION The present invention provides compositions and 5 methods for modulating the expression of C-reactive protein. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding C-reactive protein. Such compounds have been shown to modulate the 10 expression of C-reactive protein. BACKGROUND OF THE INVENTION C-reactive protein (also known as CRP and PTX1) is an essential human acute-phase reactant produced in the liver in response to a variety of inflammatory cytokines. The 15 protein, first identified in 1930, is highly conserved and considered to be an early indicator of infectious or inflammatory conditions. Plasma CRP levels increase 1,000 fold in response to infection, ischemia, trauma, burns, and inflammatory conditions. Since the biological half 20 life of CRP is not influenced by age, liver or kidney function or pharmacotherapy, it is reliable biochemical marker for tissue destruction, necrosis and inflammation and its measurement is widely used to monitor various inflammatory states, angina pectoris, vascular insults, 25 end-stage renal disease, rheumatoid arthritis, obesity and atherosclerosis (Arici and Walls, Kidney Int., 2001, 59, 407-414; Gabay and Kushner, N. Engl. J. Med., 1999, 340, 448-454; Highton et al., J. Rheumatol., 1985, 12, 871-875; Hulthe et al., Clin. Sci. (Colch), 2001, 100, 371-378; 30 Lagrand et al., Circulation, 1999, 100, 96-102; Morrow and Ridker, Med. Clin. North. Am., 2000, 84, 149-161, ix; Szalai et al., Immunol. Res., 1997, 16, 127-136; WO 03/010284 PCT/US02/22656 -2 Westhuyzen and Healy, Ann. Clin. Lab Sci., 2000, 30, 133 143; Yudkin et al., Atherosclerosis, 2000, 148, 209-214). Improved methods of quantifying CRP have led to increased application to clinical medicine including 5 diagnoses of urinary tract infections (Arici and Walls, Kidney Int., 2001, 59, 407-414), meningitis (Ruuskanen et al., J. Pediatr., 1985, 107, 97-100), neonatal sepsis, erythropoietin resistance (Barany, Nephrol. Dial. Transplant., 2001, 16, 224-227) and occult bacteremia, 10 conditions in which CRP is usually elevated. Structurally, CRP is a member of the pentraxin family of proteins, which are characterized by a cyclic pentameric structure and radial symmetry. The five identical 24-kDa protomers consist of 206 amino acids, and 15 are noncovalently linked (Lei et al., J. Biol. Chem., 1985, 260, 13377-13383; Szalai et al., Immunol. Res., 1997, 16, 127-136). The genomic DNA sequence for human CRP has been reported by Lei et al. (Lei et al., J. Biol. Chem., 1985, 260, 13377-13383) as have mutant forms of the 20 protein (Potempa et al., 1996) and methods to deliver materials into cells using the mutant protein as a carrier (Potempa et al., 2000). Polypeptides corresponding to amino acids 174-185 of CRP having immunomodulatory activity are disclosed and claimed US Patent 5,783,179 25 (Nestor et al., 1998). Peptides corresponding to positions 62-71 of human CRP have also been studied for their ability to inhibit the activity of human leukocyte elastase and/or cathepsin G for the treatment of inflammatory conditions and these are disclosed in the PCT 30 Publication WO 99/00418 (Fridkin, 1999). The CRP protein binds to a broad range of cellular substances such as phosphocholine, fibronectin, chromatin, histones, and ribonucleoprotein in a calcium-dependent WO 03/010284 PCT/US02/22656 -3 manner (Szalai et al., Immuno. Res., 1997, 16, 127-136). It is a ligand for specific receptors on phagocytic leukocytes, mediates activation reactions on monocytes and macrophages, and activates complement (Szalai et al., 5 Immunol. Res., 1997, 16, 127-136). The function of CRP is felt to be related to its role in the innate immune system. Similar to Ig (Ig)G, it activates complement, binds to Fc receptors and acts as an opsonin for various pathogens. Interaction of CRP with Fc 10 receptors leads to the generation of proinflammatory cytokines that enhance the inflammatory response. Unlike IgG, which specifically recognizes distinct antigenic epitopes, CRP recognizes altered self and foreign molecules based on pattern recognition. CRP is therefore 15 thought to act as a surveillance molecule for altered self and certain pathogens. This recognition provides early defense and leads to a proinflammatory signal and activation of the humoral, adaptive immune system. Thus, the CRP molecule has both a recognition and an effector 20 function. The pharmacological modulation of C-reactive protein activity and/or expression is therefore believed to be an appropriate point of therapeutic intervention in pathological conditions. 25 Strategies aimed at modulating C-reactive protein function by targeting protein levels have involved the use of antibodies, peptides and molecules that inhibit HMG-CoA reductase. Recently, Ridker et al. have demonstrated that 30 lovastatin, an inhibitor of the enzyme HMG-CoA reductase, is an effective agent in reducing the risk of acute coronary events in participants with elevated CRP levels but no hyperlipidemia. In this trial, the use of WO 03/010284 PCT/U S02/22656 -4 lovastatin resulted in a 14.8 percent reduction in median CRP levels after one year whereas no change was observed in the placebo group (Ridker et al., N. Engl. J. Med., 2001, 344, 1959-1965). Another statin, cerivastatin, has 5 also been demonstrated to lower CRP levels in patients with hypercholesterolemia (Ridker et al., Circulation, 2001, 103, 1191-1193). However, there are currently no known therapeutic agents which effectively inhibit CRP levels and function. 10 Consequently, there remains a long felt need for agents capable of effectively and selectively inhibiting CRP. 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 15 a number of therapeutic, diagnostic, and research applications for the modulation of CRP expression. The present invention provides compositions and methods for modulating CRP expression. SUMMARY OF THE INVENTION 20 The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding C-reactive protein, and which modulate the expression of C-reactive protein. Pharmaceutical and other compositions comprising the 25 compounds of the invention are also provided. Further provided are methods of modulating the expression of C reactive protein in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further 30 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 C-reactive protein by administering a therapeutically or prophylactically WO 03/010284 PCT/US02/22656 -5 effective amount of one or more of the antisense compounds or compositions of the invention. DETAILED DESCRIPTION OF THE INVENTION The present invention employs oligomeric compounds, 5 particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding C-reactive protein, ultimately modulating the amount of C reactive protein produced. This is accomplished by providing antisense compounds which specifically hybridize 10 with one or more nucleic acids encoding C-reactive protein. As used herein, the terms "target nucleic acid" and "nucleic acid encoding C-reactive protein" encompass DNA encoding C-reactive protein, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived 15 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 20 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 25 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 C-reactive protein. In 30 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 WO 03/010284 PCT/U S02/22656 -6 of modulation of gene expression and mRNA is a preferred target. It is preferred to target specific nucleic acids for antisense. "Targeting" an antisense compound to a 5 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 10 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 C-reactive protein. The targeting process also includes determination of a site or 15 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 20 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 25 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 30 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 WO 03/010284 PCT/US02/22656 -7 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 5 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 C-reactive protein, regardless of the sequence(s) 10 of such codons. It is also known in the art that a translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, 15 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, 20 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. 25 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' 30 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 WO 03/010284 PCT/US02/22656 -8 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 5 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 10 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. Although some eukaryotic mRNA transcripts are 15 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 20 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 25 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. Once one or more target sites have been identified, 30 oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

WO 03/010284 PCTIUS02/22656 -9 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 5 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 10 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 15 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 20 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 25 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 30 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 WO 03/010284 PCT/US02/22656 -10 assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. Antisense and other compounds of the invention which 5 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 10 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. Antisense compounds are commonly used as research 15 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 20 example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use. For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in 25 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. 30 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 WO 03/010284 PCT/U S02/22656 -11 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 5 performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns. Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and 10 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., 15 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 20 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, 25 differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Supply , 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. 30 Cancer, 1999, 35, 1895-904) and mass spectrometry methods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41). The specificity and sensitivity of antisense is also WO 03/010284 PCTIUS02/22656 -12 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, 5 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 10 treatment of cells, tissues and animals, especially humans. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or 15 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 20 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. 25 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 30 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 WO 03/010284 PCT/US02/22656 -13 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 5 hybridize to the target nucleic acid and modulate its expression. 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 10 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 15 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 20 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 25 backbone of RNA and DNA is a 3' to 5' phosphodiester linkage. Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural 30 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 WO 03/010284 PCT/US02/22656 -14 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 5 be oligonucleosides. Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates 10 including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, 15 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 20 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. 25 Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S.: 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; 30 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 WO 03/010284 PCT/US02/22656 -15 which are commonly owned with this application, and each of which i-s herein incorporated by reference. Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that 5 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 10 (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; 15 sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, 0, S and CH2 component parts. Representative United States patents that teach the 20 preparation of the above oligonucleosides include, but are not limited to, U.S.: 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; 25 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. 30 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 WO 03/010284 PCT/US02/22656 -16 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 5 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. 10 Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S.: 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., 15 Science, 1991, 254, 1497-1500. Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH 2 -NH-0-CH 2 -, -CH 2

-N(CH

3 )-0-CH 2 - [known as a 20 methylene (methylimino) or MMI backbone], -CH 2 -0-N(CH 3

)

CH

2 -, -CH 2

-N(CH

3

)-N(CH

3

)-CH

2 - and -0-N(CH 3

)-CH

2

-CH

2 - [wherein the native phosphodiester backbone is represented as -O-P 0-CH 2 -] of the above referenced U.S. patent 5,489,677, and the amide backbones of the above referenced U.S. patent 25 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. patent 5,034,506. Modified oligonucleotides may also contain one or 30 more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the WO 03/010284 PCT/US02/22656 -17 alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to Cio alkyl or C 2 to C 10 alkenyl and alkynyl. Particularly preferred are 0[(CH 2 )nOlmCH 3 ,

O(CH

2 )nOCH 3 , O(CH 2 )nNH 2 , O (CH 2 )nCH 3 , O(CH 2 )nONH 2 , and 5 O(CH 2 )nON[(CH 2 ) nCH 3

)]

2 , where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C1 to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, 0-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, 10 CF 3 , OCF 3 , SOCH 3 , SO 2

CH

3 , ON0 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 15 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, 20 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2' -dimethylaminooxyethoxy, i.e., a O(CH 2

)

2 0N(CH 3

)

2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylamino ethoxyethoxy (also known in the art as 2'-0-dimethylamino 25 ethoxyethyl or 2'-DMAEOE), i.e., 2'-0-CH 2 -0-CH 2

-N(CH

2

)

2 , also described in examples hereinbelow. A further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring 30 thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (-CH 2 -)n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 WO 03/010284 PCT/US02/22656 -18 and WO 99/14226. Other preferred modifications include 2'-methoxy (2'

O-CH

3 ), 2' -aminopropoxy (2'-OCH 2

CH

2

CH

2

NH

2 ), 2'-allyl (2'

CH

2

-CH=CH

2 ), 2'-0-allyl (2'-O-CH 2

-CH=CH

2 ) and 2'-fluoro (2' 5 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' 10 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 15 preparation of such modified sugar structures include, but are not limited to, U.S.: 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; 20 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. Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or 25 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 30 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- WO 03/010284 PCT/US02/22656 -19 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 5 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 10 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 (lH-pyrimido[5,4 15 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-blindol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2 20 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 United States 25 Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed 30 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 WO 03/010284 PCTUS02/22656 -20 the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyl adenine, 5-propynyluracil and 5-propynylcytosine. 5 5 methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 0 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 10 particularly when combined with 2'-0-methoxyethyl sugar modifications. Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, 15 but are not limited to, the above noted U.S. 3,687,808, as well as U.S.: 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; 20 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 United States patent 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference. 25 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 30 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, WO 03/010284 PCT/US02/22656 -21 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, 5 phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmaco dynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance 10 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. 15 Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed October 23, 1992 the entire disclosure of which is incor porated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a 20 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 25 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, 30 259, 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, WO 03/010284 PCT/US02/22656 -22 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., 5 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 10 invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbuta zone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+) pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodo benzoic acid, flufenamic acid, folinic acid, a 15 benzothiadiazide, chlorothiazide, a diazepine, indo methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in United States Patent Application 09/334,130 20 (filed June 15, 1999) which is incorporated herein by reference in its entirety. Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S.: 4,828,979; 4,948,882; 25 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; 30 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; WO 03/010284 PCT/U S02/22656 -23 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 5 herein incorporated by reference. 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 10 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 15 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 20 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 25 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 30 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 WO 03/010284 PCT/US02/22656 -24 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. 5 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 10 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.: 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; 15 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. The antisense compounds used in accordance with this invention may be conveniently and routinely made through 20 the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well 25 known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The antisense compounds of the invention are synthesized in vitro and do not include antisense 30 compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules. The compounds of the invention may also be admixed, WO 03/010284 PCT/US02/22656 -25 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, 5 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.: 5,108,921; 5,354,844; 5,416,016; 5,459,127; 10 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, 15 each of which is herein incorporated by reference. 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 20 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 25 prodrugs, and other bioequivalents. The term "prodrug" indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals 30 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 WO 03/010284 PCT/US02/22656 -26 al., published December 9, 1993 or in WO 94/26764 and U.S. 5,770,713 to Imbach et al. The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts 5 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. Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline 10 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, 15 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 20 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 25 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 30 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 WO 03/010284 PCT/US02/22656 -27 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, 5 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 10 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 15 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 20 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 25 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 30 ammonium cations. Carbonates or hydrogen carbonates are also possible. For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not WO 03/010284 PCT/US02/22656 -28 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 5 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, 10 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 15 chlorine, bromine, and iodine. 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 20 or disorder which can be treated by modulating the expression of C-reactive protein 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 25 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. 30 The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding C-reactive protein, enabling sandwich and other assays to easily be WO 03/010284 PCT/US02/22656 -29 constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding C-reactive protein can be detected by means known in the art. Such means may include conjugation of 5 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 C-reactive protein in a sample may also be prepared. The present invention also includes pharmaceutical 10 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 15 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 20 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 25 at least one 2'-0-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, 30 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 WO 03/010284 PCT/US02/22656 -30 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. 5 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. 10 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, 15 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, 20 linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C 1

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10 alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or 25 pharmaceutically acceptable salt thereof. Topical formulations are described in detail in United States patent application 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety. Compositions and formulations for oral administration 30 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, WO 03/010284 PCT/US02/22656 -31 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 5 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 10 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 15 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 20 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, 25 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. 30 Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, WO 03/010284 PCT/US02/22656 -32 starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, 5 N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), 10 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), 15 alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in United States applications 08/886,829 (filed July 1, 1997), 09/108,673 (filed July 1, 1998), 09/256,515 (filed February 23, 1999), 09/082,624 (filed May 21, 1998) 20 and 09/315,298 (filed May 20, 1999) each of which is incorporated herein by reference in their entirety. Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, 25 diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and 30 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.

WO 03/010284 PCT/US02/22656 -33 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. 5 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 10 finely divided solid carriers or both, and then, if necessary, shaping the product. 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, 15 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, 20 for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as 25 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 30 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.

WO 03/010284 PCT/US02/22656 -34 Emulsions The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in 5 another in the form of droplets usually exceeding 0.1 ptm 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 10 (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's Pharmaceutical Sciences, Mack Publishing Co., 15 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 20 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. 25 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 30 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 WO 03/010284 PCT/US02/22656 -35 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 5 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. 10 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 15 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. 20 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, 25 N.Y., volume 1, p. 199). 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 30 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).

WO 03/010284 PCT/US02/22656 -36 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 5 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 10 Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285). Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic 15 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 20 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 25 such as carbon or glyceryl tristearate. 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, 30 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 WO 03/010284 PCT/US02/22656 -37 Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as 5 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 10 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. 15 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 20 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 25 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. 30 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 WO 03/010284 PCT/US02/22656 -38 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 5 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, 10 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. In one embodiment of the present invention, the 15 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, 20 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, 25 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 30 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 WO 03/010284 PCT/US02/22656 -39 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 5 and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 1985, p. 271). 10 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, 15 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 20 solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously. Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non 25 ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (M0310), hexaglycerol monooleate (P0310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate 30 (M0750), decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves WO 03/010284 PCT/US02/22656 -40 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 5 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 10 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, 15 saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil. Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w 20 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 25 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, 30 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 WO 03/010284 PCT/US02/22656 -41 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 5 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 10 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. Microemulsions of the present invention may also 15 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 20 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 25 Systems, 1991, p. 92). Each of these classes has been discussed above. Liposomes There are many organized surfactant structures besides microemulsions that have been studied and used for 30 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 WO 03/010284 PCT/US02/22656 -42 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. 5 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. 10 Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each 15 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. Further advantages of liposomes include; liposomes 20 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 25 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. 30 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 WO 03/010284 PCT/U S02/22656 -43 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. 5 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 10 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. 15 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 20 applications resulted in the targeting of the upper epidermis. Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable 25 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. 30 Biophys. Res. Commun., 1987, 147, 980-985). Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly WO 03/010284 PCT/US02/22656 -44 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 5 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). One major type of liposomal composition includes phospholipids other than naturally-derived 10 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 15 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. 20 Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol. Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin 25 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 30 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 WO 03/010284 PCT/US02/22656 -45 aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265). Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the 5 skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising NovasomeTM I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and NovasometM II (glyceryl distearate/ 10 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. 15 S.T.P.Pharma. Sci., 1994, 4, 6, 466). 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 20 circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside Gm1, or (B) is 25 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 30 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., WO 03/010284 PCT/US02/22656 -46 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 Gmj, 5 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. Patent No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes 10 comprising (1) sphingomyelin and (2) the ganglioside Gm or a galactocerebroside sulfate ester. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn dimyristoylphosphatidylcholine are disclosed in WO 15 97/13499 (Lim et al.). 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 20 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 25 attachment of carboxylic groups of polyalkylene glycols (.e.g., PEG) are described by Sears (U.S. Patent Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) 30 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, WO 03/010284 PCT/US02/22656 -47 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 5 445 131 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. Patent Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Patent No. 5,213,804 10 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. Patent No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids 15 are described in WO 96/10391 (Choi et al.). U.S. Patent Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces. A limited number of liposomes comprising nucleic 20 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. Patent No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an 25 antisense RNA. U.S. Patent 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. 30 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 WO 03/010284 PCT/US02/22656 -48 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 5 (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 10 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. 15 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 20 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 25 York, NY, 1988, p. 285). 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. 30 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 WO 03/010284 PCT/US02/22656 -49 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 5 in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class. 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 10 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 15 important members of the anionic surfactant class are the alkyl sulfates and the soaps. 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 20 quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class. If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is 25 classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N alkylbetaines and phosphatides. The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in 30 Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

WO 03/010284 PCT/USO2/22656 -50 Penetration Enhancers In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, 5 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 10 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. 15 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 20 the above mentioned classes of penetration enhancers are described below in greater detail. Surfactants: In connection with the present invention, surfactants (or "surface-active agents") are chemical entities which, when dissolved in an aqueous 25 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 30 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 WO 03/010284 PCT/US02/22656 -51 emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252). Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, 5 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 10 monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, CI.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 15 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). Bile salts: The physiological role of bile includes 20 the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman'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 25 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 30 acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic WO 03/010284 PCT/US02/22656 -52 acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium 5 tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, 10 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). 15 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 20 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 25 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 30 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 WO 03/010284 PCT/US02/22656 -53 Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51). Non-chelating non-surfactants: As used herein, non chelating non-surfactant penetration enhancing compounds 5 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 10 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 15 sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626). 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, 20 cationic lipids, such as lipofectin (Junichi et al, U.S. Patent 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. 25 - 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. 30 Carriers Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a WO 03/010284 PCT/US02/22656 -54 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 5 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 10 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 15 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 & Nucl. Acid Drug Dev., 1996, 20 6, 177-183). Excipients In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically 25 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 30 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, WO 03/010284 PCT/US02/22656 -55 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 5 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 10 sulphate, etc.). 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 15 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. 20 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 25 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. 30 Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, WO 03/010284 PCT/USO2/22656 -56 magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like. Other Components The compositions of the present invention may 5 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, 10 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, 15 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 20 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. 25 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. Certain embodiments of the invention provide 30 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 WO 03/010284 PCT/US02/22656 -57 include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, 5 mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, 10 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 15 (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 20 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 25 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 30 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 WO 03/010284 PCT/US02/22656 -58 non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially. In another related embodiment, compositions of the 5 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 10 or more combined compounds may be used together or sequentially. 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 15 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 20 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 25 estimated based on EC 50 s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in 30 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 WO 03/010284 PCT/US02/22656 -59 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 5 or more daily, to once every 20 years. 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 10 same. EXAMPLES Example 1 Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2'-alkoxy amidites 15 2'-Deoxy and 2'-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham MA or Glen Research, Inc. Sterling VA). Other 2'-0-alkoxy substituted nucleoside amidites are prepared as described in U.S. Patent 20 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. 25 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 30 ChemGenes, Needham MA). 2'-Fluoro amidites 2'-Fluorodeoxyadenosine amidites 2'-fluoro oligonucleotides were synthesized as WO 03/010284 PCT/US02/22656 -60 described previously. [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and United States patent 5,670,633, herein incorporated by reference. Briefly, the protected nucleoside N6-benzoyl-2'-deoxy-2'-fluoroadenosine was 5 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 SN2-displacement of a 2' beta-trityl group. Thus N6-benzoyl-9-beta-D 10 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' 15 dimethoxytrityl-(DMT) and 5'-DMT-3'-phosphoramidite intermediates. 2'-Fluorodeoxyguanosine The synthesis of 2'-deoxy-2'-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) 20 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 25 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. 30 2'-Fluorouridine 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 WO 03/010284 PCT/US02/22656 -61 treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5'-DMT and 5'-DMT 3'phosphoramidites. 2'-Fluorodeoxycytidine 5 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. 10 2'-0-(2-Methoxyethyl) modified amidites 2'-0-Methoxyethyl-substituted nucleoside amidites are prepared as follows, or alternatively, as per the methods of Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504. 2,2'-Anhydro[1-(beta-D-arabinofuranosyl)-5 15 methyluridine] 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). 20 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 25 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 0 C at 1 30 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 WO 03/010284 PCT/US02/22656 -62 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 0 C). 5 2 '-O-Methoxyethyl-5-methyluridine 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 0 C. After 10 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 15 (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 C1 2 (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with 20 the packing solvent to give 160 g (63%) of product. Additional material was obtained by reworking impure fractions. 2' -O-Methoxyethyl-5' -O-dimethoxytrityl-5 methyluridine 25 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 30 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% WO 03/010284 PCT/US02/22656 -63 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 2x500 mL of saturated NaHCO 3 and 2x500 mL of saturated NaCl. The organic phase was dried over 5 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 10 additional was obtained from the impure fractions to give a total yield of 183 g (57%). 31-0-Acetyl-2'.-O-methoxyethyl-5'-O-dimethoxytrityl-5 methyluridine 2'-0-Methoxyethyl-5'-O-dimethoxytrityl-5 15 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 20 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 0 C. The residue was dissolved in CHC1 3 (800 mL) and extracted with 2x200 mL of saturated sodium bicarbonate and 2x200 mL of saturated NaCl. The 25 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 30 were evaporated to yield 96 g (84%). An additional 1.5 g was recovered from later fractions.

WO 03/010284 PCT/US02/22656 -64 3'-0-Acetyl-2'-0-methoxyethyl-5'-O-dimethoxytrityl-5 methyl-4-triazoleuridine A first solution was prepared by dissolving 3'-0 acetyl-2'-0-methoxyethyl-5'-0-dimethoxytrityl-5 S 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 10 stirred solution maintained at 0-10 0 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 15 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 1x300 mL of NaHCO 3 and 2x300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The 20 residue was triturated with EtOAc to give the title compound. 2'-O-Methoxyethyl-5'-0-dimethoxytrityl-5 methylcytidine A solution of 3'-0-acetyl-2'-O-methoxyethyl-5'-0 25 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 (2x200 mL). The residue was dissolved in MeOH (300 mL) and 30 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 0 C for 2 hours (TLC showed complete conversion). The vessel contents were evaporated to WO 03/010284 PCT/US02/22656 -65 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. 5 N4-Benzoyl-2'-0-methoxyethyl-5'-0-dimethoxytrityl-5 methylcytidine 2'-0-Methoxyethyl-5'-0-dimethoxytrityl-5-methyl cytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with 10 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 (2x300 mL) and saturated 15 NaCl (2x300 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. 20 N4-Benzoyl-2'-O-methoxyethyl-5'-0-dimethoxytrityl-5 methylcytidine-3'-amidite N4-Benzoyl-2'-0-methoxyethyl-5'-0-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 25 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 (1x300 mL) and 30 saturated NaCl (3x300 mL). The aqueous washes were back extracted with CH 2 C1 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 WO 03/010284 PCT/US02/22656 -66 using EtOAc/hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound. 2'-O-(Aminooxyethyl) nucleoside amidites and 2'-0 5 (dimethylaminooxyethyl) nucleoside amidites 2'-(Dimethylaminooxyethoxy) nucleoside amidites 2'-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the art as 2'-0-(dimethylaminooxyethyl) nucleoside amidites] are prepared as described in the 10 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 15 guanosine. 5'-0-tert-Butyldiphenylsilyl-0 2 -2'-anhydro-5 methyluridine 02-2'-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0g, 0.416 mmol), dimethylaminopyridine 20 (0.66g, 0.013eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert Butyldiphenylchlorosilane (125.8g, 119.OmL, 1.leq, 0.458mmol) was added in one portion. The reaction was 25 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 (2x1 L) and brine 30 (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 (600mL) and the solution was cooled to WO 03/010284 PCT/US02/22656 -67 -10*C. The resulting crystalline product was collected by filtration, washed with ethyl ether (3x200 mL) and dried (40 0 C, 1mm Hg, 24 h) to 149g (74.8%) of white solid. TLC and NMR were consistent with pure product. 5 5'-O-tert-Butyldiphenyleilyl-2'-O-(2-hydroxyethyl)-5 methyluridine 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 10 glycol (350 mL, excess) was added cautiously at first until the evolution of hydrogen gas subsided. 5'-O-tert Butyldiphenylsilyl-0 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 15 heated in an oil bath until an internal temperature of 1600 C was reached and then maintained for 16 h (pressure < 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% 20 conversion to the product. In order to avoid additional side product formation, the reaction was stopped, concentrated under reduced pressure (10 to 1mm Hg) in a warm water bath (40-100 0 C) with the more extreme conditions used to remove the ethylene glycol. [Alternatively, once 25 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 (2kg silica gel, ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate 30 fractions were combined, stripped and dried to product as a white crisp foam (84g, 50%), contaminated starting material (17.4g) and pure reusable starting material 20g. The yield based on starting material less pure recovered WO 03/010284 PCT/US02/22656 -68 starting material was 58%. TLC and NMR were consistent with 99% pure product. 2'-0-([2-phthalimidoxy)ethyl]-S'-t butyldiphenylsilyl-5-methyluridine 5 5'-0-tert-Butyldiphenylsilyl-2'-0-(2-hydroxyethyl)-5 methyluridine (20g, 36.98mmol) was mixed with triphenylphosphine (11.63g, 44.36mmol) and N hydroxyphthalimide (7.24g, 44.36mmol). It was then dried over P 2 0 5 under high vacuum for two days at 40*C. The 10 reaction mixture was flushed with argon and dry THF (369.8mL, Aldrich, sure seal bottle) was added to get a clear solution. Diethyl-azodicarboxylate (6.98mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of addition is maintained such that resulting 15 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 20 was placed on a flash column and eluted with ethyl acetate:hexane (60:40), to get 2'-0-([2 phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5 methyluridine as white foam (21.819 g, 86%). 5'-o-tert-butyldiphenylsilyl-2'-0-[(2 25 formadoximinooxy)ethyl]-5-methyluridine 2'-0-([2-phthalimidoxy)ethyl]-5'-t butyldiphenylsilyl-5-methyluridine (3.lg, 4.5mmol) was dissolved in dry CH 2 Cl 2 (4.5mL) and methylhydrazine (300mL, 4.64mmol) was added dropwise at -10 0 C to 0*C. After 1 h 30 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, WO 03/010284 PCT/US02/22656 -69 which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was stirred for 1 h. Solvent was removed under vacuum; residue chromatographed 5 to get 5'-O-tert-butyldiphenylsilyl-2'-0-[(2 formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%). 5'-0-tert-Butyldiphenylsilyl-2'-0-[N,N dimethylaminooxyethyl]-5-methyluridine 10 5'-O-tert-butyldiphenylsilyl-2'-0-[(2 formadoximinooxy)ethyl]-5-methyluridine (1.77g, 3.12mmol) was dissolved in a solution of 1M pyridinium p toluenesulfonate (PPTS) in dry MeOH (30.6mL). Sodium cyanoborohydride (0.39g, 6.13mmol) was added to this 15 solution at 10 0 C under inert atmosphere. The reaction mixture was stirred for 10 minutes at 10 0 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%, 10mL) 20 was added and extracted with ethyl acetate (2x2OmL). 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 (30.6mL). Formaldehyde (20% w/w, 30mL, 3.37mmol) was added and the reaction mixture 25 was stirred at room temperature for 10 minutes. Reaction mixture cooled to 10*C in an ice bath, sodium cyanoborohydride (0.39g, 6.13mmol) was added and reaction mixture stirred at 10 0 C for 10 minutes. After 10 minutes, the reaction mixture was removed from the ice bath and 30 stirred at room temperature for 2 hrs. To the reaction mixture 5% NaHCO 3 (25mL) solution was added and extracted with ethyl acetate (2x25mL). Ethyl acetate layer was WO 03/010284 PCT/US02/22656 -70 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 C1 2 to get 5' O-tert-butyldiphenylsilyl-2'-0-[N,N 5 dimethylaminooxyethyll-5-methyluridine as a white foam (14.6g, 80%). 2'-0-(dimethylaminooxyethyl)-5-methyluridine Triethylamine trihydrofluoride (3.91mL, 24.Ommol) was dissolved in dry THF and triethylamine (1.67mL, 12mmol, 10 dry, kept over KOH). This mixture of triethylamine-2HF was then added to 5'-0-tert-butyldiphenylsilyl-2'-0-[N,N dimethylaminooxyethyl]-5-methyluridine (1.40g, 2.4mmol) and stirred at room temperature for 24 hrs. Reaction was monitored by TLC (5% MeOH in CH 2 C1 2 ). Solvent was removed 15 under vacuum and the residue placed on a flash column and eluted with 10% MeOH in CH 2 Cl 2 to get 2'-0 (dimethylaminooxyethyl)-5-methyluridine (766mg, 92.5%). 5'-0-DMT-2'-0-(dimethylaminooxyethyl)-5-methyluridine 2'-O-(dimethylaminooxyethyl)-5-methyluridine (750mg, 20 2.17mmol) was dried over P 2 0 5 under high vacuum overnight at 40 0 C. It was then co-evaporated with anhydrous pyridine (20mL). The residue obtained was dissolved in pyridine (11mL) under argon atmosphere. 4-dimethylaminopyridine (26.5mg, 2.60mmol), 4,4'-dimethoxytrityl chloride (880mg, 25 2.60mmol) 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 C1 2 (containing a few drops of pyridine) to get 30 5'-0-DMT-2'-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13g, 80%).

WO 031010284 PCT/US02/22656 -71 5'-O-DMT-2'-0-(2-N,N-dimethylaminooxyethyl)-5 methyluridine-3'-[(2-cyanoethyl)-N,N diisopropylphosphoramidite] 5'-O-DMT-2'-0-(dimethylaminooxyethyl)-5-methyluridine 5 (1.08g, 1.67mmol) was co-evaporated with toluene (20mL). To the residue N,N-diisopropylamine tetrazonide (0.29g, 1.67mmol) was added and dried over P 2 0 5 under high vacuum overnight at 40 0 C. Then the reaction mixture was dissolved in anhydrous acetonitrile (8.4mL) and 2-cyanoethyl 10 N,N,N ,N -tetraisopropylphosphoramidite (2.12mL, 6.08mmol) 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, 15 then the residue was dissolved in ethyl acetate (70mL) and washed with 5% aqueous NaHCO 3 (40mL) . Ethyl acetate layer was dried over anhydrous Na 2

SO

4 and concentrated. Residue obtained was chromatographed (ethyl acetate as eluent) to get 5'-0-DMT-2'-0-(2-N,N-dimethylaminooxyethyl)-5 20 methyluridine-3'-[(2-cyanoethyl)-N,N diisopropylphosphoramidite] as a foam (1.04g, 74.9%). 2'-(Aminooxyethoxy) nucleoside amidites 2'-(Aminooxyethoxy) nucleoside amidites [also known in the art as 2'-0-(aminooxyethyl) nucleoside amidites) 25 are prepared as described in the following paragraphs Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly. N2-isobutyryl-6-0-diphenylcarbamoyl-2'-0-(2 ethylacetyl)-5'-0-(4,4'-dimethoxytrityl)guanosine-3' 30 [(2-cyanoethyl)-N,N-diisopropylphosphoramidite] The 2'-0-aminooxyethyl guanosine analog may be obtained by selective 2'-0-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside WO 03/010284 PCT/US02/22656 -72 may be purchased from Schering AG (Berlin) to provide 2' O-(2-ethylacetyl) diaminopurine riboside along with a minor amount of the 3'-0-isomer. 2'-0-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 5 2'-0-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 Al 940203.) Standard protection procedures should afford 2'-0-(2-ethylacetyl)-5'-0-(4,4' dimethoxytrityl)guanosine and 2-N-isobutyryl-6-0 10 diphenylcarbamoyl-2'-0-(2-ethylacetyl)-5'-0-(4,4' dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-0-diphenylcarbamoyl-2'-0-(2 hydroxyethyl)-5'-0-(4,4'-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N 15 hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-0-([2 phthalmidoxylethyl)-5'-0-(4,4'-dimethoxytrityl)guanosine 3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]. 20 2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside amidites 2'-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2'-0-dimethylaminoethoxyethyl, i.e., 2'-O-CH 2 -0-CH 2

-N(CH

2

)

2 , or 2'-DMAEOE nucleoside 25 amidites) are prepared as follows. Other nucleoside amidites are prepared similarly. 2'-0-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowly added to a solution of borane in tetra 30 hydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the solid dissolves. 02-,2' anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is sealed, WO 03/010284 PCT/US02/22656 -73 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 5 phenol is extracted into the hexane layer. The aqueous layer is extracted with ethyl acetate (3x200 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 10 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. 5'-0-dimethoxytrityl-2'-0-[2(2-N,N-dimethylaminoethoxy) 15 ethyl)]-5-methyl uridine To 0.5 g (1.3 mmol) of 2'-0-[2(2-N,N-dimethylamino ethoxy)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. 20 The reaction mixture is poured into water (200 mL) and extracted with CH 2 C1 2 (2x200 mL). The combined CH 2 C12 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 25 gel chromatography using MeOH:CH 2 Cl 2 :Et 3 N (20:1, v/v, with 1% triethylamine) gives the title compound. 5'-O-Dimethoxytrityl-2'-0-[2(2-N,N dimethylaminoethoxy)ethyl)]-5-methyl uridine-3'-0 (cyanoethyl-N,N-diisopropyl)phosphoramidite 30 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 WO 03/010284 PCT/US02/22656 -74 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 5 acetate as the eluent to give the title compound. Example 2 Oligonucleotide synthesis Unsubstituted and substituted phosphodiester (P=O) oligonucleotides are synthesized on an automated DNA 10 synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine. 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 15 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 0 C (18 20 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. Patent 5,508,270, herein incorporated by reference. 25 Alkyl phosphonate oligonucleotides are prepared as described in U.S. Patent 4,469,863, herein incorporated by reference. 3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared as described in U.S. Patents 5,610,289 or 30 5,625,050, herein incorporated by reference. Phosphoramidite oligonucleotides are prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878, herein incorporated by reference.

WO 03/010284 PCT/US02/22656 -75 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. 5 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared as described in U.S. Patent 5,476,925, herein incorporated by reference. Phosphotriester oligonucleotides are prepared as described in U.S. Patent 5,023,243, herein incorporated by 10 reference. Borano phosphate oligonucleotides are prepared as described in U.S. Patents 5,130,302 and 5,177,198, both herein incorporated by reference. Example 3 15 Oligonucleoside Synthesis Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedi methylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino 20 linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligo nucleosides, also identified as amide-4 linked oligo nucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P=O or P=S linkages are 25 prepared as described in U.S. Patents 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference. Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Patents 5,264,562 and 30 5,264,564, herein incorporated by reference. Ethylene oxide linked oligonucleosides are prepared as described in U.S. Patent 5,223,618, herein incorporated by reference.

WO 03/010284 PCT/US02/22656 -76 Example 4 PNA Synthesis Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to 5 in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Patents 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference. 10 Example 5 Synthesis of Chimeric Oligonucleotides Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type 15 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 20 type are also known in the art as "gapmers" or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as "hemimers" or "wingmers". [2'-0-Me]--[2'-deoxy]--[2'-0-Me] Chimeric Phosphorothioate Oligonucleotides 25 Chimeric oligonucleotides having 2'-0-alkyl phosphorothioate and 2'-deoxy phosphorothioate oligo nucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 380B, as above. Oligonucleotides are synthesized using the automated 30 synthesizer and 2'-deoxy-5'-dimethoxytrityl-3'-0-phosphor amidite for the DNA portion and 5'-dimethoxytrityl-2'-0 methyl-3'-0-phosphoramidite for 5' and 3' wings. The standard synthesis cycle is modified by increasing the WO 03/010284 PCT/US02/22656 -77 wait step after the delivery of tetrazole and base to 600 s repeated four times for RNA and twice for 2'-0-methyl. The fully protected oligonucleotide is cleaved from the support and the phosphate group is deprotected in 3:1 5 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 10 room temperature to deprotect the 2' positions. The reaction is then quenched with 1M TEAA and the sample is then reduced to 1/2 volume by rotovac before being desalted on a G25 size exclusion column. The oligo recovered is then analyzed spectrophotometrically for 15 yield and for purity by capillary electrophoresis and by mass spectrometry. [2'-0-(2-Methoxyethyl)]--[2'-deoxy]--[2'-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides [2'-0-(2-methoxyethyl)]--[2'-deoxy]--[-2'-0-(methoxy 20 ethyl)) chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2'-0-methyl chimeric oligonucleotide, with the substitution of 2'-0 (methoxyethyl) amidites for the 2'-0-methyl amidites. [2'-0-(2-Methoxyethyl)Phosphodiester]--[2'-deoxy 25 Phosphorothioate]--[2'-0-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides [2'-0-(2-methoxyethyl phosphodiester]--[2'-deoxy phosphorothioate]--[2'-0-(methoxyethyl) phosphodiester chimeric oligonucleotides are prepared as per the above 30 procedure for the 2'-0-methyl chimeric oligonucleotide with the substitution of 2'-0-(methoxyethyl) amidites for the 2'-0-methyl amidites, oxidization with iodine to generate the phosphodiester internucleotide linkages WO 03/010284 PCT/US02/22656 -78 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 5 gap. Other chimeric oligonucleotides, chimeric oligo nucleosides and mixed chimeric oligonucleotides/oligo nucleosides are synthesized according to United States patent 5,623,065, herein incorporated by reference. 10 Example 6 Oligonucleotide Isolation After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55 0 C for 18 hours, the 15 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 20 relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by 3P nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 25 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material. Example 7 Oligonucleotide Synthesis - 96 Well Plate Format 30 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.

WO 03/010284 PCT/US02/22656 -79 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 5 (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, CA, or Pharmacia, Piscataway, NJ). Non-standard nucleosides are synthesized as per 10 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 0H at elevated temperature 15 (55-60 0 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. 20 Example 8 Oligonucleotide Analysis - 96 Well Plate Format The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual 25 products was evaluated by capillary electrophoresis (CE) in either the 96 well format (Beckman P/ACEnd MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACETm 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the 30 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 WO 03/010284 PCT/US02/22656 -80 on the plate were at least 85% full length. Example 9 Cell culture and oligonucleotide treatment The effect of antisense compounds on target nucleic 5 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 4 cell types are provided for illustrative 10 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. 15 T-24 cells: The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, VA). T-24 cells were routinely cultured in complete McCoy's 5A basal media 20 (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 25 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. For Northern blotting or other analysis, cells may be 30 seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide. A549 cells: WO 03/010284 PCT/US02/22656 -81 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, 5 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 10 90% confluence. NHDF cells: Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville MD). NHDFs were routinely maintained in Fibroblast Growth Medium 15 (Clonetics Corporation, Walkersville MD) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier. HEK cells: Human embryonic keratinocytes (HEK) were obtained 20 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 25 supplier. HepB3 cells: HepB3: The human hepatoma cell line HepB3 (Hep3B2.1-7) was obtained from the American Type Culture Collection (ATCC 30 ATCC Catalog # HB-8064) (Manassas, VA). This cell line was initially derived from a hepatocellular carcinoma of an 8 yr-old black male. The cells are epithelial in morphology and are tumorigenic in nude mice. These cells can be WO 03/010284 PCT/US02/22656 -82 induced to produce CRP by addition of media containing 1 yM dexamethasone (Sigma-Catalog #D2915 St. Louis, MO), 400 U/ml IL1B (Sigma-Catalog #19401) and 200 U/ml IL6 (Sigma Catalog#I139), according to the protocol described by 5 Lozanski, et al., (Cytokine, vol. 8, 1996: pp.534-540). HepB3 cells were routinely cultured in Minimum Essential Medium (MEM) with Earles Balanced Salt Solution, 2mM L glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate (ATCC-ATCC Catalog 10 #20-2003) (Manassas, VA) supplemented with 10% heat inactivated fetal calf serum (Gibco/Life Technologies, Gaithersburg, MD). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. 15 In order to determine antisense oligonucleotide inhibition of induced CRP, Hep3B cells were plated at a density of 100,000 cells into each well of a 6 well plate (Primaria, Franklin N.J., Catalog# 3846) in MEM supplemented with 10% FCS and allowed to attach overnight. 20 The next day, cells were induced to produce CRP for 24 hrs in regular media supplemented with a final concentration of 1 MM dexamethasone, 400 U/ml I11B and 200 U/ml 116 as described above. At the end of this induction period, the media was removed and cells treated for 4 hrs with 50-150 25 nM of antisense oligonucleotide and 3.0-4.5 pg Lipofectin in MEM alone (minus) serum supplemented with the three cytokines. At the end of the 4 hr drug treatment, the media was removed and fresh MEM containing FCS and cytokines was added to each well and allowed to sit for an 30 additional 20 hrs. RNA was harvested 24 hrs after treatment with oligonucleotide using the Qiagen RNeasy (Qiagen Ltd, Valencia, CA) procedure and CRP RNA detected using RT-PCR analysis.

WO 03/010284 PCTIU S02/22656 -83 Treatment with antisense compounds: When cells reached 90% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 200 iL OPTI-MEMTM-1 reduced 5 serum medium (Gibco BRL) and then treated with 130 pLL of OPTI-MEMTM-1 containing 3.75 pg/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 10 oligonucleotide treatment. 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 15 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'-0-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to human H 20 ras. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2'-0-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 25 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 30 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 WO 03/010284 PCT/US02/22656 -84 cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. Example 10 5 Analysis of oligonucleotide inhibition of C-reactive protein expression Antisense modulation of C-reactive protein expression can be assayed in a variety of ways known in the art. For example, C-reactive protein mRNA levels can be quantitated 10 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, 15 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 & 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 20 Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISMTM 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, CA and used according 25 to manufacturer's instructions. Protein levels of C-reactive protein 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 30 sorting (FACS). Antibodies directed to C-reactive protein can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional WO 03/010284 PCT/US02/22656 -85 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 & Sons, Inc., 5 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 & Sons, Inc., 1997. Immunoprecipitation methods are standard in the art 10 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 & 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 15 Protocols in Molecular Biology, Volume 2, pp. 10.8.1 10.8.21, John Wiley & 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. 20 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991. Example 11 Poly(A)+ mRNA isolation Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996, 42, 1758-1764. Other methods for 25 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 & Sons, Inc., 1993. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was 30 washed with 200 pL cold PBS. 60 p±L lysis buffer (10 mM Tris-HC1, 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 WO 03/010284 PCTJUS02/22656 -86 temperature for five minutes. 55 pL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine CA). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 pL of wash 5 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 pL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70 0 C was added to each well, the plate was 10 incubated on a 90 0 C hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate. Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions. 15 Example 12 Total RNA Isolation Total RNA was isolated using an RNEASY 9 6 TM kit and buffers purchased from Qiagen Inc. (Valencia CA) following the manufacturer's recommended procedures. Briefly, for 20 cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 pL cold PBS. 100 iL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 piL of 70% ethanol was then added to each well and the contents mixed 25 by pipetting three times up and down. The samples were then transferred to the RNEASY 9 6 TM well plate attached to a QIAVACTM manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RWl was added to each well of the 30 RNEASY 9 6 TM plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well WO 03/010284 PCT/US02/22656 -87 of the RNEASY 9 6 TM 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 TM 5 manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVACTM manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 LL water into each well, incubating 1 10 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 PL water. The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia CA). Essentially, after lysing of the cells on 15 the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out. Example 13 Real-time Quantitative PCR Analysis of C-reactive protein 20 mRNA Levels Quantitation of C-reactive protein mRNA levels was determined by real-time quantitative PCR using the ABI PRISMTM 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to manufacturer's 25 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 30 completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe WO 03/010284 PCT/US02/22656 -88 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, CA or PE-Applied 5 Biosystems, Foster City, CA) is attached to the 5' end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, CA or PE-Applied Biosystems, Foster City, CA) is attached to the 3' end of the probe. When the probe and dyes are intact, reporter 10 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 15 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 20 from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISMTM 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control 25 samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples. Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated 30 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 WO 03/010284 PCT/US02/22656 -89 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 5 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 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. PCR reagents were obtained from PE-Applied 15 Biosystems, Foster City, CA. RT-PCR reactions were carried out by adding 25 pL PCR cocktail (1x TAQMANTM buffer A, 5.5 mM MgCl 2 , 300 iM each of dATP, dCTP and dGTP, 600 pM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units 20 AMPLITAQ GOLDTM, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 pL total RNA solution. The RT reaction was carried out by incubation for 30 minutes at 48 0 C. Following a 10 minute incubation at 95 0 C to activate the AMPLITAQ GOLDTM, 40 cycles of a two-step 25 PCR protocol were carried out: 95*C for 15 seconds (denaturation) followed by 60*C for 1.5 minutes (annealing/extension). Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, 30 a gene whose expression is constant, or by quantifying total RNA using RiboGreenTM (Molecular Probes, Inc. Eugene, OR). GAPDH expression is quantified by real time RT-PCR, WO 03/010284 PCT/US02/22656 -90 by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen TM RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreen T are taught in Jones, 5 L.J., et al, Analytical Biochemistry, 1998, 265, 368-374. In this assay, 175 pL of RiboGreen TM working reagent (RiboGreen reagent diluted 1:2865 in 10mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 25uL purified, cellular RNA. The plate is read in a 10 CytoFluor 4000 (PE Applied Biosystems) with excitation at 480nm and emission at 520nm. Probes and primers to human C-reactive protein were designed to hybridize to a human C-reactive protein sequence, using published sequence information (GenBank 15 accession number M11725, incorporated herein as SEQ ID NO:3). For human C-reactive protein the PCR primers were: forward primer: GCTTCCCCTCTTCCCGAA.(SEQ ID NO: 4) reverse primer: TGCGCCACTATGTAAATAATTTTCC (SEQ ID NO: 5) and the PCR probe was: FAM- TCTGACACCTGCCCCAACAAGCAATG 20 TAMRA (SEQ ID NO: 6) where FAM (PE-Applied Biosystems, Foster City, CA) is the fluorescent reporter dye) and TAMRA (PE Applied Biosystems, Foster City, CA) is the quencher dye. For human GAPDH the PCR primers were: 25 forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7) 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, CA) is the fluorescent reporter dye) and TAMRA (PE 30 Applied Biosystems, Foster City, CA) is the quencher dye. Example 14 Northern blot analysis of C-reactive protein mRNA levels Eighteen hours after antisense treatment, cell WO 03/010284 PCT/US02/22656 -91 monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOLTM (TEL-TEST "B" Inc., Friendswood, TX). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was 5 fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, OH). RNA was transferred from the gel to HYBONDTM-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ) by overnight capillary transfer 10 using a Northern/Southern Transfer buffer system (TEL-TEST "B" Inc., Friendswood, TX). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross linking using a STRATALINKERTM UV Crosslinker 2400 (Stratagene, Inc, La Jolla, CA) and then robed using 15 QUICKHYBTM hybridization solution (Stratagene, La Jolla, CA) using manufacturer's recommendations for stringent conditions. To detect human C-reactive protein, a human C reactive protein specific probe was prepared by PCR using 20 the forward primer GCTTCCCCTCTTCCCGAA (SEQ ID NO: 4) and the reverse primer TGCGCCACTATGTAAATAATTTTCC (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 25 (Clontech, Palo Alto, CA). Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGERTM and IMAGEQUANTTM Software V3.3 (Molecular Dynamics, Sunnyvale, CA). Data was normalized to GAPDH levels in untreated controls.

WO 03/010284 PCT/US02/22656 -92 Example 15 Antisense inhibition of human C-reactive protein expression by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap 5 In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human C-reactive protein RNA, using published sequences (GenBank accession number M11725, incorporated herein as SEQ ID NO: 3) . The oligonucleotides are shown 10 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 15 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. 20 All cytidine residues are 5-methylcytidines. The compounds were tested for their effect on human C-reactive protein mRNA levels in Hep3B cells after cytokine stimulation as described in other examples herein and analyzed by quantitative real-time PCR. Data are averages 25 from two experiments and are expressed as percent inhibition of cytokine-induced control. If present, "N.D." indicates "no data". Table 1 Inhibition of human C-reactive protein mRNA levels by 30 chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap ISIS # REGION TARGET TARGET SEQUENCE %INHIB SEQ ID SEQ ID SITE NO

NO

WO 03/010284 PCT/U S02/22656 -93 133709 5'UTR 3 16 GCAGGTGTCAGAGCTTCGGG 77 10 133710 5'UTR 3 71 GCAGTAAGGGAGTTTGCGCC 71 11 133711 5'UTR 3 181 GCCTGAATTCACTCCTTTGG 87 12 133712 Start 3 221 AGCTTCTCCATGGTCACGTC 92 13 Codon 133713 Coding 3 281 TGGCCCTTACCTGTCTGGCC 88 14 133714 Intron 3 311 CTCAGATCAAAACTCTCCCA 30 15 133715 Intron 3 341 TTCATGCAGTCTTAGACCCC N.D. 16 133716 Coding 3 551 GTCTGTGAGCCAGAAAAACA 77 17 133717 Coding 3 701 CGAGAAAATACTGTACCCAC 82 18 133718 Coding 3 781 GACCCACCCACTGTAAAACT 82 19 133719 Coding 3 871 CAGAACTCCACGATCCCTGA 96 20 133720 Coding 3 1091 ATTAGGACTGAAGGGCCCGC 86 21 133721 Stop 3 1171 AGCTGGCCTCAOGGCCACAG 80 22 Codon 133722 3'UTR 3 1191 GAGGTACCTTCAGGACCCAC 89 23 133723 3'UTR 3 1361f- CCCAGACCAGACACTTTACC 88 -24 133724 3'UTR 3 1391 TGGACCATTTCCCAGCATAG 67 25 133725 3'UTR 3 1631 TTCTGAGACTGAAGAGCCCT 27 26 133726 3'UTR 3 1671 GCACTCTGGACCCAAACCAG 96 27 133727 3'UTR 3 1711 CAGGAGACCTGGGCCCAGCA 85 28 133728 3'UTR 3 1918 CCCAGAAGAGCCATAAAATT 27 29 133729 3'UTR 3 1961 ATTCACAGCCCCACAAGGTT 90 30 133730 3'UTR 3 2161 AGAAGATGTCTCACTCCCAA 91 31 133731 3'UTR 3 2291 TGTTTGTCAATCCCTTGGCT 93 32 133732 3'UTR 3 2431 TTCTAAAGCAACTATCAGAA 64 33 140167 5'UTR 3 111 GCCTTAGAGCTACCTCCTCC 70 34 140168 5'UTR 3 201 CTGCTGCCAGTGATACAAGG 69 35 140169 Intron 3 317 CCATACCTCAGATCAAAACT 48 36 140170 Intron 3 451 ACCCCTTCTCCAGTTACACA 69 37 140171 Coding 3 671 CAGTTCCGTGTAGAAGTGGA 43 38 140172 Coding 3 761 GTATCCTATATCCTTAGACC N.D. 39 140173 Coding 3 821 TGGAGCTACTGTGACTTCAG 82 40 140174 Coding 3 861 CGATCCCTGAGGCGGACTCC N.D. 41 140175 Coding 3 901 CTCTTCCTCACCCTGGGCTT 84 42 140176 Coding 3 921 CAGTGTATCCCTTCTTCAGA 68 43 140177 Coding 3 951 GCCCCAAGATGATGCTTGCT 95 44 140178 Coding 3 1031 GTCCCACATGTTCACATTTC 61 45 140179 Coding 3 1111 AGTGCCCGCCAGTTCAGGAC 86 46 140180 Coding 3 1141 GTGAACACTTCGCCTTGCAC 94 47 140181 3'UTR 3 1341 TCCATTCTCAGGCGCTGAGG 85 48 140182 3'UTR 3 1461 GAAATTATCTCCAAGATCTG 33 49 140183 3'UTR 3 1551 CAGCGCTTCCTTCTCAGCTC 94 50 140184 3'UTR 3 1611 GTGAATGTGGGCAATGCTCC 58 51 140185 3'UTR 3 1651 ACACCTGGCCAGTGTCCTGA N.D. 52 140186 3'UTR 3 1771 CCTTTCCAGTGTGCTTTGAG N.D. 53 140187 3'UTR 3 1831 TAGTGCTTCATTTTGCTCTG 93 54 140188 3'UTR 3 1971 TGAAGAAAGAATTCACAGCC 58 55 140189 3'UTR 3 2041 GGCTCCTCTGACAGGACACC 86 56 140190 3'UTR 3 2101 GCTAGGAACACGTAACTATC 71 57 140191 3'UTR 3 2121 GGAAGACTGTAGTTGGTCCT 35 58 140192 3'UTR 3 2211 CTACTGGTGGTCCCAGGTTC 77 59 WO 03/010284 PCT/US02/22656 -94 140193 3'UTR 3 2271 CCTCCACTTCCAGTTTGGCT 77 60 140194 3'UTR 3 2341 CTGGTTCCAGACAAGGCTGA 92 61 140195 3'UTR 3 2402 GACTCACTCAAGTAAACAGG 71 62 140196 3'UTR 3 2461 TTCAAAGGTCATAGAGAAGT 28 63 As shown in Table 1, SEQ ID NOs 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 22, 23, 24, 27, 28, 30, 31, 32, 34, 40, 42, 44, 46, 47, 48, 50, 54, 56, 57, 59, 60, 61 and 62 5 demonstrated at least 70% inhibition of human C-reactive protein 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 10 targeting by compounds of the present invention. Example 16 Antisense inhibition of human C-reactive protein expression by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap-Dose Response Studies 15 In a further embodiment of the present invention, five oligonucleotides were selected for additional dose response studies. Cytokine-induced Hep3B cells were treated with 50, 100 and 150 nM ISIS 133712, 133719, 133726, 140180 and 140177 and mRNA levels were measured at 20 24 hours post oligonucleotide treatment as described in Example 15. Results of these studies are shown in Table 2. Data are averages from two experiments and are expressed as percent inhibition of cytokine-induced control. 25 Table 2 Inhibition of Cytokine Induced Human C-reactive protein mRNA Expression in Hep3B Cells 24 Hr After Oligonucleotide Treatment ISIS # % Inhibition SEQ ID NO 50 nM 100 nM 150 nM 133712 60 84 77 13 WO 03/010284 PCT/US02/22656 -95 133719 0 46 76 20 133726 75 85 92 27 140177 31 45 15 44 140180 26 59 91 47 As shown in Table 2, ISIS 133712, ISIS 133726 and ISIS 140180 were effective at reducing C-reactive protein mRNA levels in a dose-dependent manner and are therefore 5 preferred compounds of the present invention. Example 17 Western blot analysis of C-reactive protein protein levels Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 10 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 15 to C-reactive protein is used, with a radiolabelled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGERTM (Molecular Dynamics, Sunnyvale CA) . Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (20)

1. A compound comprising an antisense oligonucleotide 8 to 50 nucleobases in length targeted to a nucleic acid molecule encoding C-reactive protein, wherein said antisense oligonucleotide specifically hybridizes with said nucleic acid molecule encoding C-reactive protein and inhibits the expression of C-reactive protein by at least 70%.

2. The compound of claim 1, wherein said compound is targeted to: (a) a 3'-untranslated region (3' UTR) of a nucleic acid molecule encoding C-reactive protein; (b) a 5'-untranslated region (5' UTR) of a nucleic acid molecule encoding C-reactive protein; (c) a start codon region of a nucleic acid molecule encoding C-reactive protein; (d) a coding region of a nucleic acid molecule encoding C-reactive protein; or (e) a stop codon region of a nucleic acid molecule encoding C-reactive protein.

3. The compound of claim 1, wherein said compound is targeted to: (a) an intron of a nucleic acid molecule encoding C-reactive protein; or (b) a splice site of a nucleic acid molecule encoding C-reactive protein.

4. The compound of claim 1, wherein the antisense oligonucleotide comprises at least one modified intemucleoside linkage. 97

5. The compound of claim 4, wherein the modified intemucleoside linkage is a phosphorothioate linkage.

6. The compound of any one of claims 4 to 5, wherein the antisense oligonucleotide comprises at least one modified sugar moiety.

7. The compound of claim 6, wherein the modified sugar moiety is a 2'-O-methoxyethyl sugar moiety.

8. The compound of any one of claims 4 to 7, wherein the antisense oligonucleotide comprises at least one modified nucleobase.

9. The compound of claim 8, wherein the modified nucleobase is a 5-methylcyto sine.

10. The compound of anyone of claims 4 to 9, wherein the antisense oligonucleotide is a chimeric oligonucleotide.

11. The compound of anyone of claims 1 to 10, which is a chimeric oligonucleotide 20 nucleotides in length, composed of a central gap region consisting of ten 2'-deoxynucleotides, which is flanked on both sides by five nucleotide wings composed of 2'-O-methoxyethyl (2'-MOE) nucleotides, wherein the intemucleoside linkages are phosphorothioate (P=S) throughout the oligonucleotide, and all cytosine residues are 5-methylcytosines.

12. The compound of any one of claims 1 to 11, wherein said compound specifically hybridizes with at least an 8-nucleobase portion of an active site on a nucleic acid molecule encoding C-reactive protein. 98

13. A composition comprising a compound of anyone of claims 1 to 12 and a pharmaceutically acceptable carrier or diluent.

14. The composition of claim 13, further comprising a colloidal dispersion system.

15. A method of inhibiting the expression of C-reactive protein in cells or tissues comprising contacting said cells or tissues with a compound of any of one of claims 1 to 12 or a composition of claims 13 or 14, so that expression of C-reactive protein is inhibited.

16. A method of treating an animal having a disease or condition associated with C-reactive protein comprising administering to said animal a therapeutically or prophylactically effective amount of a compound of any one of claims 1 to 12 or a composition of claim 13 or 14, so that expression of C reactive protein is inhibited.

17. The method of claim 16, wherein the disease or condition is an inflammatory disease or disorder.

18. The method of claim 16, wherein the disease or condition is tumor formation.

19. The method of claim 16, wherein the disease or condition is an infection.

20. Use of a compound of any one of claims 1 to 12 for the manufacture of a medicament for the therapeutic or prophylactic treatment of a disease or condition associated with C-reactive protein. 2011203319 06 Jul 2011 2011203319 06 Jul 2011 2011203319 06 Jul 2011 2011203319 06 Jul 2011 2011203319 06 Jul 2011 2011203319 06 Jul 2011 2011203319 06 Jul 2011 2011203319 06 Jul 2011 2011203319 06 Jul 2011 2011203319 06 Jul 2011 2011203319 06 Jul 2011 2011203319 06 Jul 2011 2011203319 06 Jul 2011 2011203319 06 Jul 2011 2011203319 06 Jul 2011