US20040186069A1

US20040186069A1 - Antisense modulation of tumor necrosis factor receptor 2 expression

Info

Publication number: US20040186069A1
Application number: US10/476,021
Authority: US
Inventors: C. Bennett; Andrew Watt
Original assignee: Isis Pharmaceuticals Inc
Current assignee: Ionis Pharmaceuticals Inc
Priority date: 2001-04-27
Filing date: 2002-04-23
Publication date: 2004-09-23
Also published as: EP1390536A1; US6410324B1; WO2002088393A1

Abstract

Antisense compounds, compositions and methods are provided for modulating the expression of Tumor Necrosis Factor Receptor 2. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding Tumor Necrosis Factor Receptor 2. Methods of using these compounds for modulation of Tumor Necrosis Factor Receptor 2 expression and for treatment of diseases associated with expression of Tumor Necrosis Factor Receptor 2 are provided.

Description

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulating the expression of Tumor Necrosis Factor Receptor 2. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Tumor Necrosis Factor Receptor 2. Such compounds have been shown to modulate the expression of Tumor Necrosis Factor Receptor 2.

BACKGROUND OF THE INVENTION

One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals into intracellular signals that in turn modulate biochemical pathways. Examples of such extracellular signaling molecules include growth factors, cytokines, and chemokines. The cell surface receptors of these molecules and their associated signal transduction pathways are therefore one of the principal means by which cellular behavior is regulated. Because cellular phenotypes are largely influenced by the activity of these pathways, it is currently believed that a number of disease states and/or disorders are a result of either aberrant activation or functional mutations in the molecular components of signal transduction pathways.

For example, the polypeptide cytokine tumor necrosis factor (TNF) is normally produced during infection, injury, or invasion where it serves as a pivotal mediator of the inflammatory response. In recent years, a number of in vivo animal and human studies have demonstrated that overexpression TNF by the host in response to disease and infection is itself responsible for the pathological consequences associated with the underlying disease. For example, septic shock as a result of massive bacterial infection has been attributed to infection-induced expression of TNF. Thus, systemic exposure to TNF at levels comparable to those following massive bacterial infection has been shown to result in a spectrum of symptoms (shock, tissue injury, capillary leakage, hypoxia, pulmonary edema, multiple organ failure, and high mortality rate) that is virtually indistinguishable from septic shock syndrome (Tracey and Cerami, Annu. Rev. Med., 1994, 45, 491-503). Further evidence has been provided in animal models of septic shock, in which it has been demonstrated that systemic exposure to anti-TNF neutralizing antibodies block bacterial-induced sepsis (Tracey and Cerami, Annu. Rev. Med., 1994, 45, 491-503). In addition to these acute effects, chronic exposure to low-dose TNF, results in a syndrome of cachexia marked by anorexia, weight loss, dehydration, and depletion of whole-body protein and lipid. Chronic production of TNF has been implicated in a number of diseases including AIDS and cancer (Tracey and Cerami, Annu. Rev. Med., 1994, 45, 491-503). To date, two distinct TNF cells surface receptors, known as Tumor necrosis factor receptor 1 and Tumor necrosis factor receptor 2, have been described. Molecular analysis of Tumor necrosis factor receptor 1 and Tumor necrosis factor receptor 2 have shown that the two receptors share little homology in their intracellular domains and appear to activate distinct intracellular pathways (Tracey and Cerami, Annu. Rev. Med., 1994, 45, 491-503).

Tumor necrosis factor (TNFR2, also known as CD120b, p75 TNFR and TNFR-beta receptor) was first cloned in 1990 (Schall et al., Cell, 1990, 61, 361-370.) and mapped to chromosomal locus 1p36.2 in 1996 (Beltinger et al., Genomics, 1996, 35, 94-100).

Bruce et al. used targeted gene expression to generate mice lacking both TNFRs and concluded that drugs which target the TNF signaling pathways may prove beneficial in treating stroke or traumatic brain injury (Bruce et al., Nat. Med., 1996, 2, 788-794.). Tumor necrosis factor receptor 2 knockout mice were also used to establish a crucial role for Tumor necrosis factor receptor 2 in experimental cerebral malaria (Lucas et al., Eur. J. Immunol., 1997, 27, 1719-1725) and autoimmune encephalomyelitis (Suvannavejh et al., Cell Immunol., 2000, 205, 24-33), models for human cerebral malaria and multiple sclerosis, respectively.

Agostini et al. have determined that Tumor necrosis factor receptor 2 is present at high density on T cells and may play a role in the immune regulatory mechanists that lead to alveolitis in the pulmonary microenvironment of interstitial lung disease (Agostini et al., Am. J. Respir. Crit. Care Med., 1996, 153, 1359-1367). Tumor necrosis factor receptor 2 is implicated in human metabolic disorders of lipid metabolism and associated with obesity and insulin resistance (Fernandez-Real et al., Diabetes Care, 2000, 23, 831-837), familial combined hyperlipidemia (Geurts et al., Hum. Mol. Genet., 2000, 9, 2067-2074.; van Greevenbroek et al., Atherosclerosis, 2000, 153, 1-8), hypertension and hypercholesterolemia (Glenn et al., Hum. Mol. Genet., 2000, 9, 1943-1949). Tumor necrosis factor receptor 2 has also recently been associated with human narcolepsy (Komata et al., Tissue Antigens, 1999, 53, 527-533). In addition, Tumor necrosis factor receptor 2 polymorphism appears to lead to susceptibility to systemic lupus erythematosus (Hohjoh et al., Tissue Antigens, 2000, 56, 446-448).

An antisense oligonucelotide targeting the initiation site of the human Tumor necrosis factor receptor 2 gene was used to inhibit Tumor necrosis factor receptor 2 expression in a human neuronal cell line (Shen et al., J. Biol. Chem., 1997, 272, 3550-3553).

Disclosed and claimed in U.S. Pat. No. 5,959,094 is an isolated and purified DNA molecule comprising a promoter of the human p75 TNF-R (Tumor Necrosis Factor Receptor 2) gene having a sequence consisting of a 5′ upstream promoter sequence, an intron promoter sequence located in the first intron between the first and second exons, an isolated and purified DNA molecule containing a transcription inhibitory region of a genomic clone of human p75 TNF-R and upstream of an intron promoter sequence located in the first intron between the first and second exons, and a composition comprising said DNA molecule and a carrier. Ribozymes designed to modulate expression of Tumor Necrosis Factor Receptor 2 are generally disclosed (Wallach, et al., 1999).

Currently, there are no known therapeutic agents which effectively inhibit the synthesis of Tumor necrosis factor receptor 2 and investigative strategies aimed at modulating Tumor necrosis factor receptor 2 function have involved the use of inhibitors such as antibodies and antisense oligonucleotides.

Antisense technology is emerging as an effective means of reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic and research applications involving modulation of Tumor necrosis factor receptor 2 expression.

The present invention provides compositions and methods for modulating the expression of Tumor necrosis factor receptor 2.

SUMMARY OF THE INVENTION

The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding Tumor Necrosis Factor Receptor 2, and which modulate the expression of Tumor Necrosis Factor Receptor 2. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of Tumor Necrosis Factor Receptor 2 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of Tumor Necrosis Factor Receptor 2 by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding Tumor Necrosis Factor Receptor 2, ultimately modulating the amount of Tumor Necrosis Factor Receptor 2 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Tumor Necrosis Factor Receptor 2. As used herein, the terms “target nucleic acid” and “nucleic acid encoding Tumor Necrosis Factor Receptor 2” encompass DNA encoding Tumor Necrosis Factor Receptor 2, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of Tumor Necrosis Factor Receptor 2. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target. [0013]
It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding Tumor Necrosis Factor Receptor 2. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding Tumor Necrosis Factor Receptor 2, regardless of the sequence(s) of such codons. [0014]
It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. [0015]
The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region. [0016]
Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. 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. [0017]
Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. [0018]
In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. [0019]
Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target are identified through experimentation, and the sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The target sites to which these preferred sequences are complementary are hereinbelow referred to as “active sites” and are therefore preferred sites for targeting. Therefore another embodiment of the invention encompasses compounds which hybridize to these active sites. [0020]
Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use. [0021]
For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in combination with other antisense compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues. [0022]
Expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns. [0023]
Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, [0024] FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).
The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans. [0025]
In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. [0026]
While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 50 nucleobases (i.e. from about 8 to about 50 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression. [0027]
As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage. [0028]
Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. [0029]
Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphoro-dithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl-phosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included. [0030]
Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference. [0031]
Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH[0032] ₂component parts.
Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos.: 5,034,506; 5,166,313; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference. [0033]
In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., [0034] 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[0035] ₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)— N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C[0036] ₁to C₁₀alkyl or C₂to C₁₀alkenyl and alkynyl. Particularly preferred are O[(CH₂)_nO]_mCH₃, O(CH₂)_nOCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁to C₁₀lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.
A further prefered modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH[0037] ₂—)_ngroup bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.
Other preferred modifications include 2′-methoxy (2′-O—CH[0038] ₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH[0039] ₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. , ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference. [0040]
Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., [0041] Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.
Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference. [0042]
It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. [0043]
Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety. [0044]
The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. [0045]
The antisense compounds of the invention are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules. The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference. [0046]
The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. [0047]
The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al. [0048]
The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. [0049]
Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” [0050] J. of Pharma Sci., 1977, 66, 1-19).. The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.
For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine. [0051]
The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of Tumor Necrosis Factor Receptor 2 is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example. [0052]
The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding Tumor Necrosis Factor Receptor 2, enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding Tumor Necrosis Factor Receptor 2 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of Tumor Necrosis Factor Receptor 2 in a sample may also be prepared. [0053]
The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration. [0054]
Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Preferred topical formulations include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C[0055] _1-10alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Prefered bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate. Prefered fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also prefered are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly prefered combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673 (filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624 (filed May 21, 1998) and 09/315,298 (filed May 20, 1999) each of which is incorporated herein by reference in their entirety. [0056]
Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. [0057]
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. [0058]
The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. [0059]
The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. [0060]
In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention. [0061]
Emulsions [0062]
The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. (Idson, in [0063] Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be either water-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.
Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in [0064] Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, 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 [0065] Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate. [0066]
A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in [0067] Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase. [0068]
Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin. [0069]
The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in [0070] Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailability standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in [0071] Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are Systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
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 [0072] Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil. [0073]
Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., [0074] Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.
Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., [0075] Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
Liposomes [0076]
There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. [0077]
Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo. [0078]
In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores. [0079]
Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in [0080] Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes. As the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act. [0081]
Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin. [0082]
Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis. [0083]
Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., [0084] Biochem. 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 charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., [0085] Journal of Controlled Release, 1992, 19, 269-274).
One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol. [0086]
Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., [0087] Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. [0088] 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 circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G[0089] _M1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et. al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).
Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. ([0090] Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_M1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_M1or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphophatidylcholine are disclosed in WO 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. [0091] Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C₁₂15G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising antisense oligonucleotides targeted to the raf gene. [0092]
Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing. (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin. [0093]
Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in [0094] Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 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. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class. [0095]
If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps. [0096]
If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class. [0097]
If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides. [0098]
The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in [0099] Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
Penetration Enhancers [0100]
In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. [0101]
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., [0102] Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., [0103] Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C[0104] _1-10alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.,) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).
Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's [0105] The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).
Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, [0106] J. Chromatogr., 1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).
Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, [0107] Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides. [0108]
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. [0109]
Carriers [0110]
Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., [0111] Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).
Excipients [0112]
In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.). [0113]
Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like. [0114]
Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used. [0115]
Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like. [0116]
Other Components [0117]
The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation. [0118]
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. [0119]
Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, [0120] The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.
In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially. [0121]
The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC[0122] ₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.
While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. [0123]

EXAMPLES

Example 1

Nucleoside Phosphoramidites for Oligonucleotide Synthesis

Deoxy and 2′-alkoxy amiditeb

2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. 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. [0124]
Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C) nucleotides were synthesized according to published methods [Sanghvi, et. al., [0125] Nucleic Acids Research, 1993, 21, 3197-3203] using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes; Needham Mass.).

2′-Fluoro amidites

2′-Fluorodeoxyadenosine amidites

2′-fluoro oligonucleotides were synthesized as described previously [Kawasaki, et. al., [0126] J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2′-alpha-fluoro atom is introduced by a SN2-displacement of a 2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies and standard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites. [0127]

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 treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites. [0128]

2′-Fluorodeoxycytidine

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. [0129]

2′-O-(2-Methoxyethyl) modified amidites

2′-O-Methoxyethyl-substituted nucleoside amidites are prepared as follows, or alternatively, as per the methods of Martin, P., [0130] Helvetica Chimica Acta, 1995, 78, 486-504.

2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid that was crushed to a light tan powder (57 g, 85% crude yield). The NMR spectrum was consistent with the structure, contaminated with phenol as its sodium salt (ca. 5%). The material was used as is for further reactions (or it can be purified further by column chromatography using a gradient of methanol in ethyl acetate (10-25%) to give a white solid, mp 222-4° C.). [0131]

2′-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° C. After heating for 48 hours at 155-160° C., the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH[0132] ₃CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂(250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product. Additional material was obtained by reworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH[0133] ₃CN (200 mL). The residue was dissolved in CHCl₃(1.5 L) and extracted with 2×500 mL of saturated NaHCO₃and 2×500 mL of saturated NaCl. The organic phase was dried over Na₂SO₄, 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% Et₃NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by TLC by first quenching the TLC sample with the addition of MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl[0134] ₃(800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl₃. The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%). An additional 1.5 g was recovered from later fractions.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH[0135] ₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L), cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10° C., and the resulting mixture stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to the latter solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1×300 mL of NaHCO₃and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxy-trityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH[0136] ₄OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH3 gas was added and the vessel heated to 100° C. for 2 hours (TLC showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, TLC showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL). The residue was dissolved in CHCl[0137] ₃(700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄and 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% Et₃NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine-3′-amidite

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine (74 g, 0.10 M) was dissolved in CH[0138] ₂Cl₂(1 L). Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra(isopropyl)-phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (TLC showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO₃(1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH₂Cl₂(300 mL), and the extracts were combined, dried over MgSO₄and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound.

2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine. [0139]

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

O[0140] ²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) and the solution was cooled to −10° C. The resulting crystalline product was collected by filtration, washed with ethyl ether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMR were consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-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 glycol (350 mL, excess) was added cautiously at first until the evolution of hydrogen gas subsided. 5′-O-tert-Butyldiphenylsilyl-O[0141] ²-2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160° C. was reached and then maintained for 16 h (pressure<100 psig). The reaction vessel was cooled to ambient and opened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T side product, ethyl acetate) indicated about 70% conversion to the product. In order to avoid additional side product formation, the reaction was stopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol. [Alternatively, once the low boiling solvent is gone, the remaining solution can be partitioned between ethyl acetate and water. The product will be in the organic phase.] The residue was purified by column chromatography (2 kg silica gel, ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, stripped and dried to product as a white crisp foam (84 g, 50%), contaminated starting material (17.4 g) and pure reusable starting material 20 g. The yield based on starting material less pure recovered starting material was 58%. TLC and NMR were consistent with 99% pure product.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried over P[0142] ₂O₅under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture. The rate of addition is maintained such that resulting deep red coloration is just discharged before adding the next drop. After the addition was complete, the reaction was stirred for 4 hrs. By that time TLC showed the completion of the reaction (ethylacetate:hexane, 60:40). The solvent was evaporated in vacuum. Residue obtained was placed on a flash column and eluted with ethyl acetate:hexane (60:40), to get 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinqoxy) ethyl]-5-methyluridine

2′-O-([2-phthalimidoxy)ethyl)-5′-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH[0143] ₂Cl₂(4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h the mixture was filtered, the filtrate was washed with ice cold CH₂Cl₂and the combined organic phase was washed with water, brine and dried over anhydrous Na₂SO₄. The solution was concentrated to get 2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was stirred for 1 h. Solvent was removed under vacuum; residue chromatographed to get 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1 M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at 10° C. under inert atmosphere. The reaction mixture was stirred for 10 minutes at 10° C. After that the reaction vessel was removed from the ice bath and stirred at room temperature for 2 h, the reaction monitored by TLC (5% MeOH in CH[0144] ₂Cl₂). Aqueous NaHCO₃solution (5%, 10 mL) was added and extracted with ethyl acetate (2×20 mL). Ethyl acetate phase was dried over anhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in a solution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and the reaction mixture was stirred at room temperature for 10 minutes. Reaction mixture cooled to 10° C. in an ice bath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction mixture stirred at 10° C. for 10 minutes. After 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature for 2 hrs. To the reaction mixture 5% NaHCO₃(25 mL) solution was added and extracted with ethyl acetate (2×25 mL). Ethyl acetate layer was dried over anhydrous Na₂SO₄and evaporated to dryness. The residue obtained was purified by flash column chromatography and eluted with 5% MeOH in CH₂Cl₂to get 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). This mixture of triethylamine-2HF was then added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reaction was monitored by TLC (5% MeOH in CH[0145] ₂Cl₂). Solvent was removed under vacuum and the residue placed on a flash column and eluted with 10% MeOH in CH₂Cl₂to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P[0146] ₂O₅under high vacuum overnight at 40° C. It was then co-evaporated with anhydrous pyridine (20 mL). The residue obtained was dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the mixture and the reaction mixture was stirred at room temperature until all of the starting material disappeared. Pyridine was removed under vacuum and the residue chromatographed and eluted with 10% MeOH in CH₂Cl₂(containing a few drops of pyridine) to get 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and dried over P[0147] ₂O₅under high vacuum overnight at 40° C. Then the reaction mixture was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 hrs under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated, then the residue was dissolved in ethyl acetate (70 mL) and washed with 5% aqueous NaHCO₃(40 mL). Ethyl acetate layer was dried over anhydrous Na₂SO₄and concentrated. Residue obtained was chromatographed (ethyl acetate as eluent) to get 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy) nucleoside amidites

2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as 2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly. [0148]

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]. [0149]

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O-CH[0150] ₂—O—CH₂—N (CH₂)₂, or 2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside amidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyll-5-methyl uridine

2-[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowly added to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the solid dissolves. O[0151] ²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oil bath and heated to 155° C. for 26 hours. The bomb is cooled to room temperature and opened. The crude solution is concentrated and the residue partitioned between water (200 mL) and hexanes (200 mL). The excess phenol is extracted into the hexane layer. The aqueous layer is extracted with ethyl acetate (3×200 mL) and the combined organic layers are washed once with water, dried over anhydrous sodium sulfate and concentrated. The residue is columned on silica gel using methanol/methylene chloride 1:20 (which has 2% triethylamine) as the eluent. As the column fractions are concentrated a colorless solid forms which is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyl uridine

To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reaction mixture is poured into water (200 mL) and extracted with CH[0152] ₂Cl₂(2×200 mL). The combined CH₂Cl₂layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydrous sodium sulfate. Evaporation of the solvent followed by silica gel chromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine) gives the title compound.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

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-dimethylamino-ethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH[0153] ₂Cl₂(20 mL) under an atmosphere of argon. The reaction mixture is stirred overnight and the solvent evaporated. The resulting residue is purified by silica gel flash column chromatography with ethyl acetate as the eluent to give the title compound.

Example 2

Oligonucleotide Synthesis [0154]
Unsubstituted and substituted phosphodiester (P═O) oligo-nucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine. [0155]
Phosphorothioates (P═S) are synthesized as for the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation wait step was increased to 68 sec and was followed by the capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (18 h), the oligonucleotides were purified by precipitating.twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference. [0156]
Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference. [0157]
3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference. [0158]
Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference. [0159]
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. [0160]
3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference. [0161]
Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference. [0162]
Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference. [0163]

Example 3

Oligonucleoside Synthesis [0164]
Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethyl-hydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligo-nucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference. [0165]
Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference. [0166]
Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference. [0167]

Example 4

PNA Synthesis [0168]
Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, [0169] Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.

Example 5

Synthesis of Chimeric Oligonucleotides [0170]
Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at. either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”. [0171]

[2′-O—Me]-[2′-deoxy]-[2′-O—Me] Chimeric Phosphorothioate Oligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 380B, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for RNA and twice for 2′-O-methyl. The fully protected oligonucleotide is cleaved from the support and the phosphate group is deprotected in 3:1 ammonia/ethanol at room temperature overnight then lyophilized to dryness. Treatment in methanolic ammonia for 24 hrs at room temperature is then done to deprotect all bases and sample was again lyophilized to dryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at room temperature to deprotect the 2′ positions. The reaction is then quenched with 1M TEAA and the sample is then reduced to ½ volume by rotovac before being desalted on a G25 size exclusion column. The oligo recovered is then analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry. [0172]

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites. [0173]

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphoro-thioate]-[2′-0-(2-Methoxyethyl) Phosphodiester] Chimeric oligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxy phosphoro-thioate]-[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidization with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap. [0174]
Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference. [0175]

Example 6

Oligonucleotide Isolation [0176]
After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides or oligonucleosides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by [0177] ³¹P nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

Oligonucleotide Synthesis—96 Well Plate Format [0178]
Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a standard 96 well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per known literature or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites. [0179]
Oligonucleotides were cleaved from support and deprotected with concentrated NH[0180] ₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

Oligonucleotide Analysis—96 Well Plate Format [0181]
The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length. [0182]

Example 9

Cell Culture and Oligonucleotide Treatment [0183]
The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following 6 cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, Ribonuclease protection assays, or RT-PCR. [0184]
T-24 Cells: [0185]
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 (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis. [0186]
For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide. [0187]
A549 Cells: [0188]
The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. [0189]
NHDF Cells: [0190]
Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier. [0191]
HEK Cells: [0192]
Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier. [0193]
HUVEC Cells: [0194]
The human umbilical vein endothilial cell line HuVEC was obtained from the American Type Culure Collection (Manassas, Va.). HUVEC cells were routinely cultured in EBM (Clonetics Corporation Walkersville, Md.) supplemented with SingleQuots supplements (Clonetics Corporation, Walkersville, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence were maintained for up to 15 passages. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 10000 cells/well for use in RT-PCR analysis. [0195]
For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide. [0196]
3T3-L1 Cells: [0197]
The mouse embryonic adipocyte-like cell line 3T3-L1 was obtained from the American Type Culure Collection (Manassas, Va.). 3T3-L1 cells were routinely cultured in DMEM, high glucose (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 80% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 4000 cells/well for use in RT-PCR analysis. [0198]
For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide. [0199]
Treatment with Antisense Compounds: [0200]
When cells reached 80% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Gibco BRL) and the desired concentration of oligonucleotide. After 4-7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment. [0201]
The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to human H-ras. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. [0202]

Example 10

Analysis of Oligonucleotide Inhibition of Tumor Necrosis Factor Receptor 2 Expression [0203]
Antisense modulation of Tumor Necrosis Factor Receptor 2 expression can be assayed in a variety of ways known in the art. For example, Tumor Necrosis Factor Receptor 2 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., [0204] 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 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 PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.
Protein levels of Tumor Necrosis Factor Receptor 2 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to Tumor Necrosis Factor Receptor 2 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., [0205] Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.
Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., [0206] 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 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. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11

Poly(A)+mRNA Isolation [0207]
Poly(A)+mRNA was isolated according to Miura et al., [0208] Clin. Chem., 1996, 42, 1758-1764. Other methods for poly(A)+mRNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C. was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.
Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions. [0209]

Example 12

Total RNA Isolation [0210]
Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water. [0211]
The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out. [0212]

Example 13

Real-Time Quantitative PCR Analysis of Tumor Necrosis Factor Receptor 2 mRNA Levels [0213]
Quantitation of Tumor Necrosis Factor Receptor 2 mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE, FAM, or VIC, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples. [0214]
Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art. [0215]
PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1× TAQMAN™ buffer A, 5.5 mM MgCl[0216] ₂, 300 μM each of dATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5 Units MULV reverse transcriptase) to 96 well plates containing 25 μL total RNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).
Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, [0217] Analytical Biochemistry, 1998, 265, 368-374.
In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 25 uL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm. [0218]
Probes and primers to human Tumor Necrosis Factor Receptor 2 were designed to hybridize to a human Tumor Necrosis Factor Receptor 2 sequence, using published sequence information (GenBank accession number NM[0219] _—001066, incorporated herein as SEQ ID NO:3). For human Tumor Necrosis Factor Receptor 2 the PCR primers were: forward primer: CACTCCCCACCTTCAATTCCT (SEQ ID NO: 4) reverse primer: GCAGACACAAGACTGGCACTTG (SEQ ID NO: 5) and the PCR probe was: FAM-CCCAAACGGGCTGCCCTGC-TAMRA (SEQ ID NO: 6) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. For human GAPDH the PCR primers were:
forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7) [0220]
reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 9) [0221]
where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. [0222]
Probes and primers to mouse Tumor Necrosis Factor Receptor 2 were designed to hybridize to a mouse Tumor Necrosis Factor Receptor 2 sequence, using published sequence information (GenBank accession number M59378, incorporated herein as SEQ ID NO:10). For mouse Tumor Necrosis Factor Receptor 2 the PCR primers were: [0223]
forward primer: GTTTGCAGCCTCTGCCTTTG (SEQ ID NO:11) [0224]
reverse primer: AAGGCGTGGCCTTGGAA (SEQ ID NO: 12) and the PCR probe was: FAM-AGCTCCTCCTCCTGACCTTCTAATGAGCC-TAMRA [0225]
(SEQ ID NO: 13) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. For mouse GAPDH the PCR primers were: [0226]
forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 14) [0227]
reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 15) and the PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 16) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. [0228]

Example 14

Northern Blot Analysis of Tumor Necrosis Factor Receptor 2 mRNA Levels [0229]
Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions. [0230]
To detect human Tumor Necrosis Factor Receptor 2, a human Tumor Necrosis Factor Receptor 2 specific probe was prepared by PCR using the forward primer CACTCCCCACCTTCAATTCCT (SEQ ID NO: 4) and the reverse primer GCAGACACAAGACTGGCACTTG (SEQ ID NO: 5). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.). [0231]
To detect mouse Tumor Necrosis Factor Receptor 2, a mouse Tumor Necrosis Factor Receptor 2 specific probe was prepared by PCR using the forward primer GTTTGCAGCCTCTGCCTTTG (SEQ ID NO:11) and the reverse primer AAGGCGTGGCCTTGGAA (SEQ ID NO: 12). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.). [0232]
Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls. [0233]

Example 15

Antisense Inhibition of Human Tumor Necrosis Factor Receptor 2 Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap [0234]

In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human Tumor Necrosis Factor Receptor 2 RNA, using published sequences (GenBank accession number NM _—001066, incorporated herein as SEQ ID NO: 3, the complement of genomic sequence from GenBank accession number AL031276 containing a portion of intron 8, exon 9, intron 9 and exon 10 of human Tumor necrosis factor receptor 2, incorporated herein as SEQ ID NO: 17, GenBank accession number U52158, incorporated herein as SEQ ID NO: 18, and an mRNA variant of human Tumor necrosis factor receptor 2 contained in GenBank accession number AI379026, the complement of which is incorporated herein as SEQ ID NO: 19). The oligonucleotides are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human Tumor Necrosis Factor Receptor 2 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, “N.D.” indicates “no data”.

TABLE 1


Inhibition of human Tumor Necrosis Factor Receptor 2
mRNA levels by chimeric phosphorothioate oligonucleotides
having 2′-MOE wings and a deoxy gap

		TARGET
		SEQ ID	TARGET			SEQ ID
ISIS #	REGION	NO	SITE	SEQUENCE	% INHIB	NO

135862	5′UTR	3	4	tctccaggctccgctgcgct	89	20

135863	Intron:	18	106	ttgcatgttggcctgaggaa	85	21
	Exon
	Junction

135864	Coding	3	198	ctgagccggcatgtgctccc	0	22

135865	Coding	3	202	ttctctgagccggcatgtgc	55	23

135866	Coding	3	218	ctgtctggtcatagtattct	100	24

135867	Exon	19	227	tttaagagacaatttcatgc	74	25

135868	Coding	3	228	cacatctgagctgtctggtc	89	26

135869	Coding	3	232	gcagcacatctgagctgtct	100	27

135870	Coding	3	258	gcatgttggcccggcgagca	56	28

135871	Coding	3	268	gaagacttttgcatgttggc	83	29

135872	Coding	3	357	cagctcaagcactcgggaac	67	30

135873	Coding	3	387	tccacctggtcagagctaca	77	31

135874	Coding	3	427	ggtgcagatgcggttctgtt	88	32

135875	Coding	3	537	gtttcagttcctggtctggc	24	33

135876	Coding	3	540	gatgtttcagttcctggtct	57	34

135877	Coding	3	554	tgcacaccacgtctgatgtt	0	35

135878	Coding	3	631	cacgttacagatctggtggg	52	36

135879	Coding	3	721	gggtaagtgtactgcccctg	9	37

135880	Coding	3	747	tgttgggatcgtgtggacac	16	38

135881	Coding	3	755	gctgcgtgtgttgggatcgt	79	39

135882	Coding	3	868	cacaatcagtccaactggaa	82	40

135883	Coding	3	946	caagggcttctttttcacct	67	41

135884	Coding	3	980	gcaagtgaggcaccttggct	39	42

135885	Coding	3	998	cccgggccttatcggcaggc	48	43

135886	Intron	17	1054	gtcccatctacttgggaggc	23	44

135887	Coding	3	1066	ctccagggagctgctgctgg	73	45

135888	coding	3	1071	gagctctccagggagctgct	68	46

135889	Coding	3	1076	tggccgagctctccagggag	27	47

135890	Coding	3	1230	acgttcacgatgcaggtgac	80	48

135891	Coding	3	1246	gtcagagctgctacagacgt	77	49

135892	Coding	3	1250	tgtggtcagagctgctacag	87	50

135893	Coding	3	1293	gtgtctcccattgtggagct	71	51

135894	Coding	3	1302	ctggaatctgtgtctcccat	90	52

135895	Coding	3	1368	tgtgaccgaaaggcacattc	50	53

135896	Coding	3	1449	ggcttcatcccagcatcagg	84	54

135897	Coding	3	1453	actgggcttcatcccagcat	78	55

135898	Stop	3	1465	ccggcctggttaactgggct	17	56
	Codon

135899	3′UTR	3	1512	gtcatcctgccagggctcag	90	57

135900	3′UTR	3	1586	agaggaacttggcccagaaa	63	58

135901	Intron	17	1714	ctaagcccagcagcccagct	67	59

135902	3′UTR	3	1926	tgggtgactcaggcagcatc	88	60

135903	3′UTR	3	1963	agtctcagcctcaggctgaa	0	61

135904	3′UTR	3	2022	accccgttccctacagggct	77	62

135905	3′UTR	3	2037	gagctaacttgaaggacccc	76	63

135906	Intron	17	2354	ttagctgtgccacactggga	89	64

135907	3′UTR	3	2453	cagcactgaatatggtggcc	82	65

135908	3′UTR	3	2471	gttatcttgcccaggccaca	0	66

135909	3′UTR	3	2472	cgttatcttgcccaggccac	83	67

135910	3′UTR	3	2491	agatttctagttagaagtgc	43	68

135911	3′UTR	3	2533	gcttgttggcctgagtggta	66	69

135912	3′UTR	3	2563	tgtggctggcagagtttggc	60	70

135913	3′UTR	3	2616	gcaggcacaccggagtgaag	71	71

135914	3′UTR	3	2655	tggtgtggcctaggacagca	79	72

135915	3′UTR	3	2671	attccctgaaaggagatggt	92	73

135916	3′UTR	3	2732	tctaggctgaggtaggagta	92	74

135917	3′UTR	3	2830	ggccatgtaccaaagtggca	92	75

135918	3′UTR	3	2852	gactggcacttgggatcaca	99	76

135919	3′UTR	3	3009	ctacactggtttcccctctg	77	77

135920	3′UTR	3	3103	gacaatttcatgccttctcc	96	78

135921	3′UTR	3	3202	tggtaatggctgggctctcc	81	79

135922	3′UTR	3	3245	tctgacatcttgattccagg	69	80

135923	3′UTR	3	3428	gcagaaacctggccagtccc	65	81

135924	3′UTR	3	3439	gtccaatgtgggcagaaacc	88	82

135925	3′UTR	3	3493	tctcccaggactgtccacca	98	83

135926	3′UTR	3	3527	ggctctgccctgtgatgcca	95	84

135927	3′UTR	3	3544	caaattcatcgcttcccggc	95	85

135928	3′UTR	3	3651	aaacaggtttattgataagc	11	86

135929	Intron	17	4477	ctacagaggcaggtttgagt	77	87

135930	Intron	17	5054	agacagggttgtgctatgtt	71	88

135931	Intron	17	5190	tggcacaatcatagctgact	92	89

135932	Intron	17	5388	gatggctgaatatttgtaaa	44	90

135933	Intron	17	5431	agggagaataatagcctacc	63	91

135934	Intron	17	6640	agccttccataagtagaagc	63	92

135935	Intron	17	7126	ccaagttcatggtcattccc	87	93

135936	Intron	17	8748	tgaattgattaccaacattt	94	94

135937	Intron	17	8875	caccttgcctggccagacca	88	95

135938	Intron	17	8901	gaagtactgagattacaggc	84	96

135939	Intron	17	13529	gtccccaggagggcagagcc	51	97

As shown in Table 1, SEQ ID NOs 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 36, 39, 40, 41, 43, 45, 46, 48, 49, 50, 51, 52, 53, 54, 55, 57, 58, 59, 60, 62, 63, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 and 97 demonstrated at least 40% inhibition of human Tumor Necrosis Factor Receptor 2 expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention. [0236]

Example 16

Antisense Inhibition of Mouse Tumor Necrosis Factor Receptor 2 Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap. [0237]

In accordance with the present invention, a second series of oligonucleotides were designed to target different regions of the mouse Tumor Necrosis Factor Receptor 2 RNA, using published sequences (GenBank accession number M59378, incorporated herein as SEQ ID NO: 10, GenBank accession number Y14619, incorporated herein as SEQ ID NO: 98, and GenBank accession number Y14620, incorporated herein as SEQ ID NO: 99). The oligonucleotides are shown in Table 2. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on mouse Tumor Necrosis Factor Receptor 2 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, “N.D.” indicates “no data”.

TABLE 2


Inhibition of mouse Tumor Necrosis Factor Receptor 2
mRNA levels by chimeric phosphorothioate oligonucleotides
having 2′-MOE wings and a deoxy gap

		TARGET
		SEQ ID	TARGET			SEQ ID
ISIS #	REGION	NO	SITE	SEQUENCE	% INHIB	NO

135890	Coding	10	1189	acgttcacgatgcaggtgac	79	48

135891	Coding	10	1205	gtcagagctgctacagacgt	80	49

135892	Coding	10	1209	tgtggtcagagctgctacag	65	50

135942	5′UTR	10	1	cttgtgcctggagctctagt	85	100

135943	Coding	10	70	agctgcagttcgaagaccag	73	101

135944	Coding	10	71	cagctgcagttcgaagacca	78	102

135945	Coding	10	118	tagggtgtcaagacaacctg	43	103

135946	Coding	10	123	gtttgtagggtgtcaagaca	77	104

135947	Coding	10	136	tacccaggttccggtttgta	93	105

135948	Coding	10	201	gaggacacttagcacagcac	88	106

135949	Coding	10	214	acatattggccaggaggaca	71	107

135950	Coding	10	316	ctgcagctcaaacatgtacg	46	108

135951	Coding	10	343	tccacctggtcagtggtaca	81	109

135952	Coding	10	424	ccagaatgggttttcaaggc	90	110

135953	Coding	10	467	gccagggccgcacttgctca	95	111

135954	Coding	10	487	cttgaactggccactccgaa	90	112

135955	Coding	10	550	gatgtggtgtcagagaacgt	84	113

135956	Coding	10	556	gtggatgatgtggtgtcaga	86	114

135957	Coding	10	590	gatgctacagatgcggtggg	24	115

135958	Coding	10	601	ggaatagccaggatgctaca	31	116

135959	Coding	10	622	gcatctgtgcttgcatttcc	62	117

135960	Coding	10	689	ctctggctgagatacgtaga	87	118

135961	Coding	10	695	tgtgggctctggctgagata	95	119

135962	Coding	10	701	ggatcttgtgggctctggct	85	120

135963	Coding	10	810	ttggaagagagatgccaccc	64	121

135964	Coding	10	816	gaccaattggaagagagatg	72	122

135965	Coding	10	822	caatcagaccaattggaaga	61	123

135966	Coding	10	823	acaatcagaccaattggaag	32	124

135967	Coding	10	856	cctaacatcagcagacccag	5	125

135968	Coding	10	890	tttcctctgcaccaggatga	85	126

135969	Coding	10	896	cttctttttcctctgcacca	61	127

135970	Coding	10	901	gagggcttctttttcctctg	55	128

135971	Coding	10	902	ggagggcttctttttcctct	48	129

135972	Coding	10	909	gtaggcaggagggcttcttt	29	130

135973	Coding	10	935	cacatgaggcaccttggcat	21	131

135974	Coding	10	937	ggcacatgaggcaccttggc	75	132

135975	Coding	10	1016	ggagctgctgctggaactgg	69	133

135976	Coding	10	1144	tgggaagaatctgaaatcct	36	134

135977	Stop	10	1451	gggtcaggccactttgactg	51	135
	Codon

135978	3′UTR	10	1726	tagctccttagaaggaaaaa	91	136

135979	3′UTR	10	1778	tgcagtgtcagcattcaggc	75	137

135980	3′UTR	10	1807	ccacttgctcctacttgctg	85	138

135981	3′UTR	10	1867	agagggtacttcctaagagt	82	139

135982	3′UTR	10	1901	gattcttgcatcaaaagaat	80	140

135983	3′UTR	10	1936	cctataacagagcaactctg	59	141

135984	3′UTR	10	2004	gtgttgctgaggatcaaacc	68	142

135985	3′UTR	10	2137	tcattagaaggtcaggagga	61	143

135986	3′UTR	10	2162	aaggaaggcgtggccttgga	89	144

135987	3′UTR	10	2313	actcacagtgcctaacccgg	93	145

135988	3′UTR	10	2321	ctgttccaactcacagtgcc	74	146

135989	3′UTR	10	2371	agagctggcttcagctgttt	85	147

135990	3′UTR	10	2389	catgaatcctttggcaaaag	80	148

135991	3′UTR	10	2521	ctgccaagttcatatccagt	76	149

135992	3′UTR	10	2578	tatcttgattccagagtgct	85	150

135993	3′UTR	10	2609	gagccttaacaagtcggccc	69	151

135994	3′UTR	10	2620	ctgatgctgcagagccttaa	73	152

135995	3′UTR	10	2774	tccttagcacaccctttagg	72	153

135996	3′UTR	10	2815	atttataagcaggaattctg	69	154

135997	3′UTR	10	3253	cacatacatgcaaacatgga	63	155

135998	3′UTR	10	3314	agtaactggagagtgatcaa	70	156

135999	3′UTR	10	3323	gcccgcctcagtaactggag	41	157

136000	3′UTR	10	3346	gcaagctctgggtacagatg	58	158

136001	3′UTR	10	3398	agcactccataggcagacag	78	159

136002	3′UTR	10	3417	gcagcctgcctgtaacctga	82	160

136003	3′UTR	10	3436	taaatgtcgggcaggtatgg	44	161

136004	3′UTR	10	3489	aaaatgcaggtatacaagtg	62	162

136005	3′UTR	10	3560	gccatcttgccagttcaaaa	95	163

136006	3′UTR	10	3697	tgatgtgtgcacatatgcag	71	164

136007	3′UTR	10	3731	acatttatggtatgtgagtg	80	165

136008	Intron	98	698	gtccaatccagtgaagaatg	0	166

136009	Intron	98	902	tgatcagtacatgcctttta	59	167

136010	Intron	98	2592	agtcaagtacactagttctt	34	168

136011	Intron	98	3230	agtccataagccccacagta	25	169

136012	Intron	98	3690	agtttagttaccttcaaagt	0	170

136013	Intron	98	4366	tggattctgcccagtgggtc	60	171

136014	Intron	98	4524	caaaacctcaaccctgaaga	0	172

136015	Intron	99	599	gaggaggcttcctggcagag	72	173

136016	Intron	99	723	acatcaatataggccagccg	62	174

As shown in Table 2, SEQ ID NOs 48, 49, 50, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 117, 118, 119, 120, 121, 122, 123, 126, 127, 128, 129, 132, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 167, 171, 173 and 174 demonstrated at least 40% inhibition of mouse Tumor Necrosis Factor Receptor 2 expression in this experiment and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention. [0239]

Example 17

Western Blot Analysis of Tumor Necrosis Factor Receptor 2 Protein Levels [0240]
Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to Tumor Necrosis Factor Receptor 2 is used, with a radiolabelled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.). [0241]
1 174 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2 atgcattctg cccccaagga 20 3 3683 DNA Homo sapiens CDS (90)...(1475) 3 gcgagcgcag cggagcctgg agagaaggcg ctgggctgcg agggcgcgag ggcgcgaggg 60 cagggggcaa ccggaccccg cccgcaccc atg gcg ccc gtc gcc gtc tgg gcc 113 Met Ala Pro Val Ala Val Trp Ala 1 5 gcg ctg gcc gtc gga ctg gag ctc tgg gct gcg gcg cac gcc ttg ccc 161 Ala Leu Ala Val Gly Leu Glu Leu Trp Ala Ala Ala His Ala Leu Pro 10 15 20 gcc cag gtg gca ttt aca ccc tac gcc ccg gag ccc ggg agc aca tgc 209 Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys 25 30 35 40 cgg ctc aga gaa tac tat gac cag aca gct cag atg tgc tgc agc aaa 257 Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys Ser Lys 45 50 55 tgc tcg ccg ggc caa cat gca aaa gtc ttc tgt acc aag acc tcg gac 305 Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr Ser Asp 60 65 70 acc gtg tgt gac tcc tgt gag gac agc aca tac acc cag ctc tgg aac 353 Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn 75 80 85 tgg gtt ccc gag tgc ttg agc tgt ggc tcc cgc tgt agc tct gac cag 401 Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp Gln 90 95 100 gtg gaa act caa gcc tgc act cgg gaa cag aac cgc atc tgc acc tgc 449 Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys 105 110 115 120 agg ccc ggc tgg tac tgc gcg ctg agc aag cag gag ggg tgc cgg ctg 497 Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu 125 130 135 tgc gcg ccg ctg cgc aag tgc cgc ccg ggc ttc ggc gtg gcc aga cca 545 Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro 140 145 150 gga act gaa aca tca gac gtg gtg tgc aag ccc tgt gcc ccg ggg acg 593 Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro Gly Thr 155 160 165 ttc tcc aac acg act tca tcc acg gat att tgc agg ccc cac cag atc 641 Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln Ile 170 175 180 tgt aac gtg gtg gcc atc cct ggg aat gca agc atg gat gca gtc tgc 689 Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val Cys 185 190 195 200 acg tcc acg tcc ccc acc cgg agt atg gcc cca ggg gca gta cac tta 737 Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val His Leu 205 210 215 ccc cag cca gtg tcc aca cga tcc caa cac acg cag cca act cca gaa 785 Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr Pro Glu 220 225 230 ccc agc act gct cca agc acc tcc ttc ctg ctc cca atg ggc ccc agc 833 Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro Ser 235 240 245 ccc cca gct gaa ggg agc act ggc gac ttc gct ctt cca gtt gga ctg 881 Pro Pro Ala Glu Gly Ser Thr Gly Asp Phe Ala Leu Pro Val Gly Leu 250 255 260 att gtg ggt gtg aca gcc ttg ggt cta cta ata ata gga gtg gtg aac 929 Ile Val Gly Val Thr Ala Leu Gly Leu Leu Ile Ile Gly Val Val Asn 265 270 275 280 tgt gtc atc atg acc cag gtg aaa aag aag ccc ttg tgc ctg cag aga 977 Cys Val Ile Met Thr Gln Val Lys Lys Lys Pro Leu Cys Leu Gln Arg 285 290 295 gaa gcc aag gtg cct cac ttg cct gcc gat aag gcc cgg ggt aca cag 1025 Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg Gly Thr Gln 300 305 310 ggc ccc gag cag cag cac ctg ctg atc aca gcg ccg agc tcc agc agc 1073 Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser Ser Ser Ser 315 320 325 agc tcc ctg gag agc tcg gcc agt gcg ttg gac aga agg gcg ccc act 1121 Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg Ala Pro Thr 330 335 340 cgg aac cag cca cag gca cca ggc gtg gag gcc agt ggg gcc ggg gag 1169 Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly Ala Gly Glu 345 350 355 360 gcc cgg gcc agc acc ggg agc tca gat tct tcc cct ggt ggc cat ggg 1217 Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly Gly His Gly 365 370 375 acc cag gtc aat gtc acc tgc atc gtg aac gtc tgt agc agc tct gac 1265 Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser Ser Ser Asp 380 385 390 cac agc tca cag tgc tcc tcc caa gcc agc tcc aca atg gga gac aca 1313 His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met Gly Asp Thr 395 400 405 gat tcc agc ccc tcg gag tcc ccg aag gac gag cag gtc ccc ttc tcc 1361 Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val Pro Phe Ser 410 415 420 aag gag gaa tgt gcc ttt cgg tca cag ctg gag acg cca gag acc ctg 1409 Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro Glu Thr Leu 425 430 435 440 ctg ggg agc acc gaa gag aag ccc ctg ccc ctt gga gtg cct gat gct 1457 Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val Pro Asp Ala 445 450 455 ggg atg aag ccc agt taa ccaggccggt gtgggctgtg tcgtagccaa 1505 Gly Met Lys Pro Ser * 460 ggtgggctga gccctggcag gatgaccctg cgaaggggcc ctggtccttc caggccccca 1565 ccactaggac tctgaggctc tttctgggcc aagttcctct agtgccctcc acagccgcag 1625 cctccctctg acctgcaggc caagagcaga ggcagcgagt tggggaaagc ctctgctgcc 1685 atggtgtgtc cctctcggaa ggctggctgg gcatggacgt tcggggcatg ctggggcaag 1745 tccctgactc tctgtgacct gccccgccca gctgcacctg ccagcctggc ttctggagcc 1805 cttgggtttt ttgtttgttt gtttgtttgt ttgtttgttt ctccccctgg gctctgccca 1865 gctctggctt ccagaaaacc ccagcatcct tttctgcaga ggggctttct ggagaggagg 1925 gatgctgcct gagtcaccca tgaagacagg acagtgcttc agcctgaggc tgagactgcg 1985 ggatggtcct ggggctctgt gtagggagga ggtggcagcc ctgtagggaa cggggtcctt 2045 caagttagct caggaggctt ggaaagcatc acctcaggcc aggtgcagtg gctcacgcct 2105 atgatcccag cactttggga ggctgaggcg ggtggatcac ctgaggttag gagttcgaga 2165 ccagcctggc caacatggta aaaccccatc tctactaaaa atacagaaat tagccgggcg 2225 tggtggcggg cacctatagt cccagctact cagaagcctg aggctgggaa atcgtttgaa 2285 cccgggaagc ggaggttgca gggagccgag atcacgccac tgcactccag cctgggcgac 2345 agagcgagag tctgtctcaa aagaaaaaaa aaaaagcacc gcctccaaat gctaacttgt 2405 ccttttgtac catggtgtga aagtcagatg cccagagggc ccaggcaggc caccatattc 2465 agtgctgtgg cctgggcaag ataacgcact tctaactaga aatctgccaa ttttttaaaa 2525 aagtaagtac cactcaggcc aacaagccaa cgacaaagcc aaactctgcc agccacatcc 2585 aaccccccac ctgccatttg caccctccgc cttcactccg gtgtgcctgc agccccgcgc 2645 ctccttcctt gctgtcctag gccacaccat ctcctttcag ggaatttcag gaactagaga 2705 tgactgagtc ctcgtagcca tctctctact cctacctcag cctagaccct cctcctcccc 2765 cagaggggtg ggttcctctt ccccactccc caccttcaat tcctgggccc caaacgggct 2825 gccctgccac tttggtacat ggccagtgtg atcccaagtg ccagtcttgt gtctgcgtct 2885 gtgttgcgtg tcgtgggtgt gtgtagccaa ggtcggtaag ttgaatggcc tgccttgaag 2945 ccactgaagc tgggattcct ccccattaga gtcagccttc cccctcccag ggccagggcc 3005 ctgcagaggg gaaaccagtg tagccttgcc cggattctgg gaggaagcag gttgaggggc 3065 tcctggaaag gctcagtctc aggagcatgg ggataaagga gaaggcatga aattgtctag 3125 cagagcaggg gcagggtgat aaattgttga taaattccac tggacttgag cttggcagct 3185 gaactattgg agggtgggag agcccagcca ttaccatgga gacaagaagg gttttccacc 3245 ctggaatcaa gatgtcagac tggctggctg cagtgacgtg cacctgtact caggaggctg 3305 aggggaggat cactggagcc caggagtttg aggctgcagc gagctatgat cgcgccacta 3365 cactccagcc tgagcaacag agtgagaccc tgtctcttaa agaaaaaaaa agtcagactg 3425 ctgggactgg ccaggtttct gcccacattg gacccacatg aggacatgat ggagcgcacc 3485 tgccccctgg tggacagtcc tgggagaacc tcaggcttcc ttggcatcac agggcagagc 3545 cgggaagcga tgaatttgga gactctgtgg ggccttggtt cccttgtgtg tgtgtgttga 3605 tcccaagaca atgaaagttt gcactgtatg ctggacggca ttcctgctta tcaataaacc 3665 tgtttgtttt aaaaaaaa 3683 4 21 DNA Artificial Sequence PCR Primer 4 cactccccac cttcaattcc t 21 5 22 DNA Artificial Sequence PCR Primer 5 gcagacacaa gactggcact tg 22 6 19 DNA Artificial Sequence PCR Probe 6 cccaaacggg ctgccctgc 19 7 19 DNA Artificial Sequence PCR Primer 7 gaaggtgaag gtcggagtc 19 8 20 DNA Artificial Sequence PCR Primer 8 gaagatggtg atgggatttc 20 9 20 DNA Artificial Sequence PCR Probe 9 caagcttccc gttctcagcc 20 10 3796 DNA Mus musculus CDS (43)...(1467) 10 actagagctc caggcacaag ggcgggagcc accgctgccc ct atg gcg ccc gcc 54 Met Ala Pro Ala 1 gcc ctc tgg gtc gcg ctg gtc ttc gaa ctg cag ctg tgg gcc acc ggg 102 Ala Leu Trp Val Ala Leu Val Phe Glu Leu Gln Leu Trp Ala Thr Gly 5 10 15 20 cac aca gtg ccc gcc cag gtt gtc ttg aca ccc tac aaa ccg gaa cct 150 His Thr Val Pro Ala Gln Val Val Leu Thr Pro Tyr Lys Pro Glu Pro 25 30 35 ggg tac gag tgc cag atc tca cag gaa tac tat gac agg aag gct cag 198 Gly Tyr Glu Cys Gln Ile Ser Gln Glu Tyr Tyr Asp Arg Lys Ala Gln 40 45 50 atg tgc tgt gct aag tgt cct cct ggc caa tat gtg aaa cat ttc tgc 246 Met Cys Cys Ala Lys Cys Pro Pro Gly Gln Tyr Val Lys His Phe Cys 55 60 65 aac aag acc tcg gac acc gtg tgt gcg gac tgt gag gca agc atg tat 294 Asn Lys Thr Ser Asp Thr Val Cys Ala Asp Cys Glu Ala Ser Met Tyr 70 75 80 acc cag gtc tgg aac cag ttt cgt aca tgt ttg agc tgc agt tct tcc 342 Thr Gln Val Trp Asn Gln Phe Arg Thr Cys Leu Ser Cys Ser Ser Ser 85 90 95 100 tgt acc act gac cag gtg gag atc cgc gcc tgc act aaa cag cag aac 390 Cys Thr Thr Asp Gln Val Glu Ile Arg Ala Cys Thr Lys Gln Gln Asn 105 110 115 cga gtg tgt gct tgc gaa gct ggc agg tac tgc gcc ttg aaa acc cat 438 Arg Val Cys Ala Cys Glu Ala Gly Arg Tyr Cys Ala Leu Lys Thr His 120 125 130 tct ggc agc tgt cga cag tgc atg agg ctg agc aag tgc ggc cct ggc 486 Ser Gly Ser Cys Arg Gln Cys Met Arg Leu Ser Lys Cys Gly Pro Gly 135 140 145 ttc gga gtg gcc agt tca aga gcc cca aat gga aat gtg cta tgc aag 534 Phe Gly Val Ala Ser Ser Arg Ala Pro Asn Gly Asn Val Leu Cys Lys 150 155 160 gcc tgt gcc cca ggg acg ttc tct gac acc aca tca tcc act gat gtg 582 Ala Cys Ala Pro Gly Thr Phe Ser Asp Thr Thr Ser Ser Thr Asp Val 165 170 175 180 tgc agg ccc cac cgc atc tgt agc atc ctg gct att ccc gga aat gca 630 Cys Arg Pro His Arg Ile Cys Ser Ile Leu Ala Ile Pro Gly Asn Ala 185 190 195 agc aca gat gca gtc tgt gcg ccc gag tcc cca act cta agt gcc atc 678 Ser Thr Asp Ala Val Cys Ala Pro Glu Ser Pro Thr Leu Ser Ala Ile 200 205 210 cca agg aca ctc tac gta tct cag cca gag ccc aca aga tcc caa ccc 726 Pro Arg Thr Leu Tyr Val Ser Gln Pro Glu Pro Thr Arg Ser Gln Pro 215 220 225 ctg gat caa gag cca ggg ccc agc caa act cca agc atc ctt aca tcg 774 Leu Asp Gln Glu Pro Gly Pro Ser Gln Thr Pro Ser Ile Leu Thr Ser 230 235 240 ttg ggt tca acc ccc att att gaa caa agt acc aag ggt ggc atc tct 822 Leu Gly Ser Thr Pro Ile Ile Glu Gln Ser Thr Lys Gly Gly Ile Ser 245 250 255 260 ctt cca att ggt ctg att gtt gga gtg aca tca ctg ggt ctg ctg atg 870 Leu Pro Ile Gly Leu Ile Val Gly Val Thr Ser Leu Gly Leu Leu Met 265 270 275 tta gga ctg gtg aac tgc atc atc ctg gtg cag agg aaa aag aag ccc 918 Leu Gly Leu Val Asn Cys Ile Ile Leu Val Gln Arg Lys Lys Lys Pro 280 285 290 tcc tgc cta caa aga gat gcc aag gtg cct cat gtg cct gat gag aaa 966 Ser Cys Leu Gln Arg Asp Ala Lys Val Pro His Val Pro Asp Glu Lys 295 300 305 tcc cag gat gca gta ggc ctt gag cag cag cac ctg ttg acc aca gca 1014 Ser Gln Asp Ala Val Gly Leu Glu Gln Gln His Leu Leu Thr Thr Ala 310 315 320 ccc agt tcc agc agc agc tcc cta gag agc tca gcc agc gct ggg gac 1062 Pro Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala Gly Asp 325 330 335 340 cga agg gcg ccc cct ggg ggc cat ccc caa gca aga gtc atg gcg gag 1110 Arg Arg Ala Pro Pro Gly Gly His Pro Gln Ala Arg Val Met Ala Glu 345 350 355 gcc caa ggg ttt cag gag gcc cgt gcc agc tcc agg att tca gat tct 1158 Ala Gln Gly Phe Gln Glu Ala Arg Ala Ser Ser Arg Ile Ser Asp Ser 360 365 370 tcc cac gga agc cac ggg acc cac gtc aac gtc acc tgc atc gtg aac 1206 Ser His Gly Ser His Gly Thr His Val Asn Val Thr Cys Ile Val Asn 375 380 385 gtc tgt agc agc tct gac cac agt tct cag tgc tct tcc caa gcc agc 1254 Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser 390 395 400 gcc aca gtg gga gac cca gat gcc aag ccc tca gcg tcc cca aag gat 1302 Ala Thr Val Gly Asp Pro Asp Ala Lys Pro Ser Ala Ser Pro Lys Asp 405 410 415 420 gag cag gtc ccc ttc tct cag gag gag tgt ccg tct cag tcc ccg tgt 1350 Glu Gln Val Pro Phe Ser Gln Glu Glu Cys Pro Ser Gln Ser Pro Cys 425 430 435 gag act aca gag aca ctg cag agc cat gag aag ccc ttg ccc ctt ggt 1398 Glu Thr Thr Glu Thr Leu Gln Ser His Glu Lys Pro Leu Pro Leu Gly 440 445 450 gtg ccg gat atg ggc atg aag ccc agc caa gct ggc tgg ttt gat cag 1446 Val Pro Asp Met Gly Met Lys Pro Ser Gln Ala Gly Trp Phe Asp Gln 455 460 465 att gca gtc aaa gtg gcc tga cccctgacag gggtaacacc ctgcaaaggg 1497 Ile Ala Val Lys Val Ala 470 475 acccccgaga ccctgaaccc atggaacttc atgacttttg ctggatccat ttcccttagt 1557 ggcttccaga gccccagttg caggtcaagt gagggctgag acagctagag tggtcaaaaa 1617 ctgccatggt gttttatggg ggcagtccca ggaagttgtt gctcttccat gacccctctg 1677 gatctcctgg gctcttgcct gattcttgct tctgagaggc cccagtattt tttccttcta 1737 aggagctaac atcctcttcc atgaatagca cagctcttca gcctgaatgc tgacactgca 1797 gggcggttcc agcaagtagg agcaagtggt ggcctggtag ggcacagagg cccttcaggt 1857 tagtgctaaa ctcttaggaa gtaccctctc caagcccacc gaaattcttt tgatgcaaga 1917 atcagaggcc ccatcaggca gagttgctct gttataggat ggtagggctg taactcagtg 1977 gtccagtgtg cttttagcat gccctgggtt tgatcctcag caacacatgc aaaacgtaag 2037 tagacagcag acagcagaca gcacagccag ccccctgtgt ggtttgcagc ctctgccttt 2097 gacttttact ctggtgggca cacagagggc tggagctcct cctcctgacc ttctaatgag 2157 cccttccaag gccacgcctt ccttcaggga atctcaggga ctgtagagtt cccaggcccc 2217 tgcagccacc tgtctcttcc tacctcagcc tggagcactc cctctaactc cccaacggct 2277 tggtactgta cttgctgtga ccccaagtgc attgtccggg ttaggcactg tgagttggaa 2337 cagctgatga catcggttga aaggcccacc cggaaacagc tgaagccagc tcttttgcca 2397 aaggattcat gccggttttc taatcaacct gctcccctag catgcctgga aggaaagggt 2457 tcaggagact cctcaagaag caagttcagt ctcaggtgct tggatgccat gctcaccgat 2517 tccactggat atgaacttgg cagaggagcc tagttgttgc catggagact taaagagctc 2577 agcactctgg aatcaagata ctggacactt ggggccgact tgttaaggct ctgcagcatc 2637 agactgtaga ggggaaggaa cacgtctgcc ccctggtggc ccgtcctggg atgacctcgg 2697 gcctcctagg caacaaaaga atgaattgga aaggactgtt cctgggtgtg gcctagctcc 2757 tgtgcttgtg tggatcccta aagggtgtgc taaggagcaa ttgcactgtg tgctggacag 2817 aattcctgct tataaatgct ttttgttgtt gttttgtaca ctgagccctg gctgagccac 2877 cccaccccac ctcccatccc acctttacag ccactcttgc agagaacctg gctgtctccc 2937 acttgtagcc tgtggatgct gaggaaacac ccagccaagt agactccagg cttgccccta 2997 tctcctgctc tgagtctggc ctcctcattg tgttgtggga aggagacggg ttctgtcatc 3057 tcggaagccc acaccgtgga tgtgaacaat ggctgtacta gcttagacca gcttagggct 3117 ctgcaatcag aggaggggga gcagggaaca atttgagtgc tgacctataa cacattccta 3177 aaggatgggc agtccagaat ctccctcctt cagtgtgtgt gtgtgtgtgt gtgtgtgtgt 3237 gtgtgtgtgt gtgtgtccat gtttgcatgt atgtgtgtgc cagtgtgtgg aggcccgagg 3297 ttggctttgg gtgtgtttga tcactctcca gttactgagg cgggctctca tctgtaccca 3357 gagcttgcac attttctagt ctaacttgct tcagggatct ctgtctgcct atggagtgct 3417 caggttacag gcaggctgcc atacctgccc gacatttaca tgaatactag agatctgaat 3477 tctggtcctc acacttgtat acctgcattt tatccactaa gacatctctc caagggctcc 3537 cccttcctat ttaataagtt agttttgaac tggcaagatg gctcagtggg taaggcagtt 3597 tgcggacaaa cctgatgacc tgagttggat ccctgaccat aaggtagaag agacctgatt 3657 cctgcaagtt gtcctctgac caccacccca tacatgcttc tgcatatgtg cacacatcac 3717 attcttgcac acacactcac ataccataaa tgtaataaat ttttttaaat aaattgattt 3777 tatcttttaa aaaaaaaaa 3796 11 20 DNA Artificial Sequence PCR Primer 11 gtttgcagcc tctgcctttg 20 12 17 DNA Artificial Sequence PCR Primer 12 aaggcgtggc cttggaa 17 13 29 DNA Artificial Sequence PCR Probe 13 agctcctcct cctgaccttc taatgagcc 29 14 20 DNA Artificial Sequence PCR Primer 14 ggcaaattca acggcacagt 20 15 20 DNA Artificial Sequence PCR Primer 15 gggtctcgct cctggaagat 20 16 27 DNA Artificial Sequence PCR Probe 16 aaggccgaga atgggaagct tgtcatc 27 17 15602 DNA Homo sapiens 17 gatcttggct gggcacggtg gctcactcct gtaatcccag cactttggaa ggccgaggta 60 gatggatcac ttgaggtcag gagttcaaga ctagtctggc catcatggta aaaccccatc 120 tctactaaaa acagaaaaat tagccaggcg tggtggcacg tgcctgtagt ctcagctact 180 cgggaggctg aggcaggaga gtcgcttgaa cccgggcagt ggaggttgca atgagctgag 240 atcacaccac tgctctccag cctgggcaat agagcaagac tccatctcaa aagaaaaaat 300 aaaataaaat aaataaataa aatatcttac ttagttgtgg gggctgtccc gtgctattca 360 gcagcatctt tggtctttat ttacttacat gccagcctca ttcccactcc agtggtgaca 420 atcaaaaatt ctccagacat caccaagtgt cccctggagg gcaaaatcac ccctagtgga 480 gaaccactgc tcgagggaac agctcagccc tgggcagccc aatacccgaa gcttacacca 540 ggtgtttgga atcagataga ttgatttcat cccctgattt tggtgacaag aaaagagatg 600 cagggaggaa aactgagttg ctcaaggtcg caccgcagtg tgtctctgga ctcccatctc 660 agtgctcttt gccctgccct acagtatttc agtggggagg aaattttctc attccccggc 720 gagcgagttt cactgctccc tttctccagc ttcaattcag tgacactgac ggttgccgcc 780 tggtggcgtg cgtagagcag gtgtctcccc caagctcttc cgcagcagcc cagctgtcac 840 cttccctttc cccaccaagc gtttcccaat cctctgtgca ctgaagatgt cctgggtgtg 900 gcctagcagc ctctgcttcc cttttccatt ttttttttct ttctttcttt tttttttttt 960 gagacagcta tgtcgcccag gctgcagtgc agtggcgccc tcttggctca ctgcagcctc 1020 cgcctcccgg gttcgagtgg tcatcccacc tcagcctccc aagtagatgg gactatgggc 1080 atgcgccacc atgcctggct aatttttata tttttagtag agacggggtt ttgccatgtt 1140 ggccaggctg gtctcaaacc cctgacctca agtgatctgc ccacctcggt ctcccaaagt 1200 actgggatga gaggcataag ccaccacgcc tggccccttt cctttcactg ttgctctcag 1260 tgttcactga atgtgagtga ctcaagtatc tgttgagtgc cccagtggtg tgtctagtgc 1320 agaaaaaaac ccccaaaccc tgatttgtgg tgtttgctaa tttctgtggt ataaacactc 1380 acactgtagc cagtttccaa ctcccaagac cttcctgaat atggagttgg gaggaaatct 1440 gcacaggcct gagcttatcc cagcacagcc ctggctgagc cctgccaggc tctttgtgac 1500 tgagtcccca cgcatccctg tggggcaggg agcatcatca cccccatttt acagaggagg 1560 aaactgagac tctgggaggt tccatttttc ctgtaggatt agatcattag gtgagtggca 1620 tagcccaggt ttgaatcagg tttatgcctg tgtcccacgc tactcctcaa ccagcagcta 1680 ctcctgataa gaggccagtg gagagtcctc ttgagctggg ctgctgggct tagagtgtgg 1740 actcagcacc cacctgctcc tcctgccaca gaaagaacgt ccagggcatg ggagacagtg 1800 gcgttccttt gtttagaaaa caccaacatg tggccgggcg ccatggctca cgcctgtaat 1860 cccagcactt tgggaggccg aggggggcgg atcacgaggt caggagatcg agaccatcct 1920 ggctaacaca gtgaaacccc atctctacta aaaataaaaa aaattagccg ggcgtggtgg 1980 cgggcgcccg tagtcccagc tactccggag gctgaggcag aatagcgtga acccgggagg 2040 tggagcttgc agtgtactga gatcgcacca ctgcactcca gcctgggcga tagagcgaga 2100 ctccatctca aaaaaaaaaa aaaaaaaaac caatatgtga gcttacccac aatcaccccc 2160 atggacacgg acacactcat gcacggaccc acgtctgata acaggtgcga acatttatcg 2220 ggtgttgaac ggatgccagg cactgtgccc agcgttcttt attctcacag ccatgctgcc 2280 acacgcaggc agtatgattc ttgtccccca ttatatggcc caagaatcat gggcccagag 2340 aagttagtaa ctttcccagt gtggcacagc taatgatcac ggcctgggac acgaacctag 2400 gagtgtctct cctcgccacc ccagcgcaca cactcactac gtcactcaca gatatgcaca 2460 catgccccca acatagacac acgtgtgcac gcttcacacc ctcacgcctg tagacacagg 2520 tgtgtgagcc ttgtgaacac acaaacagac ggacacagca ggaacctgga ggacctggcc 2580 cgtggctctg ctgggtctct gcttctgccc agcagggctg ggccaggagg ggccgggagc 2640 tgagggtgct ggctggctgg ctggctggct ggaattctgt tttttttttt ttttttgaga 2700 aagagtcttg ctttgtcgcc caggctggag tgcagtggca caatcttggc tcactgcaag 2760 ctccgcctcc cgggttcaca ccattctcct gcctcagcct cccaagtagc tgggactaca 2820 ggcacccacc agctcgccca gctaattttt tgtattttta gtagagacgg ggtttcacct 2880 tgttagccag gatggtctca atctcctgac ctcgtgattc ggtgaccacc tcggcctccc 2940 aaagtgctgg gattacaggt gtgagccact gcgcccggcc ctgggatcct gtgttttgaa 3000 tgaggctcct cagtactcgg ctctactggg gtcccagccc aaggaatagg actcagcctg 3060 cttctgtgcc acctggggct gcttgaactt tgcgacttgt ggcttgggag gagggaggtg 3120 gccgtgacct ttgggggttt ttgttctgcc tggctgtagc caccagcaga gggggtgggg 3180 cacaggccag aaaaacccct tttgtggggt tgtgaggagt gacaattggc tgcttctcct 3240 ccccttccag gctcagagca gggctggggg gcagttgtgg gcagtgacca gggtcagacc 3300 acctgggcgg aggttcagca tgaacttgca atgccctcca tctctccaaa actgggggac 3360 ccagcccagg gagggtgtgg gggttcctgg ggaagctggt ctaggcttct gctcctgcca 3420 cggaccagct gtgtgatgct gggcacagga tgcactttct ctgggcctcc gtggcctctt 3480 ggggatggct tgcacgagat ccctccagtc ctgagtgaga ggctgtggcc ttggggaatt 3540 aagggtgcag gtggcgctca ggtgtccgag aagccatggg agccgggggc tgcagggatt 3600 ggacagagag gaccctggta ctcgcatctg ttctcagacc acatctggaa ttgtagctcc 3660 ctctggaggg aggcaggagg tctcagcctt tcttgggggg cggtggcacc tgcgctgctc 3720 gctccacccc tgctctcacc tcccgctgca gtgctggcga gccccatcag cccttcactc 3780 atctctaccc tccttctttc tgcctgggac acttgttttc atcctgggca ggccaggggc 3840 cagggcagct gttgggaatg tggcctgtgc catctccttt tttgctggga tcagaaaaca 3900 atcgcttaga attccaaggc aagggtgtga gcgcctggcc agccagtggg aacagacaac 3960 agcctgggag aggaatttcc agcctctctt cagtgtgcgt gtctggaaat ggggaccttg 4020 ccttgagcct ccagagttga aaccccagac acccaggaaa ggccctttgg gatttagccc 4080 agccacagta tgtcctaacc gtgaccttgg gcaagtaact caatctctcc gtgcctcagt 4140 ttccacaaag caaggataac actgggttgt tggaagaatc aatatagata ttgtctggag 4200 ggatgtaggt acagtgcatg gcattgggtg gcactcaaat gtcagctaat aatattatta 4260 ttattctacg ggaagaagac atcaggggaa gttgcagagc agcctgtggg cggactctgg 4320 aacaagaggc tgaggcagtg cagcagaggg tctcagacgt gagcgctctc tgccccggaa 4380 tgattgactg agcgcaaagg tctgcacgct ttctctgtaa agggccagat ggtaggaatt 4440 tcaggctttg tggactgtat ggtctcagtg acagctactc aaacctgcct ctgtagcaag 4500 aaagcagcta catgcataga cagcacacac ccaactgaga gtggtgtgtt cctatctaat 4560 tgtgctactg gacacccaaa cttgagcttc ccaccattcc atgtgccgca aattattctt 4620 tttttttgag atggagtttc actcttgttg cccagcctgg agtgcagtgg ctcgatctcg 4680 cctcaccaca acctctgcct cccaggttca agcgattctc ctgcctcagc ctcctgagta 4740 gctgggatta caggcatgag ccaccacgcc agctaatttt gtatttttag tagagacggg 4800 gttttgccat gttggtcagg ctggtcccca actcccttcc tcaggtgatc cgcctgcctc 4860 agcctcccaa agtgttggga ttacaggcat gagccaccaa gcctggccac aaattattct 4920 taaacatttt tttttcaacc atttaaaaca tgaaaaccag tggggtgtgg tggtgcacgc 4980 ctggtatccc agcactttgg gaggccaggg taggaggatg gcttgagccc aggagtacaa 5040 gaccagcctg ggtaacatag cacaaccctg tctctacaaa caatcgacaa caaaaaaatt 5100 agccaggagc agtgacacgt gcctgtggtc tcagctactc aggaggttga ggcaggagga 5160 tcacttgagc ctggaaaatc gagggctata gtcagctatg attgtgccac tccactcctg 5220 cctgagcaac agggtgagac cctgtctcaa aaaaaaaaaa aaaaaaaaaa agtgaaaacc 5280 attcttagtg gcaggctgta ctgggcctct gggcaatcat ttgcctagtt ctgatttaac 5340 aaactcttgt atggagttta ctatgtaata ggcattgttt taagcacttt acaaatattc 5400 agccatctaa tcttcacaac aaccctatga ggtaggctat tattctccct ttatagagta 5460 aaaaaaaaaa aaaaggcaca gagaggttaa gtaacttgtc caaggtcaca cagcaagtga 5520 gtggtagagt catgatttgc acttgtgtgg cctgggttta gagtccacac tcttggttgc 5580 taggctgggc catgtctccc tgtgcagatg gggtgaagga aagctgcttt ccttctactc 5640 ccttatgcaa aataaggatg aaaatcctgc cccacctcta agactatttg gtgaacaggg 5700 ccaggtattc tctgccctca taggacactc tagtagagca gctgggtgct aataacagaa 5760 acacagaaac cacggagaat cacacctccc aaaaagtgcc gtgtgtggaa agaacgtgca 5820 agggtgggca gaaacgtcac gtaaactgag aagtgctgat ggcaggaggt gtggatggca 5880 gtgggaagga agaggggaag aaagagctgg ctggtgggct gactgctctc ccctaccacc 5940 ccctgcccat ccagcctcac ttgcctgccg ataaggcccg gggtacacag ggccccgagc 6000 agcagcacct gctgatcaca gcgccgagct ccagcagcag ctccctggag agctcggcca 6060 gtgcgttgga cagaagggcg cccactcgga accagccaca ggcaccaggc gtggaggcca 6120 gtggggccgg ggaggcccgg gccagcaccg ggagctcagg taagaggtgg gagcacacct 6180 ggcttcttcc caagcctcct tggtctttct cacctggttt ctgtcttagc catctcctcc 6240 tgagcctccc cgcagggtgg gacgaggcct gagccacagg gaacttcctt cggttcgctg 6300 aacctaagtt ccctcccgcc tttgcccatg ctgggcctat cacctcaaaa tcctcccttc 6360 tgtgggacaa ccccagcttg tccaagtccc tggtatctgg gggaggagtt ttcctgaaac 6420 cttctccctg ctaccacccc cagctggcct ggctgctcct ccgggctcac catgccctgg 6480 cctccctttc acaggactgt cagactgcat gtacagacat tgtcttctcc tgtctcccac 6540 gccaggctgt gggacacctg gtgagcctgg atcatctcat tcatccctgt atctgcaggc 6600 ccaacacggc ccagctactg ataattaatc ataacaatcg cttctactta tggaaggcta 6660 cggaacagca ggcactgtac tgggcacttt acatgcataa aactgacctg catgtcaatg 6720 ctaagagata gttcctgttg ttatcccatt ttacagatga ggaaactgag ccccagagag 6780 gttaaacagc ttcttcaagg gcacatggct agtaaacaga agagccagac tcacccctag 6840 gctgtcgggc tccagagccc tgggattgga agatgaatga agaaatggtg gctccagggc 6900 tccactcact gcagtttgtt gctgggtctc tttaggtctg aggcactggg actgtgggga 6960 ttgtgtccca tttatgcagg ctgcattgtg ccctggacct ggtctatgac agatgtcact 7020 cctggttggc atctctaggg cccacactgg aggggcccct gtacagggtc tgctggcctg 7080 tgcctctctc cctcttacct ggcagtgcca gccagtgaga attcagggaa tgaccatgaa 7140 cttgggtcag tctgagatcc ttgcctggcc actctggtgc cataagaatt tgggtgggat 7200 gcttaaccct gccaagcctt ggttttttca cctagaggtg agctatagcg cctccttgcc 7260 agggctggtg agagatgggt gacattgtgc ccagctgggc accagaccag ggccaggctc 7320 cctctgggcc gcctccaggt ggggactgat ggctgcagcc cccaccacgc agccctcctc 7380 cagctcactc acccccagct gctccccagc tcactgcgcc cctgcctctt gcctgcactc 7440 atgccacccc tgcctgccac acctgttcct acctgccccc aactgcccac aggagctgac 7500 gtggccattt ctgtctgccc catcatagcc cccagcctcc tcggtgggag gtctcatcag 7560 tgcccctcac tccatcctga gaactcccta tgggagggcc tgggccagtg cctggaagag 7620 atcaggggcc ctacacactg ttgaatgaat gaatgaatga gtgcgctcca attataaact 7680 cttagtcttt gcccagttct tggattcgtc tgattttttt tttttaaatg gggtctcgct 7740 cgtcgcctag gctggagtgc agtggcgcag tcttggctga cttatctgct caccgcagcc 7800 tccgcctccc aggttcaagt gatcctccca cctcagcttc ccgagtaggc gggattacag 7860 gcatgtgcca tcacgcctgg ctaacttttg tacttttagt agagatggag tttcaccatg 7920 ttggccagac tggtctcgaa atcctgacct caggtgatcc atctgcctcg gcctcccgaa 7980 gggctgggat tacaggcgtg agccaacatg cgcagcccat ctttctgatt tcttagctac 8040 acctggtgtg gctccctcct tgggccaggg tggagccctg accatgtctg ccctcccctc 8100 tcccctctgc cccttctgct ctgtgctcct tctcccgagt cccccagccc gtgtccctgg 8160 cctctgtctt ctctttctct ccctcccacc cctaacacct ccctccactg tgggaacctg 8220 taaaccccag ggttgtgccc cttcatggtc ccccatccac ccccgcaatg tctcatgctc 8280 gatatacaaa ggccatggtg actttgggtg acatttgggt gctgtggagg ctcagggtgg 8340 aaatttcctt ccggccttgt gatttcaacc ctcctccccc accacatgct tggggctgtt 8400 ttgagcacag caggttgcca gctccatcca cctcccggct accctatccg agtagttgga 8460 gttagggaga accaggctgg ggtgagggca ctcagcaggc ccctgcagca acagcagcag 8520 caactctcat tttctgaggg ggctacttac tgtatgccag tcccttcata ttcatctcag 8580 caaacccacc gtccagtgcc tccccaacca gttagaaaac tcagttgccc acaggggctg 8640 ggcaggaagg tgaggcaaac cttgggctgt ccttggccgg atctcctgca tctggctccc 8700 aagggaagcc ataaatccag atttttaaat gtaaacgcct gaattttaaa tgttggtaat 8760 caattcactt aaaaacatca ccaccaccac caccaccacc accaacaaaa aaacccgtag 8820 acttgtccct gttacaggca ctaggaacac agcagggaac aatcaaaaag tccctggtct 8880 ggccaggcaa ggtggctcat gcctgtaatc tcagtacttc aggaggccaa ggcaggagga 8940 tcacttgagc ccaggagttc gagactagcc tgggcaacat agcaagaccc ccgtctctac 9000 taaaaaaata aaaaaaaaag tccctaccct cctgggttca gagtctggtt ggggacccca 9060 ggagctgggg gctctggaga tcaggagatc acagaaatgg ggagggaccc agagagtggt 9120 ggataggatg ggaagtaaat gtctctagag agggaggcca gggggtggag ggcgcttcgt 9180 ggaggaggtg gcctttgagc taaggcctga gcactagaga agagctctct aggctgaggg 9240 agcggcctgt gcaaaggccc aggggacctg aagggctcaa ggggctgtag cagggggtgg 9300 ggaatgtggc tggaaggaac cccatcaagg tcttggagcg gcaggagagg gggtgggaga 9360 aggcaggctc cagatcagac agggcctggt aggctgtagc aaggactgtg ggtttttgag 9420 cccccaagga agtgatctgc caggttcaag ggccagctct ggctgctgat gggaaacaga 9480 tttcagaggg gtggggttga agccaggaca gatggaggct gttcacaccc atccagatgg 9540 gagtgagggg aggcttccat agcccaccat gcagcagcag ggcagggtga cccttgcaga 9600 agtcatcttt tgtttttgtt tgtttttgag atggagtttg gctctttcgc ccaggctgga 9660 gtgaagtgac gtgatttcgg ctcactgcaa cctccgtagc ctgggttcaa gctattctcc 9720 tgcctcagcc tcccgagtag ctgggattat aggcacctgc caccataccc ggctaatttt 9780 tttttttgta ttttgagtag agacagagtt tcaccatgtt ggccaggctg gtctcaaact 9840 cctgacctca ggcgatccac ctgccttggc ctcccaaagt gctgggatta caggcgtgag 9900 ccaccctgcc tggtccagaa gtcatctttt gaagggagac aaggcaggaa tgatggatgg 9960 gtgtgtgata tgagagaaag atgggtccga ggctctgggc ccaagcagct gggtggatgg 10020 cagcaatggg aactgtgatg agcaggagag gttttggatg cgagatggga gtagaatcaa 10080 gagttaagtt ggaggctgag cacggtggct cacacctgta atctcagcgc tttgcgaggc 10140 tgaggtaggc agattctttg aggtcaggtg ttcgagacca acccaggcaa cctggcgaaa 10200 ccctgtctct acaaaaaatt agcagggtgc ggtggcctgt agtcccagct attcaggagg 10260 ctgatgtggg aggatcactt gaggccggga ggcagaggtc acagtgagtt gagggagtga 10320 cacagcactc ttttgagacc ctgtctcaaa aaaaaaaaaa aaaaagacag aagagacagg 10380 gtctcactat gttgcccagt ctggtcttga actcctgggc tcaagcgatc ctacaaactt 10440 ggcctcccaa gtagacatct gttttatata attggctcct cccatctctg gggtgattgg 10500 ggctgggtag gtagtgatgc tattcttatt cggcagaggg gaaaatgagg cacatgcagg 10560 ttaagtgact tgctcaaggt cacacagcag agctgggcta gaatcttggt ctcggctcct 10620 ggcccagtgc tctttcccat gtgtctgaat ctgcatcttg ggcaggggtc cctgggcccc 10680 actcctggac ccccggactg acccccaccc catcttgtgc ttagcagatt cttcccctgg 10740 tggccatggg acccaggtca atgtcacctg catcgtgaac gtctgtagca gctctgacca 10800 cagctcacag tgctcctccc aagccagctc cacaatggga gacacagatt ccagcccctc 10860 ggagtccccg aaggacgagc aggtcccctt ctccaaggag gaatgtgcct ttcggtcaca 10920 gctggagacg ccagagaccc tgctggggag caccgaagag aagcccctgc cccttggagt 10980 gcctgatgct gggatgaagc ccagttaacc aggccggtgt gggctgtgtc gtagccaagg 11040 tgggctgagc cctggcagga tgaccctgcg aaggggccct ggtccttcca ggcccccacc 11100 actaggactc tgaggctctt tctgggccaa gttcctctag tgccctccac agccgcagcc 11160 tccctctgac ctgcaggcca agagcagagg cagcgagttg tggaaagcct ctgctgccat 11220 ggcgtgtccc tctcggaagg ctggctgggc atggacgttc ggggcatgct ggggcaagtc 11280 cctgactctc tgtgacctgc cccgcccagc tgcacctgcc agcctggctt ctggagccct 11340 tgggtttttt gtttgtttgt ttgtttgttt gtttgtttct ccccctgggc tctgccccag 11400 ctctggcttc cagaaaaccc cagcatcctt ttctgcagag gggctttctg gagaggaggg 11460 atgctgcctg agtcacccat gaagacagga cagtgcttca gcctgaggct gagactgcgg 11520 gatggtcctg gggctctgtg cagggaggag gtggcagccc tgtagggaac ggggtccttc 11580 aagttagctc aggaggcttg gaaagcatca cctcaggcca ggtgcagtgg ctcacgccta 11640 tgatcccagc actttgggag gctgaggcgg gtggatcacc tgaggttagg agttcgagac 11700 cagcctggcc aacatggtaa aaccccatct ctactaaaaa tacagaaatt agccgggcgt 11760 ggtggcgggc acctatagtc ccagctactc agaagcctga ggctgggaaa tcgtttgaac 11820 ccgggaagcg gaggttgcag ggagccgaga tcacgccact gcactccagc ctgggcgaca 11880 gagcgagagt ctgtctcaaa agaaaaaaaa aagcaccgcc tccaaatgcc aacttgtcct 11940 tttgtaccat ggtgtgaaag tcagatgccc agagggccca ggcaggccac catattcagt 12000 gctgtggcct gggcaagata acgcacttct aactagaaat ctgccaattt tttaaaaaag 12060 taagtaccac tcaggccaac aagccaacga caaagccaaa ctctgccagc cacatccaac 12120 cccccacctg ccatttgcac cctccgcctt cactccggtg tgcctgcagc cccgcgcctc 12180 cttccttgct gtcctaggcc acaccatctc ctttcaggga atttcaggaa ctagagatga 12240 ctgagtcctc gtagccatct ctctactcct acctcagcct agaccctcct cctcccccag 12300 aggggtgggt tcctcttccc cactccccac cttcaattcc tgggccccaa acgggctgcc 12360 ctgccacttt ggtacatggc cagtgtgatc ccaagtgcca gtcttgtgtc tgcgtctgtg 12420 ttgcgtgtcg tgggtgtgtg tagccaaggt cggtaagttg aatggcctgc cttgaagcca 12480 ctgaagctgg gattcctccc cattagagtc agccttcccc ctcccagggc cagggccctg 12540 cagaggggaa accagtgtag ccttgcccgg attctgggag gaagcaggtt gaggggctcc 12600 tggaaaggct cagtctcagg agcatgggga taaaggagaa ggcatgaaat tgtctagcag 12660 agcaggggca gggtgataaa ttgttgataa attccactgg acttgagctt ggcagctgaa 12720 ctattggagg gtgggagagc ccagccatta ccatggagac aagaagggtt ttccaccctg 12780 gaatcaagat gtcagactgg ctggctgcag tgacgtgcac ctgtactcag gaggctgagg 12840 ggaggatcac tggagcccag gagtttgagg ctgcagcgag ctatgatcgc gccactacac 12900 tccagcctga gcaacagagt gagaccctgt ctcttaaaga aaaaaaaagt cagactgctg 12960 ggactggcca ggtttctgcc cacattggac ccacatgagg acatgatgga gcgcacctgc 13020 cccctggtgg acagtcctgg gagaacctca ggcttccttg gcatcacagg gcagagccgg 13080 gaagcgatga atttggagac tctgtggggc cttggttccc ttgtgtgtgt gtgttgatcc 13140 caagacaatg aaagtttgca ctgtatgctg gacggcattc ctgcttatca ataaacctgt 13200 ttgttttaca cgtcgacccc tggctctgcc tggggtctgg gcttgggttt gtccatgctc 13260 ctacttgtct gccacccctg tgtaagggga gatggcgtca cggtccctgg agtctggctg 13320 gcccctgttg tgactggacc acagagggac ccctgttaca gccgccccct caagcctgtg 13380 aaccataaga gaacttcctg ctcgggacca cacagctggc tgggttccaa gtgtgccctg 13440 gtctcatgcc ttcatcctcc aggtctcctg ggcctgctct caggaccggg atggggtctc 13500 tgcagatccc tagcagccta ggcagccagg ctctgccctc ctggggaccc cactcgggga 13560 gagtggttgc ccctgggata ctcagaccag tacagggttt tgggggccca gaggacatcc 13620 ctgggcccag gtaggaggtt agaacagggt tctggagatg acctctgacc tcctcctgag 13680 ggatgagcag cagttttcca gaacaaagga ttgcagggaa ctgtcaggca aaaggagttc 13740 tgagtttaaa ggccttagcc tggccagcat ggtgaaaccc catctctacc aaaaatacaa 13800 aaaattagct gggcatgacg gtatgcacct ataatcccag ctagtcagga ggccgaggca 13860 cgagaattgc ttgaatcaag gcaacagagg ttgcagtgag ctgagatcgg gccactgcac 13920 tccagcctgg gtgacagagt aagagtctgt ctccaaataa aataaataaa taaaatcaat 13980 taattagaag aaagccttgg aggggagaga gaccttggcc tgtgtattgg ttccactgac 14040 cccctgggtc accatcgtca ctccagctcc tgtgactccc tcagtgggac cattttcatt 14100 cttggtgatt ataggatctg tgattgatgg aacgccccgc ctcctggctt ctcgccctcc 14160 tctcctccat ggtcctgtcc tccagcctgt ctcagtcact caacccttga ccaaggctcc 14220 ccacacttat tccataaaga gccagggagc aaatttattt taattttttg agacaaggtc 14280 tcactctgtt acccaagctg aaatgtgatc gtggctcact gcagccctga cctccagtcg 14340 cagcctcttg agcagctagg actacaggca tggaccacta tgccgagcta aattttaaat 14400 ttttttattt ttattttttg ggtttccctg tgttgcccag gctggtctcg aactcctggg 14460 ctcaagtgat ccaccggcct gggcctcctg aactgctggg attacaggtg tgagccattg 14520 cgcctgacca gaatcaatat tttacacttg gcaggcctta cagtttctgc aacaaccact 14580 cacctgtgct gttgaagtgt gaaaacagct gtgcacagga catgaaagaa tgggcaggag 14640 gcggatgtgg ccttcggggt gtgcccatgc cagtgcctta gagcctggca ttactgataa 14700 ctgcacgcta atctcaattt caagcatccc acttcccccc acccctactt cctcctgtct 14760 ttctccctca gtggcacctg cagctgttgg ttacgttttc tcccttgtac gcacccacgg 14820 cactttctcc tggtttgttg ttctctctct ggttgaaccc agctctctgc ctgtgccaca 14880 cctgcacgtc tgcacctggc tggggcagga tgatggcctt ccaccatgct ggctggtcgc 14940 gttttcattt catgatcatt agacttggct gggcacagtg gcttatgcct gtaatcccag 15000 cactttggga ggctgaggtg ggcagactgc ttgagctcag gagttcaaga ccagcctggg 15060 gcaacatggt gaaacctcat ctccacagaa aaatacagaa actagctggg tgcggtggca 15120 cgtacctgga atcccagcta cccatgaggt tgaagtggga ggattgcttg agcccaggag 15180 gcggaggttg ttgtgagctg agatcttgcc actgcactcc agcctggggg atggaaaaaa 15240 agaataaatt ctatgggggt ccttggtgct gcccagcagt gacaacaggt cccctccccg 15300 atatattagt tcctgttgtt gctataacaa accacacaaa ctcagtggct taaaacaata 15360 caatttcatt ctcctacagc tctgggagcc agaagtataa aagcaaggtg ttgccaggcc 15420 tgctaggctc aaatggggaa tttgttcttg gaggctttag gggagaatct gattccttgc 15480 cttcagcttc cagcggtacc tgcattcctt gacttatggc cccttcgtcc atcttcagag 15540 atagcaacgg aatctcttca gaagtcagat ctccctttgc ctcccaagtg tagggacacc 15600 tg 15602 18 248 DNA Homo sapiens 18 aatagccttc ccagctgggc tttagaactc tggactttgt ggggacagtg gatgagccca 60 gggtcctggc agaaggctcg cccagctgag acctctggcc cttgtttcct caggccaaca 120 tgcaaaagtc ttctgtacca agacctcgga caccgtgtgt gactcctgtg aggacagcac 180 atacacccag ctctggaact gggttcccga gtgcttgagc tgtggctccc gctgtagctc 240 tggtgagg 248 19 519 DNA Homo sapiens 19 ctgtgttgcg tgtcatgggt gtgtgtagcc aaggtcggta agttgaatgg cctgccttga 60 agccactgaa gctgggattc ctccccatta gagtcagcct tccccctccc agggccaggg 120 ccctgcagag gggaaaccag tgtagacttg cccggattct gggaggaagc aggttgaggg 180 gctcctggaa aggctcagtc tcaggagcat ggggataaag gagaaggcat gaaattgtct 240 cttaaagaaa aaaaaagtca gactgctggg actggccagg tttctgccca cattggaccc 300 acatgaggac atgatggagc gcacctgccc cctggtggac agtcctggga gaacctcagg 360 cttccttggc atcacagggc agagccggga agcgatgaat ttggagactc tgtggggcct 420 tggttccctt gtgtgtgtgt gttgatccca agacaatgaa agtttgcact gtatgctgga 480 cggcattcct gcttatcaat aaacctgttt gttttaaaa 519 20 20 DNA Artificial Sequence Antisense Oligonucleotide 20 tctccaggct ccgctgcgct 20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21 ttgcatgttg gcctgaggaa 20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22 ctgagccggc atgtgctccc 20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23 ttctctgagc cggcatgtgc 20 24 20 DNA Artificial Sequence Antisense Oligonucleotide 24 ctgtctggtc atagtattct 20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 tttaagagac aatttcatgc 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26 cacatctgag ctgtctggtc 20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 gcagcacatc tgagctgtct 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28 gcatgttggc ccggcgagca 20 29 20 DNA Artificial Sequence Antisense Oligonucleotide 29 gaagactttt gcatgttggc 20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 cagctcaagc actcgggaac 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31 tccacctggt cagagctaca 20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 ggtgcagatg cggttctgtt 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33 gtttcagttc ctggtctggc 20 34 20 DNA Artificial Sequence Antisense Oligonucleotide 34 gatgtttcag ttcctggtct 20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 tgcacaccac gtctgatgtt 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36 cacgttacag atctggtggg 20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 gggtaagtgt actgcccctg 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38 tgttgggatc gtgtggacac 20 39 20 DNA Artificial Sequence Antisense Oligonucleotide 39 gctgcgtgtg ttgggatcgt 20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 cacaatcagt ccaactggaa 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41 caagggcttc tttttcacct 20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 gcaagtgagg caccttggct 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43 cccgggcctt atcggcaggc 20 44 20 DNA Artificial Sequence Antisense Oligonucleotide 44 gtcccatcta cttgggaggc 20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 ctccagggag ctgctgctgg 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46 gagctctcca gggagctgct 20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 tggccgagct ctccagggag 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48 acgttcacga tgcaggtgac 20 49 20 DNA Artificial Sequence Antisense Oligonucleotide 49 gtcagagctg ctacagacgt 20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 tgtggtcaga gctgctacag 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51 gtgtctccca ttgtggagct 20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 ctggaatctg tgtctcccat 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53 tgtgaccgaa aggcacattc 20 54 20 DNA Artificial Sequence Antisense Oligonucleotide 54 ggcttcatcc cagcatcagg 20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 actgggcttc atcccagcat 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 ccggcctggt taactgggct 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 gtcatcctgc cagggctcag 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58 agaggaactt ggcccagaaa 20 59 20 DNA Artificial Sequence Antisense Oligonucleotide 59 ctaagcccag cagcccagct 20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 tgggtgactc aggcagcatc 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 agtctcagcc tcaggctgaa 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 accccgttcc ctacagggct 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63 gagctaactt gaaggacccc 20 64 20 DNA Artificial Sequence Antisense Oligonucleotide 64 ttagctgtgc cacactggga 20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 cagcactgaa tatggtggcc 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 gttatcttgc ccaggccaca 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 cgttatcttg cccaggccac 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68 agatttctag ttagaagtgc 20 69 20 DNA Artificial Sequence Antisense Oligonucleotide 69 gcttgttggc ctgagtggta 20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 tgtggctggc agagtttggc 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 gcaggcacac cggagtgaag 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 tggtgtggcc taggacagca 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73 attccctgaa aggagatggt 20 74 20 DNA Artificial Sequence Antisense Oligonucleotide 74 tctaggctga ggtaggagta 20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 ggccatgtac caaagtggca 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 gactggcact tgggatcaca 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 ctacactggt ttcccctctg 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78 gacaatttca tgccttctcc 20 79 20 DNA Artificial Sequence Antisense Oligonucleotide 79 tggtaatggc tgggctctcc 20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 tctgacatct tgattccagg 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 gcagaaacct ggccagtccc 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 gtccaatgtg ggcagaaacc 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83 tctcccagga ctgtccacca 20 84 20 DNA Artificial Sequence Antisense Oligonucleotide 84 ggctctgccc tgtgatgcca 20 85 20 DNA Artificial Sequence Antisense Oligonucleotide 85 caaattcatc gcttcccggc 20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86 aaacaggttt attgataagc 20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87 ctacagaggc aggtttgagt 20 88 20 DNA Artificial Sequence Antisense Oligonucleotide 88 agacagggtt gtgctatgtt 20 89 20 DNA Artificial Sequence Antisense Oligonucleotide 89 tggcacaatc atagctgact 20 90 20 DNA Artificial Sequence Antisense Oligonucleotide 90 gatggctgaa tatttgtaaa 20 91 20 DNA Artificial Sequence Antisense Oligonucleotide 91 agggagaata atagcctacc 20 92 20 DNA Artificial Sequence Antisense Oligonucleotide 92 agccttccat aagtagaagc 20 93 20 DNA Artificial Sequence Antisense Oligonucleotide 93 ccaagttcat ggtcattccc 20 94 20 DNA Artificial Sequence Antisense Oligonucleotide 94 tgaattgatt accaacattt 20 95 20 DNA Artificial Sequence Antisense Oligonucleotide 95 caccttgcct ggccagacca 20 96 20 DNA Artificial Sequence Antisense Oligonucleotide 96 gaagtactga gattacaggc 20 97 20 DNA Artificial Sequence Antisense Oligonucleotide 97 gtccccagga gggcagagcc 20 98 5874 DNA Mus musculus unsure 1188 unknown 98 tggatccctg cttttcagta ttacctaaac catacatgct ctagttacct taatgtgaaa 60 aagtgtaaag cttttggggt aacacacact tttgcttgta gactttgagc catctttata 120 tggtacttct aataccttta tgagtttaga gaataaacaa taatgtcgag aaaaaaagtc 180 tgtatatgtt caatacgcac actcaatgta tttttcttgt ttgcttccta gaacatgtct 240 gatccctttt actgttgagt aaaccgtaga tatggagccc acagatctga ggcctccctg 300 aaatcctctg tgttattttt catactaagg cagaaaatag tctagagaaa cgttaaactc 360 aaagcagttg tccttgggaa gaaattgcat gagtgagaac tgtagcttag aggcaaagac 420 atctagttta atgataaagt gaaagagtgt ggaataaggg gctggagaga tgtcttggcg 480 tttgttaaga gtgtttgcca ctcattcaat tcaaaggact cgagttcagt tcctagcacc 540 tataacatta gccttagagg gatgtgtggg ccactcgtgc acatgcgcat gtatatgtat 600 atgtgtctat gtatgtgtat gtctgtgtga atgtgtattt gaatgtgtgt cggggatgta 660 gaggtccaag gtggtaatag gatgagtttt ctgcagccat tcttcactgg attggactat 720 ctggctagct agcttgccct caggctctgt tgtttccacc tctcacatgc tgggagaaca 780 aattggctac cacacccaca cagcatctgg tgagtgctgg ggatcagggt tctagtgttc 840 aagctcttgg ggaaaacact ttacccattg aactctgtct atctatttat ttatttctct 900 ttaaaaggca tgtactgatc atacatttaa aagcctataa cacagctata cacaatgtac 960 agtaatctgt tccttagatg ccagtccagc aaaggctctt agataatgat ttagcaataa 1020 atactggcca aacagctgtt tttttttttt tttttttttt agagaccggg aaaagacaac 1080 tcagaataga aattttgaat gtctcctaac aaccttctcc caaatgactg ggtcctcttt 1140 gaggcccaga ggtgggaaga tttgtgcccc taagcagggg tgaccctntt cataaggaga 1200 tgaggatgga gacactgttt tggggggtca acactccacc tcccagcatg aatgctcagc 1260 gtacgccaag agatgcatgt tgtgtgtgtg tggtgtgcat gtgcacctca gaggcataca 1320 tgttcatcga gacaagaaga gatgcatgtt gtgtgtgtgt ggtgtgcatg tgcacctgag 1380 aagcacacac atgttcatca agaccagaag ttaatgtcaa gtgtcttcct caattctcaa 1440 tattttgagg tacagtctct cacttgagcc cagagctcac ccatttgact ggttctaact 1500 agcacacttc acagattctg tctctacctc ccgagtctaa gatacaggag gaccacaata 1560 tccactatgg ttttttagcc ctccccgatg ggtgctgggg atgcagacat gggtctcatg 1620 cttgcatact tgcatgcttg catggcaagt attcactgta ccatcttacc agaccctaga 1680 tgacctgaca tttttctttt tctctctctc ttttttttaa gatttactta tttatcttta 1740 tatatgagta cactgtagct gtcttcacac acaccagaag agggcatcag aacccatccc 1800 attagagatg gttgagagcc accatgtggt tttggggaat tgaactcagg acctctggaa 1860 gaacaatcag tgctcttaac cgctgagcca actctccagc ccctgactgt tattttgttt 1920 gtttgtttgt ttgtttgttt tgttttgttt tgtttttttt atgataccaa atacgcacgc 1980 acaccttgac tgaagaaacc atcagagggt tgaggtgcag cagagagtgg cttgtgtgct 2040 gggggaggca gcttctacta agagagagaa tctctgatgt tcttttctga atgaactcct 2100 aggaacagcc acaaagcaag ctctttccaa catggcctct agaggcccaa gatattataa 2160 ctgcttctag gcaatgagat tcccttgttc ctccttctcc agtggtccca gccagacctt 2220 tattgcctga ctctttgcct gtgcttatct ctacaccttg tgctgggcca gccccttggt 2280 gagtcacaga ctaaaatcac gagcaggaag ctgtgttagc cacagactca caactgtctc 2340 ccctggttgt cctcccacct gtatccccag acacatacac gcacatgcac acacaccaca 2400 ccacacggta gtagaaggct ttctttctag aaaacaacgc agctgagaca aaaagagtcc 2460 accacagcaa ggtcaatgga cagttacatt tccattaatg gaaatggggt ggggaaggct 2520 cacctgactc tccagccact cctccaagag ctctatccaa attgctcctg ggggactcgc 2580 tgtgtccctg gaagaactag tgtacttgac tgtggacgac tataagagga actctgtgca 2640 attgcatgga agccactaac cttgctgcgt gaagactttc cggtgcagct gccttggggt 2700 caccatgctc tctgtaggaa agtccaacaa acttgttggt tccccagggt aaacttggtt 2760 agaatcatgc tttggtttgt cattagaacc ttatgaggag tgggataggc caacacaggc 2820 acatcacatg aacaagccca ataaaataaa ataaaagtca taggatgctg gcactggggt 2880 tttgtaaata gagtttctgg cacattctct ctggttgtat acaggtggcc atgtcagaag 2940 tgaagcctgt gttggtgaag ctggagtctt cctccagagg cttgccaggc caactgcata 3000 cagtaagtgc tcagttagca gcctgagcac ccctgcttgc tttgcctttc aaaggtgagt 3060 cttctgtttg cagaagcaga gatgtcagct tgttggagtg tgagatgata gggttgggac 3120 tgtcctggga agtggggagg taccagatta agatgatccg tctgcgaccc ttatgaaatc 3180 caccatccta tctctgctga ctgatataag aacaatggga ccttttctgt actgtggggc 3240 ttatggacta aaaggtgaat ggtggggttg atagagccaa gcactgactg gattagactg 3300 gattccaacc ctgatatata cctagccatt cgttaatgtt ccccacttgg tgttttgaga 3360 caaggtttct ctatatagcc ctggctgtcc tgatattcgc tatatatatt cactatatac 3420 atcaagctgg ccttgaactc tctgagatct tcctccctag ggctggaatt aaaggcatgc 3480 accaccacgt ctggttaaca ttcatctctt tattgtctct tgtgtatact tagcagaagt 3540 cctcacttac ttccatttta ttgagctgtc cttattgtgt gttccctagg caccaaggga 3600 atagccagaa caggatggtg tacctgtgtc ctagaggagt atataatgtg acagagtgaa 3660 cagtgactgg gctgtgtagg agctccttga ctttgaaggt aactaaactg ggtttgaatc 3720 aacatactcc ctcctagctg tgaaaggtcc tataaaactt catctccccc cacccctgct 3780 cctgcaagac cacatgtacc agcagacata tgttgctgac cctaagtagt agtcttccag 3840 taactgctgg tttcacaact actaagtgtt atcagtgtga atagcaagtg atccctggtg 3900 aagatttgca aggcacctgc acaggtgcta agttagctgt cagggattgg gggggggggg 3960 gggttggggg gtggcaggtg ggctgtcagg ctgtactatt ttcacagaat aacagctgat 4020 gctatatcgt aagtagcatt ttaaaaaaat catcagggta gagctgctgt gattgctatt 4080 ggcggcttta taagaagaaa aagagaaaca caaacacaca cacacacaca cacacacacc 4140 atccttatct cttactatgt agtttttgcc accaaggcta tcgtcagcta tgttccatta 4200 acgttggatg agaaccatga aactaagtca cccccttctc ttcaaagtgt atgaggtgtg 4260 tgatgctgaa ccacagaata cagactaagt taggaaccag cctttcgtct cccacatgtg 4320 attcttggtg gcattgttac ctggctggag agaatgagta aattggaccc actgggcaga 4380 atccagggca gaccactgaa tcttgcttct gacagatgac actggctttc agctttctca 4440 gatagaccag ggcacctcct ccacatgtct tccacaggag gcttaaagac cagaaccctc 4500 cctgcctgct cagtggctac ccctcttcag ggttgaggtt ttggagctga gacctatgtc 4560 tcagaaacat gtgagaacca ggtgatttcc caggctcctg accagcaggt gggattttcc 4620 atcctcccaa gggtcacttc ccagtttctc ccccagcacc taaaagtacc gtccttgagg 4680 tggccctaca aggaccagtg gctgcagggt ctctcagtat ctctatcagc ctacagtgag 4740 agtgtgtgtg tccatgaagg agggacagaa tagttgcctg cctccctacc caccctacct 4800 ctccaaatac tccatattca gggttgtagg gatatcgact gtccttggga gcttgctgat 4860 gaggtcagtt ctaggaagca agactttctg gatggttttc ttcctcgatt tctgtttacc 4920 tatcagccaa gagaactcag aaaaagagga ctgctcttac ttagggttcc aagtatgttt 4980 aaggaaaaaa tgaaacatat taaaaagagc tatacctatg caatgctgct tatactatgc 5040 ctgtacactc cgagtaagca gacatatgct gcacgttata ggagccatat tatatagctc 5100 atattaataa gctccatgag aagatgcaaa cattcatgtg gagttagcct ggctctagaa 5160 ggctttggtt ctcctgagcg gtgccaactt tgggactgct ttccccccat gtcctaccac 5220 gaagagtagg aagtccatga gatctgggac gggctgatgg tgacaaatta gctgggggag 5280 gcccaatcag ggcacccagt cacgaatata gtggacacct gcgccagtct cttccccacc 5340 actggaaggg tgtctcgaag aggaaggagt gatagaagct cattcattcc cggaagagat 5400 gctggagaaa gaggcccaga gatgccaggg aatcacaggt ggaggtgtcc tgcgaggggc 5460 gaggactctg tgtaaagagg cgtgtcctca gggcgcggcc ccgcccattc ccgccctccc 5520 cccaccccct ggtctgccct agctcctggc ctgagggttt cgctttcagt caccagctag 5580 agcgcagctg aggcactaga gctccaggca caagggcggg agccaccgct gcccctatgg 5640 cgcccgccgc cctctgggtc gcgctggtct tcgaactgca gctgtgggcc accgggcaca 5700 cagtgcccgc ccaggtgggt gactcttggg gtcacggggg acagctgcgc atcacaaagt 5760 gcccattcca gctactgcta ctgcacaatt ccgggacagc atgagaggcc atcacgtccc 5820 cagcaaacac gccacgngcc tttggggacc actggggacc cgaggtctgg ccgt 5874 99 1003 DNA Mus musculus 99 tttgtgtgga ggtctttgtc acagtgagtc aagccactgt cttaaaaaaa ctacatcttc 60 tcagagcctt gctgggtctc acccagcagg caggagggaa gccctaaagt aacccacttc 120 ctggcccagc aaactgcaga cacagcgtgc acctgaagag gagcagagga aagctgctct 180 actctctcag ctctcgtctc cacgaaacca tcatgagctg gtgacagtat gctggagccc 240 aagagtcata ctgatggctg cctcccttgt tccttccagg ttgtcttgac accctacaaa 300 ccggaacctg ggtacgagtg ccagatctca caggaatact atgacaggaa ggctcagatg 360 tgctgtgcta agtgtcctcc tggtgagagg cagctgctgg ggctttggaa gctggtgcat 420 ggagggcatg cttgtctggg aatgagggcc ttcagctctc acttggctgc tttatacatg 480 ctagggttca tgattcatct tgccctgggc cctgggtctc gcaagtgctt gtccctccac 540 tgagcacact tctcagtgtc ttctcctggt tactgcctac ctacattttg accctatcct 600 ctgccaggaa gcctcctcaa tactgcaatg atgtccctag cactttcata gcccactatg 660 tgcctatgtc ctcaccctat tgagtagtga gtgtagcttg gtccctgtga gttccaagca 720 cacggctggc ctatattgat gtcctgtact taattgtcaa gtaaaatgaa tggatagcca 780 tatcatagat ggcggatctg agccctggcc tcattggcga ggactgagta gctgccccag 840 tgccgagtag cacaatcaag tgtagcattc aattaagtcg tatttataat gcatctactc 900 tgtgctcaac ctgctgagag gaaagccaca caaacacggg atacagcagg ctgggaagta 960 agtgcaaagc cctaaagcag gggcctgttt taatgggtcc aaa 1003 100 20 DNA Artificial Sequence Antisense Oligonucleotide 100 cttgtgcctg gagctctagt 20 101 20 DNA Artificial Sequence Antisense Oligonucleotide 101 agctgcagtt cgaagaccag 20 102 20 DNA Artificial Sequence Antisense Oligonucleotide 102 cagctgcagt tcgaagacca 20 103 20 DNA Artificial Sequence Antisense Oligonucleotide 103 tagggtgtca agacaacctg 20 104 20 DNA Artificial Sequence Antisense Oligonucleotide 104 gtttgtaggg tgtcaagaca 20 105 20 DNA Artificial Sequence Antisense Oligonucleotide 105 tacccaggtt ccggtttgta 20 106 20 DNA Artificial Sequence Antisense Oligonucleotide 106 gaggacactt agcacagcac 20 107 20 DNA Artificial Sequence Antisense Oligonucleotide 107 acatattggc caggaggaca 20 108 20 DNA Artificial Sequence Antisense Oligonucleotide 108 ctgcagctca aacatgtacg 20 109 20 DNA Artificial Sequence Antisense Oligonucleotide 109 tccacctggt cagtggtaca 20 110 20 DNA Artificial Sequence Antisense Oligonucleotide 110 ccagaatggg ttttcaaggc 20 111 20 DNA Artificial Sequence Antisense Oligonucleotide 111 gccagggccg cacttgctca 20 112 20 DNA Artificial Sequence Antisense Oligonucleotide 112 cttgaactgg ccactccgaa 20 113 20 DNA Artificial Sequence Antisense Oligonucleotide 113 gatgtggtgt cagagaacgt 20 114 20 DNA Artificial Sequence Antisense Oligonucleotide 114 gtggatgatg tggtgtcaga 20 115 20 DNA Artificial Sequence Antisense Oligonucleotide 115 gatgctacag atgcggtggg 20 116 20 DNA Artificial Sequence Antisense Oligonucleotide 116 ggaatagcca ggatgctaca 20 117 20 DNA Artificial Sequence Antisense Oligonucleotide 117 gcatctgtgc ttgcatttcc 20 118 20 DNA Artificial Sequence Antisense Oligonucleotide 118 ctctggctga gatacgtaga 20 119 20 DNA Artificial Sequence Antisense Oligonucleotide 119 tgtgggctct ggctgagata 20 120 20 DNA Artificial Sequence Antisense Oligonucleotide 120 ggatcttgtg ggctctggct 20 121 20 DNA Artificial Sequence Antisense Oligonucleotide 121 ttggaagaga gatgccaccc 20 122 20 DNA Artificial Sequence Antisense Oligonucleotide 122 gaccaattgg aagagagatg 20 123 20 DNA Artificial Sequence Antisense Oligonucleotide 123 caatcagacc aattggaaga 20 124 20 DNA Artificial Sequence Antisense Oligonucleotide 124 acaatcagac caattggaag 20 125 20 DNA Artificial Sequence Antisense Oligonucleotide 125 cctaacatca gcagacccag 20 126 20 DNA Artificial Sequence Antisense Oligonucleotide 126 tttcctctgc accaggatga 20 127 20 DNA Artificial Sequence Antisense Oligonucleotide 127 cttctttttc ctctgcacca 20 128 20 DNA Artificial Sequence Antisense Oligonucleotide 128 gagggcttct ttttcctctg 20 129 20 DNA Artificial Sequence Antisense Oligonucleotide 129 ggagggcttc tttttcctct 20 130 20 DNA Artificial Sequence Antisense Oligonucleotide 130 gtaggcagga gggcttcttt 20 131 20 DNA Artificial Sequence Antisense Oligonucleotide 131 cacatgaggc accttggcat 20 132 20 DNA Artificial Sequence Antisense Oligonucleotide 132 ggcacatgag gcaccttggc 20 133 20 DNA Artificial Sequence Antisense Oligonucleotide 133 ggagctgctg ctggaactgg 20 134 20 DNA Artificial Sequence Antisense Oligonucleotide 134 tgggaagaat ctgaaatcct 20 135 20 DNA Artificial Sequence Antisense Oligonucleotide 135 gggtcaggcc actttgactg 20 136 20 DNA Artificial Sequence Antisense Oligonucleotide 136 tagctcctta gaaggaaaaa 20 137 20 DNA Artificial Sequence Antisense Oligonucleotide 137 tgcagtgtca gcattcaggc 20 138 20 DNA Artificial Sequence Antisense Oligonucleotide 138 ccacttgctc ctacttgctg 20 139 20 DNA Artificial Sequence Antisense Oligonucleotide 139 agagggtact tcctaagagt 20 140 20 DNA Artificial Sequence Antisense Oligonucleotide 140 gattcttgca tcaaaagaat 20 141 20 DNA Artificial Sequence Antisense Oligonucleotide 141 cctataacag agcaactctg 20 142 20 DNA Artificial Sequence Antisense Oligonucleotide 142 gtgttgctga ggatcaaacc 20 143 20 DNA Artificial Sequence Antisense Oligonucleotide 143 tcattagaag gtcaggagga 20 144 20 DNA Artificial Sequence Antisense Oligonucleotide 144 aaggaaggcg tggccttgga 20 145 20 DNA Artificial Sequence Antisense Oligonucleotide 145 actcacagtg cctaacccgg 20 146 20 DNA Artificial Sequence Antisense Oligonucleotide 146 ctgttccaac tcacagtgcc 20 147 20 DNA Artificial Sequence Antisense Oligonucleotide 147 agagctggct tcagctgttt 20 148 20 DNA Artificial Sequence Antisense Oligonucleotide 148 catgaatcct ttggcaaaag 20 149 20 DNA Artificial Sequence Antisense Oligonucleotide 149 ctgccaagtt catatccagt 20 150 20 DNA Artificial Sequence Antisense Oligonucleotide 150 tatcttgatt ccagagtgct 20 151 20 DNA Artificial Sequence Antisense Oligonucleotide 151 gagccttaac aagtcggccc 20 152 20 DNA Artificial Sequence Antisense Oligonucleotide 152 ctgatgctgc agagccttaa 20 153 20 DNA Artificial Sequence Antisense Oligonucleotide 153 tccttagcac accctttagg 20 154 20 DNA Artificial Sequence Antisense Oligonucleotide 154 atttataagc aggaattctg 20 155 20 DNA Artificial Sequence Antisense Oligonucleotide 155 cacatacatg caaacatgga 20 156 20 DNA Artificial Sequence Antisense Oligonucleotide 156 agtaactgga gagtgatcaa 20 157 20 DNA Artificial Sequence Antisense Oligonucleotide 157 gcccgcctca gtaactggag 20 158 20 DNA Artificial Sequence Antisense Oligonucleotide 158 gcaagctctg ggtacagatg 20 159 20 DNA Artificial Sequence Antisense Oligonucleotide 159 agcactccat aggcagacag 20 160 20 DNA Artificial Sequence Antisense Oligonucleotide 160 gcagcctgcc tgtaacctga 20 161 20 DNA Artificial Sequence Antisense Oligonucleotide 161 taaatgtcgg gcaggtatgg 20 162 20 DNA Artificial Sequence Antisense Oligonucleotide 162 aaaatgcagg tatacaagtg 20 163 20 DNA Artificial Sequence Antisense Oligonucleotide 163 gccatcttgc cagttcaaaa 20 164 20 DNA Artificial Sequence Antisense Oligonucleotide 164 tgatgtgtgc acatatgcag 20 165 20 DNA Artificial Sequence Antisense Oligonucleotide 165 acatttatgg tatgtgagtg 20 166 20 DNA Artificial Sequence Antisense Oligonucleotide 166 gtccaatcca gtgaagaatg 20 167 20 DNA Artificial Sequence Antisense Oligonucleotide 167 tgatcagtac atgcctttta 20 168 20 DNA Artificial Sequence Antisense Oligonucleotide 168 agtcaagtac actagttctt 20 169 20 DNA Artificial Sequence Antisense Oligonucleotide 169 agtccataag ccccacagta 20 170 20 DNA Artificial Sequence Antisense Oligonucleotide 170 agtttagtta ccttcaaagt 20 171 20 DNA Artificial Sequence Antisense Oligonucleotide 171 tggattctgc ccagtgggtc 20 172 20 DNA Artificial Sequence Antisense Oligonucleotide 172 caaaacctca accctgaaga 20 173 20 DNA Artificial Sequence Antisense Oligonucleotide 173 gaggaggctt cctggcagag 20 174 20 DNA Artificial Sequence Antisense Oligonucleotide 174 acatcaatat aggccagccg 20

Claims

What is claimed is:

1. A compound 8 to 50 nucleobases in length targeted to a nucleic acid molecule encoding Tumor Necrosis Factor Receptor 2, wherein said compound specifically hybridizes with and inhibits the expression of Tumor Necrosis Factor Receptor 2.

2. The compound of claim 1 which is an antisense oligonucleotide.

3. The compound of claim 2 wherein the antisense oligonucleotide has a sequence comprising SEQ ID NO: 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 36, 39, 40, 41, 43, 45, 46, 48, 49, 50, 51, 52, 53, 54, 55, 57, 58, 59, 60, 62, 63, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 117, 118, 119, 120, 121, 122, 123, 126, 127, 128, 129, 132, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 167, 171, 173 or 174.

4. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified internucleoside linkage.

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

6. The compound of claim 2 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 claim 2 wherein the antisense oligonucleotide comprises at least one modified nucleobase.

9. The compound of claim 8 wherein the modified nucleobase is a 5-methylcytosine.

10. The compound of claim 2 wherein the antisense oligonucleotide is a chimeric oligonucleotide.

11. A compound 8 to 50 nucleobases in length which specifically hybridizes with at least an 8-nucleobase portion of an active site on a nucleic acid molecule encoding Tumor Necrosis Factor Receptor 2.

12. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier or diluent.

13. The composition of claim 12 further comprising a colloidal dispersion system.

14. The composition of claim 12 wherein the compound is an antisense oligonucleotide.

15. A method of inhibiting the expression of Tumor Necrosis Factor Receptor 2 in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of Tumor Necrosis Factor Receptor 2 is inhibited.

16. A method of treating an animal having a disease or condition associated with Tumor Necrosis Factor Receptor 2 comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of Tumor Necrosis Factor Receptor 2 is inhibited.

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

18. The method of claim 16 wherein the disease or condition is a neurodegenerative disorder.

19. The method of claim 16 wherein the disease or condition is diabetes.

20. The method of claim 16 wherein the disease or condition is a pulmonary disorder.