Antisense oligonucleotide modulation of tumor necrosis factor-(α) (TNF-α) expression

Abstract

Compositions and methods are provided for inhibiting the expression of human tumor necrosis factor-α (TNF-α). Antisense oligonucleotides targeted to nucleic acids encoding TNF-α are preferred. Methods of using these oligonucleotides for inhibition of TNF-α expression and for treatment of diseases, particularly inflammatory and autoimmune diseases, associated with overexpression of TNF-α are provided.

Description

FIELD OF THE INVENTION

This invention relates to compositions and methods for modulating expression of the human tumor necrosis factor-α (TNF-α) gene, which encodes a naturally present cytokine involved in regulation of immune function and implicated in infectious and inflammatory disease. This invention is also directed to methods for inhibiting TNF-α mediated immune responses; these methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of conditions associated with expression of the human TNF-α gene.

BACKGROUND OF THE INVENTION

Tumor necrosis factor α (TNF-α also cachectin) is an important cytokine that plays a role in host defense. The cytokine is produced primarily in macrophages and monocytes in response to infection, invasion, injury, or inflammation. Some examples of inducers of TNF-α include bacterial endotoxins, bacteria, viruses, lipopolysaccharide (LPS) and cytokines including GM-CSF, IL-1, IL-2 and IFN-γ.

TNF-α interacts with two different receptors, TNF receptor I (TNFRI, p55) and TNFRII (p75), in order to transduce its effects, the net result of which is altered gene expression. Cellular factors induced by TNF-α include interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-γ (IFN-γ), platelet derived growth factor (PDGF) and epidermal growth factor (EGF), and endothelial cell adhesion molecules including endothelial leukocyte adhesion molecule 1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (Tracey, K. J., et al., Annu. Rev. Cell Biol., 1993, 9, 317-343; Arvin, B., et al., Ann. NY Acad. Sci., 1995, 765, 62-71).

Despite the protective effects of the cytokine, overexpression of TNF-α often results in disease states, particularly in infectious, inflammatory and autoimmune diseases. This process may involve the apoptotic pathways (Ksontini, R., et al., J. Immunol., 1998, 160, 4082-4089). High levels of plasma TNF-α have been found in infectious diseases such as sepsis syndrome, bacterial meningitis, cerebral malaria, and AIDS; autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease (including Crohn's disease), sarcoidosis, multiple sclerosis, Kawasaki syndrome, graft-versus-host disease and transplant (allograft) rejection; and organ failure conditions such as adult respiratory distress syndrome, congestive heart failure, acute liver failure and myocardial infarction (Eigler, A., et al., Immunol. Today, 1997, 18, 487-492). Other diseases in which TNF-α is involved include asthma (Shah, A., et al., Clinical and Experimental Allergy, 1995, 25, 1038-1044), brain injury following ischemia (Arvin, B., et al., Ann. NY Acad. Sci., 1995, 765, 62-71), non-insulin-dependent diabetes mellitus (Hotamisligil, G. S., et al., Science, 1993, 259, 87-90), insulin-dependent diabetes mellitus (Yang, X.-D., et al., J. Exp. Med., 1994, 180, 995-1004), hepatitis (Ksontini, R., et al., J. Immunol., 1998, 160, 4082-4089), atopic dermatitis (Sumimoto, S., et al., Arch. Dis. Child., 1992, 67, 277-279), and pancreatitis (Norman, J. G., et al., Surgery, 1996, 120, 515-521). Further, inhibitors of TNF-α have been suggested to be useful for cancer prevention (Suganuma, M., et al. ( Cancer Res., 1996, 56, 3711-3715). Elevated TNF-α expression may also play a role in obesity (Kern, P. A., J. Nutr., 1997, 127, 1917S-1922S). TNF-α was found to be expressed in human adipocytes and increased expression, in general, correlated with obesity.

There are currently several approaches to inhibiting TNF-α expression. Approaches used to treat rheumatoid arthritis include a chimeric anti-TNF-α antibody, a humanized monoclonal anti-TNF-α antibody, and recombinant human soluble TNF-α receptor (Camussi, G., Drugs, 1998, 55, 613-620). Other examples are indirect TNF-α inhibitors including phosphodiesterase inhibitors (e.g. pentoxifylline) and metalloprotease inhibitors (Eigler, A., et al., Immunol. Today, 1997, 18, 487-492). An additional class of direct TNF-α inhibitors is oligonucleotides, including triplex-forming oligonucleotides, ribozymes, and antisense oligonucleotides.

Several publications describe the use of oligonucleotides targeting TNF-α by non-antisense mechanisms. U.S. Pat. No. 5,650,316, WO 95/33493 and Aggarwal, B. B. et al. ( Cancer Research, 1996, 56, 5156-5164) disclose triplex-forming oligonucleotides targeting TNF-α. WO 95/32628 discloses triplex-forming oligonucleotides especially those possessing one or more stretches of guanosine residues capable of forming secondary structure. WO 94/10301 discloses ribozyme compounds active against TNF-α mRNA. WO 95/23225 discloses enzymatic nucleic acid molecules active against TNF-α mRNA.

A number of publications have described the use of antisense oligonucleotides targeting nucleic acids encoding TNF-α. The TNF-α gene has four exons and three introns. WO 93/09813 discloses TNF-α antisense oligonucleotides conjugated to a radioactive moiety, including sequences targeted to the 5′-UTR, AUG start site, exon 1, and exon 4 including the stop codon of human TNF-α. EP 0 414 607 B1 discloses antisense oligonucleotides targeting the AUG start codon of human TNF-α. WO 95/00103 claims antisense oligonucleotides to human TNF-α including sequences targeted to exon 1 including the AUG start site. Hartmann, G. et al. ( Mol. Med., 1996, 2, 429-438) disclose uniform phosphorothioates and mixed backbone phosphorothioate/phosphodiester oligonucleotides targeted to the AUG start site of human TNF-α. Hartmann, G. et al. ( Antisense Nucleic Acid Drug Devel., 1996, 6, 291-299) disclose antisense phosphorothioate oligonucleotides targeted to the AUG start site, the exon 1/intron 1 junction, and exon 4 of human TNF-α. d'Hellencourt, C. F. et al. ( Biochim. Biophys. Acta, 1996, 1317, 168-174) designed and tested a series of unmodified oligonucleotides targeted to the 5′-UTR, and exon 1, including the AUG start site, of human TNF-α. Additionally, one oligonucleotide each was targeted to exon 4 and the 3′-UTR of human TNF-α and one oligonucleotide was targeted to the AUG start site of mouse TNF-α. Rojanasakul, Y. et al. ( J. Biol. Chem., 1997, 272, 3910-3914) disclose an antisense phosphorothioate oligonucleotide targeted to the AUG start site of mouse TNF-α. Taylor, M. F. et al. ( J. Biol. Chem., 1996, 271, 17445-17452 and Antisense Nucleic Acid Drug Devel., 1998, 8, 199-205) disclose morpholino, methylmorpholino, phosphodiester and phosphorothioate oligonucleotides targeted to the 5′-UTR and AUG start codon of mouse TNF-α. Tu, G.-C. et al. ( J. Biol. Chem., 1998, 273, 25125-25131) designed and tested 42 phosphorothioate oligonucleotides targeting sequences throughout the rat TNF-α gene.

Interestingly, some phosphorothioate oligodeoxynucleotides have been found to enhance lipopolysaccharide-stimulated TNF-α synthesis up to four fold due to nonspecific immunostimulatory effects (Hartmann et al. Mol. Med., 1996, 2, 429-438).

Accordingly, there remains an unmet need for therapeutic compositions and methods for inhibiting expression of TNF-α, and disease processes associated therewith.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides oligonucleotides which are targeted to nucleic acids encoding TNF-α and are capable of modulating TNF-α expression. The present invention also provides chimeric oligonucleotides targeted to nucleic acids encoding human TNF-α. The oligonucleotides of the invention are believed to be useful both diagnostically and therapeutically, and are believed to be particularly useful in the methods of the present invention.

The present invention also comprises methods of modulating the expression of human TNF-α, in cells and tissues, using the oligonucleotides of the invention. Methods of inhibiting TNF-α expression are provided; these methods are believed to be useful both therapeutically and diagnostically. These methods are also useful as tools, for example, for detecting and determining the role of TNF-α in various cell functions and physiological processes and conditions and for diagnosing conditions associated with expression of TNF-α.

The present invention also comprises methods for diagnosing and treating infectious and inflammatory diseases, particularly diabetes, rheumatoid arthritis, Crohn's disease, pancreatitis, multiple sclerosis, atopic dermatitis and hepatitis. These methods are believed to be useful, for example, in diagnosing TNF-α-associated disease progression. These methods employ the oligonucleotides of the invention. These methods are believed to be useful both therapeutically, including prophylactically, and as clinical research and diagnostic tools.

DETAILED DESCRIPTION OF THE INVENTION

TNF-α plays an important regulatory role in the immune response to various foreign agents. Overexpression of TNF-α results in a number of infectious and inflammatory diseases. As such, this cytokine represents an attractive target for treatment of such diseases. In particular, modulation of the expression of TNF-α may be useful for the treatment of diseases such as Crohn's disease, diabetes mellitus, multiple sclerosis, rheumatoid arthritis, hepatitis, pancreatitis and asthma.

The present invention employs antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding TNF-α, ultimately modulating the amount of TNF-α produced. This is accomplished by providing oligonucleotides which specifically hybridize with nucleic acids, preferably mRNA, encoding TNF-α.

This relationship between an antisense compound such as an oligonucleotide and its complementary nucleic acid target, to which it hybridizes, is commonly referred to as “antisense”. “Targeting” an oligonucleotide to a chosen nucleic acid target, in the context of this invention, is a multistep process. The process usually begins with identifying a nucleic acid sequence whose function is to be modulated. This may be, as examples, a cellular gene (or mRNA made from the gene) whose expression is associated with a particular disease state, or a foreign nucleic acid from an infectious agent. In the present invention, the targets are nucleic acids encoding TNF-α; in other words, a gene encoding TNF-α, or mRNA expressed from the TNF-α gene. mRNA which encodes TNF-α is presently the preferred target. The targeting process also includes determination of a site or sites within the nucleic acid sequence for the antisense interaction to occur such that modulation of gene expression will result.

In accordance with this invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5′-untranslated region, the 3′-untranslated region, the 5′ cap region and intron/exon junction ribonucleotides. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides. The oligonucleotide may therefore be specifically hybridizable with a transcription initiation site region, a translation initiation codon region, a 5′ cap region, an intron/exon junction, coding sequences, a translation termination codon region or sequences in the 5′- or 3′-untranslated region. 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 (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 TNF-α, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region,” “AUG 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. This region is a preferred target region. 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. This region is a preferred target region. 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 preferred 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.

Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a pre-mRNA transcript to yield one or more mature mRNAs. 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., exon—exon or 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. Targeting particular exons in alternatively spliced mRNAs may also be preferred. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

Once the target site or 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 modulation.

“Hybridization”, in the context of this invention, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them.

“Specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide.

It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide 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 or, in the case of in vitro assays, under conditions in which the assays are conducted.

Hybridization of antisense oligonucleotides with mRNA interferes with one or more of the normal functions of mRNA. The functions of mRNA 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 by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.

The overall effect of interference with mRNA function is modulation of expression of TNF-α. In the context of this invention “modulation” means either inhibition or stimulation; i.e., either a decrease or increase in expression. This modulation can be measured in ways which are routine in the art, for example by Northern blot assay of mRNA expression, or reverse transcriptase PCR, as taught in the examples of the instant application or by Western blot or ELISA assay of protein expression, or by an immunoprecipitation assay of protein expression. Effects of antisense oligonucleotides of the present invention on TNF-α expression can also be determined as taught in the examples of the instant application. Inhibition is presently a preferred form of modulation.

The oligonucleotides of this invention can be used in diagnostics, therapeutics, prophylaxis, and as research reagents and in kits. Since the oligonucleotides of this invention hybridize to nucleic acids encoding TNF-α, sandwich, colorimetric and other assays can easily be constructed to exploit this fact. Provision of means for detecting hybridization of oligonucleotides with the TNF-α gene or mRNA can routinely be accomplished. Such provision may include enzyme conjugation, radiolabelling or any other suitable detection systems. Kits for detecting the presence or absence of TNF-α may also be prepared.

The present invention is also suitable for diagnosing abnormal inflammatory states in tissue or other samples from patients suspected of having an inflammatory disease such as rheumatoid arthritis. The ability of the oligonucleotides of the present invention to inhibit inflammatory processes may be employed to diagnose such states. A number of assays may be formulated employing the present invention, which assays will commonly comprise contacting a tissue sample with an oligonucleotide of the invention under conditions selected to permit detection and, usually, quantitation of such inhibition. In the context of this invention, to “contact” tissues or cells with an oligonucleotide or oligonucleotides means to add the oligonucleotide(s), usually in a liquid carrier, to a cell suspension or tissue sample, either in vitro or ex vivo, or to administer the oligonucleotide(s) to cells or tissues within an animal.

The oligonucleotides of this invention may also be used for research purposes. Thus, the specific hybridization exhibited by the oligonucleotides may be used for assays, purifications, cellular product preparations and in other methodologies which may be appreciated by persons of ordinary skill in the art.

In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intersugar (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 binding to target and increased stability in the presence of nucleases.

The antisense compounds in accordance with this invention preferably comprise from about 5 to about 50 nucleobases. Particularly preferred are antisense oligonucleotides comprising from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides). 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.

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.

Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

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; and 5,625,050.

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; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.

Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

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. Further teaching of PNA compounds can be found in Nielsen et al. ( Science, 1991, 254, 1497-1500).

Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH 2 —NH—O—CH 2 —, —CH 2 —N(CH 3 )—O—CH 2 — [known as a methylene (methylimino) or MMI backbone], —CH 2 O——N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 — and —O—N(CH 3 )—CH 2 —CH 2 — [wherein the native phosphodiester backbone is represented as —O—P—O—CH 2 —] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

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-alkyl-O-alkyl, O—, S—, or N-alkenyl, or O—, S— or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl. Particularly preferred are O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) 2 ON(CH 3 ) 2 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta 1995, 78, 486-504) i.e., an alkoxyalkoxy group.

Other preferred modifications include 2′-methoxy (2′-O—CH 3 ), 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 ) and 2′-fluoro (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 sugars 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; and 5,700,920.

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 or m5c), 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 uracil and cytosine, 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, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering 1990, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, those disclosed by Englisch et al. ( Angewandte Chemie, International Edition 1991, 30, 613-722), and those disclosed by Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications 1993, CRC Press, Boca Raton, pages 289-302. 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 1993, CRC Press, Boca Raton, pages 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. Nos. 3,687,808, as well as U.S. Pat. No. 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; and 5,681,941.

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. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett. 1994, 4, 1053-1059), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci. 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let. 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J. 1991, 10, 1111-1118; Kabanov et al., FEBS Lett. 1990, 259, 327-330; Svinarchuk et al., Biochimie 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res. 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & 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).

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.

The present invention also includes oligonucleotides which are chimeric oligonucleotides. “Chimeric” oligonucleotides or “chimeras,” in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. 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 antisense inhibition of gene expression. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. This RNAse H-mediated cleavage of the RNA target is distinct from the use of ribozymes to cleave nucleic acids. Ribozymes are not comprehended by the present invention.

Examples of chimeric oligonucleotides include but are not limited to “gapmers,” in which three distinct regions are present, normally with a central region flanked by two regions which are chemically equivalent to each other but distinct from the gap. A preferred example of a gapmer is an oligonucleotide in which a central portion (the “gap”) of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2′-deoxynucleotides, while the flanking portions (the 5′ and 3′ “wings”) are modified to have greater affinity for the target RNA molecule but are unable to support nuclease activity (e.g., fluoro- or 2′-O-methoxyethyl-substituted). Chimeric oligonucleotides are not limited to those with modifications on the sugar, but may also include oligonucleosides or oligonucleotides with modified backbones, e.g., with regions of phosphorothioate (P═S) and phosphodiester (P═O) backbone linkages or with regions of MMI and P═S backbone linkages. Other chimeras include “wingmers,” also known in the art as “hemimers,” that is, oligonucleotides with two distinct regions. In a preferred example of a wingmer, the 5′ portion of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2′-deoxynucleotides, whereas the 3′ portion is modified in such a fashion so as to have greater affinity for the target RNA molecule but is unable to support nuclease activity (e.g., 2′-fluoro- or 2′-O-methoxyethyl-substituted), or vice-versa. In one embodiment, the oligonucleotides of the present invention contain a 2′-O-methoxyethyl (2′-O—CH 2 CH 2 OCH) 3 modification on the sugar moiety of at least one nucleotide. This modification has been shown to increase both affinity of the oligonucleotide for its target and nuclease resistance of the oligonucleotide. According to the invention, one, a plurality, or all of the nucleotide subunits of the oligonucleotides of the invention may bear a 2′-O-methoxyethyl (—O—CH 2 CH 2 OCH 3 ) modification. Oligonucleotides comprising a plurality of nucleotide subunits having a 2′-O-methoxyethyl modification can have such a modification on any of the nucleotide subunits within the oligonucleotide, and may be chimeric oligonucleotides. Aside from or in addition to 2′-O-methoxyethyl modifications, oligonucleotides containing other modifications which enhance antisense efficacy, potency or target affinity are also preferred. Chimeric oligonucleotides comprising one or more such modifications are presently preferred.

The oligonucleotides 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 Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of the routineer. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and 2′-alkoxy or 2′-alkoxyalkoxy derivatives, including 2′-O-methoxyethyl oligonucleotides (Martin, P., Helv. Chim. Acta 1995, 78, 486-504). It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling, Va.) to synthesize fluorescently labeled, biotinylated or other conjugated oligonucleotides.

The antisense compounds of the present invention include bioequivalent compounds, including pharmaceutically acceptable salts and prodrugs. This is intended to 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 pharmaceutically acceptable salts of the nucleic acids of the invention and prodrugs of such nucleic acids. “Pharmaceutically acceptable salts” are physiologically and pharmaceutically acceptable salts of the nucleic acids of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci. 1977, 66, 1-19).

For oligonucleotides, 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; © 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.

The oligonucleotides of the invention may additionally or alternatively be prepared to be delivered in a “prodrug” form. 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.

For therapeutic or prophylactic treatment, oligonucleotides are administered in accordance with this invention. Oligonucleotide compounds of the invention may be formulated in a pharmaceutical composition, which may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients and the like in addition to the oligonucleotide. Such compositions and formulations are comprehended by the present invention.

Pharmaceutical compositions comprising the oligonucleotides of the present invention may include penetration enhancers in order to enhance the alimentary delivery of the oligonucleotides. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., fatty acids, bile salts, chelating agents, surfactants and non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, 8, 91-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1-33). One or more penetration enhancers from one or more of these broad categories may be included. Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a. 1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono- and di-glycerides and physiologically acceptable salts thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1; El-Hariri et al., J. Pharm. Pharmacol. 1992 44, 651-654).

The physiological roles of bile include the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term “bile salt” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.

Complex formulations comprising one or more penetration enhancers may be used. For example, bile salts may be used in combination with fatty acids to make complex formulations.

Chelating agents 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). Chelating agents have the added advantage of also serving as DNase inhibitors.

Surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Phamacol. 1988, 40, 252-257).

Non-surfactants 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).

As used herein, “carrier compound” refers 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. I n contrast to a carrier compound, a “pharmaceutically acceptable carrier” (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 pharmaceutically acceptable carrier 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 pharmaceutically acceptable 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.); disintegrates (e.g., starch, sodium starch glycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate, etc.). Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are described in U.S. Pat. Nos. 4,704,295; 4,556,552; 4,309,406; and 4,309,404.

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, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of 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 invention.

Regardless of the method by which the oligonucleotides of the invention are introduced into a patient, colloidal dispersion systems may be used as delivery vehicles to enhance the in vivo stability of the oligonucleotides and/or to target the oligonucleotides to a particular organ, tissue or cell type. Colloidal dispersion systems include, but are not limited to, macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid:oligonucleotide complexes of uncharacterized structure. A preferred colloidal dispersion system is a plurality of liposomes. Liposomes are microscopic spheres having an aqueous core surrounded by one or more outer layers made up of lipids arranged in a bilayer configuration (see, generally, Chonn et al., Current Op. Biotech. 1995, 6, 698-708).

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, vaginal, rectal, intranasal, epidermal, and transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g., by inhalation or insufflation, 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.

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.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. In some cases it may be more effective to treat a patient with an oligonucleotide of the invention in conjunction with other traditional therapeutic modalities in order to increase the efficacy of a treatment regimen. In the context of the invention, the term “treatment regimen” is meant to encompass therapeutic, palliative and prophylactic modalities. For example, a patient may be treated with conventional chemotherapeutic agents such as those used for tumor and cancer treatment. When used with the compounds of the invention, such chemotherapeutic agents may be used individually, sequentially, or in combination with one or more other such chemotherapeutic agents.

The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC 50 s found to be effective in vitro and in in vivo animal models. In general, dosage is from 0.01 μg 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 μg to 100 g per kg of body weight, once or more daily, to once every 20 years.

Thus, in the context of this invention, by “therapeutically effective amount” is meant the amount of the compound which is required to have a therapeutic effect on the treated individual. This amount, which will be apparent to the skilled artisan, will depend upon the age and weight of the individual, the type of disease to be treated, perhaps even the gender of the individual, and other factors which are routinely taken into consideration when designing a drug treatment. A therapeutic effect is assessed in the individual by measuring the effect of the compound on the disease state in the animal.

The following examples illustrate the present invention and are not intended to limit the same.

EXAMPLES

Example 1

Synthesis of Oligonucleotides

Unmodified oligodeoxynucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine. β-cyanoethyldiisopropyl-phosphoramidites are purchased from Applied Biosystems (Foster City, Calif.). For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a 0.2 M solution of 3 H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation cycle wait step was increased to 68 seconds and was followed by the capping step. Cytosines may be 5-methyl cytosines. (5-methyl deoxycytidine phosphoramidites available from Glen Research, Sterling, Va. or Amersham Pharmacia Biotech, Piscataway, N.J.)

2′-methoxy oligonucleotides are synthesized using 2′-methoxy β-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham, Mass.) and the standard cycle for unmodified oligonucleotides, except the wait step after pulse delivery of tetrazole and base is increased to 360 seconds. Other 2′-alkoxy oligonucleotides are synthesized by a modification of this method, using appropriate 2′-modified amidites such as those available from Glen Research, Inc., Sterling, Va.

2′-fluoro oligonucleotides are synthesized as described in Kawasaki et al. ( J. Med. Chem. 1993, 36, 831-841). Briefly, the protected nucleoside N 6 -benzoyl-2′-deoxy-2′-fluoroadenosine is synthesized utilizing commercially available 9-β-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2′-α-fluoro atom is introduced by a S N 2-displacement of a 2′-β-O-trifyl group. Thus N 6 -benzoyl-9-β-D-arabinofuranosyladenine is selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N 6 -benzoyl groups is accomplished using standard methodologies. Standard methods are also used to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

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

Synthesis of 2′-deoxy-2′-fluorouridine is accomplished by the modification of a known procedure in which 2,2′-anhydro-1-β-D-arabinofuranosyluracil is treated with 70% hydrogen fluoride-pyridine. Standard procedures are used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-deoxy-2′-fluorocytidine is synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N 4 -benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures are used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-(2-methoxyethyl)-modified amidites were synthesized according to Martin, P. ( Helv. Chim. Acta 1995, 78, 486-506). For ease of synthesis, the last nucleotide may be a deoxynucleotide. 2′-O—CH 2 CH 2 OCH 3− cytosines may be 5-methyl cytosines.

Synthesis of 5-Methyl cytosine monomers:

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

5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). 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 hours) to give a solid which was crushed to a light tan powder (57 g, 85% crude yield). The material was used as is for further reactions.

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160° C. After heating for 48 hours at 155-160° C., the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH 3 CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH 2 Cl 2 /acetone/MeOH (20:5:3) containing 0.5% Et 3 NH. The residue was dissolved in CH 2 Cl 2 (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product.

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 3 CN (200 mL). The residue was dissolved in CHCl 3 (1.5 L) and extracted with 2×500 mL of saturated NaHCO 3 and 2×500 mL of saturated NaCl. The organic phase was dried over Na 2 SO 4 , filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et 3 NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).

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 tic by first quenching the tlc sample with the addition of MeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl 3 (800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl 3 . The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/Hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%).

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

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

A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH 4 0H (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH 3 gas was added and the vessel heated to 100° C. for 2 hours (tlc showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.

N 4 -Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, tlc showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL). The residue was dissolved in CHCl 3 (700 mL) and extracted with saturated NaHCO 3 (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO 4 and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica column using EtOAc/Hexane (1:1) containing 0.5% Et 3 NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.

N 4 -Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N 4 -Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH 2 Cl 2 (1 L). Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (tlc showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO 3 (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH 2 Cl 2 (300 mL), and the extracts were combined, dried over MgSO 4 and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound.

5-methyl-2′-deoxycytidine (5-me-C) containing oligonucleotides were synthesized according to published methods (Sanghvi et al., Nucl. Acids Res. 1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.).

Oligonucleotides having methylene(methylimino) (MMI) backbones were synthesized according to U.S. Pat. No. 5,378,825, which is coassigned to the assignee of the present invention and is incorporated herein in its entirety. For ease of synthesis, various nucleoside dimers containing MMI linkages were synthesized and incorporated into oligonucleotides. Other nitrogen-containing backbones are synthesized according to WO 92/20823 which is also coassigned to the assignee of the present invention and incorporated herein in its entirety.

Oligonucleotides having amide backbones are synthesized according to De Mesmaeker et al. ( Acc. Chem. Res. 1995, 28, 366-374). The amide moiety is readily accessible by simple and well-known synthetic methods and is compatible with the conditions required for solid phase synthesis of oligonucleotides.

Oligonucleotides with morpholino backbones are synthesized according to U.S. Pat. No. 5,034,506 (Summerton and Weller).

Peptide-nucleic acid (PNA) oligomers are synthesized according to P. E. Nielsen et al. ( Science 1991, 254, 1497-1500).

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

Example 2

Human TNF-α Oligodeoxynucleotide Sequences

Antisense oligonucleotides were designed to target human TNF-α. Target sequence data are from the TNF-α cDNA sequence published by Nedwin, G. E. et al. ( Nucleic Acids Res. 1985, 13, 6361-6373); Genbank accession number X02910, provided herein as SEQ ID NO: 1. Oligodeoxynucleotides were synthesized primarily with phosphorothioate linkages. Oligonucleotide sequences are shown in Table 1. Oligonucleotide 14640 (SEQ ID NO. 2) is a published TNF-α antisense oligodeoxynucleotide targeted to the start site of the TNF-α gene (Hartmann, G., et al., Antisense Nucleic Acid Drug Dev., 1996, 6, 291-299). Oligonucleotide 2302 (SEQ ID NO. 41) is an antisense oligodeoxynucleotide targeted to the human intracellular adhesion molecule-1 (ICAM-1) and was used as an unrelated (negative) target control. Oligonucleotide 13664 (SEQ ID NO. 42) is an antisense oligodeoxynucleotide targeted to the Herpes Simplex Virus type 1 and was used as an unrelated target control.

NeoHK cells, human neonatal foreskin keratinocytes (obtained from Cascade Biologicals, Inc., Portland, Oreg.) were cultured in Keratinocyte medium containing the supplied growth factors (Life Technologies, Rockville, Md.).

At assay time, the cells were between 70% and 90% confluent. The cells were incubated in the presence of Keratinocyte medium, without the supplied growth factors added, and the oligonucleotide formulated in LIPOFECTIN® (Life Technologies), a 1:1 (w/w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA), and dioleoyl phosphotidylethanolamine (DOPE) in membrane filtered water. For an initial screen, the oligonucleotide concentration was 300 nM in 9 μg/mL LIPOFECTIN®. Treatment was for four hours. After treatment, the medium was removed and the cells were further incubated in Keratinocyte medium containing the supplied growth factors and 100 nM phorbol 12-myristate 13-acetate (PMA, Sigma, St. Louis, Mo.). mRNA was analyzed 2 hours post-induction with PMA. Protein levels were analyzed 12 to 20 hours post-induction.

Total mRNA was isolated using the RNEASY® Mini Kit (Qiagen, Valencia, Calif.; similar kits from other manufacturers may also be used), separated on a 1% agarose gel, transferred to HYBOND™N+ membrane (Amersham Pharmacia Biotech, Piscataway, N.J.), a positively charged nylon membrane, and probed. A TNF-α probe consisted of the 505 bp EcoRI-HindIII fragment from BBG 18 (R&D Systems, Minneapolis, Minn.), a plasmid containing human TNF-α cDNA. A glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe consisted of the 1.06 kb HindIII fragment from pHcGAP (American Type Culture Collection, Manassas, Va.), a plasmid containing human G3PDH cDNA. The restriction fragments were purified from low-melting temperature agarose, as described in Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, 1989 and labeled with REDIVUE™ 32 P-dCTP (Amersham Pharmacia Biotech, Piscataway, N.J.) and PRIME-A-GENE® labeling kit (Promega, Madison, Wis.). mRNA was quantitated by a PhosphoImager (Molecular Dynamics, Sunnyvale, Calif.).

Secreted TNF-α protein levels were measured using a human TNF-α ELISA kit (R&D Systems, Minneapolis, Minn. or Genzyme, Cambridge, Mass.).

TABLE 1
Nucleotide Sequences of Human TNF-α
Phosphorothioate Oligodeoxynucleotides
		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE 1	ID	NUCLEOTIDE	TARGET
NO.	(5′ -> 3′)	NO:	CO-ORDINATES 2	REGION
14640	C ATG C TTT C AGTG C T C AT	2	0796-0813	AUG
14641	TGAGGGAG C GT C TG C TGG C T	3	0615-0634	5′-UTR
14642	GTG C T C ATGGTGT CC TTT CC	4	0784-0803	AUG
14643	TAAT C A C AAGTG C AAA C ATA	5	3038-3057	3′-UTR
14644	TA CCCC GGT C T CCC AAATAA	6	3101-3120	3′-UTR
14810	GTGCTCATGGTGTCCTTTCC	4	0784-0803	AUG
14811	AGCACCGCCTGGAGCCCT	7	0869-0886	coding
14812	GCTGAGGAACAAGCACCGCC	8	0878-0897	coding
14813	AGGCAGAAGAGCGTGGTGGC	9	0925-0944	coding
14814	AAAGTGCAGCAGGCAGAAGA	10	0935-0954	coding
14815	TTAGAGAGAGGTCCCTGG	11	1593-1610	coding
14816	TGACTGCCTGGGCCAGAG	12	1617-1634	junc-
				tion
14817	GGGTTCGAGAAGATGATC	13	1822-1839	junc-
				tion
14818	GGGCTACAGGCTTGTCACTC	14	1841-1860	coding
14820	CCCCTCAGCTTGAGGGTTTG	15	2171-2190	junc-
				tion
14821	CCATTGGCCAGGAGGGCATT	16	2218-2237	coding
14822	ACCACCAGCTGGTTATCTCT	17	2248-2267	coding
14823	CTGGGAGTAGATGAGGTACA	18	2282-2301	coding
14824	CCCTTGAAGAGGACCTGGGA	19	2296-2315	coding
14825	GGTGTGGGTGAGGAGCACAT	20	2336-2355	coding
14826	GTCTGGTAGGAGACGGCGAT	21	2365-2384	coding
14827	GCAGAGAGGAGGTTGACCTT	22	2386-2405	coding
14828	GCTTGGCCTCAGCCCCCTCT	23	2436-2455	coding
14829	CCTCCCAGATAGATGGGCTC	24	2464-2483	coding
14830	CCCTTCTCCAGCTGGAAGAC	25	2485-2504	coding
14831	ATCTCAGCGCTGAGTCGGTC	26	2506-2525	coding
14832	TCGAGATAGTCGGGCCGATT	27	2527-2546	coding
14833	AAGTAGACCTGCCCAGACTC	28	2554-2573	coding
14834	GGATGTTCGTCCTCCTCACA	29	2588-2607	STOP
14835	ACCCTAAGCCCCCAATTCTC	30	2689-2708	3′-UTR
14836	CCACACATTCCTGAATCCCA	31	2758-2777	3′-UTR
14837	AGGCCCCAGTGAGTTCTGGA	32	2825-2844	3′-UTR
14838	GTCTCCAGATTCCAGATGTC	33	2860-2879	3′-UTR
14839	CTCAAGTCCTGCAGCATTCT	34	2902-2921	3′-UTR
14840	TGGGTCCCCCAGGATACCCC	35	3115-3134	3′-UTR
14841	ACGGAAAACATGTCTGAGCC	36	3151-3170	3′-UTR
14842	CTCCGTTTTCACGGAAAACA	37	3161-3180	3′-UTR
14843	GCCTATTGTTCAGCTCCGTT	38	3174-3193	3′-UTR
14844	GGTCACCAAATCAGCATTGT	39	3272-3292	3′-UTR
14845	GAGGCTCAGCAATGAGTGAC	40	3297-3316	3′-UTR
2302	G CCC AAG C TGG C AT CC GT C A	41	target control
13664	GCCGAGGTCCATGTCGTACGC	42	target control
1 “C” residues are 5-methy1-cytosines except “ C ” residues are unmodified cytidines; all linkages are phosphorothioate linkages.
2 Co-ordinates from Genbank Accession No. X02910, locus name “HSTNFA”, SEQ ID NO. 1.

Results are shown in Table 2. Oligonucleotides 14828 (SEQ ID NO. 23), 14829 (SEQ ID NO. 24), 14832 (SEQ ID NO. 27), 14833 (SEQ ID NO. 28), 14834 (SEQ ID NO. 29), 14835 (SEQ ID NO. 30), 14836 (SEQ ID NO. 31), 14839 (SEQ ID NO. 34), 14840 (SEQ ID NO. 35), and 14844 (SEQ ID NO. 39) inhibited TNF-α expression by approximately 50% or more.

Oligonucleotides 14828 (SEQ ID NO. 23), 14834 (SEQ ID NO. 29), and 14840 (SEQ ID NO. 35) gave better than 70% inhibition.

TABLE 2
Inhibition of Human TNF-α mRNA Expression by
Phosphorothioate Oligodeoxynucleotides
	SEQ	GENE
ISIS	ID	TARGET	% mRNA	% mRNA
No.:	NO:	REGION	EXPRESSION	INHIBITION
basal	—	—	16%	—
induced	—	—	100%	0%
13664	42	control	140%	—
14640	2	AUG	61%	39%
14641	3	5′-UTR	95%	5%
14642	4	AUG	131%	—
14810	4	AUG	111%	—
14815	11	coding	85%	15%
14816	12	junction	106%	—
14817	13	junction	97%	3%
14818	14	coding	64%	36%
14820	15	junction	111%	—
14821	16	coding	91%	9%
14822	17	coding	57%	43%
14827	22	coding	67%	33%
14828	23	coding	27%	73%
14829	24	coding	33%	67%
14830	25	coding	71%	29%
14831	26	coding	62%	38%
14832	27	coding	40%	60%
14833	28	coding	43%	57%
14834	29	STOP	26%	74%
14835	30	3′-UTR	32%	68%
14836	31	3′-UTR	40%	60%
14837	32	3′-UTR	106%	—
14838	33	3′-UTR	70%	30%
14839	34	5′-UTR	49%	51%
14840	35	3′-UTR	28%	72%
14841	36	3′-UTR	60%	40%
14842	37	3′-UTR	164%	—
14843	38	3′-UTR	67%	33%
14844	39	3′-UTR	46%	54%
14845	40	3′-UTR	65%	35%

Example 3

Dose Response of Antisense Phosphorothioate Oligonucleotide Effects on Human TNF-α mRNA Levels in NeoHK Cells

Four of the more active oligonucleotides from the initial screen were chosen for dose response assays. These include oligonucleotides 14828 (SEQ ID NO. 23), 14833 (SEQ ID NO. 28), 14834 (SEQ ID NO. 29) and 14839 (SEQ ID NO. 34). NeoHK cells were grown, treated and processed as described in Example 2. LIPOFECTIN® was added at a ratio of 3 μg/mL per 100 nM of oligonucleotide. The control included LIPOFECTIN® at a concentration of 9 μg/mL. The effect of the TNF-α antisense oligonucleotides was normalized to the non-specific target control. Results are shown in Table 3. Each oligonucleotide showed a dose response effect with maximal inhibition greater than 70%. Oligonucleotides 14828 (SEQ ID NO. 23) had an IC 50 of approximately 185 nM. Oligonucleotides 14833 (SEQ ID NO. 28) had an IC 50 of approximately 150 nM. Oligonucleotides 14834 (SEQ ID NO. 29) and 14839 (SEQ ID NO. 34) had an IC 50 of approximately 140 nM.

TABLE 3
Dose Response of NeoHK Cells to TNF-α
Antisense Phosphorothioate Oligodeoxynucleotides (ASOs)
	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition
2302	41	control	25	nM	100%	—
″	″	″	50	nM	100%	—
″	″	″	100	nM	100%	—
″	″	″	200	nM	100%	—
″	″	″	300	nM	100%	—
14828	23	coding	25	nM	122%	—
″	″	″	50	nM	97%	3%
″	″	″	100	nM	96%	4%
″	″	″	200	nM	40%	60%
″	″	″	300	nM	22%	78%
14833	28	coding	25	nM	89%	11%
″	″	″	50	nM	78%	22%
″	″	″	100	nM	64%	36%
″	″	″	200	nM	36%	64%
″	″	″	300	nM	25%	75%
14834	29	STOP	25	nM	94%	6%
″	″	″	50	nM	69%	31%
″	″	″	100	nM	65%	35%
″	″	″	200	nM	26%	74%
″	″	″	300	nM	11%	89%
14839	34	31′-UTR	25	nM	140%	—
″	″	″	50	nM	112%	—
″	″	″	100	nM	65%	35%
″	″	″	200	nM	29%	71%
″	″	″	300	nM	22%	78%

Example 4

Design and Testing of Chimeric (Deoxy Gapped) 2′-O-methoxyethyl TNF-α Antisense Oligonucleotides on TNF-α Levels in NeoHK Cells

Oligonucleotides having SEQ ID NO: 28 and SEQ ID NO: 29 were synthesized as uniformly phosphorothioate or mixed phosphorothioate/phosphodiester chimeric oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. The sequences and the oligonucleotide chemistries are shown in Table 4. All 2′-MOE cytosines were 5-methyl-cytosines.

Dose response experiments, as discussed in Example 3, were performed using these chimeric oligonucleotides. The effect of the TNF-α antisense oligonucleotides was normalized to the non-specific target control. Results are shown in Table 5. The activities of the chimeric oligonucleotides tested were comparable to the parent phosphorothioate oligonucleotide.

TABLE 4
Nucleotide Sequences of TNF-α
Chimeric (deoxy gapped) 2′-O-methoxyethyl Oligonucleotides
		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE	ID	NUCLEOTIDE	TARGET
NO.	(5′ ->3′)	NO:	CO-ORDINATES 1	REGION
14833	AsAsGsTsAsGsAsCsCsTsGsCsCsCsAsGsAsCsTsC	28	2554-2573	coding
16467	A o A o G o T o A sGsAsCsCsTsGsCsCsCsAs G o A o C o T o C	28	2554-2573	coding
16468	A s A s G s T s A sGsAsCsCsTsGsCsCsCsAs G s A s C s T s C	28	2554-2573	coding
16469	A s A s G s T s A s G sAsCsCsTsGsCsCsCs A s G s A s C s T s C	28	2554-2573	coding
16470	AsAsGsTsAsGsAsCsCsTsGs C s C s C s A s G s A s C s T s C	28	2554-2573	coding
16471	A s A s G s T s A s G s A s C s C sTsGsCsCsCsAsGsAsCsTsC	28	2554-2573	coding
14834	GsGsAsTsGsTsTsCsGsTsCsCsTsCsCsTsCsAsCsA	29	2588-2607	STOP
16472	G o G o A o T o G sTsTsCsGsTsCsCsTsCsCs T o C o A o C o A	29	2588-2607	STOP
16473	G s G s A s T s G sTsTsCsGsTsCsCsTsCsCs T s C s A s C s A	29	2588-2607	STOP
16474	G s G s A s T s G s T sTsCsGsTsCsCsTsCs C s T s C s A s C s A	29	2588-2607	STOP
16475	G s G s A s T s G s T s T s C s G sTsCsCsTsCsCsTsCsAsCsA	29	2588-2607	STOP
16476	GsGsAsTsGsTsTsCsGsTsCs C s T s C s C s T s C s A s C s A	29	2588-2607	STOP
1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethoxy cytidines are 5-methyl-cytidines; “s” linkages are phosphorothioate linkages, “o” linkages are phosphodiester linkages.
2 Co-ordinates from Genbank Accession No. X02910, locus name “HSTNFA”, SEQ ID NO. 1.

TABLE 5
Dose Response of NeoHK Cells to TNF-α
Chimeric (deoxy gapped) 2′-O-methoxyethyl Antisense
Oligonucleotides
	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition
13664	42	control	50	nM	100%	—
″	″	″	100	nM	100%	—
″	″	″	200	nM	100%	—
″	″	″	300	nM	100%	—
14833	28	coding	50	nM	69%	31%
″	″	″	100	nM	64%	36%
″	″	″	200	nM	56%	44%
″	″	″	300	nM	36%	64%
16468	28	coding	50	nM	66%	34%
″	″	″	100	nM	53%	47%
″	″	″	200	nM	34%	66%
″	″	″	300	nM	25%	75%
16471	28	coding	50	nM	77%	23%
″	″	″	100	nM	56%	44%
″	″	″	200	nM	53%	47%
″	″	″	300	nM	31%	69%
14834	29	STOP	50	nM	74%	26%
″	″	″	100	nM	53%	47%
″	″	″	200	nM	24%	76%
″	″	″	300	nM	11%	89%
16473	29	STOP	50	nM	71%	29%
″	″	″	100	nM	51%	49%
″	″	″	200	nM	28%	72%
″	″	″	300	nM	23%	77%
16476	29	STOP	50	nM	74%	26%
″	″	″	100	nM	58%	42%
″	″	″	200	nM	32%	68%
″	″	″	300	nM	31%	69%

Example 5

Design and Testing of Chimeric Phosphorothioate/MMI TNF-α Antisense Oligodeoxynucleotides on TNF-α Levels in NeoHK Cells

Oligonucleotides having SEQ ID NO. 29 were synthesized as mixed phosphorothioate/methylene(methylimino) (MMI) chimeric oligodeoxynucleotides. The sequences and the oligonucleotide chemistries are shown in Table 6. Oligonucleotide 13393 (SEQ ID NO. 49) is an antisense oligonucleotide targeted to the human intracellular adhesion molecule-1 (ICAM-1) and was used as an unrelated target control. All cytosines were 5-methyl-cytosines.

Dose response experiments were performed using these chimeric oligonucleotides, as discussed in Example 3 except quantitation of TNF-α mRNA levels was determined by real-time PCR (RT-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 RT-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 or FAM, PE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, 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 (six-second) 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.

RT-PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif. RT-PCR reactions were carried out by adding 25 μl PCR cocktail (1× TAQMAN® buffer A, 5.5 mM MgCl 2 , 300 μM each of dATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 U RNAse inhibitor, 1.25 units AMPLITAQ GOLD®, and 12.5 U MuLV reverse transcriptase) to 96 well plates containing 25 μl poly(A) mRNA 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).

For TNF-α the PCR primers were:

Forward: 5′-CAGGCGGTGCTTGTTCCT-3′ SEQ ID NO. 43

Reverse: 5′-GCCAGAGGGCTGATTAGAGAGA-3′ SEQ ID NO. 44

and the PCR probe was: FAM-CTTCTCCTTCCTGATCGTGGCAGGC-TAMRA (SEQ ID NO. 45) where FAM or JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

For GAPDH the PCR primers were:

Forward primer: 5′-GAAGGTGAAGGTCGGAGTC-3′ SEQ ID NO. 46

Reverse primer: 5′-GAAGATGGTGATGGGATTTC-3′ SEQ ID NO. 47

and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO. 48) where FAM or JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Results are shown in Table 7. The oligonucleotide containing MMI linkages was more effective in reducing TNF-α mRNA levels than the uniformly phosphorothioate oligonucleotide. The IC 50 value was reduced from approximately 75 nM, for oligonucleotide 14834 (SEQ ID NO: 29), to approximately 30 nM for oligonucleotide 16922 (SEQ ID NO: 29).

Dose response experiments were also performed measuring the effect on TNF-α protein levels. Protein levels were measured as described in Example 2. Results are shown in Table 8. The oligonucleotide containing four MMI linkages on each end was more effective in reducing protein levels than the uniformly phosphorothioate oligonucleotide. The IC 50 value was reduced from approximately 90 nM, for oligonucleotide 14834 (SEQ ID NO: 29), to approximately 45 nM for oligonucleotide 16922 (SEQ ID NO: 29).

TABLE 6
Nucleotide Sequences of Human TNF-α
Chimeric Phosphorothioate/MMI Oligodeoxynucleotides
		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE	ID	NUCLEOTIDE	TARGET
NO.	(5′ -> 3′)	NO:	CO-ORDINATES 1	REGION
14834	GsGsAsTsGsTsTsCsGsTsCsCsTsCsCsTsCsAsCsA	29	2588-2607	STOP
16922	GmGmAmTmGsTsTsCsGsTsCsCsTsCsCsTmCmAmCmA	29	2588-2607	STOP
16923	GmGmAmTmGmTmTsCsGsTsCsCsTsCmCmTmCmAmCmA	29	2588-2607	STOP
13393	TsCsTsGsAsGsTsAsGsCsAsGsAsGsGsAsGsCsTsC	49	target control
1 All cytosine residues are 5-methyl-cytosines; “s” linkages are phosphorothioate linkages, “m” linkages are methylene (methylimino) (MMI).
2 Co-ordinates from Genbank Accession No. X0291Q, locus name “HSTNFA”, SEQ ID NO. 1.

TABLE 7
Dose Response of Chimeric Phosphorothioate/MMI TNF-α
Antisense Oligodeoxynucleotides on TNF-α mRNA Levels in
PMA-Induced NeoHK Cells
	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition
induced	—	—	—	100%	—
13393	49	control	25	nM	87.3%	12.7%
″	″	″	50	nM	98.5%	1.5%
″	″	″	100	nM	133.1%	—
″	″	″	200	nM	139.6%	—
14834	29	STOP	25	nM	98.7%	1.3%
″	″	″	50	nM	70.8%	29.2%
″	″	″	100	nM	36.0%	64.0%
″	″	″	200	nM	38.2%	61.8%
16922	29	STOP	25	nM	58.9%	41.1%
″	″	″	50	nM	28.2%	71.8%
″	″	″	100	nM	22.2%	77.8%
″	″	″	200	nM	18.9%	81.1%

TABLE 7
Dose Response of Chimeric Phosphorothioate/MMI TNF-α
Antisense Oligodeoxynucleotides on TNF-α mRNA Levels in
PMA-Induced NeoHK Cells
	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition
induced	—	—	—	100%	—
13393	49	control	25	nM	87.3%	12.7%
″	″	″	50	nM	98.5%	1.5%
″	″	″	100	nM	133.1%	—
″	″	″	200	nM	139.6%	—
14834	29	STOP	25	nM	98.7%	1.3%
″	″	″	50	nM	70.8%	29.2%
″	″	″	100	nM	36.0%	64.0%
″	″	″	200	nM	38.2%	61.8%
16922	29	STOP	25	nM	58.9%	41.1%
″	″	″	50	nM	28.2%	71.8%
″	″	″	100	nM	22.2%	77.8%
″	″	″	200	nM	18.9%	81.1%

Example 6

Additional Human TNF-α Antisense Oligonucleotide Sequences

A second screening of human TNF-α antisense oligonucleotides was performed. Oligonucleotides were designed specifically against specific regions of the TNF-α gene. A series of oligonucleotides was designed to target introns 1 and 3, and exon 4. Sequences targeting introns 1 or 3 were synthesized as uniformly phosphorothioate oligodeoxynucleotides or mixed phosphorothioate/phosphodiester chimeric backbone oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. Sequences targeting exon 4 were synthesized as mixed phosphorothioate/phosphodiester chimeric backbone oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. The sequences of the chimeric oligonucleotides are shown in Table 9. Sequences of the uniformly phosphorothioate oligodeoxynucleotides are shown in Table 11.

These oligonucleotides were screened at 50 nM and 200 nM for their ability to inhibit TNF-α protein secretion, essentially as described in Example 2. Results for the chimeric backbone oligonucleotides are shown in Table 10; results for the uniformly phosphorothioate oligodeoxynucleotides are shown in Table 12.

For the chimeric backbone oligonucleotides targeting introns 1 or 3, oligonucleotide 21688 (SED ID NO. 69) gave 60% inhibition or greater. For chimeric backbone oligonucleotides targeting exon 4, two-thirds of the oligonucleotides gave nearly 60% inhibition or greater (SEQ ID NOs. 88, 90, 91, 92, 93, 94, 97, and 98). See Table 10. For the uniformly phosphorothioate oligodeoxynucleotides, five of nine oligonucleotides targeting intron 3 were effective in reducing TNF-α expression by nearly 60% or greater (SEQ ID NOs. 79, 80, 81, 82, and 84). See Table 12.

Oligonucleotides having SEQ ID NO. 91 and SEQ ID NO. 98 were synthesized as a uniformly phosphorothioate oligodeoxynucleotides or mixed phosphorothioate/phosphodiester chimeric backbone oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. The sequences and the oligonucleotide chemistries are shown in Table 13. All 2′-MOE cytosines and 2′-deoxy cytosines were 5-methyl-cytosines.

Dose response experiments, as discussed in Example 3, were performed using these oligonucleotides. Included in this experiment were two oligonucleotides targeting intron 1 and two oligonucleotides targeting intron 3. Results are shown in Tables 14 and 15. The oligonucleotides targeting exon 4 with variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides and/or uniformly phosphorothioate or mixed phosphorothioate/phosphodiester were, in general, comparable to the parent compound.

Oligonucleotides targeting introns 1 or 3 having SEQ ID NOs 66, 69 and 80 were effective in reducing TNF-α mRNA levels by greater than 80% and showed a dose response effect with an IC 50 approximately 110 nM. See Tables 14 and 15.

TABLE 9
Nucleotide Sequences of TNF-α
Chimeric Backbone (deoxy gapped) 2′-O-methoxyethyl Oligonucleotides
		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE	ID	NUCLEOTIDE	TARGET
NO.	(5′ -> 3′)	NO:	CO-ORDINATES 1	REGION
21669	T o G o C o G o T sCsTsCsTsCsAsTsTsTsCs C o C o C o T o T	50	1019-1038	intron 1
21670	T o C o C o C o A sTsCsTsCsTsCsTsCsCsCs T o C o T o C o T	51	1039-1058	intron 1
21671	C o A o G o C o G sCsAsCsAsTsCsTsTsTsCs A o C o C o C o A	52	1059-1078	intron 1
21672	T o C o T o C o T sCsTsCsAsTsCsCsCsTsCs C o C o T o A o T	53	1079-1098	intron 1
21673	C o G o T o C o T sTsTsCsTsCsCsAsTsGsTs T o T o T o T o T	54	1099-1118	intron 1
21674	C o A o C o A o T sCsTsCsTsTsTsCsTsGsCs A o T o C o C o C	55	1119-1138	intron 1
21675	C o T o C o T o C sTsTsCsCsCsCsAsTsCsTs C o T o T o G o C	56	1139-1158	intron 1
21676	G o T o C o T o C sTsCsCsAsTsCsTsTsTsCs C o T o T o C o T	57	1159-1178	intron 1
21677	T o T o C o C o A sTsGsTsGsCsCsAsGsAsCs A o T o C o C o T	58	1179-1198	intron 1
21678	A o T o A o C o A sCsAsCsTsTsAsGsTsGsAs G o C o A o C o C	59	1199-1218	intron 1
21679	T o T o C o A o T sTsCsAsTsTsCsAsTsTsCs A o C o T o C o C	60	1219-1238	intron 1
21680	T o A o T o A o T sCsTsGsCsTsTsGsTsTsCs A o T o T o C o A	61	1239-1258	intron 1
21681	C o T o G o T o C sTsCsCsAsTsAsTsCsTsTs A o T o T o T o A	62	1259-1278	intron 1
21682	T o C o T o C o T sTsCsTsCsAsCsAsCsCsCs C o A o C o A o T	63	1279-1298	intron 1
21683	C o A o C o T o T sGsTsTsTsCsTsTsCsCsCs C o C o A o T o C	64	1299-1318	intron 1
21684	C o T o C o A o C sCsAsTsCsTsTsTsAsTsTs C o A o T o A o T	65	1319-1338	intron 1
21685	A o T o A o T o T sTsCSCsCsGsCsTsCsTsTs T o C o T o G o T	66	1339-1358	intron 1
21686	C o A o T o C o T sCsTsCsTsCsCsTsTsAsGs C o T o G o T o C	67	1359-1378	intron 1
21687	T o C o T o T o C sTsCsTsCsCsTsTsAsTsCs T o C o C o C o C	68	1379-1398	intron 1
21688	G o T o G o T o G sCsCsAsGsAsCsAsCsCsCs T o A o T o C o T	69	1399-1418	intron 1
21689	T o C o T o T o T sCsCsCsTsGsAsGsTsGsTs C o T o T o C o T	70	1419-1438	intron 1
21690	A o C o C o T o T sCsCsAsGsCsAsTsTsCsAs A o C o A o G o C	71	1439-1458	intron 1
21691	C o T o C o C o A sTsTsCsAsTsCsTsGsTsGs T o A o T o T o C	72	1459-1478	intron 1
21692	T o G o A o G o G sTsGsTsCsTsGsGsTsTsTs T o C o T o C o T	73	1479-1498	intron 1
21693	A o C o A o C o A sTsCsCsTsCsAsGsAsGsCs T o C o T o T o A	74	1871-1890	intron 3
21694	C o T o A o G o C sCsCsTsCsCsAsAsGsTsTs C o C o A o A o G	75	1891-1910	intron 3
21695	C o G o G o G o C sTsTsCsAsAsTsCsCsCsCs A o A o A o T o C	76	1911-1930	intron 3
21696	A o A o G o T o T sCsTsGsCsCsTsAsCsCsAs T o C o A o G o C	77	1931-1950	intron 3
21697	G o T o C o C o T sTsCsTsCsAsCsAsTsTsGs T o C o T o C o C	78	1951-1970	intron 3
21698	C o C o T o T o C sCsCsTsTsGsAsGsCsTsCs A o G o C o G o A	79	1971-1990	intron 3
21699	G o G o C o C o T sGsTsGsCsTsGsTsTsCsCs T o C o C o A o C	80	1991-2010	intron 3
21700	C o G o T o T o C sTsGsAsGsTsAsTsCsCsCs A o C o T o A o A	81	2011-2030	intron 3
21701	C o A o C o A o T sCsCsCsAsCsCsTsGsGsCs C o A o T o G o A	82	2031-2050	intron 3
21702	G o T o C o C o T sCsTsCsTsGsTsCsTsGsTs C o A o T o C o C	83	2051-2070	intron 3
21703	C o C o A o C o C sCsCsAsCsAsTsCsCsGsGo T o T o C o C o T	84	2071-2090	intron 3
21704	T o C o C o T o G sGsCsCsCsTsCsGsAsGsCs T o C o T o G o C	85	2091-2110	intron 3
21705	A o T o G o T o C sGsGsTsTsCsAsCsTsCsTs C o C o A o C o A	86	2111-2130	intron 3
21706	A o G o A o G o G sAsGsAsGsTsCsAsGsTsGs T o G o G o C o C	87	2131-2150	intron 3
21722	G o A o T o C o C sCsAsAsAsGsTsAsGsAsCs C o T o G o C o C	88	2561-2580	exon 4
21723	C o A o G o A o C sTsCsGsGsCsAsAsAsGsTs C o G o A o G o A	89	2541-2560	exon 4
21724	T o A o G o T o C sGsGsGsCsCsGsAsTsTsGs A o T o C o T o C	90	2521-2540	exon 4
21725	A o G o C o G o C sTsGsAsGsTsCsGsGsTsCs A o C o C o C o T	91	2501-2520	exon 4
21726	T o C o T o C o C sAsGsCsTsGsGsAsAsGsAs C o C o C o C o T	92	2481-25OO	exon 4
21727	C o C o C o A o G sAsTsAsGsAsTsGsGsGsCs T o C o A o T o A	93	2461-2480	exon 4
21728	C o C o A o G o G sGsCsTsTsGsGsCsCsTsCs A o G o C o C o C	94	2441-2460	exon 4
21729	C o C o T o C o T sGsGsGsGsTsCsTsCsCsCs T o C o T o G o G	95	2421-2440	exon 4
21730	C o A o G o G o G sGsCsTsCsTsTsGsAsTsGs G o C o A o G o A	96	2401-2420	exon 4
21731	G o A o G o G o A sGsGsTsTsGsAsCsCsTsTs G o G o T o C o T	97	2381-2400	exon 4
21732	G o G o T o A o G sGsAsGsAsCsGsGsCsGsAs T o G o C o G o G	98	2361-2380	exon 4
21733	C o T o G o A o T sGsGsTsGsTsGsGsGsTsGs A o G o G o A o G	99	2341-2360	exon 4
1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethoxy cytidines and 2′-deoxycytidines are 5-methyl-cytidines; “s” linkages are phosphorothioate linkages, “o” linkages are phosphodiester linkages.
2 Co-ordinates from Genbank Accession No. X02910, locus name “HSTNFA”, SEQ ID NO. 1.

TABLE 10
Dose Response of PMA-Induced neoHK Cells to Chimeric
Backbone (deoxy gapped) 2′-O-methoxyethyl TNF-α Antisense
Oligonucleotides
	SEQ ID	ASO Gene		% protein	% protein
ISIS #	NO:	Target	Dose	Expression	Inhibition
induced	—	—	—	100%	—
14834	29	STOP	50	nM	76%	24%
″	″	″	200	nM	16%	84%
21669	50	intron 1	50	nM	134%	—
″	″	″	200	nM	114%	—
21670	51	intron 1	50	nM	122%	—
″	″	″	200	nM	101%	—
21671	52	intron 1	50	nM	90%	10%
″	″	″	200	nM	58%	42%
21672	53	intron 1	50	nM	122%	—
″	″	″	200	nM	131%	—
21673	54	intron 1	50	nM	102%	—
″	″	″	200	nM	110%	—
21674	55	intron 1	50	nM	111%	—
″	″	″	200	nM	96%	4%
21675	56	intron 1	50	nM	114%	—
″	″	″	200	nM	99%	1%
21676	57	intron 1	50	nM	107%	—
″	″	″	200	nM	96%	4%
21677	58	intron 1	50	nM	86%	14%
″	″	″	200	nM	95%	5%
21678	59	intron 1	50	nM	106%	—
″	″	″	200	nM	107%	—
21679	60	intron 1	50	nM	75%	25%
″	″	″	200	nM	73%	27%
21680	61	intron 1	50	nM	76%	24%
″	″	″	200	nM	80%	20%
21681	62	intron 1	50	nM	79%	21%
″	″	″	200	nM	82%	18%
21682	63	intron 1	50	nM	102%	—
″	″	″	200	nM	88%	12%
21683	64	intron 1	50	nM	80%	20%
″	″	″	200	nM	66%	34%
21684	65	intron 1	50	nM	91%	9%
″	″	″	200	nM	69%	31%
21685	66	intron i	50	nM	98%	2%
″	″	″	200	nM	90%	10%
21686	67	intron 1	50	nM	97%	3%
″	″	″	200	nM	72%	28%
21687	68	intron 1	50	nM	103%	—
″	″	″	200	nM	64%	36%
21688	69	intron 1	50	nM	87%	13%
″	″	″	200	nM	40%	60%
21689	70	intron 1	50	nM	78%	22%
″	″	″	200	nM	74%	26%
21690	71	intron 1	50	nM	84%	16%
″	″	″	200	nM	8Q%	20%
21691	72	intron 1	50	nM	86%	14%
″	″	″	200	nM	75%	25%
21692	73	intron 1	50	nM	85%	15%
″	″	″	200	nM	61%	39%
21693	74	intron 3	50	nM	81%	19%
″	″	″	200	nM	83%	17%
21694	75	intron 3	50	nM	99%	1%
″	″	″	200	nM	56%	44%
21695	76	intron 3	50	nM	87%	13%
″	″	″	200	nM	84%	16%
21696	77	intron 3	50	nM	103%	—
″	″	″	200	nM	86%	14%
21697	78	intron 3	50	nM	99%	1%
″	″	″	200	nM	52%	48%
21698	79	intron 3	50	nM	96%	4%
″	″	″	200	nM	47%	53%
21699	80	intron 3	50	nM	73%	27%
″	″	″	200	nM	84%	16%
21700	81	intron 3	50	nM	80%	20%
″	″	″	200	nM	53%	47%
21701	82	intron 3	50	nM	94%	6%
″	″	″	200	nM	56%	44%
21702	83	intron 3	50	nM	86%	14%
″	″	″	200	nM	97%	3%
21703	84	intron 3	50	nM	88%	12%
″	″	″	200	nM	74%	26%
21704	85	intron 3	50	nM	69%	31%
″	″	″	200	nM	65%	35%
21705	86	intron 3	50	nM	92%	8%
″	″	″	200	nM	77%	23%
21706	87	intron 3	50	nM	95%	5%
″	″	″	200	nM	82%	18%
21722	88	exon 4	50	nM	81%	19%
″	″	″	200	nM	41%	59%
21723	89	exon 4	50	nM	87%	13%
″	″	″	200	nM	74%	26%
21724	90	exon 4	50	nM	68%	32%
″	″	″	200	nM	33%	67%
21725	91	exon 4	50	nM	55%	45%
″	″	″	200	nM	30%	70%
21726	92	exon 4	50	nM	72%	28%
″	″	″	200	nM	40%	60%
21727	93	exon 4	50	nM	67%	33%
″	″	″	200	nM	40%	60%
21728	94	exon 4	50	nM	62%	38%
″	″	″	200	nM	41%	59%
21729	95	exon 4	50	nM	78%	22%
″	″	″y	200	nM	53%	47%
21730	96	exon 4	50	nM	68%	32%
″	″	″	200	nM	48%	52%
21731	97	exon 4	50	nM	77%	23%
″	″	″	200	nM	41%	59%
21732	98	exon 4	50	nM	62%	38%
″	″	″	200	nM	28%	72%
21733	99	exon 4	50	nM	92%	8%
″	″	″	200	nM	74%	26%

TABLE 11
Nucleotide Sequences of Additional Human TNF-α
Phosphorothioate Oligodeoxynucleotides
		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE 1	ID	NUCLEOTIDE	TARGET
NO.	(5′ -> 3′)	NO:	CO-ORDINATES 2	REGION
21804	TGCGTCTCTCATTTCCCCTT	50	1019-1038	intron
				1
21805	TCCCATCTCTCTCCCTCTCT	51	1039-1058	intron
				1
21806	CAGCGCACATCTTTCACCCA	52	1059-1078	intron
				1
21807	TCTCTCTCATCCCTCCCTAT	53	1079-1098	intron
				1
21808	CGTCTTTCTCCATGTTTTTT	54	1099-1118	intron
				1
21809	CACATCTCTTTCTGCATCCC	55	1119-1138	intron
				1
21810	CTCTCTTCCCCATCTCTTGC	56	1139-1158	intron
				1
21811	GTCTCTCCATCTTTCCTTCT	57	1159-1178	intron
				1
21812	TTCCATGTGCCAGACATCCT	58	1179-1198	intron
				1
21813	ATACACACTTAGTGAGCACC	59	1199-1218	intron
				1
21814	TTCATTCATTCATTCACTCC	60	1219-1238	intron
				1
21815	TATATCTGCTTGTTCATTCA	61	1239-1258	intron
				1
21816	CTGTCTCCATATCTTATTTA	62	1259-1278	intron
				1
21817	TCTCTTCTCACACCCCACAT	63	1279-1298	intron
				1
21818	CACTTGTTTCTTCCCCCATC	64	1299-1318	intron
				1
21819	CTCACCATCTTTATTCATAT	65	1319-1338	intron
				1
21820	ATATTTCCCGCTCTTTCTGT	66	1339-1358	intron
				1
21821	CATCTCTCTCCTTAGCTGTC	67	1359-1378	intron
				1
21822	TCTTCTCTCCTTATCTCCCC	68	1379-1398	intron
				1
21823	GTGTGCCAGACACCCTATCT	69	1399-1418	intron
				1
21824	TCTTTCCCTGAGTGTCTTCT	70	1419-1438	intron
				1
21825	ACCTTCCAGCATTCAACAGC	71	1439-1458	intron
				1
21826	CTCCATTCATCTGTGTATTC	72	1459-1478	intron
				1
21827	TGAGGTGTCTGGTTTTCTCT	73	1479-1498	intron
				1
21828	ACACATCCTCAGAGCTCTTA	74	1871-1890	intron
				3
21829	CTAGCCCTCCAAGTTCCAAG	75	1891-1910	intron
				3
21830	CGGGCTTCAATCCCCAAATC	76	1911-1930	intron
				3
21831	AAGTTCTGCCTACCATCAGC	77	1931-1950	intron
				3
21832	GTCCTTCTCACATTGTCTCC	78	1951-1970	intron
				3
21833	CCTTCCCTTGAGCTCAGCGA	79	1971-1990	intron
				3
21834	GGCCTGTGCTGTTCCTCQAC	80	1991-2010	intron
				3
21835	CGTTCTGAGTATCCCACTAA	81	2011-2030	intron
				3
21836	CACATCCCACCTGGCCATGA	82	2031-2050	intron
				3
21837	GTCCTCTCTGTCTGTCATCC	83	2051-2070	intron
				3
21838	CCACCCCACATCCGGTTCCT	84	2071-2090	intron
				3
21839	TCCTGGCCCTCGAGCTCTGC	85	2091-2110	intron
				3
21840	ATGTCGGTTCACTCTCCACA	86	2111-2130	intron
				3
21841	AGAGGAGAGTCAGTGTGGCC	87	2131-2150	intron
				3
1 All “C” residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages.
2 Co-ordinates from Genbank Accession No. X02910, locus name “HSTNFA”, SEQ ID NO. 1.

TABLE 12
Dose Response of PMA-Induced neoHK Cells to TNF-α
Antisense Phosphorothioate Oligodeoxynucleotides
	SEQ ID	ASO Gene		% protein	% protein
ISIS #	NO:	Target	Dose	Expression	Inhibition
induced	—	—	—	100%	—
14834	29	STOP	50	nM	80%	20%
″	″	″	200	nM	13%	87%
21812	58	intron 1	50	nM	110%	—
″	″	″	200	nM	193%	—
21833	79	intron 3	50	nM	88%	12%
″	″	″	200	nM	8%	92%
21834	80	intron 3	50	nM	70%	30%
″	″	″	200	nM	18%	82%
21835	81	intron 3	50	nM	106%	—
″	″	″	200	nM	42%	58%
21836	82	intron 3	50	nM	71%	29%
″	″	″	200	nM	12%	88%
21837	83	intron 3	50	nM	129%	—
″	″	″	200	nM	74%	26%
21838	84	intron 3	50	nM	85%	15%
″	″	″	200	nM	41%	59%
21839	85	intron 3	50	nM	118%	—
″	″	″	200	nM	58%	42%
21840	86	intron 3	50	nM	120%	—
″	″	″	200	nM	96%	4%
21841	87	intron 3	50	nM	117%	—
″	″	″	200	nM	78%	22%

TABLE 13
Nucleotide Sequences of TNF-α Chimeric (deoxy gapped)
2′-O-methoxyethyl Oligonucleotides
		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE	ID	NUCLEOTIDE	TARGET
NO.	(5′ -> 3′)	NO:	CO-ORDINATES 1	REGION
21725	A o G o C o G o C sTsGsAsGsTsCsGsGsTsCs A o C o C o C o T	91	2501-2520	exon 4
25655	A s G s C s G sCsTsGsAsGsTsCsGsGsTsCsAs C s C s C s T	″	″	″
25656	A s G s C sGsCsTsGsAsGsTsCsGsGsTsCsAsCs C s C s T	″	″	″
25660	A o G o C o G sCsTsGsAsGsTsCsGsGsTsCsAs C o C o C o T	″	″	″
21732	G o G o T o A o G sGsAsGsAsCsGsGsCsGsAs T o G o C o G o G	98	2361-2380	exon 4
25657	G s G s T s A sGsGsAsGsAsCsGsGsCsGsAsTs G s C s G s G	″	″	″
25658	G s G s T sAsGsGsAsGsAsCsGsGsCsGsAsTsGs C s G s G	″	″	″
25661	G o G o T o A sGsGsAsGsAsCsGsGsCsGsAsTs G o C o G o G	″	″	″
1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-) . All 2′-methoxyethoxy cytidines and 2′-deoxycytidines are 5-methyl-cytidines; “s” linkages are phosphorothioate linkages, “o” linkages are phosphodiester linkages.
2 Co-ordinates from Genbank Accession No. X02910, locus name “HSTNFA”, SEQ ID NO. 1.

TABLE 14
Dose Response of 20 Hour PMA-Induced neoHK Cells to TNF-α
Antisense Oligonucleotides (ASOs)
	SEQ ID	ASO Gene		% protein	% protein
ISIS #	NO:	Target	Dose	Expression	Inhibition
induced	—	—	—	100%	—
14834	29	STOP	75	nM	91.2%	8.8%
″	″	″	150	nM	42.0%	58.0%
″	″	″	300	nM	16.9%	83.1%
21820	66	intron 1	75	nM	79.0%	21.0%
″	″	″	150	nM	34.5%	65.5%
″	″	″	300	nM	15.6%	84.4%
21823	69	intron 1	75	nM	79.5%	20.5%
″	″	″	150	nM	31.8%	68.2%.
″	″	″	300	nM	16.2%	83.8%
21725	91	exon 4	75	nM	74.8%	25.2%
″	″	″	150	nM	58.4%	41.6%
″	″	″	300	nM	45.2%	54.8%
25655	91	exon 4	75	nM	112.0%	—
″	″	″	150	nM	55.0%	45.0%
″	″	″	300	nM	39.3%	60.7%
25656	91	exon 4	75	nM	108.3%	—
″	″	″	150	nM	60.7%	39.3%
″	″	″	300	nM	42.8%	57.2%
25660	91	exon 4	75	nM	93.2%	6.8%
″	″	″	150	nM	72.8%	27.2%
″	″	″	300	nM	50.3%	49.7%

TABLE 15
Dose Response of 20 Hour PMA-Induced neoHK Cells to TNF-α
Antisense Oligonucleotides (ASOs)
	SEQ ID	ASO Gene		% protein	% protein
ISIS #	NO:	Target	Dose	Expression	Inhibition
induced	—	—	—	100%	—
14834	29	STOP	75	nM	44.9%	55.1%
″	″	″	150	nM	16.3%	83.7%
″	″	″	300	nM	2.2%	97.8%
21834	80	intron 3	75	nM	102.9%	—
″	″	″	150	nM	24.5%	75.5%
″	″	″	300	nM	19.1%	80.9%
21836	82	intron 3	75	nM	70.8%	29.2%
″	″	″	150	nM	55.9%	44.1%
″	″	″	300	nM	32.7%	67.3%
21732	98	exon 4	75	nM	42.4%	57.6%
″	″	″	150	nM	34.9%	65.1%
″	″	″	300	nM	15.4%	84.6%
25657	98	exon 4	75	nM	46.7%	53.3%
″	″	″	150	nM	72.0%	28.0%
″	″	″	300	nM	50.6%	49.4%
25658	98	exon 4	75	nM	83.7%	16.3%
″	″	″	150	nM	56.6%	43.4%
″	″	″	300	nM	36.9%	63.1%
25661	98	exon 4	75	nM	54.9%	45.1%
″	″	″	150	nM	34.4%	65.6%
″	″	″	300	nM	8.6%	91.4%

Example 7

Activity of Fully 2′-MOE Modified TNF-α Antisense Oligonucleotides

A series of antisense oligonucleotides were synthesized targeting the terminal twenty nucleotides of each exon at every exon-intron junction of the TNF-α gene. These oligonucleotides were synthesized as fully 2′-methoxyethoxy modified oligonucleotides. The oligonucleotide sequences are shown in Table 16. Oligonucleotide 12345 (SEQ ID NO. 106) is an antisense oligonucleotide targeted to the human intracellular adhesion molecule-1 (ICAM-1) and was used as an unrelated target control.

The oligonucleotides were screened at 50 nM and 200 nM for their ability to inhibit TNF-α mRNA levels, as described in Example 3. Results are shown in Table 17. Oligonucleotide 21794 (SEQ ID NO. 102) showed an effect at both doses, with greater than 75% inhibition at 200 nM.

TABLE 16
Nucleotide Sequences of Human TNF-α Uniform 2′-MOE
Oligonucleotides
			TARGET GENE
		SEQ	NUCLEOTIDE	GENE
ISIS	NUCLEOTIDE SEQUENCE 1	ID	CO-	TARGET
NO.	(5′ -> 3′)	NO:	ORDINATES 2	REGION 3
21792	AGGCACTCACCTCTTCCCTC	100	0972-0991	E1/I1
21793	CCCTGGGGAACTGTTGGGGA	101	1579-1598	I1/E2
21794	AGACACTTACTGACTGCCTG	102	1625-1644	E2/I2
21795	GAAGATGATCCTGAAGAGGA	103	1812-1831	I2/E3
21796	GAGCTCTTACCTACAACATG	104	1860-1879	E3/I3
21797	TGAGGGTTTGCTGGAGGGAG	105	2161-2180	I3/E4
12345	GATCGCGTCGGACTATGAAG	106	target control
1 Emboldened residues are 2′-methoxyethoxy residues, 2′-methoxyethoxy cytosine residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages.
2 Co-ordinates from Genbank Accession No. X02910, locus name “HSTNFA”, SEQ ID NO. 1.
3 Each target region is an exon-intron junction and is represented in the form, for example, I1/E2, where I, followed by a number, refers to the intron number and E, followed by a number, refers to the exon number.

TABLE 17
Dose Response of neoHK Cells to TNF-α
Antisense 2′-MOE Oligonucleotides
	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition
induced	—	—	—	100%	—
12345	106	control	50	nM	121%	—
″	″	″	200	nM	134%	—
13393	49	control	50	nM	110%	—
″	″	″	200	nM	112%	—
14834	29	STOP	50	nM	92%	8%
″	″	″	200	nM	17%	83%
21792	100	E1/I1	50	nM	105%	—
″	″	″	200	nM	148%	—
21793	101	I1/E2	50	nM	106%	—
″	″	″	200	nM	172%	—
21794	102	E2/I2	50	nM	75%	25%
″	″	″	200	nM	23%	77%
21795	103	I2/E3	50	nM	79%	21%
″	″	″	200	nM	125%	—
21796	104	E3/I3	50	nM	56%	44%
″	″	″	200	nM	150%	—
21797	105	I3/E4	50	nM	90%	10%
″	″	″	200	nM	128%	—

Example 8

Mouse TNF-α Oligonucleotide Sequences

Antisense oligonucleotides were designed to target mouse TNF-α. Target sequence data are from the TNF-α cDNA sequence published by Semon, D. et al. ( Nucleic Acids Res. 1987, 15, 9083-9084); Genbank accession number Y00467, provided herein as SEQ ID NO: 107. Oligonucleotides were synthesized primarily as phosphorothioate oligodeoxynucleotides. Oligonucleotide sequences are shown in Table 18. Oligonucleotide 3082 (SEQ ID NO. 141) is an antisense oligodeoxynucleotide targeted to the human intracellular adhesion molecule-1 (ICAM-1) and was used as an unrelated target control. Oligonucleotide 13108 (SEQ ID NO. 142) is an antisense oligodeoxynucleotide targeted to the herpes simplex virus type 1 and was used as an unrelated target control.

P388D1, mouse macrophage cells (obtained from American Type Culture Collection, Manassas, Va.) were cultured in RPMI 1640 medium with 15% fetal bovine serum (FBS) (Life Technologies, Rockville, Md.).

At assay time, cell were at approximately 90% confluency. The cells were incubated in the presence of OPTI-MEM® medium (Life Technologies, Rockville, Md.), and the oligonucleotide formulated in LIPOFECTIN® (Life Technologies), a 1:1 (w/w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA), and dioleoyl phosphotidylethanolamine (DOPE) in membrane filtered water. For an initial screen, the oligonucleotide concentration was 100 nM in 3 μg/ml LIPOFECTIN®. Treatment was for four hours. After treatment, the medium was removed and the cells were further incubated in RPMI medium with 15% FBS and induced with 10 ng/ml LPS. mRNA was analyzed 2 hours post-induction with PMA.

Total mRNA was isolated using the TOTALLY RNA™ kit (Ambion, Austin, Tex.), separated on a 1% agarose gel, transferred to HYBOND™-N+ membrane (Amersham, Arlington Heights, Ill.), a positively charged nylon membrane, and probed. A TNF-α probe consisted of the 502 bp EcoRI-HindIII fragment from BBG 56 (R&D Systems, Minneapolis, Minn.), a plasmid containing mouse TNF-α cDNA. A glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe consisted of the 1.06 kb HindIII fragment from pHcGAP (American Type Culture Collection, Manassas, Va.), a plasmid containing human G3PDH cDNA. The fragments were purified from low-melting temperature agarose, as described in Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, 1989 and labeled with REDIVUE™ 32 P-dCTP (Amersham Pharmacia Biotech, Piscataway, N.J.) and PRIME-A-GENE® labelling kit (Promega, Madison, Wis.). mRNA was quantitated by a PhosphoImager (Molecular Dynamics, Sunnyvale, Calif.).

Secreted TNF-α protein levels were measured using a mouse TNF-α ELISA kit (R&D Systems, Minneapolis, Minn. or Genzyme, Cambridge, Mass.).

TABLE 18
Nucleotide Sequences of Mouse TNF-α
phosphorothioate Oligodeoxynucleotides
			TARGET
			GENE
		SEQ	NUCLEOTIDE	GENE
ISIS	NUCLEOTIDE SEQUENCE1	ID	CO-	TARGET
NO.	(5′->3′)	NO:	ORDINATES 2	REGION
14846	GAGCTTCTGCTGGCTGGCTG	108	4351-4370	5′-UTR
14847	CCTTGCTGTCCTCGCTGAGG	109	4371-4390	5′-UTR
14848	TCATGGTGTCTTTTCTGGAG	110	4511-4530	AUG
14849	CTTTCTGTGCTCATGGTGTC	111	4521-4540	AUG
14850	GCGGATCATGCTTTCTGTGC	112	4531-4550	coding
14851	GGGAGGCCATTTGGGAACTT	113	5225-5244	junction
14852	CGAATTTTGAGAAGATGATC	114	5457-5476	junction
14846	GAGCTTCTGCTGGCTGGCTG	108	4351-437C	5′-UTR
14853	CTCCTCCACTTGGTGGTTTG	115	5799-5818	junction
14854	CCTGAGATCTTATCCAGCCT	116	6540-6559	3′-UTR
14855	CAATTACAGTCACGGCTCCC	117	6927-6946	3′-UTR
15921	CCCTTCATTCTCAAGGCACA	118	5521-5540	junction
15922	CACCCCTCAACCCGCCCCCC	119	5551-5570	intron
15923	AGAGCTCTGTCTTTTCTCAG	120	5581-5600	intron
15924	CACTGCTCTGACTCTCACGT	121	5611-5630	intron
15925	ATGAGGTCCCGGGTGGCCCC	122	5651-5670	intron
15926	CACCCTCTGTCTTTCCACAT	123	5681-5700	intron
15927	CTCCACATCCTGAGCCTCAG	124	5731-5750	intron
15928	ATTGAGTCAGTGTCACCCTC	125	5761-5780	intron
15929	GCTGGCTCAGCCACTCCAGC	126	5821-5840	coding
15930	TCTTTGAGATCCATGCCGTT	127	5861-5880	coding
15931	AACCCATCGGCTGGCACCAC	128	5891-5910	coding
15932	GTTTGAGCTCAGCCCCCTCA	129	6061-6080	coding
15933	CTCCTCCCAGGTATATGGGC	130	6091-6110	coding
15934	TGAGTTGGTCCCCCTTCTCC	131	6121-6140	coding
15935	CAAAGTAGACCTGCCCGGAC	132	6181-6200	coding
15936	ACACCCATTCCCTTCACAGA	133	6211-6230	STOP
15937	CATAATCCCCTTTCTAAGTT	134	6321-6340	3′-UTR
15938	CACAGAGTTGGACTCTGAGC	135	6341-6360	3′-UTR
15939	CAGCATCTTGTGTTTCTGAG	136	6381-6400	3′-UTR
15940	CACAGTCCAGGTCACTGTCC	137	6401-6420	3′-UTR
15941	TGATGGTGGTGCATGAGAGG	138	6423-6442	3′-UTR
15942	GTGAATTCGGAAAGCCCATT	139	6451-6470	3′-UTR
15943	CCTGACCACTCTCCCTTTGC	140	6501-6520	3′-UTR
3082	TG C AT CCCCC AGG CC A CC AT	141	target control
13108	GCCGAGGTCCATGTCGTACGC	142	target control
1 All “C” residues are 5-methyl-cytosines except underlined “ C ”residues are unmodified cytosines; all linkages are phosphorothioate linkages.
2 Co-ordinates from Genbank Accession No. Y00467, locus name “MMTNFAB”, SEQ ID NO. 107.

Results are shown in Table 19. Oligonucleotides 14853 (SEQ ID NO. 115), 14854 (SEQ ID NO. 116), 14855 (SEQ ID NO. 117), 15921 (SEQ ID NO. 118), 15923 (SEQ ID NO. 120), 15924 (SEQ ID NO. 121), 15925 (SEQ ID NO. 122), 15926 (SEQ ID NO. 123), 15929 (SEQ ID NO. 126), 15930 (SEQ ID NO. 127), 15931 (SEQ ID NO. 128), 15932 (SEQ ID NO. 129), 15934 (SEQ ID NO. 131), 15935 (SEQ ID NO. 132), 15936 (SEQ ID NO. 133), 15937 (SEQ ID NO. 134), 15939 (SEQ ID NO. 136), 15940 (SEQ ID NO. 137), 15942 (SEQ ID NO. 139), and 15943 (SEQ ID NO. 140) gave better than 50% inhibition. Oligonucleotides 15931 (SEQ ID NO. 128), 15932 (SEQ ID NO. 129), 15934 (SEQ ID NO. 131), and 15943 (SEQ ID NO. 140) gave 75% inhibition or better.

TABLE 19
Inhibition of Mouse TNF-α mRNA expression in P388D1 Cells
by Phosphorothioate Oligodeoxynucleotides
	SEQ	GENE
ISIS	ID	TARGET	% mRNA	% mRNA
No:	NO:	REGION	EXPRESSION	INHIBITION
induced	—	—	100%	0%
3082	141	control	129%	—
13664	42	control	85%	15%
14846	108	5′-UTR	84%	16%
14847	109	5′-UTR	88%	12%
14848	110	AUG	60%	40%
14849	111	AUG	75%	25%
14850	112	coding	67%	33%
14851	113	junction	62%	38%
14852	114	junction	69%	31%
14853	115	junction	49%	51%
14854	116	3′-UTR	31%	69%
14855	117	3′-UTR	39%	61%
15921	118	junction	42%	58%
15922	119	intron	64%	36%
15923	120	intron	31%	69%
15924	121	intron	29%	71%
15925	122	intron	30%	70%
15926	123	intron	29%	71%
15928	125	intron	59%	41%
15929	126	coding	38%	62%
15930	127	coding	43%	57%
15931	128	coding	23%	77%
15932	129	coding	25%	75%
15933	130	coding	52%	48%
15934	131	coding	21%	79%
15935	132	coding	39%	61%
15936	133	STOP	35%	65%
15937	134	3′-UTR	45%	55%
15938	135	3′-UTR	76%	24%
15939	136	3′-UTR	33%	67%
15940	137	3′-UTR	38%	62%
15941	138	3′-UTR	54%	46%
15942	139	3′-UTR	42%	58%
15943	140	3′-UTR	25%	75%

Example 9

Dose Response of Antisense Phosphorothiaote Oligodeoxynucleotide Effects on Mouse TNF-α mRNA Levels in P388D1 Cells

Four of the more active oligonucleotides from the initial screen were chosen for dose response assays. These include oligonucleotides 15924 (SEQ ID NO. 121), 15931 (SEQ ID NO. 128), 15934 (SEQ ID NO. 131) and 15943 (SEQ ID NO. 140). P388D1 cells were grown, treated and processed as described in Example 8. LIPOFECTIN® was added at a ratio of 3 μg/ml per 100 nM of oligonucleotide. The control included LIPOFECTIN® at a concentration of 6 μg/ml. Results are shown in Table 20. Each oligonucleotide tested showed a dose response effect with maximal inhibition about 70% or greater and IC 50 values less than 50 nM.

TABLE 20
Dose Response of LPS-Induced P388D1 Cells to TNF-α
Antisense Phosphorothioate Oligodeoxynucleotides (ASOs)
	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition
induced	—	—	—	100%	—
13108	142	control	25 nM	68%	32%
″	″	″	50 nM	71%	29%
″	″	″	100 nM	64%	36%
″	″	″	200 nM	75%	25%
15924	121	intron	25 nM	63%	37%
″	″	″	50 nM	49%	51%
″	″	″	100 nM	36%	64%
″	″	″	200 nM	31%	69%
15931	128	coding	25 nM	42%	58%
″	″	″	50 nM	30%	70%
″	″	″	100 nM	17%	83%
″	″	″	200 nM	16%	84%
15934	131	coding	25 nM	37%	63%
″	″	″	50 nM	26%	74%
″	″	″	100 nM	13%	87%
″	″	″	200 nM	13%	87%
15943	140	3′-UTR	25 nM	38%	62%
″	″	″	50 nM	38%	62%
″	″	″	100 nM	16%	84%
″	″	″	200 nM	16%	84%

Example 10

Design and Testing of 2′-O-methoxyethyl (Deoxy Gapped) TNF-α Antisense Oligonucleotides on TNF-α Levels in P388D1 Cells

Oligonucleotides having SEQ ID NO: 128, SEQ ID NO: 131, and SEQ ID NO: 140 were synthesized as uniformly phosphorothioate oligodeoxynucleotides or mixed phosphorothioate/phosphodiester chimeric oligonucleotides having variable regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. The sequences and the oligonucleotide chemistries are shown in Table 21. All 2′-MOE cytosines were 5-methyl-cytosines.

Oligonucleotides were screened as described in Example 8. Results are shown in Table 22. All the oligonucleotides tested, except oligonucleotide 16817 (SEQ ID NO. 140) showed 44% or greater inhibition of TNF-α mRNA expression. Oligonucleotides 16805 (SEQ ID NO: 131), 16813 (SEQ ID NO: 140), and 16814 (SEQ ID NO: 140) showed greater than 70% inhibition.

TABLE 21
Nucleotide Sequences of Mouse 2′-O-methoxyethyl (deoxy gapped)
TNF-αOligonucleotides
		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE 1	ID	NUCLEOTIDE	TARGET
NO.	(5′ -> 3′)	NO:	CO-ORDINATES 2	REGION
15931	AsAsCsCsCsAsTsCsGsGsCsTsGsGsCsAsCsCsAsC	128	5891-5910	coding
16797	A o A o C o C sCsAsTsCsGsGsCsTsGsGsCsAs C o C o A o C	″	5891-5910	coding
16798	A s A s C s C   sCsAsTsCsGsGsCsTsGsGsCsAs C s C s A s C	″	5891-5910	coding
16799	A o A o C o C o C sAsTsCsGsGsCsTsGsGsCs A o C o C o A o C	″	5891-5910	coding
16800	A s A s C s C s C sAsTsCsGsGsCsTsGsGsCs A s C s C s A s C	″	5891-5910	coding
16801	A o A o C o C o C o A o T o C o G sGsCsTsGsGsCsAsCsCsAsC	″	5891-5910	coding
16802	A s A s C s C s C s A s T s C s G sGsCsTsGsGsCsAsCsCsAsC	″	5891-5910	coding
16803	AsAsCsCsCsAsTsCsGsGsCs T o G o G o C o A o C o C o A o C	″	5891-5910	coding
16804	AsAsCsCsCsAsTsCsGsGsCs T s G s G s C s A s C s C s A s C	″	5891-5910	coding
15934	TsGsAsGsTsTsGsGsTsCsQsCsCsCsTsTsCsTsCsC	131	6121-6140	coding
16805	T o G o A o G sTsTsGsGsTsCsCsCsCsCsTsTs C o T o C o C	″	6121-6140	coding
16806	T s G s A s G sTsTsGsGsTsCsCsCsCsCsTsTs C s T s C s C	″	6121-6140	coding
16807	T o G o A o G o T sTsGsGsTsCsCsCsCsCsTs T o C o T o C o C	″	6121-6140	coding
16808	T s G s A s G s T sTsGsGsTsCsCsCsCsCsTs T s C s T s C s C	″	6121-6140	coding
16809	T o G o A o G o T o T o G o G o T sCsCsCsCsCsTsTsCsTsCsC	″	6121-6140	coding
16810	T s G s A s G s T s T s G s G s T sCsCsCsCsCsTsTsCsTsCsC	″	6121-6140	coding
16811	TsGsAsGsTsTsGsGsTsCsCs C o C o C o T o T o C o T o C o C	″	6121-6140	coding
16812	TsGsAsGsTsTsGsGsTsCsCs C s C s C s T s T s C s T s C s C	″	6121-6140	coding
15943	CsCsTsGsAsCsCsAsCsTsCsTsCsCsCsTsTsTsGsC	140	6501-6520	3′-UTR
16813	C o C o T o G sAsCsCsAsCsTsCsTsCsCsCsTs T o T o G o C	″	6501-6520	3′-UTR
16814	C s C s T s G sAsCsCsAsCsTsCsTsCsCsCsTs T s T s G s C	″	6501-6520	3′-UTR
16815	C o C o T o G o A sCsCsAsCsTsCsTsCsCsCs T o T o T o G o C	″	6501-6520	3′-UTR
16816	C s C s T s G s A sCsCsAsCsTsCsTsCsCsCs T s T s T s G s C	″	6501-6520	3′-UTR
16817	C o C o T o G o A o C o C o A o C sTsCsTsCsCsCsTsTsTsGsC	″	6501-6520	3′-UTR
16818	C s C s T s G s A s C s C s A s C sTsCsTsCsCsCsTsTsTsGsC	″	6501-6520	3′-UTR
16819	CsCsTsGsAsCsCsAsCsTsCs T o C o C o C o T o T o T o G o C	″	6501-6520	3′-UTR
16820	CsCsTsGsAsCsCsAsCsTsCs T s C s C s C s T s T s T s G s C	″	6501-6520	3′-UTR
1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethoxy cytidines are 5-methyl-cytidines; “s” linkages are phosphorothioate linkages, “o ” linkages are phosphodiester linkages,“o” linkages are phosphodiester linkages.
2 Co-ordinates from Genbank Accession No. Y00467, locus name “MMTNFAB”, SEQ ID NO. 107.

TABLE 22
Inhibition of mouse TNF-α mRNA expression in P388D1 Cells
by 2′-O-methoxyethyl (deoxy gapped) Oligonucleotides
	SEQ	GENE
ISIS	ID	TARGET	% mRNA	% mRNA
No:	NO:	REGION	EXPRESSION	INHIBITION
induced	—	13	100%	0%
13108	142	control	87%	13%
15934	131	coding	28%	72%
16797	128	coding	33%	67%
16798	″	coding	34%	66%
16799	″	coding	56%	44%
16800	″	coding	35%	65%
16801	″	coding	34%	66%
16802	″	coding	38%	62%
16803	″	coding	35%	65%
16804	″	coding	39%	61%
16805	131	coding	29%	71%
16806	″	coding	31%	69%
16807	″	coding	46%	54%
16808	″	coding	43%	57%
16809	″	coding	33%	67%
16810	″	coding	37%	63%
16811	″	coding	40%	60%
16812	″	coding	31%	69%
16813	140	3′-UTR	28%	72%
16814	″	3′-UTR	28%	72%
16815	″	3′-UTR	46%	54%
16816	″	3′-UTR	49%	51%
16817	″	3′-UTR	172%	—
16818	″	3′-UTR	34%	66%
16819	″	3′-UTR	51%	49%
16820	″	3′-UTR	44%	56%

Example 11

Effect of TNF-α Antisense Oligonucleotides in a Murine Model for Non-Insulin-dependent Diabetes Mellitus

The db/db mouse model, a standard model for non-insulin-dependent diabetes mellitus (NIDDM; Hotamisligil, G. S., et al., Science, 1993, 259, 87-90), was used to assess the activity of TNF-α antisense oligonucleotides on blood glucose levels and TNF-α mRNA levels in whole mice. These mice have elevated blood glucose levels and TNF-α mRNA levels compared to wild type mice. Female db/db mice and wild-type littermates were purchased from Jackson Laboratories (Bar Harbor, Me.). The effect on oligonucleotide 15931 (SEQ ID NO. 128) on blood glucose levels was determined. For determination of TNF-α mRNA levels, oligonucleotide 15931 (SEQ ID NO. 128), a uniformly modified phosphorothioate oligodeoxynucleotide, was compared to oligonucleotide 25302 (SEQ ID NO. 128), a mixed phosphorothioate/phosphodiester chimeric oligonucleotide having regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides. The sequences and chemistries are shown in Table 23. Oligonucleotide 18154 (SEQ ID NO. 143) is an antisense mixed phosphorothioate/phosphodiester chimeric oligonucleotide, having regions of 2′-O-methoxyethyl (2′-MOE) nucleotides and deoxynucleotides, targeted to the human vascular cell adhesion molecule-1 (VCAM-1) and was used as an unrelated target control.

TABLE 23
Nucleotide Sequence of TNF-α Antisense
Oligonucleotide
		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE 1	ID	NUCLEOTIDE	TARGET
NO.	(5′ -> 3′)	NO:	CO-ORDINATES 2	REGION
15931	AACCCATCGGCTGGCACCAC	128	5891-5910	coding
25302	AACCC ATCGGCTGGC ACCAC	128	5891-5910	coding
18154	TCAAG CAGTGCCACC GATCC	143	target control
1 All 2′-methoxyethyl cytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages.
2 Co-ordinates from Genbank Accession No. Y00467, locus name “MNTNFAB”, SEQ ID NO. 107.

db/db mice, six to ten weeks old, were dosed intraperitoneally with oligonucleotide every other day for 2 weeks at 10 mg/kg. The mice were fasted for seven hours prior to administration of the oligonucleotide. The mice were bled via retro orbital sinus every other day, and glucose measurements were performed on the blood. Results are shown in Table 24. Oligonucleotide 15931 (SEQ ID NO. 128) was able to reduce blood glucose levels in db/db mice to levels comparable with wild type mice. Food intake between wild type mice, treated and untreated, did not differ. Food intake between db/db mice, treated and untreated, although higher than wild type mice, did not differ significantly.

Samples of the fat (adipose) tissue from the inguinal fat pads were taken for RNA extraction. RNA was extracted according to Current Protocols in Molecular Biology, 1997, Ausubel, F., et al. ed., John Wiley & Sons. RNA was purified using the RNA clean up procedure of the RNEASY® Mini kit (Qiagen, Valencia, Calif.). TNF-α mRNA levels were measured using the RIBOQUANT® kit (PharMingen, San Diego, Calif.) with 15 μg of RNA per lane. The probe used was from the mCK-3b Multi-Probe Template set (PharMingen, San Diego, Calif.) labeled with [α 32 P]UTP (Amersham Pharmacia Biotech, Piscataway, N.J.). Results are shown in Table 25. Both oligonucleotide 15931 (SEQ ID NO. 128) and 25302 (SEQ ID NO. 128) were able to reduce TNF-α levels in fat, with 25302 (SEQ ID NO. 128) reducing TNF-α to nearly wild-type levels.

TABLE 24
Level of Blood Glucose in Normal and db/db Mice After
Treatment with TNF-α Antisense Oligonucleotides
			ASO		blood
Mouse		SEQ ID	Gene	Time	glucose
Strain	ISIS #	NO:	Target	(days)	(mg/dL)
wild type	—	—	—	1	140
″	15931	128	coding	″	138
db/db	—	—	—	1	260
″	15931	128	coding	″	254
wild type	—	—	—	9	175
″	15931	128	coding	″	163
db/db	—	—	—	9	252
″	15931	128	coding	″	128

TABLE 24
Level of Blood Glucose in Normal and db/db Mice After
Treatment with TNF-α Antisense Oligonucleotides
			ASO		blood
Mouse		SEQ ID	Gene	Time	glucose
Strain	ISIS #	NO:	Target	(days)	(mg/dL)
wild type	—	—	—	1	140
″	15931	128	coding	″	138
db/db	—	—	—	1	260
″	15931	128	coding	″	254
wild type	—	—	—	9	175
″	15931	128	coding	″	163
db/db	—	—	—	9	252
″	15931	128	coding	″	128

Example 12

Effect of TNF-α Antisense Oligonucleotides in a Murine Model for Rheumatoid Arthritis

Collagen-induced arthritis (CIA) was used as a murine model for arthritis (Mussener, A., et al., Clin. Exp. Immunol., 1997, 107, 485-493). Female DBA/1LacJ mice (Jackson Laboratories, Bar Harbor, Me.) between the ages of 6 and 8 weeks were used to assess the activity of TNF-α antisense oligonucleotides.

On day 0, the mice were immunized at the base of the tail with 100 μg of bovine type II collagen which is emulsified in Complete Freund's Adjuvant (CFA). On day 7, a second booster dose of collagen was administered by the same route. On day 14, the mice were injected subcutaneously with 100 μg of LPS. Oligonucleotide was administered intraperitoneally daily (10 mg/kg bolus) starting on day −3 ( three days before day 0) and continuing for the duration of the study.

Weights were recorded weekly. Mice were inspected daily for the onset of CIA. Paw widths are rear ankle widths of affected and unaffected joints were measured three times a week using a constant tension caliper. Limbs were clinically evaluated and graded on a scale from 0-4 (with 4 being the highest).

Oligonucleotide 25302 (SEQ ID NO. 128) was compared to a saline control. The antisense TNF-α oligonucleotide reduced the incidence of CIA from 70% for the saline control to 40% for the oligonucleotide. The severity of the disease (based on the mean score of the limbs) was also reduced from 3.2 for the saline control to 2.1 for the oligonucleotide.

Example 13

Effect of TNF-α Antisense Oligonucleotides in a Murine Model for Contact Sensitivity

Contact sensitivity is a type of immune response resulting from contact of the surface of the skin with a sensitizing chemical. A murine model for contact sensitivity is widely used to develop therapies for chronic inflammation, autoimmune disorder, and organ transplant rejection (Goebeler, M., et al., Int Arch. Allergy Appl. Immunol., 1990, 93, 294-299). One example of such a disease is atopic dermatitis. Female Balb/c mice between the ages of 8 and 12 weeks are used to assess the activity of TNF-α antisense oligonucleotides in a contact sensitivity model.

Balb/c mice receive injections of oligonucleotide drug in saline via i.v. injection into the tail vein. The abdomen of the mice is shaved using an Oster hair clipper. The animals are anesthesized using isoflurane, and 25 μl of 0.2% 2,4-dinitrofluorobenzene (DNFB) in 4:1 acetone:olive oil is applied to the shaved abdomen two days in a row. After five days, 10 ml of 0.2% DNFB in the same vehicle is applied to the right ear. After each exposure, the mouse is suspended in air for two minutes to allow the DNFB to absorb into the skin. 24 and 48 hours after application of DNFB to the ear, the ear thickness is measured using a micrometer. Inflammation (dermatitis) is indicated by a ranked thickening of the ear. Thickness of the treated ear is compared to untreated (contralateral) ear thickness.

Example 14

Effect of TNF-α Antisense Oligonucleotides in a Murine Model for Crohn's Disease

C3H/HeJ, SJL/JK and IL10−/− mice are used in a TNBS (2,4,5,-trinitrobenzene sulfonic acid) induced colitis model for Crohn's disease (Neurath, M. F., et al., J. Exp. Med., 1995, 182, 1281-1290). Mice between the ages of 6 weeks and 3 months are used to assess the activity of TNF-α antisense oligonucleotides.

C3H/HeJ, SJL/JK and IL10−/− mice are fasted overnight prior to administration of TNBS. A thin, flexible polyethylene tube is slowly inserted into the colon of the mice so that the tip rests approximately 4 cm proximal to the anus. 0.5 mg of the TNBS in 50% ethanol is slowly injected from the catheter fitted onto a 1 ml syringe. Animals are held inverted in a vertical position for approximately 30 seconds. TNF-α antisense oligonucleotides are administered either at the first sign of symptoms or simultaneously with induction of disease.

Animals, in most cases, are dosed every day. Administration is by i.v., i.p., s.q., minipumps or intracolonic injection. Experimental tissues are collected at the end of the treatment regimen for histochemical evaluation.

Example 15

Effect of TNF-α Antisense Oligonucleotides in a Murine Model for Multiple Sclerosis

Experimental autoimmune encephalomyelitis (EAE) is a commonly accepted murine model for multiple sclerosis (Myers, K. J., et al., J. Neuroimmunol., 1992, 41, 1-8). SJL/H, PL/J, (SJLxPL/J)F1, (SJLxBalb/c)F1 and Balb/c female mice between the ages of 6 and 12 weeks are used to test the activity of TNF-α antisense oligonucleotides.

The mice are immunized in the two rear foot pads and base of the tail with an emulsion consisting of encephalitogenic protein or peptide (according to Myers, K. J., et al., J. of Immunol., 1993, 151, 2252-2260) in Complete Freund's Adjuvant supplemented with heat killed Mycobacterium tuberculosis. Two days later, the mice receive an intravenous injection of 500 ng Bordatella pertussis toxin and additional adjuvant.

Alternatively, the disease may also be induced by the adoptive transfer of T-cells. T-cells are obtained from the draining of the lymph nodes of mice immunized with encephalitogenic protein or peptide in CFA. The T cells are grown in tissue culture for several days and then injected intravenously into naive syngeneic recipients.

Mice are monitored and scored daily on a 0-5 scale for signals of the disease, including loss of tail muscle tone, wobbly gait, and various degrees of paralysis.

Example 16

Effect of TNF-α Antisense Oligonucleotides in a Murine Model for Pancreatitis

Swiss Webster, C57BL/56, C57BL/6 lpr and gld male mice are used in an experimental pancreatitis model (Niederau, C., et al., Gastroenterology, 1985, 88, 1192-1204). Mice between the ages of 4 and 10 weeks are used to assess the activity of TNF-α antisense oligonucleotides.

Caerulin (5-200 μg/kg) is administered i.p. every hour for one to six hours. At varying time intervals, the mice are given i.p. injection of avertin and bled by cardiac puncture. The pancreas and spleen are evaluated for histopathology and increased levels of IL-1β, IL-6, and TNF-α. The blood is analyzed for increased levels of serum amylase and lipase. TNF-α antisense oligonucleotides are administered by intraperitoneal injection at 4 hours pre-caerulin injections.

Example 17

Effect of TNF-α Antisense Oligonucleotides in a Murine Model for Hepatitis

Concanavalin A-induced hepatitis is used as a murine model for hepatitis (Mizuhara, H., et al., J. Exp. Med., 1994, 179, 1529-1537). It has been shown that this type of liver injury is mediated by Fas (Seino, K., et al., Gastroenterology 1997, 113, 1315-1322). Certain types of viral hepatitis, including Hepatitis C, are also mediated by Fas ( J. Gastroenterology and Hepatology, 1997, 12, S223-S226). Female Balb/c and C57BL/6 mice between the ages of 6 weeks and 3 months are used to assess the activity of TNF-α antisense oligonucleotides.

Mice are intravenenously injected with oligonucleotide. The pretreated mice are then intravenously injected with 0.3 mg concanavalin A (Con A) to induce liver injury. Within 24 hours following Con A injection, the livers are removed from the animals and analyzed for cell death (apoptosis) by in vitro methods. In some experiments, blood is collected from the retro-orbital vein.

Example 18

Effect of Antisense Oligonucleotide Targeted to TNF-α on Survival in Murine Heterotopic Heart Transplant Model

To determine the therapeutic effects of TNF-α antisense oligonucleotides in preventing allograft rejection, murine TNF-α-specific oligonucleotides are tested for activity in a murine vascularized heterotopic heart transplant model. Hearts from Balb/c mice are transplanted into the abdominal cavity of C3H mice as primary vascularized grafts essentially as described by Isobe et al., Circulation 1991, 84, 1246-1255. Oligonucleotide is administered by continuous intravenous administration via a 7-day Alzet pump. The mean survival time for untreated mice is usually approximately 9-10 days. Treatment of the mice for 7 days with TNF-α antisense oligonucleotides is expected to increase the mean survival time.

Example 19

Optimization of Human TNF-α Antisense Oligonucleotide

Additional antisense oligonucleotides targeted to intron 1 of human TNF-α were designed. These are shown in Table 26. Oligonucleotides are screened by RT-PCR as described in Example 5 hereinabove.

TABLE 26
Nucleotide Sequences of Human TNF-αIntron 1
Antisense Oligonucleotides
			TARGET GENE
		SEQ	NUCLEOTIDE	GENE
ISIS	NUCLEOTIDE SEQUENCE 1	ID	CO-	TARGET
NO.	(5′ -> 3′)	NO	NUCLEOTIDE	REGION
100181	AG TGTCTTCTGTGTGCCA GA	144	1409-1428	intron 1
100201	AG TGTCTTCTGTGTGC CAGA	″	″	intron 1
100230	AGTG TCTTCTGTGTGCCA GA	″	″	intron 1
100250	AGTG TCTTCTGTGTGC CAGA	″	″	intron 1
100182	GT GTCTTCTGTGTGCCAG AC	145	1408-1427	intron 1
100202	GT GTCTTCTGTGTGCC AGAC	″	″	intron 1
100231	GTGT CTTCTGTGTGCCAG AC	″	″	intron 1
100251	GTGT CTTCTGTGTGCC AGAC	″	″	intron 1
100183	TG TCTTCTGTGTGCCAGA CA	146	1407-1426	intron 1
100203	TG TCTTCTGTGTGCCA GACA	″	″	intron 1
100232	TGTC TTCTGTGTGCCAGA CA	″	″	intron 1
100252	TGTC TTCTGTGTGCCA GACA	″	″	intron 1
100184	GT CTTCTGTGTGCCAGAC AC	147	1406-1425	intron 1
100204	GT CTTCTGTGTGCCAG ACAC	″	″	intron 1
100233	GTCT TCTGTGTGCCAGAC AC	″	″	intron 1
100253	GTCT TCTGTGTGCCAG ACAC	″	″	intron 1
100185	TC TTCTGTGTGCCAGACA CC	148	1405-1424	intron 1
100205	TC TTCTGTGTGCCAGA CACC	″	″	intron 1
100234	TCTT CTGTGTGCCAGACA CC	″	″	intron 1
100254	TCTT CTGTGTGCCAGA CACC	″	″	intron 1
100186	CT TCTGTGTGCCAGACAC CC	149	1404-1423	intron 1
100206	CT TCTGTGTGCCAGAC ACCC	″	″	intron 1
100235	CTTC TGTGTGCCAGACAC CC	″	″	intron 1
100255	CTTC TGTGTGCCAGAC ACCC	″	″	intron 1
100187	TT CTGTGTGCCAGACACC CT	150	1403-1422	intron 1
100207	TT CTGTGTGCCAGACA CCCT	″	″	intron 1
100236	TTCT GTGTGCCAGACACC CT	″	″	intron 1
100256	TTCT GTGTGCCAGACA CCCT	″	″	intron 1
100188	TC TGTGTGCCAGACACCC TA	151	1402-1421	intron 1
100208	TC TGTGTGCCAGACAC CCTA	″	″	intron 1
100237	TCTG TGTGCCAGACACCC TA	″	″	intron 1
100257	TCTG TGTGCCAGACAC CCTA	″	″	intron 1
100189	CT GTGTGCCAGACACCCT AT	152	1401-1420	intron 1
100209	CT GTGTGCCAGACACC CTAT	″	″	intron 1
100238	CTGT GTGCCAGACACCCT AT	″	″	intron 1
100258	CTGT GTGCCAGACACC CTAT	″	″	intron 1
100190	TG TGTGCCAGACACCCTA TC	153	1400-1419	intron 1
100210	TG TGTGCCAGACACCC TATC	″	″	intron 1
100239	TGTG TGCCAGACACCCTA TC	″	″	intron 1
100259	TGTG TGCCAGACACCC TATC	″	″	intron 1
100191	TG TGCCAGACACCCTATC TT	154	1398-1417	intron 1
100211	TG TGCCAGACACCCTA TCTT	″	″	intron 1
100240	TGTG CCAGACACCCTATC TT	″	″	intron 1
100260	TGTG CCAGACACCCTA TCTT ″	″	intron 1
100192	GT GCCAGACACCCTATCT TC	155	1397-1416	intron 1
100212	GT GCCAGACACCCTAT CTTC	″	″	intron 1
100241	GTGC CAGACACCCTATCT TC	″	″	intron 1
100261	GTGC CAGACACCCTAT CTTC	″	″	intron 1
100193	TG CCAGACACCCTATCTT CT	156	1396-1415	intron 1
100213	TG CCAGACACCCTATC TTCT	″	″	intron 1
100242	TGCC AGACACCCTATCTT CT	″	″	intron 1
100262	TGCC AGACACCCTATC TTCT	″	″	intron 1
100194	GC CAGACACCCTATCTTC TT	157	1395-1414	intron 1
100214	GC CAGACACCCTATCT TCTT	″	″	intron	1
100243	GCCA GACACCCTATCTTC TT	″	″	intron 1
100263	GCCA GACACCCTATCT TCTT	″	″	intron 1
100195	CC AGACACCCTATCTTCT TC	158	1394-1413	intron 1
100215	CC AGACACCCTATCTT CTTC	″	″	intron 1
100244	CCAG ACACCCTATCTTCT TC	″	″	intron 1
100264	CCAG ACACCCTATCTT CTTC	″	″	intron 1
100196	CA GACACCCTATCTTCTT CT	159	1393-1412	intron 1
100216	CA GACACCCTATCTTC TTCT	″	″	intron 1
100245	CAGA CACCCTATCTTCTT CT	″	″	intron 1
100265	CAGA CACCCTATCTTC TTCT	″	″	intron 1
100197	AG ACACCCTATCTTCTTC TC	160	1392-1411	intron 1
100217	AG ACACCCTATCTTCT TCTC	″	″	intron 1
100246	AGAC ACCCTATCTTCTTC TC	″	″	intron 1
100266	AGAC ACCCTATCTTCT TCTC	″	″	intron 1
100198	GA CACCCTATCTTCTTCT CT	161	1391-1410	intron 1
100218	GA CACCCTATCTTCTT CTCT	″	″	intron 1
100247	GACA CCCTATCTTCTTCT CT	″	″	intron 1
100267	GACA CCCTATCTTCTT CTCT	″	″	intron 1
100199	AC ACCCTATCTTCTTCTC TC	162	1390-1409	intron 1
100219	AC ACCCTATCTTCTTCTC TC	″	″	intron 1
100248	ACAC CCTATCTTCTTCTC TC	″	″	intron 1
100268	ACAC CCTATCTTCTTC TCTC	″	″	intron 1
100200	CA CCCTATCTTCTTCTCT CC	163	1389-1408	intron 1
100220	CA CCCTATCTTCTTCT CTCC	″	″	intron 1
100249	CACC CTATCTTCTTCTCT CC	″	″	intron 1
100269	CACC CTATCTTCTTCT CTCC	″	″	intron 1
100270	GTCTTCTGTGTGCCA GAC	164	1408-1425	intron 1
100271	TCT TCTGTGTGCCAG ACA	165	1407-1424	intron 1
100272	CTT CTGTGTGCCAGA CAC	166	1406-1423	intron 1
100273	TTC TGTGTGCCAGAC ACC	167	1405-1422	intron 1
100274	TCT GTGTGCCAGACA CCC	168	1404-1421	intron 1
100275	CTG TGTGCCAGACAC CCT	169	1403-1420	intron 1
100276	TGT GTGCCAGACACC CTA	170	1402-1419	intron 1
100277	GTG TGCCAGACACCC TAT	171	1401-1418	intron 1
100278	TGT GCCAGACACCCT ATC	172	1400-1417	intron 1
100279	TGC CAGACACCCTAT CTT	173	1398-1415	intron 1
100280	GCC AGACACCCTATC TTC	174	1397-1414	intron 1
100281	CCA GACACCCTATCT TCT	175	1396-1413	intron 1
100282	CAG ACACCCTATCTT CTT	176	1395-1412	intron 1
100283	AGA CACCCTATCTTC TTC	177	1394-1411	intron 1
100284	GAC ACCCTATCTTCT TCT	178	1393-1410	intron 1
100285	ACA CCCTATCTTCTT CTC	179	1392-1409	intron 1
1 Emboldened residues are 2′-methoxyethoxy residue (others are 2′-deoxy-). All 2′methoxyethyl cytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages
2 Co-ordinates from Genbank Accession No. X02910, locus name “HSTNEA”, SEQ ID NO. 1.

Example 20

Design of Antisense Oligonucleotides Targeting Human TNF-α Intron 2

Additional antisense oligonucleotides targeted to intron 2 and coding regions of human TNF-α were designed. These are shown in Table 27. Oligonucleotides are screened by RT-PCR as described in Example 5 hereinabove.

TABLE 27
Nucleotide Sequences of Human TNF-αIntron 2
Antisense Oligonucleotides
			TARGET GENE
		SEQ	NUCLEOTIDE	GENE
ISIS	NUCLEOTIDE SEQUENCE 1	ID	CO-	TARGET
NO.	(5′ -> 3′)	NO	NUCLEOTIDE	REGION
100549	AGAGGTTTGGAGACACTTAC	180	1635-1654	intron 2
100566	AG AGGTTTGGAGACACTT AC	″	″	intron 2
100550	GAATTAGGAAAGAGGTTTGG	181	1645-1664	intron 2
100567	GA ATTAGGAAAGAGGTTT GG	″	″	intron 2
100551	CCCAAACCCAGAATTAGGAA	182	1655-1674	intron 2
100568	CC CAAACCCAGAATTAGG AA	″	″	intron 2
100552	TACCCCCAAACCCAAACCCA	183	1665-1684	intron 2
100569	TA CCCCCAAACCCAAACC CA	″	″	intron 2
100553	GTACTAACCCTACCCCCAAA	184	1675-1694	intron 2
100570	GT ACTAACCCTACCCCCA AA	″	″	intron 2
100554	TTCCATACCGGTACTAACCC	185	1685-1704	intron 2
100571	TT CCATACCGGTACTAAC CC	″	″	intron 2
100555	CCCCCACTGCTTCCATACCG	186	1695-1714	intron 2
100572	CC CCCACTGCTTCCATAC CG	″	″	intron 2
100556	CTTTAAATTTCCCCCACTGC	187	1705-1724	intron 2
100573	CT TTAAATTTCCCCCACT GC	″	″	intron 2
100557	AAGACCAAAACTTTAAATTT	188	1715-1734	intron 2
100571	AA GACCAAAACTTTAAAT TT	″	″	intron 2
100558	ATCCTCCCCCAAGACCAAAA	189	1725-1744	intron 2
100640	AT CCTCCCCCAAGACCAA AA	″	″	intron 2
100559	ACCTCCATCCATCCTCCCCC	190	1735-1754	intron 2
100641	AC CTCCATCCATCCTCCC CC	″	″	intron 2
100560	CCCTACTTTCACCTCCATCC	191	1745-1764	intron 2
100642	CC CTACTTTCACCTCCAT CC	″	″	intron 2
100561	GAAAATACCCCCCTACTTTC	192	1755-1774	intron 2
100643	GA AAATACCCCCCTACTT TC	″	″	intron 2
100562	AAACTTCCTAGAAAATACCC	193	1765-1784	intron 2
100644	AA ACTTCCTAGAAAATAC CC	″	″	intron 2
100563	TGAGACCCTTAAACTTCCTA	194	1775-1794	intron 2
100645	TG AGACCCTTAAACTTCC TA	″	″	intron 2
100564	AAGAAAAAGCTGAGACCCTT	195	1785-1804	intron 2
100646	AA GAAAAAGCTGAGACCC TT	″	″	intron 2
100565	GGAGAGAGAAAAGAAAAAGC	196	1795-1814	intron 2
100647	GG AGAGAGAAAAGAAAAA GC	″	″	intron 2
100575	TGAGCCAGAAGAGGTTGAGG	197	2665-2684	coding
100576	ATTCTCTTTTTGAGCCAGAA	198	2675-2694	coding
100577	TAAGCCCCCAATTCTCTTTT	199	2685-2704	coding
100578	GTTCCGACCCTAAGCCCCCA	200	2695-2714	coding
100579	CTAAGCTTGGGTTCCGACCC	201	2705-2724	coding
100580	GCTTAAAGTTCTAAGCTTGG	202	2715-2734	coding
100581	TGGTCTTGTTGCTTAAAGTT	203	2725-2744	coding
100582	TTCGAAGTGGTGGTCTTGTT	204	2735-2754	coding
100583	AATCCCAGGTTTCGAAGTGG	205	2745-2764	coding
100584	CACATTCCTGAATCCCAGGT	206	2755-2774	coding
100585	GTGCAGGCCACACATTCCTG	207	2765-2784	coding
100586	GCACTTCACTGTGCAGGCCA	208	2775-2794	coding
100587	GTGGTTGCCAGCACTTCACT	209	2785-2804	coding
100588	TGAATTCTTAGTGGTTGCCA	210	2795-2814	coding
100589	GGCCCCAGTTTGAATTCTTA	211	2805-2824	coding
100590	GAGTTCTGGAGGCCCCAGTT	212	2815-2834	coding
100591	AGGCCCCAGTGAGTTCTGGA	32	2825-2844	coding
100592	TCAAAGCTGTAGGCCCCAGT	214	2835-2854	coding
100593	ATGTCAGGGATCAAAGCTGT	215	2845-2864	coding
100594	CAGATTCCAGATGTCAGGGA	216	2855-2874	coding
100595	CCCTGGTCTCCAGATTCCAG	217	2865-2884	coding
100596	ACCAAAGGCTCCCTGGTCTC	218	2875-2894	coding
100597	TCTGGCCAGAACCAAAGGCT	219	2885-2904	coding
100598	CCTGCAGCATTCTGGCCAGA	220	2895-2914	coding
100599	CTTCTCAAGTCCTGCAGCAT	221	2905-2924	coding
100600	TAGGTGAGGTCTTCTCAAGT	222	2915-2934	coding
100601	TGTCAATTTCTAGGTGAGGT	223	2925-2944	coding
100602	GGTCCACTTGTGTCAATTTC	224	2935-2954	coding
100603	GAAGGCCTAAGGTCCACTTG	225	2945-2964	coding
100604	CTGGAGAGAGGAAGGCCTAA	226	2955-2974	coding
100605	CTGGAAACATCTGGAGAGAG	227	2965-2984	coding
100606	TCAAGGAAGTCTGGAAACAT	228	2975-2994	coding
100607	GCTCCGTGTCTCAAGGAAGT	229	2985-3004	coding
100608	ATAAATACATTCATCTGTAA	230	3085-3104	coding
100609	GGTCTCCCAAATAAATACAT	231	3095-3114	coding
100610	AGGATACCCCGGTCTCCCAA	232	3105-3124	coding
100611	TGGGTCCCCCAGGATACCCC	35	3115-3134	coding
100612	GCTCCTACATTGGGTCCCCC	234	3125-3144	coding
100613	AGCCAAGGCAGCTCCTACAT	235	3135-3154	coding
100614	AACATGTCTGAGCCAAGGCA	236	3145-3164	coding
100615	TTTCACGGAAAACATGTCTG	237	3155-3174	coding
100616	TCAGCTCCGTTTTCACGGAA	238	3165-3184	coding
100617	AGCCTATTGTTCAGCTCCGT	239	3175-3194	coding
100618	ACATGGGAACAGCCTATTGT	240	3185-3204	coding
100619	ATCAAAAGAAGGCACAGAGG	241	3215-3234	coding
100620	GTTTAGACAACTTAATCAGA	242	3255-3274	coding
100621	AATCAGCATTGTTTAGACAA	243	3265-3284	coding
100622	TTGGTCACCAAATCAGCATT	244	3275-3294	coding
100623	TGAGTGACAGTTGGTCACCA	245	3285-3304	coding
100624	GGCTCAGCAATGAGTGACAG	246	3295-3314	coding
100625	ATTACAGACACAACTCCCCT	247	3325-3344	coding
100626	TAGTAGGGCGATTACAGACA	248	3335-3354	coding
100627	CGCCACTGAATAGTAGGGCG	249	3345-3364	coding
100628	CTTTATTTCTCGCCACTGAA	250	3355-3374	coding
1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethyl cytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages.
2 Co-ordinates from Genbank Accession No. X02910, locus name “HSTNFA”, SEQ ID NO.1.

Several of these oligonucleotides were chosen for dose response studies. Cells were grown and treated as described in Example 3. Results are shown in Table 28. Each oligonucleotide tested showed a dose response curve with maximum inhibition greater than 75%.

TABLE 28
Dose Response of PMA-Induced neoHK Cells to TNF-α
Antisense Oligonucleotides (ASOs)
	SEQ ID	ASO Gene		% protein	% protein
ISIS #	NO:	Target	Dose	Expression	Inhibition
induced	—	—	—	100%	—
100235	149	intron 1	75 nM	77%	23%
″	″	″	150 nM	25%	75%
″	″	″	300 nM	6%	94%
100243	157	intron 1	75 nM	68%	32%
″	″	″	150 nM	15%	85%
″	″	″	300 nM	6%	94%
100263	157	intron 1	75 nM	79%	21%
″	″	″	150 nM	30%	70%
″	″	″	300 nM	23%	77%

Example 21

Optimization of Human TNF-α Antisense Oligonucleotide Chemistry

Analogs of oligonucleotides 21820 (SEQ ID NO. 66) and 21823 (SEQ ID NO. 69) were designed and synthesized to find an optimum gap size. The sequences and chemistries are shown in Table 29.

Dose response experiments were performed as described in Example 3. Results are shown in Table 30.

TABLE 29
Nucleotide Sequences of TNF-αChimeric Backbone
(deoxy gapped) Oligonucleotides
			TARGET GENE
		SEQ	NUCLEOTIDE	GENE
ISIS	NUCLEOTIDE SEQUENCE 1	ID	CO-	TARGET
NO.	(5′ -> 3′)	NO	NUCLEOTIDE	REGION
21820	ATATTTCCCGCTCTTTCTGT	66	1339-1358	intron 1
28086	AT ATTTCCCGCTCTTTCT GT	″	″	″
28087	ATAT TTCCCGCTCTTT CTGT	″	″	″
21823	GTGTGCCAGACACCCTATCT	69	1399-1418	intron 1
28088	GT GTGCCAGACACCCTAT CT	″	″	″
28089	GTGT GCCAGACACCCT ATCT	″	″	″
1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethoxy cytidines and 2′-deoxycytidines are 5-methyl-cytidines; all linkages are phosphorothioate linkages.
2 Co-ordinates from Genbank Accession No. X02910, locus name “HSTNFA ”, SEQ ID NO.1.

TABLE 29
Nucleotide Sequences of TNF-αChimeric Backbone
(deoxy gapped) Oligonucleotides
			TARGET GENE
		SEQ	NUCLEOTIDE	GENE
ISIS	NUCLEOTIDE SEQUENCE 1	ID	CO-	TARGET
NO.	(5′ -> 3′)	NO	NUCLEOTIDE	REGION
21820	ATATTTCCCGCTCTTTCTGT	66	1339-1358	intron 1
28086	AT ATTTCCCGCTCTTTCT GT	″	″	″
28087	ATAT TTCCCGCTCTTT CTGT	″	″	″
21823	GTGTGCCAGACACCCTATCT	69	1399-1418	intron 1
28088	GT GTGCCAGACACCCTAT CT	″	″	″
28089	GTGT GCCAGACACCCT ATCT	″	″	″
1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethoxy cytidines and 2′-deoxycytidines are 5-methyl-cytidines; all linkages are phosphorothioate linkages.
2 Co-ordinates from Genbank Accession No. X02910, locus name “HSTNFA ”, SEQ ID NO.1.

Example 22

Screening of Additional TNF-α Chimeric (Deoxy Gapped) Antisense Oligonucleotides

Additional oligonucleotides targeting the major regions of TNF-α were synthesized. Oligonucleotides were synthesized as uniformly phosphorothioate chimeric oligonucleotides having regions of five 2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and a central region of ten deoxynucleotides. Oligonucleotide sequences are shown in Table 31.

Oligonucleotides were screened as described in Example 5. Results are shown in Table 32.

TABLE 31
Nucleotide Sequence of Additional Human TNF-α
Chimeric (deoxy gapped) Antisense Oligonucleotides
	NUCLEOTIDE SEQUENCE 1		TARGET GENE NUCLEOTIDE	GENE TARGET
ISIS NO.	(5′->3′)	SEQ ID NO:	CO-ORDINATES 2	REGION
104649	CTGAG GGAGCGTCTG CTGGC	251	0616-0635	5′-UTR
104650	CCTTG CTGAGGGAGC GTCTG	252	0621-0640	5′-UTR
104651	CTGGT CCTCTGCTGT CCTTG	253	0636-0655	5′-UTR
104652	CCTCT GCTGTCCTTG CTGAG	254	0631-0650	5′-UTR
104653	TTCTC TCCCTCTTAG CTGGT	255	0651-0670	5′-UTR
104654	TCCCT CTTAGCTGGT CCTCT	256	0646-0665	5′-UTR
104655	TCTGA GGGTTGTTTT CAGGG	257	0686-0705	5′-UTR
104656	CTGTA GTTGCTTCTC TCCCT	258	0661-0680	5′-UTR
104657	ACCTG CCTGGCAGCT TGTCA	259	0718-0737	5′-UTR
104658	GGATG TGGCGTCTGA GGGTT	260	0696-0715	5′-UTR
104659	TGTGA GAGGAAGAGA ACCTG	261	0733-0752	5′-UTR
104660	GAGGA AGAGAACCTG CCTGG	262	0728-0747	5′-UTR
104661	AGCCG TGGGTCAGTA TGTGA	263	0748-0767	5′-UTR
104662	TGGGT CAGTATGTGA GAGGA	264	0743-0762	5′-UTR
104663	GAGAG GGTGAAGCCG TGGGT	265	0758-0777	5′-UTR
104664	TCATG GTGTCCTTTC CAGGG	266	0780-0799	AUG
104665	CTTTC AGTGCTCATG GTGTC	267	0790-0809	AUG
104666	TCATG CTTTCAGTGC TCATG	268	0795-0814	AUG
104667	ACGTC CCGGATCATG CTTTC	269	0805-0824	coding
104668	GCTCC ACGTCCCGGA TCATG	270	0810-0829	coding
104669	TCCTC GGCCAGCTCC ACGTC	271	0820-0839	coding
104670	GCGCC TCCTCGGCCA GCTCC	272	0825-0844	coding
104671	AGGAA CAAGCACCGC CTGGA	273	0874-0893	coding
104672	CAAGC ACCGCCTGGA GCCCT	274	0869-0888	coding
104673	AAGGA GAAGAGGCTG AGGAA	275	0889-0908	coding
104674	GAAGA GGCTGAGGAA CAAGC	276	0884-0903	coding
104675	CCTGC CACGATCAGG AAGGA	277	0904-0923	coding
104676	CACGA TCAGGAAGGA GAAGA	278	0899-0918	coding
104677	AAGAG CGTGGTGGCG CCTGC	279	0919-0938	coding
104678	CGTGG TGGCGCCTGC CACGA	280	0914-0933	coding
104679	AAGTG CAGCAGGCAG AAGAG	281	0934-0953	coding
104680	CAGCA GGCAGAAGAG CGTGG	282	0929-0948	coding
104681	GATCA CTCCAAAGTG CAGCA	283	0944-0963	coding
104682	GGGCC GATCACTCCA AAGTG	284	0949-0968	coding
104683	GGGCC AGAGGGCTGA TTAGA	285	1606-1625	coding
104684	AGAGG GCTGATTAGA GAGAG	286	1601-1620	coding
104685	GCTAC AGGCTTGTCA CTCGG	287	1839-1858	coding
104686	CTGAC TGCCTGGGCC AGAGG	288	1616-1635	E2/I2 3
104687	TACAA CATGGGCTAC AGGCT	289	1849-1868	coding
104688	AGCCA CTGGAGCTGC CCCTC	290	2185-2204	coding
104689	CTGGA GCTGCCCCTC AGCTT	291	2180-2199	coding
104690	TTGGC CCGGCGGTTC AGCCA	292	2200-2219	coding
104691	TTGGC CAGGAGGGCA TTGGC	293	2215-2234	coding
104692	CCGGC GGTTCAGCCA CTGGA	294	2195-2214	coding
104693	CTCAG CTCCACGCCA TTGGC	295	2230-2249	coding
104694	CAGGA GGGCATTGGC CCGGC	296	2210-2229	coding
104695	CTCCA CGCCATTGGC CAGGA	297	2225-2244	coding
104696	ACCAG CTGGTTATCT CTCAG	298	2245-2264	coding
104697	CTGGT TATCTCTCAG CTCCA	299	2240-2259	coding
104698	CCCTC TGATGGCACC ACCAG	300	2260-2279	coding
104699	TGATG GCACCACCAG CTGGT	301	2255-2274	coding
104700	TAGAT GAGGTACAGG CCCTC	302	2275-2294	coding
104701	AAGAG GACCTGGGAG TAGAT	303	2290-2309	coding
104702	GAGGT ACAGGCCCTC TGATG	304	2270-2289	coding
104703	CAGCC TTGGCCCTTG AAGAG	305	2305-2324	coding
104704	GACCT GGGAGTAGAT GAGGT	306	2285-2304	coding
104705	TTGGC CCTTGAAGAG GACCT	307	2300-2319	coding
104706	TGGTG TGGGTGAGGA GCACA	308	2337-2356	coding
104707	CGGCG ATGCGGCTGA TGGTG	309	2352-2371	coding
104708	TGGGT GAGGAGCACA TGGGT	310	2332-2351	coding
104709	TGGTC TGGTAGGAGA CGGCG	311	2367-2386	coding
104710	ATGCG GCTGATGGTG TGGGT	312	2347-2366	coding
104711	AGAGG AGGTTGACCT TGGTC	313	2382-2401	coding
104712	TGGTA GGAGACGGCG ATGCG	314	2362-2381	coding
104713	AGGTT GACCTTGGTC TGGTA	315	2377-2396	coding
104714	GGCTC TTGATGGCAG AGAGG	316	2397-2416	coding
104715	TCATA CCAGGGCTTG GCCTC	317	2446-2465	coding
104716	TTGAT GGCAGAGAGG AGGTT	318	2392-2411	coding
104717	CCCAG ATAGATGGGC TCATA	93	2461-2480	coding
104718	CCAGG GCTTGGCCTC AGCCC	94	2441-2460	coding
104719	AGCTG GAAGACCCCT CCCAG	319	2476-2495	coding
104720	ATAGA TGGGCTCATA CCAGG	320	2456-2475	coding
104721	CGGTC ACCCTTCTCC AGCTG	321	2491-2510	coding
104722	GAAGA CCCCTCCCAG ATAGA	322	2471-2490	coding
104723	ATCTC AGCGCTGAGT CGGTC	26	2506-2525	coding
104724	ACCCT TCTCCAGCTG GAAGA	323	2486-2505	coding
104725	TAGTC GGGCCGATTG ATCTC	90	2521-2540	coding
104726	AGCGC TGAGTCGGTC ACCCT	91	2501-2520	coding
104727	TCGGC AAAGTCGAGA TAGTC	324	2536-2554	coding
104728	GGGCC GATTGATCTC AGCGC	325	2516-2535	coding
104729	TAGAC CTGCCCAGAC TCGGC	326	2551-2570	coding
104730	AAAGT CGAGATAGTC GGGCC	327	2531-2550	coding
104731	GCAAT GATCCCAAAG TAGAC	328	2566-2585	coding
104732	CTGCC CAGACTCGGC AAAGT	329	2546-2565	coding
104733	CGTCC TCCTCACAGG GCAAT	330	2581-2600	stop
104734	GATCC CAAAGTAGAC CTGCC	88	2561-2580	coding
104735	GGAAG GTTGGATGTT CGTCC	331	2596-2615	3′-UTR
104736	TCCTC ACAGGGCAAT GATCC	332	2576-2595	stop
104737	GTTGA GGGTGTCTGA AGGAG	333	2652-2671	3′-UTR
104738	GTTGG ATGTTCGTCC TCCTC	334	2591-2610	stop
104739	TTTGA GCCAGAAGAG GTTGA	335	2667-2686	3′-UTR
104740	GAGGC GTTTGGGAAG GTTGG	336	2606-2625	3′-UTR
104741	GCCCC CAATTCTCTT TTTGA	337	2682-2701	3′-UTR
104742	GCCAG AAGAGGTTGA GGGTG	338	2662-2681	3′-UTR
104743	GGGTT CCGACCCTAA GCCCC	339	2697-2716	3′-UTR
104744	CAATT CTCTTTTTGA GCCAG	340	2677-2696	3′-UTR
104745	TAAAG TTCTAAGCTT GGGTT	341	2712-2731	3′-UTR
104746	CCGAC CCTAAGCCCC CAATT	342	2692-2711	3′-UTR
104747	GGTGG TCTTGTTGCT TAAAG	343	2727-2746	3′-UTR
104748	TTCTA AGCTTGGGTT CCGAC	344	2707-2726	3′-UTR
104749	CCCAG GTTTCGAAGT GGTGG	345	2742-2761	3′-UTR
104750	TCTTG TTGCTTAAAG TTCTA	346	2722-2741	3′-UTR
104751	CACAC ATTCCTGAAT CCCAG	347	2757-2776	3′-UTR
104752	GTTTC GAAGTGGTGG TCTTG	348	2737-2756	3′-UTR
104753	CTTCA CTGTGCAGGC CACAC	349	2772-2791	3′-UTR
104754	ATTCC TGAATCCCAG GTTTC	350	2752-2771	3′-UTR
104755	TAGTG GTTGCCAGCA CTTCA	351	2787-2806	3′-UTR
104756	CCCAG TTTGAATTCT TAGTG	352	2802-2821	3′-UTR
104757	CTGTG CAGGCCACAC ATTCC	353	2767-2786	3′-UTR
104758	GTGAG TTCTGGAGGC CCCAG	354	2817-2836	3′-UTR
104759	GTTGC CAGCACTTCA CTGTG	355	2782-2801	3′-UTR
104760	TTTGA ATTCTTAGTG GTTGC	356	2797-2816	3′-UTR
104761	AAGCT GTAGGCCCCA GTGAG	357	2832-2851	3′-UTR
104762	TTCTG GAGGCCCCAG TTTGA	358	2812-2831	3′-UTR
104763	AGATG TCAGGGATCA AAGCT	359	2847-2866	3′-UTR
104764	TGGTC TCCAGATTCC AGATG	360	2862-2881	3′-UTR
104765	GTAGG CCCCAGTGAG TTCTG	361	2827-2846	3′-UTR
104766	GAACC AAAGGCTCCC TGGTC	362	2877-2896	3′-UTR
104767	TCAGG GATCAAAGCT GTAGG	363	2842-2861	3′-UTR
104768	TCCAG ATTCCAGATG TCAGG	364	2857-2876	3′-UTR
104769	GCAGC ATTCTGGCCA GAACC	365	2892-2911	3′-UTR
104770	GTCTT CTCAAGTCCT GCAGC	366	2907-2926	3′-UTR
104771	AAAGG CTCCCTGGTC TCCAG	367	2872-2891	3′-UTR
104772	CAATT TCTAGGTGAG GTCTT	368	2922-2941	3′-UTR
104773	ATTCT GGCCAGAACC AAAGG	369	2887-2906	3′-UTR
104774	CTCAA GTCCTGCAGC ATTCT	34	2902-2921	3′-UTR
104775	AAGGT CCACTTGTGT CAATT	370	2937-2956	3′-UTR
104776	GAGAG AGGAAGGCCT AAGGT	371	2952-2971	3′-UTR
104777	TCTAG GTGAGGTCTT CTCAA	372	2917-2936	3′-UTR
104778	CCACT TGTGTCAATT TCTAG	373	2932-2951	3′-UTR
104779	GTCTG GAAACATCTG GAGAG	374	2967-2986	3′-UTR
104780	CCGTG TCTCAAGGAA GTCTG	375	2982-3001	3′-UTR
104781	AGGAA GGCCTAAGGT CCACT	376	2947-2966	3′-UTR
104782	GAGGG AGCTGGCTCC ATGGG	377	3014-3033	3′-UTR
104783	GAAAC ATCTGGAGAG AGGAA	378	2962-2981	3′-UTR
104784	GTGCA AACATAAATA GAGGG	379	3029-3048	3′-UTR
104785	TCTCA AGGAAGTCTG GAAAC	380	2977-2996	3′-UTR
104786	AATAA ATAATCACAA GTGCA	381	3044-3063	3′-UTR
104787	GGGCT GGGCTCCGTG TCTCA	382	2992-3011	3′-UTR
104788	TACCC CGGTCTCCCA AATAA	383	3101-3120	3′-UTR
104789	AACAT AAATAGAGGG AGCTG	384	3024-3043	3′-UTR
104790	TTGGG TCCCCCAGGA TACCC	385	3116-3135	3′-UTR
104791	ATAAT CACAAGTGCA AACAT	386	3039-3058	3′-UTR
104792	AAGGC AGCTCCTACA TTGGG	387	3131-3150	3′-UTR
104793	CGGTC TCCCAAATAA ATACA	388	3096-3115	3′-UTR
104794	AAACA TGTCTGAGCC AAGGC	389	3146-3165	3′-UTR
104795	TCCCC CAGGATACCC CGGTC	390	3111-3130	3′-UTR
104796	AGCTC CTACATTGGG TCCCC	391	3126-3145	3′-UTR
104797	CTCCG TTTTCACGGA AAACA	37	3161-3180	3′-UTR
104798	TGTCT GAGCCAAGGC AGCTC	392	3141-3160	3′-UTR
104799	CAGCC TATTGTTCAG CTCCG	393	3176-3195	3′-UTR
104800	AGAAG GCACAGAGGC CAGGG	394	3209-3228	3′-UTR
104801	TTTTC ACGGAAAACA TGTCT	395	3156-3175	3′-UTR
104802	TATTG TTCAGCTCCG TTTTC	396	3171-3190	3′-UTR
104803	AAAAA CATAATCAAA AGAAG	397	3224-3243	3′-UTR
104804	CAGAT AAATATTTTA AAAAA	398	3239-3258	3′-UTR
104805	TACAT GGGAACAGCC TATTG	399	3186-3205	3′-UTR
104806	TTTAG ACAACTTAAT CAGAT	400	3254-3273	3′-UTR
104807	CATAA TCAAAAGAAG GCACA	401	3219-3238	3′-UTR
104808	ACCAA ATCAGCATTG TTTAG	402	3269-3288	3′-UTR
104809	AAATA TTTTAAAAAA CATAA	403	3234-3253	3′-UTR
104810	GAGTG ACAGTTGGTC ACCAA	404	3284-3303	3′-UTR
104811	ACAAC TTAATCAGAT AAATA	405	3249-3268	3′-UTR
104812	CAGAG GCTCAGCAAT GAGTG	406	3299-3318	3′-UTR
104813	ATCAG CATTGTTTAG ACAAC	407	3264-3283	3′-UTR
104814	AGGGC GATTACAGAC ACAAC	408	3331-3350	3′-UTR
104815	ACAGT TGGTCACCAA ATCAG	409	3279-3298	3′-UTR
104816	TCGCC ACTGAATAGT AGGGC	410	3346-3365	3′-UTR
104817	GCTCA GCAATGAGTG ACAGT	411	3294-3313	3′-UTR
104818	AGCAA ACTTTATTTC TCGCC	412	3361-3380	3′-UTR
104819	GATTA CAGACACAAC TCCCC	413	3326-3345	3′-UTR
104820	ACTGA ATAGTAGGGC GATTA	414	3341-3360	3′-UTR
104821	ACTTT ATTTCTCGCC ACTGA	415	3356-3375	3′-UTR
104822	GCTGT CCTTGCTGAG GGAGC	416	0626-0645	5′-UTR
104823	CTTAG CTGGTCCTCT GCTGT	417	0641-0660	5′-UTR
104824	GTTGC TTCTCTCCCT CTTAG	418	0656-0675	5′-UTR
104825	TGGCG TCTGAGGGTT GTTTT	419	0691-0710	5′-UTR
104826	AGAGA ACCTGCCTGG CAGCT	420	0723-0742	5′-UTR
104827	CAGTA TGTGAGAGGA AGAGA	421	0738-0757	5′-UTR
104828	GGTGA AGCCGTGGGT CAGTA	422	0753-0772	5′-UTR
104829	AGTGC TCATGGTGTC CTTTC	423	0785-0804	AUG
104830	CCGGA TCATGCTTTC AGTGC	424	0800-0819	coding
104831	GGCCA GCTCCACGTC CCGGA	425	0815-0834	coding
104832	GGCCC CCCTGTCTTC TTGGG	426	0847-0866	coding
104833	GGCTG AGGAACAAGC ACCGC	427	0879-0898	coding
104834	TCAGG AAGGAGAAGA GGCTG	428	0894-0913	coding
104835	TGGCG CCTGCCACGA TCAGG	429	0909-0918	coding
104836	GGCAG AAGAGCGTGG TGGCG	430	0924-0943	coding
104837	CTCCA AAGTGCAGCA GGCAG	431	0939-0958	coding
104838	GCTGA TTAGAGAGAG GTCCC	432	1596-1615	coding
104839	TGCCT GGGCCAGAGG GCTGA	433	1611-1630	coding
104840	GCTGC CCCTCAGCTT GAGGG	434	2175-2194	coding
104841	GGTTC AGCCACTGGA GCTGC	435	2190-2209	coding
104842	GGGCA TTGGCCCGGC GGTTC	436	2205-2224	coding
104843	CGCCA TTGGCCAGGA GGGCA	437	2220-2239	coding
104844	TATCT CTCAGCTCCA CGCCA	438	2235-2254	coding
104845	GCACC ACCAGCTGGT TATCT	439	2250-2269	coding
104846	ACAGG CCCTCTGATG GCACC	440	2265-2284	coding
104847	GGGAG TAGATGAGGT ACAGG	441	2280-2299	coding
104848	CCTTG AAGAGGACCT GGGAG	442	2295-2314	coding
104849	GAGGA GCACATGGGT GGAGG	443	2327-2346	coding
104850	GCTGA TGGTGTGGGT GAGGA	444	2342-2361	coding
104851	GGAGA CGGCGATGCG GCTGA	445	2357-2376	coding
104852	GACCT TGGTCTGGTA GGAGA	446	2372-2391	coding
104853	GGCAG AGAGGAGGTT GACCT	447	2387-2406	coding
104854	GCTTG GCCTCAGCCC CCTCT	23	2436-2455	coding
104855	TGGGC TCATACCAGG GCTTG	448	2451-2470	coding
104856	CCCCT CCCAGATAGA TGGGC	449	2466-2485	coding
104857	TCTCC AGCTGGAAGA CCCCT	92	2481-2500	coding
104858	TGAGT CGGTCACCCT TCTCC	450	2496-2515	coding
104859	GATTG ATCTCAGCGC TGAGT	451	2511-2530	coding
104860	CGAGA TAGTCGGGCC GATTG	452	2526-2545	coding
104861	CAGAC TCGGCAAAGT CGAGA	89	2541-2560	coding
104862	CAAAG TAGACCTGCC CAGAC	453	2556-2575	coding
104863	ACAGG GCAATGATCC CAAAG	454	2571-2590	stop
104864	ATGTT CGTCCTCCTC ACAGG	455	2586-2605	stop
104865	GTTTG GGAAGGTTGG ATGTT	456	2601-2620	3′-UTR
104866	AAGAG GTTGAGGGTG TCTGA	457	2657-2676	3′-UTR
104867	CTCTT TTTGAGCCAG AAGAG	458	2672-2691	3′-UTR
104868	CCTAA GCCCCCAATT CTCTT	459	2687-2706	3′-UTR
104869	AGCTT GGGTTCCGAC CCTAA	460	2702-2721	3′-UTR
104870	TTGCT TAAAGTTCTA AGCTT	461	2717-2736	3′-UTR
104871	GAAGT GGTGGTCTTG TTGCT	462	2732-2751	3′-UTR
104872	TGAAT CCCAGGTTTC GAAGT	463	2747-2766	3′-UTR
104873	CAGGC CACACATTCC TGAAT	464	2762-2781	3′-UTR
104874	CAGCA CTTCACTGTG CAGGC	465	2777-2796	3′-UTR
104875	ATTCT TAGTGGTTGC CAGCA	466	2792-2811	3′-UTR
104876	GAGGC CCCAGTTTGA ATTCT	467	2807-2826	3′-UTR
104877	CCCCA GTGAGTTCTG GAGGC	468	2822-2841	3′-UTR
104878	GATCA AAGCTGTAGG CCCCA	469	2837-2856	3′-UTR
104879	ATTCC AGATGTCAGG GATCA	470	2852-2871	3′-UTR
104880	CTCCC TGGTCTCCAG ATTCC	471	2867-2886	3′-UTR
104881	GGCCA GAACCAAAGG CTCCC	472	2882-2901	3′-UTR
104882	GTCCT GCAGCATTCT GGCCA	473	2897-2916	3′-UTR
104883	GTGAG GTCTTCTCAA GTCCT	474	2912-2931	3′-UTR
104884	TGTGT CAATTTCTAG GTGAG	475	2927-2946	3′-UTR
104885	GGCCT AAGGTCCACT TGTGT	476	2942-2961	3′-UTR
104886	ATCTG GAGAGAGGAA GGCCT	477	2957-2976	3′-UTR
104887	AGGAA GTCTGGAAAC ATCTG	478	2972-2991	3′-UTR
104888	GGGCT CCGTGTCTCA AGGAA	479	2987-3006	3′-UTR
104889	AAATA GAGGGAGCTG GCTCC	480	3019-3038	3′-UTR
104890	CACAA GTGCAAACAT AAATA	481	3034-3053	3′-UTR
104891	TCCCA AATAAATACA TTCAT	482	3091-3110	3′-UTR
104892	CAGGA TACCCCGGTC TCCCA	483	3106-3125	3′-UTR
104893	CTACA TTGGGTCCCC CAGGA	484	3121-3140	3′-UTR
104894	GAGCC AAGGCAGCTC CTACA	485	3136-3155	3′-UTR
104895	ACGGA AAACATGTCT GAGCC	486	3151-3170	3′-UTR
104896	TTCAG CTCCGTTTTC ACGGA	487	3166-3185	3′-UTR
104897	GGGAA CAGCCTATTG TTCAG	488	3181-3200	3′-UTR
104898	TCAAA AGAAGGCACA GAGGC	489	3214-3233	3′-UTR
104899	TTTTA AAAAACATAA TCAAA	490	3229-3248	3′-UTR
104900	TTAAT CAGATAAATA TTTTA	491	3244-3263	3′-UTR
104901	CATTG TTTAGACAAC TTAAT	492	3259-3278	3′-UTR
104902	TGGTC ACCAAATCAG CATTG	493	3274-3293	3′-UTR
104903	GCAAT GAGTGACAGT TGGTC	494	3289-3308	3′-UTR
104904	GGGAG CAGAGGCTCA GCAAT	495	3304-3323	3′-UTR
104905	ATAGT AGGGCGATTA CAGAC	496	3336-3355	3′-UTR
104906	ATTTC TCGCCACTGA ATAGT	497	3351-3370	3′-UTR
1 Emboldened residues are 2′-O-methoxyethyl residues (others are 2′-deoxy-). All 2′-O-methoxyethyl cytosines and 2′-deoxy cytosines residues are 5-methyl-cytosines; all linkages are phosphorothioate linkages.
2 Co-ordinates from Genbank Accession No. X02910, locus name “HSTNFA”, SEQ ID NO. 1.
3 This target region is an exon-intron junction and is represented in the form, for example, I1/E2, where I, followed by a number, refers to the intron number and E, followed by a number, refers to the exon number.

TABLE 32
Inhibition of Human TNF-α mRNA Expression by Chimeric
(deoxy gapped) Phosphorothioate Oligodeoxynucleotides
	SEQ	GENE
ISIS	ID	TARGET	% mRNA	% mRNA
No:	NO:	REGION	EXPRESSION	INHIBITION
basal	—	—	0.0%	—
induced	—	—	100.0%	0.0%
28089	69	intron 1	42.3%	57.7%
104649	251	5′-UTR	165.6%	—
104650	252	5′-UTR	75.8%	24.2%
104651	253	5′-UTR	58.2%	41.8%
104652	254	5′-UTR	114.5%	—
104653	255	5′-UTR	84.9%	15.1%
104654	256	5′-UTR	80.8%	19.2%
104655	257	5′-UTR	94.3%	5.7%
104656	258	5′-UTR	78.4%	21.6%
104657	259	5′-UTR	87.4%	12.6%
104658	260	5′-UTR	213.4%	—
104659	261	5′-UTR	96.3%	3.7%
104660	262	5′-UTR	153.1%	—
104661	263	5′-UTR	90.0%	10.0%
104662	264	5′-UTR	33.3%	66.7%
104663	265	5′-UTR	144.2%	—
104664	266	AUG	76.3%	23.7%
104665	267	AUG	185.3%	—
104666	268	AUG	67.4%	32.6%
104667	269	coding	94.3%	5.7%
104668	270	coding	63.1%	36.9%
104669	271	coding	50.8%	49.2%
104670	272	coding	43.7%	56.3%
104671	273	coding	52.2%	47.8%
104672	274	coding	51.8%	48.2%
104673	275	coding	102.3%	—
104674	276	coding	135.4%	—
104675	277	coding	83.1%	16.9%
104676	278	coding	87.5%	12.5%
104677	279	coding	53.6%	46.4%
104678	280	coding	75.2%	24.8%
104679	281	coding	114.0%	—
104680	282	coding	142.5%	—
104681	283	coding	58.5%	41.5%
104682	284	coding	101.9%	—
104683	285	coding	77.1%	22.9%
104684	286	coding	61.0%	39.0%
104685	287	coding	65.9%	34.1%
104686	288	E2/I2	59.2%	40.8%
104687	289	coding	77.0%	23.0%
104688	290	coding	40.1%	59.9
104689	291	coding	78.6%	21.4%
104690	292	coding	90.9%	9.1%
104691	293	coding	107.6%	—
104692	294	coding	63.4%	36.6%
104693	295	coding	74.1%	25.9%
104694	296	coding	108.3%	—
104695	297	coding	48.2%	51.8%
104696	298	coding	120.3%	—
104697	299	coding	45.0%	55.0%
104698	300	coding	77.1%	22.9%
104699	301	coding	143.7%	—
104700	302	coding	96.1%	3.9%
104701	303	coding	106.8%	—
104702	304	coding	157.4%	—
104703	305	coding	84.3%	15.7%
104704	306	coding	182.8%	—
104705	307	coding	125.1%	—
104706	308	coding	81.8%	18.2%
104707	309	coding	104.8%	—
104708	310	coding	163.0%	—
104709	311	coding	95.0%	5.0%
104710	312	coding	182.1%	—
104711	313	coding	82.1%	17.9%
104712	314	coding	118.1%	—
104713	315	coding	31.1%	68.9%
104714	316	coding	90.5%	9.5%
104715	317	coding	96.7%	3.3%
104716	318	coding	180.7%	—
104717	93	coding	71.6%	28.4%
104718	94	coding	187.0%	—
104719	319	coding	88.8%	11.2%
104720	320	coding	166.5%	—
104721	321	coding	65.0%	35.0%
104722	322	coding	59.6%	40.4%
104723	26	coding	90.1%	9.9%
104724	323	coding	88.7%	11.3%
104725	90	coding	94.7%	5.3%
104726	91	coding	84.1%	15.9%
104727	324	coding	125.3%	—
104728	325	coding	221.7%	—
104729	326	coding	102.4%	—
104730	327	coding	151.6%	—
104731	328	coding	102.2%	—
104732	329	coding	53.2%	46.8%
104733	330	stop	57.0%	43.0%
104734	88	coding	119.2%	—
104735	331	3′-UTR	71.2%	28.8%
104736	332	stop	79.0%	21.0%
104737	333	3′-UTR	87.4%	12.6%
104738	334	stop	36.8%	63.2%
104739	335	3′-UTR	106.0%	—
104740	336	3′-UTR	130.9%	—
104741	337	3′-UTR	79.2%	20.8%
104742	338	3′-UTR	159.0%	—
104743	339	3′-UTR	96.1%	3.9%
104744	340	3′-UTR	129.9%	—
104745	341	3′-UTR	80.2%	19.8%
104746	342	3′-UTR	168.8%	—
104747	343	3′-UTR	89.2%	10.8%
104748	344	3′-UTR	103.4%	—
104749	345	3′-UTR	89.0%	11.0%
104750	346	3′-UTR	160.0%	—
104751	347	3′-UTR	60.1%	39.9%
104752	348	3′-UTR	72.4%	27.6%
104753	349	3′-UTR	70.0%	30.0%
104754	350	3′-UTR	115.6%	—
104755	351	3′-UTR	71.7%	28.3%
104756	352	3′-UTR	91.5%	8.5%
104757	353	3′-UTR	85.6%	14.4%
104758	354	3′-UTR	97.6%	2.4%
104759	355	3′-UTR	68.6%	31.4%
104760	356	3′-UTR	182.4%	—
104761	357	3′-UTR	110.9%	—
104762	358	3′-UTR	161.4%	—
104763	359	3′-UTR	102.0%	—
104764	360	3′-UTR	113.5%	—
104765	361	3′-UTR	154.8%	—
104766	362	3′-UTR	126.4%	—
104767	363	3′-UTR	116.1%	—
104768	364	3′-UTR	177.7%	—
104769	365	3′-UTR	89.8%	10.2%
104770	366	3′-UTR	94.3%	5.7%
104771	367	3′-UTR	191.2%	—
104772	368	3′-UTR	80.3%	19.7%
104773	369	3′-UTR	133.9%	—
104774	34	3′-UTR	94.8%	5.2%
104775	370	3′-UTR	80.6%	19.4%
104776	371	3′-UTR	90.1%	9.9%
104777	372	3′-UTR	84.7%	15.3%
104778	373	3′-UTR	121.3%	—
104779	374	3′-UTR	97.8%	2.2%
104780	375	3′-UTR	67.6%	32.4%
104781	376	3′-UTR	141.5%	—
104782	377	3′-UTR	96.5%	3.5%
104783	378	3′-UTR	153.2%	—
104784	379	3′-UTR	85.4%	14.6%
104785	380	3′-UTR	163.9%	—
104786	381	3′-UTR	82.9%	17.1%
104787	382	3′-UTR	89.7%	10.3%
104788	383	3′-UTR	103.9%	—
104789	384	3′-UTR	75.8%	24.2%
104790	385	3′-UTR	106.3%	—
104791	386	3′-UTR	165.3%	—
104792	387	3′-UTR	71.8%	28.2%
104793	388	3′-UTR	101.9%	—
104794	389	3′-UTR	70.7%	29.3%
104795	390	3′-UTR	68.8%	31.2%
104796	391	3′-UTR	93.4%	6.6%
104797	37	3′-UTR	131.7%	—
104798	392	3′-UTR	89.4%	10.6%
104799	393	3′-UTR	89.6%	10.4%
104800	394	3′-UTR	89.0%	11.0%
104801	395	3′-UTR	196.8%	—
104802	396	3′-UTR	189.3%	—
104803	397	3′-UTR	119.7%	—
104804	398	3′-UTR	102.4%	—
104805	399	3′-UTR	90.6%	9.4%
104806	400	3′-UTR	89.1%	10.9%
104807	401	3′-UTR	152.6%	—
104808	402	3′-UTR	96.8%	3.2%
104809	403	3′-UTR	178.8%	—
104810	404	3′-UTR	94.9%	5.1%
104811	405	3′-UTR	234.4%	—
104812	406	3′-UTR	114.3%	—
104813	407	3′-UTR	153.7%	—
104814	408	3′-UTR	86.3%	13.7%
104815	409	3′-UTR	153.9%	—
104816	410	3′-UTR	79.9%	20.1%
104817	411	3′-UTR	196.5%	—
104818	412	3′-UTR	94.3%	5.7%
104819	413	3′-UTR	143.3%	—
104820	414	3′-UTR	123.8%	—
104821	415	3′-UTR	129.2%	—
104822	416	5′-UTR	76.6%	23.4%
104823	417	5′-UTR	63.9%	36.1%
104824	418	5′-UTR	22.0%	78.0%
104825	419	5′-UTR	109.4%	—
104826	420	5′-UTR	45.2%	54.8%
104827	421	5′-UTR	68.9%	31.1%
104828	422	5′-UTR	70.9%	29.1%
104829	423	AUG	46.6%	53.4%
104830	424	coding	55.0%	45.0%
104831	425	coding	49.5%	50.5%
104832	426	coding	106.0%	—
104833	427	coding	23.7%	76.3%
104834	428	coding	91.8%	8.2%
104835	429	coding	72.3%	27.7%
104836	430	coding	63.4%	36.6%
104837	431	coding	31.0%	69.0%
104838	432	coding	18.0%	82.0%
104839	433	coding	67.9%	32.1%
104840	434	coding	93.8%	6.2%
104841	435	coding	43.0%	57.0%
104842	436	coding	73.2%	26.8%
104843	437	coding	48.1%	51.9%
104844	438	coding	39.2%	60.8%
104845	439	coding	37.6%	62.4%
104846	440	coding	81.7%	18.3%
104847	441	coding	50.8%	49.2%
104848	442	coding	56.7%	43.3%
104849	443	coding	51.8%	48.2%
104850	444	coding	91.8%	8.2%
104851	445	coding	93.9%	6.1%
104852	446	coding	100.9%	—
104853	447	coding	67.7%	32.3%
104854	23	coding	11.0%	89.0%
104855	448	coding	62.5%	37.5%
104856	449	coding	67.8%	32.2%
104857	92	coding	28.1%	71.9%
104858	450	coding	76.2%	23.8%
104859	451	coding	52.3%	47.7%
104860	452	coding	93.6%	6.4%
104861	89	coding	79.3%	20.7%
104862	453	coding	63.1%	36.9%
104863	454	stop	64.5%	35.5%
104864	455	stop	43.2%	56.8%
104865	456	3′-UTR	83.1%	16.9%
104866	457	3′-UTR	49.4%	50.6%
104867	458	3′-UTR	49.5%	50.5%
104868	459	3′-UTR	89.6%	10.4%
104869	460	3′-UTR	21.4%	78.6%
104870	461	3′-UTR	118.0%	—
104871	462	3′-UTR	55.8%	44.2%
104872	463	3′-UTR	49.0%	51.0%
104873	464	3′-UTR	92.6%	7.4%
104874	465	3′-UTR	33.4%	66.6%
104875	466	3′-UTR	36.2%	63.8%
104876	467	3′-UTR	73.4%	26.6%
104877	468	3′-UTR	40.9%	59.1%
104878	469	3′-UTR	78.7%	21.3%
104879	470	3′-UTR	75.4%	24.6%
104880	471	3′-UTR	50.2%	49.8%
104881	472	3′-UTR	47.0%	53.0%
104882	473	3′-UTR	82.7%	17.3%
104883	474	3′-UTR	46.4%	53.6%
104884	475	3′-UTR	46.1%	53.9%
104885	476	3′-UTR	156.9%	—
104886	477	3′-UTR	102.4%	—
104887	478	3′-UTR	59.1%	40.9%
104888	479	3′-UTR	64.7%	35.3%
104889	480	3′-UTR	83.7%	16.3%
104890	481	3′-UTR	52.9%	47.1%
104891	482	3′-UTR	87.9%	12.1%
104892	483	3′-UTR	39.8%	60.2%
104893	484	3′-UTR	71.1%	28.9%
104894	485	3′-UTR	34.0%	66.0%
104895	486	3′-UTR	129.8%	—
104896	487	3′-UTR	57.6%	42.4%
104897	488	3′-UTR	49.6%	50.4%
104898	489	3′-UTR	71.7%	28.3%
104899	490	3′-UTR	101.5%	—
104900	491	3′-UTR	142.1%	—
104901	492	3′-UTR	55.9%	44.1%
104902	493	3′-UTR	85.3%	14.7%
104903	494	3′-UTR	46.0%	54.0%
104904	495	3′-UTR	59.9%	40.1%
104905	496	3′-UTR	47.2%	52.8%
104906	497	3′-UTR	56.3%	43.7%

Oligonucleotides 104662 (SEQ ID NO: 264), 104669 (SEQ ID NO: 271), 104670 (SEQ ID NO: 272), 104688 (SEQ ID NO: 290), 104695 (SEQ ID NO: 297), 104697 (SEQ ID NO: 299), 104713 (SEQ ID NO: 315), 104738 (SEQ ID NO:334), 104824 (SEQ ID NO: 418), 104826 (SEQ ID NO: 420), 104829 (SEQ ID NO: 423), 104831 (SEQ ID NO: 425), 104833 (SEQ ID NO: 427), 104837 (SEQ ID NO: 431), 104838 (SEQ ID NO: 432), 104841 (SEQ ID NO: 435), 104843 (SEQ ID NO: 437), 104844 (SEQ ID NO: 438), 104845 (SEQ ID NO: 439), 104847 (SEQ ID NO: 441), 104854 (SEQ ID NO: 23), 104857 (SEQ ID NO: 92), 104864 (SEQ ID NO: 455), 104866 (SEQ ID NO: 457), 104867 (SEQ ID NO: 458), 104869 (SEQ ID NO: 460), 104872 (SEQ ID NO: 463), 104874 (SEQ ID NO: 465), 104875 (SEQ ID NO: 466), 104877 (SEQ ID NO: 468), 104880 (SEQ ID NO: 471), 104881 (SEQ ID NO: 472), 104883 (SEQ ID NO: 474), 104884 (SEQ ID NO: 475), 104892 (SEQ ID NO: 483), 104894 (SEQ ID NO: 485), 104897 (SEQ ID NO: 488), 104903 (SEQ ID NO: 494) and 104905 (SEQ ID NO: 496) gave approximately 50% or greater reduction in TNF-α mRNA expression in this assay. Oligonucleotides 104713 (SEQ ID NO: 315), 104824 (SEQ ID NO: 418), 104833 (SEQ ID NO: 427), 104837 (SEQ ID NO: 431), 104838 (SEQ ID NO: 432), 104854 (SEQ ID NO: 23), 104857 (SEQ ID NO: 92), and 104869 (SEQ ID NO: 460) gave approximately 70% or greater reduction in TNF-α mRNA expression in this assay.

Example 23

Dose Response of Chimeric (Deoxy Gapped) Antisense Phosphorothioate Oligodeoxynucleotide Effects on TNF-α mRNA and Protein Levels

Several oligonucleotides from the initial screen were chosen for dose response assays. NeoHk cells were grown, treated and processed as described in Example 3. LIPOFECTIN® was added at a ratio of 3 μg/ml per 100 nM of oligonucleotide. The control included LIPOFECTIN® at a concentration of 9 μg/ml.

The human promonocytic leukaemia cell line, THP-1 (American Type Culture Collection, Manassas, Va.) was maintained in RPMI 1640 growth media supplemented with 10% fetal calf serum (FCS; Life Technologies, Rockville, Md.). A total of 8×10 5 cells were employed for each treatment by combining 50 μl of cell suspension in OPTIMEM™, 1% FBS with oligonucleotide at the indicated concentrations to reach a final volume of 100 μl with OPTIMEM™, 1% FBS. Cells were then transferred to a 1 mm electroporation cuvette and electroporated using an Electrocell Manipulator 600 instrument (Biotechnologies and Experimental Research, Inc.) employing 90 V, 1000 μF, at 13Ω. Electroporated cells were then transferred to 24 well plates. 400 μl of RPMI 1640, 10% FCS was added to the cells and the cells were allowed to recover for 6 hrs. Cells were then induced with LPS at a final concentration of 100 ng/ml for 2 hours. RNA was isolated and processed as described in Example 3.

Results with NeoHK cells are shown in Table 33 for mRNA, and Table 34 for protein. Results with THP-1 cells are shown in Table 35.

Most of the oligonucleotides tested showed dose response effects with a maximum inhibition of mRNA greater than 70% and a maximum inhibition of protein greater than 85%.

TABLE 33
Dose Response of NeoHK Cells to TNF-α
Chimeric (deoxy gapped) Antisense Oligonucleotides
	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition
induced	—	—	—	100%	—
16798	128	coding	30 nM	87%	13%
″	″	″	100 nM	129%	—
″	″	″	300 nM	156%	—
21823	69	intron 1	30 nM	82%	18%
″	″	″	100 nM	90%	10%
″	″	″	300 nM	59%	41%
28088	68	intron 1	30 nM	68%	32%
″	″	″	100 nM	43%	57%
″	″	″	300 nM	42%	58%
28089	69	intron 1	30 nM	59%	41%
″	″	″	100 nM	44%	56%
″	″	″	300 nM	38%	62%
104697	299	coding	30 nM	60%	40%
″	″	″	100 nM	45%	55%
″	″	″	300 nM	27%	73%
104777	372	3″-UTR	30 nM	66%	34%
″	″	″	100 nM	55%	45%
″	″	″	300 nM	43%	57%

TABLE 34
Dose Response of NeoHK Cells to TNF-α
Chimeric (deoxy gapped) Antisense Oligonucleotides
	SEQ ID	ASO Gene		% Protein	% Protein
ISIS #	NO:	Target	Dose	Expression	Inhibition
induced	—	—	—	100.0%	—
16798	128	coding	30 nM	115.0%	—
″	″	″	100 nM	136.0%	—
″	″	″	300 nM	183.0%	—
28089	69	intron 1	30 nM	87.3%	12.7%
″	″	″	100 nM	47.4%	52.6%
″	″	″	300 nM	22.8%	77.2%
104681	283	coding	30 nM	91.3%	8.7%
″	″	″	100 nM	62.0%	38.0%
″	″	″	300 nM	28.5%	71.5%
104697	299	coding	30 nM	87.1%	12.9%
″	″	″	100 nM	59.6%	40.4%
″	″	″	300 nM	29.1%	70.9%
104838	432	coding	30 nM	91.9%	8.1%
″	″	″	100 nM	56.9%	43.1%
″	″	″	300 nM	14.8%	85.2%
104854	23	coding	30 nM	64.4%	35.6%
″	″	″	100 nM	42.3%	57.7%
″	″	″	300 nM	96.1%	3.9%
104869	460	3′-UTR	30 nM	88.9%	11.1%
″	″	″	100 nM	56.8%	43.2%
″	″	″	300 nM	42.3%	57.7%

TABLE 35
Dose Response of LPS-Induced THP-1 Cells to Chimeric (deoxy
gapped) TNF-α Antisense Phosphorothioate
Oligodeoxynucleotides (ASOs)
	SEQ ID	ASO Gene		% mRNA	% mRNA
ISIS #	NO:	Target	Dose	Expression	Inhibition
induced	—	—	—	100%	—
16798	128	coding	1 μM	102%	—
″	″	″	3 μM	87%	13%
″	″	″	10 μM	113%	—
″	″	″	30 μM	134%	—
28089	69	intron 1	1 μM	39%	61%
″	″	″	3 μM	79%	21%
″	″	″	10 μM	91%	9%
″	″	″	30 μM	63%	37%
104697	299	coding	1 μM	99%	1%
″	″	″	3 μM	96%	4%
″	″	″	10 μM	92%	8%
″	″	″	30 μM	52%	48%
104838	432	coding	1 μM	31%	69%
″	″	″	3 μM	20%	80%
″	″	″	10 μM	15%	85%
″	″	″	30 μM	7%	93%
104854	23	coding	1 μM	110%	—
″	″	″	3 μM	90%	10%
″	″	″	10 μM	95%	5%
″	″	″	30 μM	61%	39%

Example 24

Further Optimization of Human TNF-α Antisense Oligonucleotide Chemistry

Additional analogs of TNF-α oligonucleotides were designed and synthesized to find an optimum gap size. The sequences and chemistries are shown in Table 36.

Dose response experiments are performed as described in Example 3.

TABLE 29
Nucleotide Sequences of TNF-αChimeric Backbone
(deoxy gapped) Oligonucleotides
		SEQ	TARGET GENE	GENE
ISIS	NUCLEOTIDE SEQUENCE 1	ID	NUCLEOTIDE	TARGET
NO.	(5′->3′)	NO:	CO-ORDINATES 2	REGION
110554	GCTG ATTAGAGAGA GGTCCC	432	104838 analog
110555	GCT GATTAGAGAG AGGTCCC	″	″
110556	GC TGATTAGAGA GAGGTCCC	″	″
110557	G CTGATTAGAG AGAGGTCCC	″	″
110583	GCTGATTAGA GAGAGGTCCC	″	″
110558	CTGA TTAGAGAGAG GTCCC	498	1596-1614	coding
110559	CTG ATTAGAGAGAG GTCCC	″	″	″
110560	CTG ATTAGAGAGA GGTCCC	″	″	″
110561	CT GATTAGAGAGAG GTCCC	″	″	″
110562	CT GATTAGAGAGA GGTCCC	″	″	″
110563	CT GATTAGAGAG AGGTCCC	″	″	″
110564	C TGATTAGAGAGAG GTCCC	″	″	″
110565	C TGATTAGAGAGA GGTCCC	″	″	″
110566	C TGATTAGAGAG AGGTCCC	″	″	″
110567	C TGATTAGAGA GAGGTCCC	″	″	″
110584	CTGATTAGAG AGAGGTCCC	″	″	″
108371	CTGA TTAGAGAGAG GTCC	499	1597-1614	coding
110568	CTG ATTAGAGAGA GGTCC	″	″	″
110569	CT GATTAGAGAG AGGTCC	″	″	″
110570	C TGATTAGAGA GAGGTCC	″	″	″
110585	CTGATTAGAG AGAGGTCC	″	″	″
110571	CTGGTTA TCTCTCAGCT CCA	299	104697 analog
110572	CTGGTTAT CTCTCAGCTC CA	″	″
110573	CTG GTTATCTCTC AGCTCCA	″	″
110586	CTGGTTATCT CTCAGCTCCA	″	″
110574	GATCACT CCAAAGTGCA GCA	283	104681 analog
110575	GATCACTC CAAAGTGCAG CA	″	″
110576	GAT CACTCCAAAG TGCAGCA	″	″
110587	GATCACTCCA AAGTGCAGCA	″	″
110577	AGCTTGG GTTCCGACCC TAA	460	104689 analog
110578	AGCTTGGG TTCCGACCCT AA	″	″
110579	AGC TTGGGTTCCG ACCCTAA	″	″
110588	AGCTTGGGTT CCGACCCTAA	″	″
110580	AGGTTGA CCTTGGTCTG GTA	315	104713 analog
110581	AGGTTGAC CTTGGTCTGG TA	″	″
110582	AGG TTGACCTTGG TCTGGTA	″	″
110589	AGGTTGACCT TGGTCTGGTA	″	″
110637	GTGTG CCAGACACCC TATCT	69	21823 analog
110651	GTGT GCCAGACACC CTATCT	″	″
110665	GTG TGCCAGACAC CCTATCT	″	″
110679	GT GTGCCAGACA CCCTATCT	″	″
110693	G TGTGCCAGAC ACCCTATCT	″	″
110707	GTGTGCCAGA CACCCTATCT	″	″
110590	TGAGT GTCTTCTGTG TGCCA	500	1411-1430	intron
				1
110597	TGAG TGTCTTCTGT GTGCCA	″	″	intron
				1
110604	TGA GTGTCTTCTG TGTGCCA	″	″	intron
				1
110611	TG AGTGTCTTCT GTGTGCCA	″	″	intron
				1
110618	T GAGTGTCTTC TGTGTGCCA	″	″	intron
				1
110625	TGAGTGTCTT CTGTGTGCCA	″	″	intron
				1
110591	GAGTG TCTTCTGTGT GCCAG	501	1410-1429	intron
				1
110598	GAGT GTCTTCTGTG TGCCAG	″	″	intron
				1
110605	GAG TGTCTTCTGT GTGCCAG	″	″	intron
				1
110612	GA GTGTCTTCTG TGTGCCAG	″	″	intron
				1
110619	G AGTGTCTTCT GTGTGCCAG	″	″	intron
				1
110626	GAGTGTCTTC TGTGTGCCAG	″	″	intron
				1
110592	AGTGT CTTCTGTGTG CCAGA	144	100181 analog
110599	AGTG TCTTCTGTGT GCCAGA	″	″
110606	AGT GTCTTCTGTG TGCCAGA	″	″
110613	AG TGTCTTCTGT GTGCCAGA	″	″
110620	A GTGTCTTCTG TGTGCCAGA	″	″
110627	AGTGTCTTCT GTGTGCCAGA	″	″
110593	GTGTC TTCTGTGTGC CAGAC	145	100182 analog
110600	GTGT CTTCTGTGTG CCAGAC	″	″
110607	GTG TCTTCTGTGT GCCAGAC	″	″
110614	GT GTCTTCTGTG TGCCAGAC	″	″
110621	G TGTCTTCTGT GTGCCAGAC	″	″
110628	GTGTCTTCTG TGTGCCAGAC	″	″
110594	TGTCT TCTGTGTGCC AGACA	146	100183 analog
110601	TGTC TTCTGTGTGC CAGACA	″	″
110608	TGT CTTCTGTGTG CCAGACA	″	″
110615	TG TCTTCTGTGT GCCAGACA	″	″
110622	T GTCTTCTGTG TGCCAGACA	″	″
110629	TGTCTTCTGT GTGCCAGACA	″	″
110595	GTCTT CTGTGTGCCA GACAC	147	100184 analog
110602	GTCT TCTGTGTGCC AGACAC	″	″
110609	GTC TTCTGTGTGC CAGACAC	″	″
110616	GT CTTCTGTGTG CCAGACAC	″	″
110623	G TCTTCTGTGT GCCAGACAC	″	″
110630	GTCTTCTGTG TGCCAGACAC	″	″
110596	TCTTC TGTGTGCCAG ACACC	148	100185 analog
110603	TCTT CTGTGTGCCA GACACC	″	″
110610	TCT TCTGTGTGCC AGACACC	″	″
110617	TC TTCTGTGTGC CAGACACC	″	″
110624	T CTTCTGTGTG CCAGACACC	″	″
110631	TCTTCTGTGT GCCAGACACC	″	″
110632	CTTCT GTGTGCCAGA CACCC	149	100186 analog
110646	CTTC TGTGTGCCAG ACACCC	″	″
110660	CTT CTGTGTGCCA GACACCC	″	″
110674	CT TCTGTGTGCC AGACACCC	″	″
110688	C TTCTGTGTGC CAGACACCC	″	″
110702	CTTCTGTGTG CCAGACACCC	″	″
110633	TTCTG TGTGCCAGAC ACCCT	150	100187 analog
110647	TTCT GTGTGCCAGA CACCCT	″	″
110661	TTC TGTGTGCCAG ACACCCT	″	″
110675	TT CTGTGTGCCA GACACCCT	″	″
110689	T TCTGTGTGCC AGACACCCT	″	″
110703	TTCTGTGTGC CAGACACCCT	″	″
110634	TCTGT GTGCCAGACA CCCTA	151	100188 analog
110648	TCTG TGTGCCAGAC ACCCTA	″	″
110662	TCT GTGTGCCAGA CACCCTA	″	″
110676	TC TGTGTGCCAG ACACCCTA	″	″
110690	T CTGTGTGCCA GACACCCTA	″	″
110704	TCTGTGTGCC AGACACCCTA	″	″
110635	CTGTG TGCCAGACAC CCTAT	152	100189 analog
110649	CTGT GTGCCAGACA CCCTAT	″	″
110663	CTG TGTGCCAGAC ACCCTAT	″	″
110677	CT GTGTGCCAGA CACCCTAT	″	″
110691	C TGTGTGCCAG ACACCCTAT	″	″
110705	CTGTGTGCCA GACACCCTAT	″	″
110636	TGTGT GCCAGACACC CTATC	153	100190 analog
110650	TGTG TGCCAGACAC CCTATC	″	″
110664	TGT GTGCCAGACA CCCTATC	″	″
110678	TG TGTGCCAGAC ACCCTATC	″	″
110692	T GTGTGCCAGA CACCCTATC	″	″
110706	TGTGTGCCAG ACACCCTATC	″	″
110638	TGTGC CAGACACCCT ATCTT	154	100191 analog
110652	TGTG CCAGACACCC TATCTT	″	″
110666	TGT GCCAGACACC CTATCTT	″	″
110680	TG TGCCAGACAC CCTATCTT	″	″
110694	T GTGCCAGACA CCCTATCTT	″	″
110708	TGTGCCAGAC ACCCTATCTT	″	″
110639	GTGCC AGACACCCTA TCTTC	155	100192 analog
110653	GTGC CAGACACCCT ATCTTC	″	″
110667	GTG CCAGACACCC TATCTTC	″	″
110681	GT GCCAGACACC CTATCTTC	″	″
110695	G TGCCAGACAC CCTATCTTC	″	″
110709	GTGCCAGACA CCCTATCTTC	″	″
110640	TGCCA GACACCCTAT CTTCT	156	100193 analog
110654	TGCC AGACACCCTA TCTTCT	″	″
110668	TGC CAGACACCCT ATCTTCT	″	″
110682	TG CCAGACACCC TATCTTCT	″	″
110696	T GCCAGACACC CTATCTTCT	″	″
110710	TGCCAGACAC CCTATCTTCT	″	″
110641	GCCAG ACACCCTATC TTCTT	157	100194 analog
110655	GCCA GACACCCTAT CTTCTT	″	″
110669	GCC AGACACCCTA TCTTCTT	″	″
110683	GC CAGACACCCT ATCTTCTT	″	″
110697	G CCAGACACCC TATCTTCTT	″	″
110711	GCCAGACACC CTATCTTCTT	″	″
110642	CCAGA CACCCTATCT TCTTC	158	100195 analog
110656	CCAG ACACCCTATC TTCTTC	″	″
110670	CCA GACACCCTAT CTTCTTC	″	″
110684	CC AGACACCCTA TCTTCTTC	″	″
110698	C CAGACACCCT ATCTTCTTC	″	″
110712	CCAGACACCC TATCTTCTTC	″	″
110643	CAGAC ACCCTATCTT CTTCT	159	100196 analog
110657	CAGA CACCCTATCT TCTTCT	″	″
110671	CAG ACACCCTATC TTCTTCT	″	″
110685	CA GACACCCTAT CTTCTTCT	″	″
110699	C AGACACCCTA TCTTCTTCT	″	″
110713	CAGACACCCT ATCTTCTTCT	″	″
110644	AGACA CCCTATCTTC TTCTC	160	100197 analog
110658	AGAC ACCCTATCTT CTTCTC	″	″
110672	AGA CACCCTATCT TCTTCTC	″	″
110686	AG ACACCCTATC TTCTTCTC	″	″
110700	A GACACCCTAT CTTCTTCTC	″	″
110714	AGACACCCTA TCTTCTTCTC	″	″
110645	GACAC CCTATCTTCT TCTCT	161	100198 analog
110659	GACA CCCTATCTTC TTCTCT	″	″
110673	GAC ACCCTATCTT CTTCTCT	″	″
110687	GA CACCCTATCT TCTTCTCT	″	″
110701	G ACACCCTATC TTCTTCTCT	″	″
110715	GACACCCTAT CTTCTTCTCT	″	″
1 Emboldened residues are 2′-methoxyethoxy residues (others are 2′-deoxy-). All 2′-methoxyethoxy cytidines and 2′-deoxycytidines are 5-methyl-cytidines; all linkages are phosphorothioate linkages.
2 Co-ordinates from Genbank Accession No. X02910, locus name “HSTNFA”, SEQ ID NO. 1.

Example 25

Effect of TNF-α Antisense Oligonucleotides in TNF-α Transgenic Mouse Models

The effect of TNF-α antisense oligonucleotides is studied in transgenic mouse models of human diseases. Such experiments can be performed through contract laboratories (e.g. The Laboratory of Molecular Genetics at The Hellenic Pasteur Institute, Athens, Greece) where such transgenic mouse models are available. Such models are available for testing human oligonucleotides in arthritis (Keffer, J., et al., EMBO J., 1991, 10, 4025-4031) and multiple sclerosis (Akassoglou, K., et al., J. Immunol., 1997, 158, 438-445) models. A model for inflammatory bowel disease is available for testing mouse oligonucleotides (Kontoyiannis, D., et al., Immunity, 1999, 10, 387-398).

Briefly, litters of the appropriate transgenic mouse strain are collected and weighed individually. Twice weekly from birth, oligonucleotide in saline is administered intraperitoneally or intravenously. Injections continue for 7 weeks. Each week the animals are scored for manifestations of the appropriate disease. After the final treatment, the mice are sacrificed and histopathology is performed for indicators of disease as indicated in the references cited for each model.

SEQUENCE LISTING
NUMBER OF SEQ ID NOS: 501
SEQ ID NO: 1
LENGTH: 3634
TYPE: DNA
ORGANISM: Homo sapiens
FEATURE:
NAME/KEY: CDS
LOCATION: (796..981,1589..1634,1822..1869,2171..2592)
FEATURE:
NAME/KEY: exon
LOCATION: (615)..(981)
FEATURE:
NAME/KEY: intron
LOCATION: (982)..(1588)
FEATURE:
NAME/KEY: exon
LOCATION: (1589)..(1634)
FEATURE:
NAME/KEY: intron
LOCATION: (1635)..(1821)
FEATURE:
NAME/KEY: exon
LOCATION: (1822)..(1869)
FEATURE:
NAME/KEY: intron
LOCATION: (1870)..(2070)
FEATURE:
NAME/KEY: exon
LOCATION: (2171)..(3381)
PUBLICATION INFORMATION:
AUTHORS: Nedwin, G.E.
Naylor, S.L.
Sakaguchi, A.Y.
Smith, D.
Jarrett-Nedwin, J.
Pennica, D.
Goeddel, D.V.
Gray, P.W.
TITLE: Human lymphotoxin and tumor necrosis factor genes:
TITLE: structure, homology and chromosomal l ocalization
JOURNAL: Nucleic Acids Res.
VOLUME: 13
ISSUE: 17
PAGES: 6361-6373
DATE: 1985-09-11
DATABASE ACCESSION NUMBER: X02910 Genbank
DATABASE ENTRY DATE: 1997-02-17
SEQUENCE: 1
gaattccggg tgatttcact cccggctgtc caggcttgtc ctgctacccc ac ccagcctt     60
tcctgaggcc tcaagcctgc caccaagccc ccagctcctt ctccccgcag ga cccaaaca    120
caggcctcag gactcaacac agcttttccc tccaacccgt tttctctccc tc aacggact    180
cagctttctg aagcccctcc cagttctagt tctatctttt tcctgcatcc tg tctggaag    240
ttagaaggaa acagaccaca gacctggtcc ccaaaagaaa tggaggcaat ag gttttgag    300
gggcatgggg acggggttca gcctccaggg tcctacacac aaatcagtca gt ggcccaga    360
agacccccct cggaatcgga gcagggagga tggggagtgt gaggggtatc ct tgatgctt    420
gtgtgtcccc aactttccaa atccccgccc ccgcgatgga gaagaaaccg ag acagaagg    480
tgcagggccc actaccgctt cctccagatg agctcatggg tttctccacc aa ggaagttt    540
tccgctggtt gaatgattct ttccccgccc tcctctcgcc ccagggacat at aaaggcag    600
ttgttggcac acccagccag cagacgctcc ctcagcaagg acagcagagg ac cagctaag    660
agggagagaa gcaactacag accccccctg aaaacaaccc tcagacgcca ca tcccctga    720
caagctgcca ggcaggttct cttcctctca catactgacc cacggcttca cc ctctctcc    780
cctggaaagg acacc atg agc act gaa agc atg atc cgg gac gtg gag ctg     831
Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu L eu
1               5                  10
gcc gag gag gcg ctc ccc aag aag aca ggg gg g ccc cag ggc tcc agg      879
Ala Glu Glu Ala Leu Pro Lys Lys Thr Gly Gl y Pro Gln Gly Ser Arg
15                  20                  25
cgg tgc ttg ttc ctc agc ctc ttc tcc ttc ct g atc gtg gca ggc gcc      927
Arg Cys Leu Phe Leu Ser Leu Phe Ser Phe Le u Ile Val Ala Gly Ala
30                  35                  40
acc acg ctc ttc tgc ctg ctg cac ttt gga gt g atc ggc ccc cag agg      975
Thr Thr Leu Phe Cys Leu Leu His Phe Gly Va l Ile Gly Pro Gln Arg
45                  50                  55                  60
gaa gag gtgagtgcct ggccagcctt catccactct cccacccaag gg gaaatgag      1031
Glu Glu
agacgcaaga gagggagaga gatgggatgg gtgaaagatg tgcgctgata gg gagggatg   1091
agagagaaaa aaacatggag aaagacgggg atgcagaaag agatgtggca ag agatgggg   1151
aagagagaga gagaaagatg gagagacagg atgtctggca catggaaggt gc tcactaag   1211
tgtgtatgga gtgaatgaat gaatgaatga atgaacaagc agatatataa at aagatatg   1271
gagacagatg tggggtgtga gaagagagat gggggaagaa acaagtgata tg aataaaga   1331
tggtgagaca gaaagagcgg gaaatatgac agctaaggag agagatgggg ga gataagga   1391
gagaagaaga tagggtgtct ggcacacaga agacactcag ggaaagagct gt tgaatgct   1451
ggaaggtgaa tacacagatg aatggagaga gaaaaccaga cacctcaggg ct aagagcgc   1511
aggccagaca ggcagccagc tgttcctcct ttaagggtga ctccctcgat gt taaccatt   1571
ctccttctcc ccaacag ttc ccc agg gac ctc tct cta atc agc cct ctg      1621
Phe Pro Arg Asp Leu Ser Leu Ile Ser Pro Leu
65                  70
gcc cag gca gtc agtaagtgtc tccaaacctc tttcctaatt ct gggtttgg         1673
Ala Gln Ala Val
75
gtttgggggt agggttagta ccggtatgga agcagtgggg gaaatttaaa gt tttggtct   1733
tgggggagga tggatggagg tgaaagtagg ggggtatttt ctaggaagtt ta agggtctc   1793
agctttttct tttctctctc ctcttca gga tca tct tct cga acc ccg agt gac   1847
Arg Ser Ser Se r Arg Thr Pro Ser Asp
80                  85
aag cct gta gcc cat gtt gta ggtaagagct ctgaggatg t gtcttggaac        1898
Lys Pro Val Ala His Val Val
90
ttggagggct aggatttggg gattgaagcc cggctgatgg taggcagaac tt ggagacaa   1958
tgtgagaagg actcgctgag ctcaagggaa gggtggagga acagcacagg cc ttagtggg   2018
atactcagaa cgtcatggcc aggtgggatg tgggatgaca gacagagagg ac aggaaccg   2078
gatgtggggt gggcagagct cgagggccag gatgtggaga gtgaaccgac at ggccacac   2138
tgactctcct ctccctctct ccctccctcc a gca aac cct caa gct gag ggg      2190
Ala  Asn Pro Gln Ala Glu Gly
95                 100
cag ctc cag tgg ctg aac cgc cgg gcc aat gc c ctc ctg gcc aat ggc     2238
Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Al a Leu Leu Ala Asn Gly
105                 110                 115
gtg gag ctg aga gat aac cag ctg gtg gtg cc a tca gag ggc ctg tac     2286
Val Glu Leu Arg Asp Asn Gln Leu Val Val Pr o Ser Glu Gly Leu Tyr
120                 125                 130
ctc atc tac tcc cag gtc ctc ttc aag ggc ca a ggc tgc ccc tcc acc     2334
Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gl n Gly Cys Pro Ser Thr
135                 140                 145
cat gtg ctc ctc acc cac acc atc agc cgc at c gcc gtc tcc tac cag     2382
His Val Leu Leu Thr His Thr Ile Ser Arg Il e Ala Val Ser Tyr Gln
150                 155                 160
acc aag gtc aac ctc ctc tct gcc atc aag ag c ccc tgc cag agg gag     2430
Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Se r Pro Cys Gln Arg Glu
165                 1 70                 1 75                 1 80
acc cca gag ggg gct gag gcc aag ccc tgg ta t gag ccc atc tat ctg     2478
Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Ty r Glu Pro Ile Tyr Leu
185                 190                 195
gga ggg gtc ttc cag ctg gag aag ggt gac cg a ctc agc gct gag atc     2526
Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Ar g Leu Ser Ala Glu Ile
200                 205                 210
aat cgg ccc gac tat ctc gac ttt gcc gag tc t ggg cag gtc tac ttt     2574
Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Se r Gly Gln Val Tyr Phe
215                 220                 225
ggg atc att gcc ctg tga ggaggacgaa catccaacct tc ccaaacgc            2622
Gly Ile Ile Ala Leu
230
ctcccctgcc ccaatccctt tattaccccc tccttcagac accctcaacc tc ttctggct   2682
caaaaagaga attgggggct tagggtcgga acccaagctt agaactttaa gc aacaagac   2742
caccacttcg aaacctggga ttcaggaatg tgtggcctgc acagtgaagt gc tggcaacc   2802
actaagaatt caaactgggg cctccagaac tcactggggc ctacagcttt ga tccctgac   2862
atctggaatc tggagaccag ggagcctttg gttctggcca gaatgctgca gg acttgaga   2922
agacctcacc tagaaattga cacaagtgga ccttaggcct tcctctctcc ag atgtttcc   2982
agacttcctt gagacacgga gcccagccct ccccatggag ccagctccct ct atttatgt   3042
ttgcacttgt gattatttat tatttattta ttatttattt atttacagat ga atgtattt   3102
atttgggaga ccggggtatc ctgggggacc caatgtagga gctgccttgg ct cagacatg   3162
ttttccgtga aaacggagct gaacaatagg ctgttcccat gtagccccct gg cctctgtg   3222
ccttcttttg attatgtttt ttaaaatatt tatctgatta agttgtctaa ac aatgctga   3282
tttggtgacc aactgtcact cattgctgag cctctgctcc ccaggggagt tg tgtctgta   3342
atcgccctac tattcagtgg cgagaaataa agtttgctta gaaaagaaac at ggtctcct   3402
tcttggaatt aattctgcat ctgcctcttc ttgtgggtgg gaagaagctc cc taagtcct   3462
ctctccacag gctttaagat ccctcggacc cagtcccatc cttagactcc ta gggccctg   3522
gagaccctac ataaacaaag cccaacagaa tattccccat cccccaggaa ac aagagcct   3582
gaacctaatt acctctccct cagggcatgg gaatttccaa ctctgggaat tc            3634
SEQ ID NO: 2
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 2
catgctttca gtgctcat                                                   18
SEQ ID NO: 3
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 3
tgagggagcg tctgctggct                                                 20
SEQ ID NO: 4
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 4
gtgctcatgg tgtcctttcc                                                 20
SEQ ID NO: 5
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 5
taatcacaag tgcaaacata                                                 20
SEQ ID NO: 6
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 6
taccccggtc tcccaaataa                                                 20
SEQ ID NO: 7
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 7
agcaccgcct ggagccct                                                   18
SEQ ID NO: 8
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 8
gctgaggaac aagcaccgcc                                                 20
SEQ ID NO: 9
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 9
aggcagaaga gcgtggtggc                                                 20
SEQ ID NO: 10
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 10
aaagtgcagc aggcagaaga                                                 20
SEQ ID NO: 11
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 11
ttagagagag gtccctgg                                                   18
SEQ ID NO: 12
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 12
tgactgcctg ggccagag                                                   18
SEQ ID NO: 13
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 13
gggttcgaga agatgatc                                                   18
SEQ ID NO: 14
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 14
gggctacagg cttgtcactc                                                 20
SEQ ID NO: 15
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 15
cccctcagct tgagggtttg                                                 20
SEQ ID NO: 16
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 16
ccattggcca ggagggcatt                                                 20
SEQ ID NO: 17
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 17
accaccagct ggttatctct                                                 20
SEQ ID NO: 18
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 18
ctgggagtag atgaggtaca                                                 20
SEQ ID NO: 19
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 19
cccttgaaga ggacctggga                                                 20
SEQ ID NO: 20
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 20
ggtgtgggtg aggagcacat                                                 20
SEQ ID NO: 21
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 21
gtctggtagg agacggcgat                                                 20
SEQ ID NO: 22
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 22
gcagagagga ggttgacctt                                                 20
SEQ ID NO: 23
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 23
gcttggcctc agccccctct                                                 20
SEQ ID NO: 24
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 24
cctcccagat agatgggctc                                                 20
SEQ ID NO: 25
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 25
cccttctcca gctggaagac                                                 20
SEQ ID NO: 26
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 26
atctcagcgc tgagtcggtc                                                 20
SEQ ID NO: 27
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 27
tcgagatagt cgggccgatt                                                 20
SEQ ID NO: 28
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 28
aagtagacct gcccagactc                                                 20
SEQ ID NO: 29
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 29
ggatgttcgt cctcctcaca                                                 20
SEQ ID NO: 30
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 30
accctaagcc cccaattctc                                                 20
SEQ ID NO: 31
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 31
ccacacattc ctgaatccca                                                 20
SEQ ID NO: 32
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 32
aggccccagt gagttctgga                                                 20
SEQ ID NO: 33
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 33
gtctccagat tccagatgtc                                                 20
SEQ ID NO: 34
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 34
ctcaagtcct gcagcattct                                                 20
SEQ ID NO: 35
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 35
tgggtccccc aggatacccc                                                 20
SEQ ID NO: 36
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 36
acggaaaaca tgtctgagcc                                                 20
SEQ ID NO: 37
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 37
ctccgttttc acggaaaaca                                                 20
SEQ ID NO: 38
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 38
gcctattgtt cagctccgtt                                                 20
SEQ ID NO: 39
LENGTH: 21
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 39
ggtcaccaaa tcagcattgt t                                                        21
SEQ ID NO: 40
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 40
gaggctcagc aatgagtgac                                                          20
SEQ ID NO: 41
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: control sequence
SEQUENCE: 41
gcccaagctg gcatccgtca                                                 20
SEQ ID NO: 42
LENGTH: 21
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: control seqeuence
SEQUENCE: 42
gccgaggtcc atgtcgtacg c                                                        21
SEQ ID NO: 43
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: PCR primer
SEQUENCE: 43
caggcggtgc ttgttcct                                                   18
SEQ ID NO: 44
LENGTH: 22
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: PCR primer
SEQUENCE: 44
gccagagggc tgattagaga ga                                              22
SEQ ID NO: 45
LENGTH: 25
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: PCR probe
SEQUENCE: 45
cttctccttc ctgatcgtgg caggc                                           25
SEQ ID NO: 46
LENGTH: 19
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: PCR primer
SEQUENCE: 46
gaaggtgaag gtcggagtc                                                  19
SEQ ID NO: 47
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: PCR primer
SEQUENCE: 47
gaagatggtg atgggatttc                                                 20
SEQ ID NO: 48
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: PCR probe
SEQUENCE: 48
caagcttccc gttctcagcc                                                 20
SEQ ID NO: 49
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: control sequence
SEQUENCE: 49
tctgagtagc agaggagctc                                                 20
SEQ ID NO: 50
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 50
tgcgtctctc atttcccctt                                                 20
SEQ ID NO: 51
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 51
tcccatctct ctccctctct                                                 20
SEQ ID NO: 52
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 52
cagcgcacat ctttcaccca                                                 20
SEQ ID NO: 53
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 53
tctctctcat ccctccctat                                                 20
SEQ ID NO: 54
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 54
cgtctttctc catgtttttt                                                 20
SEQ ID NO: 55
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 55
cacatctctt tctgcatccc                                                 20
SEQ ID NO: 56
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 56
ctctcttccc catctcttgc                                                 20
SEQ ID NO: 57
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 57
gtctctccat ctttccttct                                                 20
SEQ ID NO: 58
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 58
ttccatgtgc cagacatcct                                                 20
SEQ ID NO: 59
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 59
atacacactt agtgagcacc                                                 20
SEQ ID NO: 60
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 60
ttcattcatt cattcactcc                                                 20
SEQ ID NO: 61
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 61
tatatctgct tgttcattca                                                 20
SEQ ID NO: 62
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 62
ctgtctccat atcttattta                                                 20
SEQ ID NO: 63
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 63
tctcttctca caccccacat                                                 20
SEQ ID NO: 64
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 64
cacttgtttc ttcccccatc                                                 20
SEQ ID NO: 65
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 65
ctcaccatct ttattcatat                                                 20
SEQ ID NO: 66
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 66
atatttcccg ctctttctgt                                                 20
SEQ ID NO: 67
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 67
catctctctc cttagctgtc                                                 20
SEQ ID NO: 68
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 68
tcttctctcc ttatctcccc                                                 20
SEQ ID NO: 69
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 69
gtgtgccaga caccctatct                                                 20
SEQ ID NO: 70
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 70
tctttccctg agtgtcttct                                                 20
SEQ ID NO: 71
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 71
accttccagc attcaacagc                                                 20
SEQ ID NO: 72
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 72
ctccattcat ctgtgtattc                                                 20
SEQ ID NO: 73
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 73
tgaggtgtct ggttttctct                                                 20
SEQ ID NO: 74
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 74
acacatcctc agagctctta                                                 20
SEQ ID NO: 75
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 75
ctagccctcc aagttccaag                                                 20
SEQ ID NO: 76
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 76
cgggcttcaa tccccaaatc                                                 20
SEQ ID NO: 77
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 77
aagttctgcc taccatcagc                                                 20
SEQ ID NO: 78
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 78
gtccttctca cattgtctcc                                                 20
SEQ ID NO: 79
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 79
ccttcccttg agctcagcga                                                 20
SEQ ID NO: 80
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 80
ggcctgtgct gttcctccac                                                 20
SEQ ID NO: 81
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 81
cgttctgagt atcccactaa                                                 20
SEQ ID NO: 82
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 82
cacatcccac ctggccatga                                                 20
SEQ ID NO: 83
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 83
gtcctctctg tctgtcatcc                                                 20
SEQ ID NO: 84
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 84
ccaccccaca tccggttcct
SEQ ID NO: 85
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 85
tcctggccct cgagctctgc
SEQ ID NO: 86
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 86
atgtcggttc actctccaca
SEQ ID NO: 87
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 87
agaggagagt cagtgtggcc
SEQ ID NO: 88
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 88
gatcccaaag tagacctgcc
SEQ ID NO: 89
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 89
cagactcggc aaagtcgaga
SEQ ID NO: 90
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 90
tagtcgggcc gattgatctc
SEQ ID NO: 91
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 91
agcgctgagt cggtcaccct
SEQ ID NO: 92
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 92
tctccagctg gaagacccct
SEQ ID NO: 93
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 93
cccagataga tgggctcata
SEQ ID NO: 94
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 94
ccagggcttg gcctcagccc
SEQ ID NO: 95
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 95
cctctggggt ctccctctgg
SEQ ID NO: 96
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 96
caggggctct tgatggcaga
SEQ ID NO: 97
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 97
gaggaggttg accttggtct
SEQ ID NO: 98
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 98
ggtaggagac ggcgatgcgg
SEQ ID NO: 99
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 99
ctgatggtgt gggtgaggag
SEQ ID NO :100
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 100
aggcactcac ctcttccctc
SEQ ID NO :101
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 101
ccctggggaa ctgttgggga
SEQ ID NO :102
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 102
agacacttac tgactgcctg
SEQ ID NO :103
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 103
gaagatgatc ctgaagagga
SEQ ID NO :104
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 104
gagctcttac ctacaacatg
SEQ ID NO :105
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 105
tgagggtttg ctggagggag                                                 20
SEQ ID NO :106
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: control sequence
SEQUENCE: 106
gatcgcgtcg gactatgaag                                                 20
SEQ ID NO: 107
LENGTH: 7208
TYPE: DNA
ORGANISM: Mus musculus
FEATURE:
NAME/KEY: CDS
LOCATION: (4527..4712,5225..5279,5457..5504,5799..6217)
FEATURE:
NAME/KEY: exon
LOCATION: (4371)..(4712)
FEATURE:
NAME/KEY: intron
LOCATION: (4713)..(5224)
FEATURE:
NAME/KEY: exon
LOCATION: (5225)..(5279)
FEATURE:
NAME/KEY: intron
LOCATION: (5280)..(5456)
FEATURE:
NAME/KEY: exon
LOCATION: (5457)..(5504)
FEATURE:
NAME/KEY: intron
LOCATION: (5505)..(5798)
FEATURE:
NAME/KEY: exon
LOCATION: (5799)..(>6972)
PUBLICATION INFORMATION:
AUTHORS: Semon, D.
Kawashima, E.
Jongeneel, C.V.
Shakhov, A.N.
Nedospasov, S.A.
TITLE: Nucleotide sequence of the murine TNF locus, including the
TITLE: TNF-alpha (tumor necrosis factor) and  TNF-beta
(lymphotoxin)
TITLE: genes
JOURNAL: Nucleic Acids Res.
VOLUME: 15
ISSUE: 21
PAGES: 9083-9084
DATE: 1987-11-11
DATABASE ACCESSION NUMBER: Y00467 Genbank
DATABASE ENTRY DATE: 1993-05-11
SEQUENCE: 107
gaattctgaa gctccctctg tacagagcat tggaagcctg gggtgtacat tt ggggttac     60
atgatcttgg ggttctaaga gaataccccc aaatcatctt ccagacctgg aa cattctag    120
gacagggttc tcaaccttcc taactccatg accctttaat acagttcctc at gttgtggt    180
gaccccaacc atacaattat tttcgttgct atttcataac tgtaatttcg ct gctattat    240
gaatcataat gtaaatattt gttttaaata gaggtttgcc aaagggacct tg cccacagg    300
ttgagaactg ccgctccaga gagtaagggg acacagttaa gattgttaca ca ccaggatg    360
ccccagattt ggggagaggg cactgtaatg gaacttcttg acatgaaact gg cagatgaa    420
actggcagaa aaaaaaaaaa aagctgggca gtggtggcac acacctttaa tc ccagcact    480
tgggaggcag aggcaggcgg atttctgagt tctaggccag cctggtctac ag agtgagtt    540
tcaggacagc cagggctaca cagagaaacc ctgtctcgaa aaaagcaaaa aa aaaaaaaa    600
aaaaaaaaaa aaactggcag atgaccagaa aatacagata tattggaata ac tgtgactt    660
gaacccccaa agacaagaga ggaaataggc ctgaaggggc ggcaggcatg tc aagcatcc    720
agagccctgg gttcgaacct gaaaaaacaa aggtgccgct aaccacatgt gg cttcggag    780
ccctccagac atgaccatga tcgacagaga gggaaatgtg cagagaagcc tg tgagcagt    840
caagggtgca gaagtgatat aaaccatcac tcttcaggga accaggcttc ca gtcacagc    900
ccagctgcac cctctccacg aattgctcgg ccgttcactg gaactcctgg gc ctgaccca    960
gctccctgct agtccctgcg gcccacagtt ccccggaccc gactcccttt cc cagaacgc   1020
agtagtctaa gcccttagcc tgcggttctc tcctaggccc cagcctttcc tg ccttcgac   1080
tgaaacagca gcatcttcta agccctgggg gcttccccaa gccccagccc cg acctagaa   1140
cccgcccgct gcctgccaca ctgccgcttc ctctataaag ggacccgagc gc cagcgccc   1200
aggaccccgc acagcaggtg agcctctcct accctgtctc cttgggctta cc ctggtatc   1260
aggcatccct caggatccta cctcctttct tgagccacag ccttttctat ac aacctgcc   1320
tggatcccca gccttaatgg gtctggtcct cctgtcgtgg ctttgatttt tg gtctgttc   1380
ctgtggcggc cttatcagtc tctctctctc tctctctctc tctctctctc tc tctctctc   1440
tctctctctc tctccctctc tctctctctc tctctctctc ttctctctct ct gcctctgt   1500
tagccattgt ctgattctat ggtggagctt tcctcttccc ctctgtctct cc ttatccct   1560
gctcacttca gggttcccct gcctgtcccc ttttctgtct gtcgccctgt ct ctcagggt   1620
ggctgtctca gctgggaggt aaggtctgtc ttccgctgtg tgccccgcct cc gctacaca   1680
cacacactct ctctctctct ctcagcaggt tctccacatg acactgctcg gc cgtctcca   1740
cctcttgagg gtgcttggca cccctcctgt cttcctcctg gggctgctgc tg gccctgcc   1800
tctaggggcc caggtgaggc agcaagagat tgggggtgct ggggtggcct ag ctaactca   1860
gagtcctaga gtcctctcca ctctcttctg tcccagggac tctctggtgt cc gcttctcc   1920
gctgccagga cagcccatcc actccctcag aagcacttga cccatggcat cc tgaaacct   1980
gctgctcacc ttgttggtaa acttctgcct ccagaggaga ggtccagtcc ct gccttttg   2040
tcctacttgc ccaggggctc aggcgatctt cccatctccc cacaccaact tt tcttaccc   2100
ctaagggcag gcaccccact cccatctccc taccaaccat cccacttgtc ca gtgcctgc   2160
tcctcaggga tggggacctc tgatcttgat agccccccaa tgtcttgtgc ct cttcccag   2220
ggtaccccag caagcagaac tcactgctct ggagagcaag cacggatcgt gc ctttctcc   2280
gacatggctt ctctttgagc aacaactccc tcctgatccc caccagtggc ct ctactttg   2340
tctactccca ggtggttttc tctggagaaa gctgctcccc cagggccatt cc cactccca   2400
tctacctggc acacgaggtc cagctctttt cctcccaata ccccttccat gt gcctctcc   2460
tcagtgcgca gaagtctgtg tatccgggac ttcaaggacc gtgggtgcgc tc aatgtacc   2520
agggggctgt gttcctgctc agtaagggag accagctgtc cacccacacc ga cggcatct   2580
cccatctaca cttcagcccc agcagtgtat tctttggagc ctttgcactg ta gattctaa   2640
agaaacccaa gaattggatt ccaggcctcc atcctgaccg ttgtttcaag gg tcacatcc   2700
ccacagtctc cagccttccc cactaaaata acctggagct ctcacgggag tc tgagacac   2760
ttcaggggac tacatcttcc ccagggccac tccagatgct caggggacga ct caagccta   2820
cctagaagtt cctgcacaga gcagggtttt tgtgggtcta ggtcggacag ag acctggac   2880
atgaaggagg gacagacatg ggagaggtgg ctgggaacag gggaaggttg ac tatttatg   2940
gagagaaaag ttaagttatt tatttataga gaatagaaag aggggaaaaa ta gaaagccg   3000
tcagatgaca actaggtccc agacacaaag gtgtctcacc tcagacagga cc catctaag   3060
agagagatgg cgagagaatt agatgtgggt gaccaagggg ttctagaaga aa gcacgaag   3120
ctctaaaagc cagccactgc ttggctagac atccacaggg accccctgca cc atctgtga   3180
aacccaataa acctcttttc tctgagattc tgtctgcttg tgtctgtctt gc gttggggg   3240
agaaacttcc tggtctcttt aaggagtgga gcaggggaca gaggcctcag tt ggtccatg   3300
ggatccgggc agagcaaaga gacatgagga gcaggcagct cccagagaca tg gtggattc   3360
acgggagtga ggcagcttaa ctgccgagag acccaaagga tgagctaggg ag atccatcc   3420
aagggtggag agagatgagg gttctgggga gaagtgactc cactggaggg tg ggagagtg   3480
tttaggagtg ggagggtggg ggaggggaat ccttggaaga ccggggagtc at acggattg   3540
ggagaaatcc tggaagcagg gctgtgggac ctaaatgtct gagttgatgt ac cgcagtca   3600
agatatggca gaggctccgt ggaaaactca cttgggagca gggacccaaa gc agcagcct   3660
gagctcatga tcagagtgaa aggagaaggc ttgtgaggtc cgtgaattcc ca gggctgag   3720
ttcattccct ctgggctgcc ccatactcat cccattaccc cccccaccag cc ctcccaaa   3780
gcccatgcac acttcccaac tctcaagctg ctctgccttc agccacttcc tc caagaact   3840
caaacagggg gctttccctc ctcaatatca tgtctccccc cttatgcacc ca gctttcag   3900
aagcaccccc ccatgctaag ttctccccca tggatgtccc atttagaaat ca aaaggaaa   3960
tagacacagg catggtcttt ctacaaagaa acagacaatg attagctctg ga ggacagag   4020
aagaaatggg tttcagttct cagggtccta tacaacacac acacacacac ac acacacac   4080
acacacacac acacaccctc ctgattggcc ccagattgcc acagaatcct gg tggggacg   4140
acgggggaga gattccttga tgcctgggtg tccccaactt tccaaaccct ct gcccccgc   4200
gatggagaag aaaccgagac agaggtgtag ggccactacc gcttcctcca ca tgagatca   4260
tggttttctc caccaaggaa gttttccgag ggttgaatga gagcttttcc cc gccctctt   4320
ccccaagggc tataaaggcg gccgtctgca cagccagcca gcagaagctc cc tcagcgag   4380
gacagcaagg gactagccag gagggagaac agaaactcca gaacatcttg ga aatagctc   4440
ccagaaaagc aagcagccaa ccaggcaggt tctgtccctt tcactcactg gc ccaaggcg   4500
ccacatctcc ctccagaaaa gacacc atg agc aca gaa agc atg atc cgc gac    4553
Met Ser Thr Glu Ser Met Ile Arg Asp
1               5
gtg gaa ctg gca gaa gag gca ctc ccc caa aa g atg ggg ggc ttc cag     4601
Val Glu Leu Ala Glu Glu Ala Leu Pro Gln Ly s Met Gly Gly Phe Gln
10                  15                  20                  25
aac tcc agg cgg tgc cta tgt ctc agc ctc tt c tca ttc ctg ctt gtg     4649
Asn Ser Arg Arg Cys Leu Cys Leu Ser Leu Ph e Ser Phe Leu Leu Val
30                  35                  40
gca ggg gcc acc acg ctc ttc tgt cta ctg aa c ttc ggg gtg atc ggt     4697
Ala Gly Ala Thr Thr Leu Phe Cys Leu Leu As n Phe Gly Val Ile Gly
45                  50                  55
ccc caa agg gat gag gtgagtgtct gggcaaccct tattctcgc t cacaagcaaa     4752
Pro Gln Arg Asp Glu
60
acgggttagg agggcaagaa ggacagtgtg agggaaagaa gtgggctaat gg gcagggca   4812
aggtggagga gagtgtggag gggacagagt caggacctcg gacccatgcg tc cagctgac   4872
taaacatcct tcgtcggatg cacagagaga tgaatgaacg aacaagtgtg tt cacacgtg   4932
gagagatctg gaaagatgtg gccaggggaa gaggggataa gcaagagata aa actcagag   4992
acagaaatga gagaggcatg agagataagg aggaagatga aggggagata ac gggagatc   5052
aagcacagag ggcaccgcag aaagaagccg tgggttggac agatgaatga at gaagaaga   5112
aaacacaaag tggggggtgg gtggggcaaa gaggaactgt aagcggggca at cagccggg   5172
agcttctcct ttggggtgag tctgtcttaa ctaacctcct tttcctacac ag  aag ttc   5230
Lys Phe
cca aat ggc ctc cct ctc atc agt tct atg gc c cag acc ctc aca ctc     5278
Pro Asn Gly Leu Pro Leu Ile Ser Ser Met Al a Gln Thr Leu Thr Leu
65                  70                  75                  80
agtaagtgtt cccacacctc tctcttaatt taagatggag aagggcagtt ag gcatggga   5338
Arg
tgagatgggg tggggggaaa acttaaagct ttggtttggg aggaaagggg tc taagtgca   5398
tagatgcttg ctgggaagcc taaaaggctc atccttgcct ttgtctcttc cc ctcca      5455
gga tca tct tct caa aat tcg agt gac aag cc t gta gcc cac gtc gta     5503
Ser Ser Ser Gln Asn Ser Ser Asp Lys Pro Val Ala His Val Val
85                  90                  95
ggtaagattt ctttacatgt gccttgagaa tgaaggggca tgattttggg gg gcgggttg   5563
aggggtgtcg agccaggctg agaaaagaca gagctcttag agacagcacg tg agagtcag   5623
agcagtgact caaaagcaag gcatcagggg gccacccggg acctcatagc ca atgggatg   5683
tggaaagaca gagggtgcag gaaccggaag tgaagtgtgg gtagctgctg ag gctcagga   5743
tgtggagtgt gaactaagag ggtgacactg actcaatcct cccccccccc ct ca gca     5800
Ala
aac cac caa gtg gag gag cag ctg gag tgg ct g agc cag cgc gcc aac     5848
Asn His Gln Val Glu Glu Gln Leu Glu Trp Le u Ser Gln Arg Ala Asn
100                 105                 110
gcc ctc ctg gcc aac ggc atg gat ctc aaa ga c aac caa cta gtg gtg     5896
Ala Leu Leu Ala Asn Gly Met Asp Leu Lys As p Asn Gln Leu Val Val
115                 120                 125
cca gcc gat ggg ttg tac ctt gtc tac tcc ca g gtt ctc ttc aag gga     5944
Pro Ala Asp Gly Leu Tyr Leu Val Tyr Ser Gl n Val Leu Phe Lys Gly
130                 1 35                 1 40                 1 45
caa ggc tgc ccc gac tac gtg ctc ctc acc ca c acc gtc agc cga ttt     5992
Gln Gly Cys Pro Asp Tyr Val Leu Leu Thr Hi s Thr Val Ser Arg Phe
150                 155                 160
gct atc tca tac cag gag aaa gtc aac ctc ct c tct gcc gtc aag agc     6040
Ala Ile Ser Tyr Gln Glu Lys Val Asn Leu Le u Ser Ala Val Lys Ser
165                 170                 175
ccc tgc ccc aag gac acc cct gag ggg gct ga g ctc aaa ccc tgg tat     6088
Pro Cys Pro Lys Asp Thr Pro Glu Gly Ala Gl u Leu Lys Pro Trp Tyr
180                 185                 190
gag ccc ata tac ctg gga gga gtc ttc cag ct g gag aag ggg gac caa     6136
Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Le u Glu Lys Gly Asp Gln
195                 200                 205
ctc agc gct gag gtc aat ctg ccc aag tac tt a gac ttt gcg gag tcc     6184
Leu Ser Ala Glu Val Asn Leu Pro Lys Tyr Le u Asp Phe Ala Glu Ser
210                 2 15                 2 20                 2 25
ggg cag gtc tac ttt gga gtc att gct ctg tg a agggaatggg tgttcatcca   6237
Gly Gln Val Tyr Phe Gly Val Ile Ala Leu
230                 235
ttctctaccc agcccccact ctgacccctt tactctgacc cctttattgt ct actcctca   6297
gagcccccag tctgtgtcct tctaacttag aaaggggatt atggctcaga gt ccaactct   6357
gtgctcagag ctttcaacaa ctactcagaa acacaagatg ctgggacagt ga cctggact   6417
gtgggcctct catgcaccac catcaaggac tcaaatgggc tttccgaatt ca ctggagcc   6477
tcgaatgtcc attcctgagt tctgcaaagg gagagtggtc aggttgcctc tg tctcagaa   6537
tgaggctgga taagatctca ggccttccta ccttcagacc tttccagact ct tccctgag   6597
gtgcaatgca cagccttcct cacagagcca gcccccctct atttatattt gc acttatta   6657
tttattattt atttattatt tatttatttg cttatgaatg tatttatttg ga aggccggg   6717
gtgtcctgga ggacccagtg tgggaagctg tcttcagaca gacatgtttt ct gtgaaaac   6777
ggagctgagc tgtccccacc tggcctctct accttgttgc ctcctctttt gc ttatgttt   6837
aaaacaaaat atttatctaa cccaattgtc ttaataacgc tgatttggtg ac caggctgt   6897
cgctacatca ctgaacctct gctccccacg ggagccgtga ctgtaattgc cc tacagtca   6957
attgagagaa ataaagatcg cttggaaaag aaatgtgatt tctgtcttgg ga tgaagtct   7017
gcatccatct ctttgcggag gcctaaagtc tctgggtcca gatctcagtc tt tatacccc   7077
tgggccatta agacccccaa gacccccgtg gaacaaaagg cagccaacat cc ctacctct   7137
cccccggaaa caggagccta accctaatta cctttgccct ggggcatggg aa tttcccac   7197
tctgggaatt c                                                        7208
SEQ ID NO: 108
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 108
gagcttctgc tggctggctg                                                 20
SEQ ID NO: 109
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 109
ccttgctgtc ctcgctgagg                                                 20
SEQ ID NO: 110
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 110
tcatggtgtc ttttctggag                                                 20
SEQ ID NO: 111
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 111
ctttctgtgc tcatggtgtc                                                 20
SEQ ID NO: 112
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 112
gcggatcatg ctttctgtgc                                                 20
SEQ ID NO: 113
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 113
gggaggccat ttgggaactt                                                 20
SEQ ID NO: 114
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 114
cgaattttga gaagatgatc                                                 20
SEQ ID NO: 115
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 115
ctcctccact tggtggtttg                                                 20
SEQ ID NO: 116
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 116
cctgagatct tatccagcct                                                 20
SEQ ID NO: 117
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 117
caattacagt cacggctccc                                                 20
SEQ ID NO: 118
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
SEQUENCE: 118
cccttcattc tcaaggcaca                                                 20
SEQ ID NO: 119
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 119
cacccctcaa cccgcccccc                                                 20
SEQ ID NO: 120
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 120
agagctctgt cttttctcag                                                 20
SEQ ID NO: 121
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 121
cactgctctg actctcacgt                                                 20
SEQ ID NO: 122
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 122
atgaggtccc gggtggcccc                                                 20
SEQ ID NO: 123
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 123
caccctctgt ctttccacat                                                 20
SEQ ID NO: 124
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 124
ctccacatcc tgagcctcag                                                 20
SEQ ID NO: 125
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 125
attgagtcag tgtcaccctc                                                 20
SEQ ID NO: 126
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 126
gctggctcag ccactccagc                                                 20
SEQ ID NO: 127
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 127
tctttgagat ccatgccgtt                                                 20
SEQ ID NO: 128
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 128
aacccatcgg ctggcaccac                                                 20
SEQ ID NO: 129
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 129
gtttgagctc agccccctca                                                 20
SEQ ID NO: 130
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 130
ctcctcccag gtatatgggc                                                 20
SEQ ID NO: 131
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 131
tgagttggtc ccccttctcc                                                 20
SEQ ID NO: 132
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 132
caaagtagac ctgcccggac                                                 20
SEQ ID NO: 133
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 133
acacccattc ccttcacaga                                                 20
SEQ ID NO: 134
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 134
cataatcccc tttctaagtt                                                 20
SEQ ID NO: 135
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 135
cacagagttg gactctgagc                                                 20
SEQ ID NO: 136
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 136
cagcatcttg tgtttctgag                                                 20
SEQ ID NO: 137
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 137
cacagtccag gtcactgtcc                                                 20
SEQ ID NO: 138
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 138
tgatggtggt gcatgagagg                                                 20
SEQ ID NO: 139
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 139
gtgaattcgg aaagcccatt                                                 20
SEQ ID NO: 140
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 140
cctgaccact ctccctttgc                                                 20
SEQ ID NO: 141
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 141
tgcatccccc aggccaccat                                                 20
SEQ ID NO: 142
LENGTH: 21
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 142
gccgaggtcc atgtcgtacg c                                               21
SEQ ID NO: 143
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 143
tcaagcagtg ccaccgatcc                                                 20
SEQ ID NO: 144
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 144
agtgtcttct gtgtgccaga                                                 20
SEQ ID NO: 145
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 145
gtgtcttctg tgtgccagac                                                 20
SEQ ID NO: 146
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 146
tgtcttctgt gtgccagaca                                                 20
SEQ ID NO: 147
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 147
gtcttctgtg tgccagacac                                                 20
SEQ ID NO: 148
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 148
tcttctgtgt gccagacacc                                                 20
SEQ ID NO: 149
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 149
cttctgtgtg ccagacaccc                                                 20
SEQ ID NO: 150
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 150
ttctgtgtgc cagacaccct                                                 20
SEQ ID NO: 151
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 151
tctgtgtgcc agacacccta                                                 20
SEQ ID NO: 152
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 152
ctgtgtgcca gacaccctat                                                 20
SEQ ID NO: 153
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 153
tgtgtgccag acaccctatc                                                 20
SEQ ID NO: 154
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 154
tgtgccagac accctatctt                                                 20
SEQ ID NO: 155
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 155
gtgccagaca ccctatcttc                                                 20
SEQ ID NO: 156
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 156
tgccagacac cctatcttct                                                 20
SEQ ID NO: 157
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 157
gccagacacc ctatcttctt                                                 20
SEQ ID NO: 158
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 158
ccagacaccc tatcttcttc                                                 20
SEQ ID NO: 159
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 159
cagacaccct atcttcttct                                                 20
SEQ ID NO: 160
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 160
agacacccta tcttcttctc                                                 20
SEQ ID NO: 161
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 161
gacaccctat cttcttctct                                                 20
SEQ ID NO: 162
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 162
acaccctatc ttcttctctc                                                 20
SEQ ID NO: 163
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 163
caccctatct tcttctctcc                                                 20
SEQ ID NO: 164
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 164
gtcttctgtg tgccagac                                                   18
SEQ ID NO: 165
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 165
tcttctgtgt gccagaca                                                   18
SEQ ID NO: 166
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 166
cttctgtgtg ccagacac                                                   18
SEQ ID NO: 167
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 167
ttctgtgtgc cagacacc                                                   18
SEQ ID NO: 168
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 168
tctgtgtgcc agacaccc                                                   18
SEQ ID NO: 169
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 169
ctgtgtgcca gacaccct                                                   18
SEQ ID NO: 170
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 170
tgtgtgccag acacccta                                                   18
SEQ ID NO: 171
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 171
gtgtgccaga caccctat                                                   18
SEQ ID NO: 172
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 172
tgtgccagac accctatc                                                   18
SEQ ID NO: 173
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 173
tgccagacac cctatctt                                                   18
SEQ ID NO: 174
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 174
gccagacacc ctatcttc                                                   18
SEQ ID NO: 175
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 175
ccagacaccc tatcttct                                                   18
SEQ ID NO: 176
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 176
cagacaccct atcttctt                                                   18
SEQ ID NO: 177
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 177
agacacccta tcttcttc                                                   18
SEQ ID NO: 178
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 178
gacaccctat cttcttct                                                   18
SEQ ID NO: 179
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 179
acaccctatc ttcttctc                                                   18
SEQ ID NO: 180
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 180
agaggtttgg agacacttac                                                 20
SEQ ID NO: 181
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 181
gaattaggaa agaggtttgg                                                 20
SEQ ID NO: 182
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 182
cccaaaccca gaattaggaa                                                 20
SEQ ID NO: 183
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 183
tacccccaaa cccaaaccca                                                 20
SEQ ID NO: 184
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 184
gtactaaccc tacccccaaa                                                 20
SEQ ID NO: 185
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 185
ttccataccg gtactaaccc                                                 20
SEQ ID NO: 186
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 186
cccccactgc ttccataccg                                                 20
SEQ ID NO: 187
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 187
ctttaaattt cccccactgc                                                 20
SEQ ID NO: 188
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 188
aagaccaaaa ctttaaattt                                                 20
SEQ ID NO: 189
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 189
atcctccccc aagaccaaaa                                                 20
SEQ ID NO: 190
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 190
acctccatcc atcctccccc                                                 20
SEQ ID NO: 191
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 191
ccctactttc acctccatcc                                                 20
SEQ ID NO: 192
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 192
gaaaataccc ccctactttc                                                 20
SEQ ID NO: 193
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 193
aaacttccta gaaaataccc                                                 20
SEQ ID NO: 194
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 194
tgagaccctt aaacttccta                                                 20
SEQ ID NO: 195
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 195
aagaaaaagc tgagaccctt                                                 20
SEQ ID NO: 196
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 196
ggagagagaa aagaaaaagc                                                 20
SEQ ID NO: 197
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 197
tgagccagaa gaggttgagg                                                 20
SEQ ID NO: 198
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 198
attctctttt tgagccagaa                                                 20
SEQ ID NO: 199
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 199
taagccccca attctctttt                                                 20
SEQ ID NO: 200
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 200
gttccgaccc taagccccca                                                 20
SEQ ID NO: 201
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 201
ctaagcttgg gttccgaccc                                                 20
SEQ ID NO: 202
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 202
gcttaaagtt ctaagcttgg                                                 20
SEQ ID NO: 203
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 203
tggtcttgtt gcttaaagtt                                                 20
SEQ ID NO: 204
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 204
ttcgaagtgg tggtcttgtt                                                 20
SEQ ID NO: 205
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 205
aatcccaggt ttcgaagtgg                                                 20
SEQ ID NO: 206
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 206
cacattcctg aatcccaggt                                                 20
SEQ ID NO: 207
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 207
gtgcaggcca cacattcctg                                                 20
SEQ ID NO: 208
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 208
gcacttcact gtgcaggcca                                                 20
SEQ ID NO: 209
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 209
gtggttgcca gcacttcact                                                 20
SEQ ID NO: 210
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 210
tgaattctta gtggttgcca                                                 20
SEQ ID NO: 211
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 211
ggccccagtt tgaattctta                                                 20
SEQ ID NO: 212
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 212
gagttctgga ggccccagtt                                                 20
SEQ ID NO: 213
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 213
aggccccagt gagttctgga                                                 20
SEQ ID NO: 214
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 214
tcaaagctgt aggccccagt                                                 20
SEQ ID NO: 215
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 215
atgtcaggga tcaaagctgt                                                 20
SEQ ID NO: 216
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 216
cagattccag atgtcaggga                                                 20
SEQ ID NO: 217
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 217
ccctggtctc cagattccag                                                 20
SEQ ID NO: 218
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 218
accaaaggct ccctggtctc                                                 20
SEQ ID NO: 219
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 219
tctggccaga accaaaggct                                                 20
SEQ ID NO: 220
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 220
cctgcagcat tctggccaga                                                 20
SEQ ID NO: 221
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 221
cttctcaagt cctgcagcat                                                 20
SEQ ID NO: 222
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 222
taggtgaggt cttctcaagt                                                 20
SEQ ID NO: 223
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 223
tgtcaatttc taggtgaggt                                                 20
SEQ ID NO: 224
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 224
ggtccacttg tgtcaatttc                                                 20
SEQ ID NO: 225
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 225
gaaggcctaa ggtccacttg                                                 20
SEQ ID NO: 226
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 226
ctggagagag gaaggcctaa                                                 20
SEQ ID NO: 227
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 227
ctggaaacat ctggagagag                                                 20
SEQ ID NO: 228
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 228
tcaaggaagt ctggaaacat                                                 20
SEQ ID NO: 229
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 229
gctccgtgtc tcaaggaagt                                                 20
SEQ ID NO: 230
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 230
ataaatacat tcatctgtaa                                                 20
SEQ ID NO: 231
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 231
ggtctcccaa ataaatacat                                                 20
SEQ ID NO: 232
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 232
aggatacccc ggtctcccaa                                                 20
SEQ ID NO: 233
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 233
tgggtccccc aggatacccc                                                 20
SEQ ID NO: 234
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 234
gctcctacat tgggtccccc                                                 20
SEQ ID NO: 235
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 235
agccaaggca gctcctacat                                                 20
SEQ ID NO: 236
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 236
aacatgtctg agccaaggca                                                 20
SEQ ID NO: 237
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 237
tttcacggaa aacatgtctg                                                 20
SEQ ID NO: 238
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 238
tcagctccgt tttcacggaa                                                 20
SEQ ID NO: 239
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 239
agcctattgt tcagctccgt                                                 20
SEQ ID NO: 240
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 240
acatgggaac agcctattgt                                                 20
SEQ ID NO: 241
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 241
atcaaaagaa ggcacagagg                                                 20
SEQ ID NO: 242
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 242
gtttagacaa cttaatcaga                                                 20
SEQ ID NO: 243
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 243
aatcagcatt gtttagacaa                                                 20
SEQ ID NO: 244
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 244
ttggtcacca aatcagcatt                                                 20
SEQ ID NO: 245
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 245
tgagtgacag ttggtcacca                                                 20
SEQ ID NO: 246
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 246
ggctcagcaa tgagtgacag                                                 20
SEQ ID NO: 247
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 247
attacagaca caactcccct                                                 20
SEQ ID NO: 248
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 248
tagtagggcg attacagaca                                                 20
SEQ ID NO: 249
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 249
cgccactgaa tagtagggcg                                                 20
SEQ ID NO: 250
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 250
ctttatttct cgccactgaa                                                 20
SEQ ID NO: 251
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 251
ctgagggagc gtctgctggc                                                 20
SEQ ID NO: 252
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 252
ccttgctgag ggagcgtctg                                                 20
SEQ ID NO: 253
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 253
ctggtcctct gctgtccttg                                                 20
SEQ ID NO: 254
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 254
cctctgctgt ccttgctgag                                                 20
SEQ ID NO: 255
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 255
ttctctccct cttagctggt                                                 20
SEQ ID NO: 256
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 256
tccctcttag ctggtcctct                                                 20
SEQ ID NO: 257
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 257
tctgagggtt gttttcaggg                                                 20
SEQ ID NO: 258
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 258
ctgtagttgc ttctctccct                                                 20
SEQ ID NO: 259
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 259
acctgcctgg cagcttgtca                                                 20
SEQ ID NO: 260
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 260
ggatgtggcg tctgagggtt                                                 20
SEQ ID NO: 261
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 261
tgtgagagga agagaacctg                                                 20
SEQ ID NO: 262
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 262
gaggaagaga acctgcctgg                                                 20
SEQ ID NO: 263
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 263
agccgtgggt cagtatgtga                                                 20
SEQ ID NO: 264
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 264
tgggtcagta tgtgagagga                                                 20
SEQ ID NO: 265
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 265
gagagggtga agccgtgggt                                                 20
SEQ ID NO: 266
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 266
tcatggtgtc ctttccaggg                                                 20
SEQ ID NO: 267
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 267
ctttcagtgc tcatggtgtc                                                 20
SEQ ID NO: 268
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 268
tcatgctttc agtgctcatg                                                 20
SEQ ID NO: 269
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 269
acgtcccgga tcatgctttc                                                 20
SEQ ID NO: 270
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 270
gctccacgtc ccggatcatg                                                 20
SEQ ID NO: 271
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 271
tcctcggcca gctccacgtc                                                 20
SEQ ID NO: 272
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 272
gcgcctcctc ggccagctcc                                                 20
SEQ ID NO: 273
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 273
aggaacaagc accgcctgga                                                 20
SEQ ID NO: 274
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 274
caagcaccgc ctggagccct                                                 20
SEQ ID NO: 275
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 275
aaggagaaga ggctgaggaa                                                 20
SEQ ID NO: 276
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 276
gaagaggctg aggaacaagc                                                 20
SEQ ID NO: 277
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 277
cctgccacga tcaggaagga                                                 20
SEQ ID NO: 278
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 278
cacgatcagg aaggagaaga                                                 20
SEQ ID NO: 279
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 279
aagagcgtgg tggcgcctgc                                                 20
SEQ ID NO: 280
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 280
cgtggtggcg cctgccacga                                                 20
SEQ ID NO: 281
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 281
aagtgcagca ggcagaagag                                                 20
SEQ ID NO: 282
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 282
cagcaggcag aagagcgtgg                                                 20
SEQ ID NO: 283
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 283
gatcactcca aagtgcagca                                                 20
SEQ ID NO: 284
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 284
gggccgatca ctccaaagtg                                                 20
SEQ ID NO: 285
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 285
gggccagagg gctgattaga                                                 20
SEQ ID NO: 286
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 286
agagggctga ttagagagag                                                 20
SEQ ID NO: 287
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 287
gctacaggct tgtcactcgg                                                 20
SEQ ID NO: 288
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 288
ctgactgcct gggccagagg                                                 20
SEQ ID NO: 289
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 289
tacaacatgg gctacaggct                                                 20
SEQ ID NO: 290
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 290
agccactgga gctgcccctc                                                 20
SEQ ID NO: 291
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 291
ctggagctgc ccctcagctt                                                 20
SEQ ID NO: 292
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 292
ttggcccggc ggttcagcca                                                 20
SEQ ID NO: 293
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 293
ttggccagga gggcattggc                                                 20
SEQ ID NO: 294
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 294
ccggcggttc agccactgga                                                 20
SEQ ID NO: 295
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 295
ctcagctcca cgccattggc                                                 20
SEQ ID NO: 296
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 296
caggagggca ttggcccggc                                                 20
SEQ ID NO: 297
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 297
ctccacgcca ttggccagga                                                 20
SEQ ID NO: 298
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 298
accagctggt tatctctcag                                                 20
SEQ ID NO: 299
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 299
ctggttatct ctcagctcca                                                 20
SEQ ID NO: 300
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 300
ccctctgatg gcaccaccag                                                 20
SEQ ID NO: 301
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 301
tgatggcacc accagctggt                                                 20
SEQ ID NO: 302
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 302
tagatgaggt acaggccctc                                                 20
SEQ ID NO: 303
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 303
aagaggacct gggagtagat                                                 20
SEQ ID NO: 304
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 304
gaggtacagg ccctctgatg                                                 20
SEQ ID NO: 305
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 305
cagccttggc ccttgaagag                                                 20
SEQ ID NO: 306
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 306
gacctgggag tagatgaggt                                                 20
SEQ ID NO: 307
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 307
ttggcccttg aagaggacct                                                 20
SEQ ID NO: 308
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 308
tggtgtgggt gaggagcaca                                                 20
SEQ ID NO: 309
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 309
cggcgatgcg gctgatggtg                                                 20
SEQ ID NO: 310
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 310
tgggtgagga gcacatgggt                                                 20
SEQ ID NO: 311
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 311
tggtctggta ggagacggcg                                                 20
SEQ ID NO: 312
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 312
atgcggctga tggtgtgggt                                                 20
SEQ ID NO: 313
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 313
agaggaggtt gaccttggtc                                                 20
SEQ ID NO: 314
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 314
tggtaggagac ggcgatgcg                                                 20
SEQ ID NO: 315
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 315
aggttgacct tggtctggta                                                 20
SEQ ID NO: 316
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 316
ggctcttgat ggcagagagg                                                 20
SEQ ID NO: 317
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 317
tcataccaggg cttggcctc                                                 20
SEQ ID NO: 318
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 318
ttgatggcag agaggaggtt                                                 20
SEQ ID NO: 319
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 319
agctggaaga cccctcccag                                                 20
SEQ ID NO: 320
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 320
atagatgggc tcataccagg                                                 20
SEQ ID NO: 321
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 321
cggtcaccct tctccagctg                                                 20
SEQ ID NO: 322
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 322
gaagacccct cccagataga                                                 20
SEQ ID NO: 323
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 323
acccttctcc agctggaaga                                                 20
SEQ ID NO: 324
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 324
tcggcaaagt cgagatagtc                                                 20
SEQ ID NO: 325
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 325
gggccgattg atctcagcgc                                                 20
SEQ ID NO: 326
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 326
tagacctgcc cagactcggc                                                 20
SEQ ID NO: 327
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 327
aaagtcgaga tagtcgggcc                                                 20
SEQ ID NO: 328
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 328
gcaatgatcc caaagtagac                                                 20
SEQ ID NO: 329
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 329
ctgcccagac tcggcaaagt                                                 20
SEQ ID NO: 330
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 330
cgtcctcctc acagggcaat                                                 20
SEQ ID NO: 331
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 331
ggaaggttgg atgttcgtcc                                                 20
SEQ ID NO: 332
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 332
tcctcacagg gcaatgatcc                                                 20
SEQ ID NO: 333
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 333
gttgagggtg tctgaaggag                                                 20
SEQ ID NO: 334
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 334
gttggatgtt cgtcctcctc                                                 20
SEQ ID NO: 335
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 335
tttgagccag aagaggttga                                                 20
SEQ ID NO: 336
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 336
gaggcgtttg ggaaggttgg                                                 20
SEQ ID NO: 337
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 337
gcccccaatt ctctttttga                                                 20
SEQ ID NO: 338
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 338
gccagaagag gttgagggtg                                                 20
SEQ ID NO: 339
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 339
gggttccgac cctaagcccc                                                 20
SEQ ID NO: 340
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 340
caattctctt tttgagccag                                                 20
SEQ ID NO: 341
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 341
taaagttcta agcttgggtt                                                 20
SEQ ID NO: 342
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 342
ccgaccctaa gcccccaatt                                                 20
SEQ ID NO: 343
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 343
ggtggtcttg ttgcttaaag                                                 20
SEQ ID NO: 344
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 344
ttctaagctt gggttccgac                                                 20
SEQ ID NO: 345
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 345
cccaggtttc gaagtggtgg                                                 20
SEQ ID NO: 346
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 346
tcttgttgct taaagttcta                                                 20
SEQ ID NO: 347
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 347
cacacattcc tgaatcccag                                                 20
SEQ ID NO: 348
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 348
gtttcgaagt ggtggtcttg                                                 20
SEQ ID NO: 349
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 349
cttcactgtg caggccacac                                                 20
SEQ ID NO: 350
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 350
attcctgaat cccaggtttc                                                 20
SEQ ID NO: 351
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 351
tagtggttgc cagcacttca                                                 20
SEQ ID NO: 352
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 352
cccagtttga attcttagtg                                                 20
SEQ ID NO: 353
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 353
ctgtgcaggc cacacattcc                                                 20
SEQ ID NO: 354
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 354
gtgagttctg gaggccccag                                                 20
SEQ ID NO: 355
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 355
gttgccagca cttcactgtg                                                 20
SEQ ID NO: 356
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 356
tttgaattct tagtggttgc                                                 20
SEQ ID NO: 357
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 357
aagctgtagg ccccagtgag                                                 20
SEQ ID NO: 358
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 358
ttctggaggc cccagtttga                                                 20
SEQ ID NO: 359
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 359
agatgtcagg gatcaaagct                                                 20
SEQ ID NO: 360
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 360
tggtctccag attccagatg                                                 20
SEQ ID NO: 361
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 361
gtaggcccca gtgagttctg                                                 20
SEQ ID NO: 362
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 362
gaaccaaagg ctccctggtc                                                 20
SEQ ID NO: 363
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 363
tcagggatca aagctgtagg                                                 20
SEQ ID NO: 364
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 364
tccagattcc agatgtcagg                                                 20
SEQ ID NO: 365
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 365
gcagcattct ggccagaacc                                                 20
SEQ ID NO: 366
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 366
gtcttctcaa gtcctgcagc                                                 20
SEQ ID NO: 367
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 367
aaaggctccc tggtctccag                                                 20
SEQ ID NO: 368
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 368
caatttctag gtgaggtctt                                                 20
SEQ ID NO: 369
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 369
attctggcca gaaccaaagg                                                 20
SEQ ID NO: 370
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 370
aaggtccact tgtgtcaatt                                                 20
SEQ ID NO: 371
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 371
gagagaggaa ggcctaaggt                                                 20
SEQ ID NO: 372
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 372
tctaggtgag gtcttctcaa                                                 20
SEQ ID NO: 373
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 373
ccacttgtgt caatttctag                                                 20
SEQ ID NO: 374
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 374
gtctggaaac atctggagag                                                 20
SEQ ID NO: 375
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 375
ccgtgtctca aggaagtctg                                                 20
SEQ ID NO: 376
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 376
aggaaggcct aaggtccact                                                 20
SEQ ID NO: 377
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 377
gagggagctg gctccatggg                                                 20
SEQ ID NO: 378
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 378
gaaacatctg gagagaggaa                                                 20
SEQ ID NO: 379
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 379
gtgcaaacat aaatagaggg                                                 20
SEQ ID NO: 380
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 380
tctcaaggaa gtctggaaac                                                 20
SEQ ID NO: 381
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 381
aataaataat cacaagtgca                                                 20
SEQ ID NO: 382
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 382
gggctgggct ccgtgtctca                                                 20
SEQ ID NO: 383
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 383
taccccggtc tcccaaataa                                                 20
SEQ ID NO: 384
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 384
aacataaata gagggagctg                                                 20
SEQ ID NO: 385
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 385
ttgggtcccc caggataccc                                                 20
SEQ ID NO: 386
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 386
ataatcacaa gtgcaaacat                                                 20
SEQ ID NO: 387
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 387
aaggcagctc ctacattggg                                                 20
SEQ ID NO: 388
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 388
cggtctccca aataaataca                                                 20
SEQ ID NO: 389
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 389
aaacatgtct gagccaaggc                                                 20
SEQ ID NO: 390
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 390
tcccccagga taccccggtc                                                 20
SEQ ID NO: 391
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 391
agctcctaca ttgggtcccc                                                 20
SEQ ID NO: 392
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 392
tgtctgagcc aaggcagctc                                                 20
SEQ ID NO: 393
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 393
cagcctattg ttcagctccg                                                 20
SEQ ID NO: 394
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 394
agaaggcaca gaggccaggg                                                 20
SEQ ID NO: 395
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 395
ttttcacgga aaacatgtct                                                 20
SEQ ID NO: 396
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 396
tattgttcag ctccgttttc                                                 20
SEQ ID NO: 397
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 397
aaaaacataa tcaaaagaag                                                 20
SEQ ID NO: 398
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 398
cagataaata ttttaaaaaa                                                 20
SEQ ID NO: 399
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 399
tacatgggaa cagcctattg                                                 20
SEQ ID NO: 400
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 400
tttagacaac ttaatcagat                                                 20
SEQ ID NO: 401
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 401
cataatcaaa agaaggcaca                                                 20
SEQ ID NO: 402
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 402
accaaatcag cattgtttag                                                 20
SEQ ID NO: 403
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 403
aaatatttta aaaaacataa                                                 20
SEQ ID NO: 404
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 404
gagtgacagt tggtcaccaa                                                 20
SEQ ID NO: 405
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 405
acaacttaatc agataaata                                                 20
SEQ ID NO: 406
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 406
cagaggctca gcaatgagtg                                                 20
SEQ ID NO: 407
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 407
atcagcattg tttagacaac                                                 20
SEQ ID NO: 408
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 408
agggcgatta cagacacaac                                                 20
SEQ ID NO: 409
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 409
acagttggtc accaaatcag                                                 20
SEQ ID NO: 410
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 410
tcgccactga atagtagggc                                                 20
SEQ ID NO: 411
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 411
gctcagcaat gagtgacagt                                                 20
SEQ ID NO: 412
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 412
agcaaacttt atttctcgcc                                                 20
SEQ ID NO: 413
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 413
gattacagac acaactcccc                                                 20
SEQ ID NO: 414
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 414
actgaatagt agggcgatta                                                 20
SEQ ID NO: 415
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 415
actttatttc tcgccactga                                                 20
SEQ ID NO: 416
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 416
gctgtccttg ctgagggagc                                                 20
SEQ ID NO: 417
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 417
cttagctggt cctctgctgt                                                 20
SEQ ID NO: 418
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 418
gttgcttctc tccctcttag                                                 20
SEQ ID NO: 419
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 419
tggcgtctga gggttgtttt                                                 20
SEQ ID NO: 420
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 420
agagaacctg cctggcagct                                                 20
SEQ ID NO: 421
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 421
cagtatgtga gaggaagaga                                                 20
SEQ ID NO: 422
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 422
ggtgaagccg tgggtcagta                                                 20
SEQ ID NO: 423
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 423
agtgctcatg gtgtcctttc                                                 20
SEQ ID NO: 424
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 424
ccggatcatg ctttcagtgc                                                 20
SEQ ID NO: 425
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 425
ggccagctcc acgtcccgga                                                 20
SEQ ID NO: 426
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 426
ggcccccctg tcttcttggg                                                 20
SEQ ID NO: 427
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 427
ggctgaggaa caagcaccgc                                                 20
SEQ ID NO: 428
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 428
tcaggaagga gaagaggctg                                                 20
SEQ ID NO: 429
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 429
tggcgcctgc cacgatcagg                                                 20
SEQ ID NO: 430
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 430
ggcagaagag cgtggtggcg                                                 20
SEQ ID NO: 431
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 431
ctccaaagtg cagcaggcag                                                 20
SEQ ID NO: 432
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 432
gctgattaga gagaggtccc                                                 20
SEQ ID NO: 433
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 433
tgcctgggcc agagggctga                                                 20
SEQ ID NO: 434
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 434
gctgcccctc agcttgaggg                                                 20
SEQ ID NO: 435
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 435
ggttcagcca ctggagctgc                                                 20
SEQ ID NO: 436
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 436
gggcattggc ccggcggttc                                                 20
SEQ ID NO: 437
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 437
cgccattggc caggagggca                                                 20
SEQ ID NO: 438
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 438
tatctctcag ctccacgcca                                                 20
SEQ ID NO: 439
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 439
gcaccaccag ctggttatct                                                 20
SEQ ID NO: 440
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 440
acaggccctc tgatggcacc                                                 20
SEQ ID NO: 441
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 441
gggagtagat gaggtacagg                                                 20
SEQ ID NO: 442
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 442
ccttgaagag gacctgggag                                                 20
SEQ ID NO: 443
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 443
gaggagcaca tgggtggagg                                                 20
SEQ ID NO: 444
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 444
gctgatggtg tgggtgagga                                                 20
SEQ ID NO: 445
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 445
ggagacggcg atgcggctga                                                 20
SEQ ID NO: 446
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 446
gaccttggtc tggtaggaga                                                 20
SEQ ID NO: 447
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 447
ggcagagagg aggttgacct                                                 20
SEQ ID NO: 448
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 448
tgggctcata ccagggcttg                                                 20
SEQ ID NO: 449
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 449
cccctcccag atagatgggc                                                 20
SEQ ID NO: 450
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 450
tgagtcggtc acccttctcc                                                 20
SEQ ID NO: 451
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 451
gattgatctc agcgctgagt                                                 20
SEQ ID NO: 452
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 452
cgagatagtc gggccgattg                                                 20
SEQ ID NO: 453
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 453
caaagtagac ctgcccagac                                                 20
SEQ ID NO: 454
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 454
acagggcaat gatcccaaag                                                 20
SEQ ID NO: 455
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 455
atgttcgtcc tcctcacagg                                                 20
SEQ ID NO: 456
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 456
gtttgggaag gttggatgtt                                                 20
SEQ ID NO: 457
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 457
aagaggttga gggtgtctga                                                 20
SEQ ID NO: 458
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 458
ctctttttga gccagaagag                                                 20
SEQ ID NO: 459
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 459
cctaagcccc caattctctt                                                 20
SEQ ID NO: 460
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 460
agcttgggtt ccgaccctaa                                                 20
SEQ ID NO: 461
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 461
ttgcttaaag ttctaagctt                                                 20
SEQ ID NO: 462
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 462
gaagtggtgg tcttgttgct                                                 20
SEQ ID NO: 463
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 463
tgaatcccag gtttcgaagt                                                 20
SEQ ID NO: 464
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 464
caggccacac attcctgaat                                                 20
SEQ ID NO: 465
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 465
cagcacttca ctgtgcaggc                                                 20
SEQ ID NO: 466
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 466
attcttagtg gttgccagca                                                 20
SEQ ID NO: 467
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 467
gaggccccag tttgaattct                                                 20
SEQ ID NO: 468
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 468
ccccagtgag ttctggaggc                                                 20
SEQ ID NO: 469
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 469
gatcaaagct gtaggcccca                                                 20
SEQ ID NO: 470
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 470
attccagatg tcagggatca                                                 20
SEQ ID NO: 471
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 471
ctccctggtc tccagattcc                                                 20
SEQ ID NO: 472
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 472
ggccagaacc aaaggctccc                                                 20
SEQ ID NO: 473
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 473
gtcctgcagc attctggcca                                                 20
SEQ ID NO: 474
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 474
gtgaggtctt ctcaagtcct                                                 20
SEQ ID NO: 475
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 475
tgtgtcaatt tctaggtgag                                                 20
SEQ ID NO: 476
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 476
ggcctaaggt ccacttgtgt                                                 20
SEQ ID NO: 477
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 477
atctggagag aggaaggcct                                                 20
SEQ ID NO: 478
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 478
aggaagtctg gaaacatctg                                                 20
SEQ ID NO: 479
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 479
gggctccgtg tctcaaggaa                                                 20
SEQ ID NO: 480
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 480
aaatagaggg agctggctcc                                                 20
SEQ ID NO: 481
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 481
cacaagtgca aacataaata                                                 20
SEQ ID NO: 482
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 482
tcccaaataa atacattcat                                                 20
SEQ ID NO: 483
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 483
caggataccc cggtctccca                                                 20
SEQ ID NO: 484
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 484
ctacattggg tcccccagga                                                 20
SEQ ID NO: 485
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 485
gagccaaggc agctcctaca                                                 20
SEQ ID NO: 486
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 486
acggaaaaca tgtctgagcc                                                 20
SEQ ID NO: 487
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 487
ttcagctccg ttttcacgga                                                 20
SEQ ID NO: 488
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 488
gggaacagcc tattgttcag                                                 20
SEQ ID NO: 489
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 489
tcaaaagaag gcacagaggc                                                 20
SEQ ID NO: 490
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 490
ttttaaaaaa cataatcaaa                                                 20
SEQ ID NO: 491
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 491
ttaatcagat aaatatttta                                                 20
SEQ ID NO: 492
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 492
cattgtttag acaacttaat                                                 20
SEQ ID NO: 493
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 493
tggtcaccaa atcagcattg                                                 20
SEQ ID NO: 494
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 494
gcaatgagtg acagttggtc                                                 20
SEQ ID NO: 495
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 495
gggagcagag gctcagcaat                                                 20
SEQ ID NO: 496
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 496
atagtagggc gattacagac                                                 20
SEQ ID NO: 497
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 497
atttctcgcc actgaatagt                                                 20
SEQ ID NO: 498
LENGTH: 19
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 498
ctgattagag agaggtccc                                                  19
SEQ ID NO: 499
LENGTH: 18
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 499
ctgattagag agaggtcc                                                   18
SEQ ID NO: 500
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 500
tgagtgtctt ctgtgtgcca                                                 20
SEQ ID NO: 501
LENGTH: 20
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Synthetic
SEQUENCE: 501
gagtgtcttc tgtgtgccag                                                 20

Claims

1. An oligonucleotide up to 30 nucleotides in length complementary to a nucleic acid molecule encoding human tumor necrosis factor-α, wherein said oligonucleotide inhibits the expression of said human tumor necrosis factor-α and comprises at least an 8 nucleobase portion of SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 39, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 149, SEQ ID NO: 157, SEQ ID NO: 264, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 290, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 315, SEQ ID NO: 334, SEQ ID NO: 418, SEQ ID NO: 423, SEQ ID NO: 425, SEQ ID NO: 427, SEQ ID NO: 431, SEQ ID NO: 432, SEQ ID NO: 435, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 441, SEQ ID NO: 455, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 460, SEQ ID NO: 463, SEQ ID NO: 465, SEQ ID NO: 466, SEQ ID NO: 468, SEQ ID NO: 472, SEQ ID NO: 474, SEQ ID NO: 475, SEQ ID NO: 483, SEQ ID NO: 485, SEQ ID NO: 494 or SEQ ID NO: 496.

2. The oligonucleotide of claim 1 which contains at least one phosphorothioate intersugar linkage.

3. The oligonucleotide of claim 1 which has at least one 2′-O-methoxyethyl modification.

4. The oligonucleotide of claim 1 which contains at least one 5-methyl cytidine.

5. The oligonucleotide of claim 3 in which every 2′-O-methoxyethyl modified cytidine residue is a 5-methyl cytidine.

6. The oligonucleotide of claim 4 in which every cytidine residue is a 5-methyl cytidine.

7. The oligonucleotide of claim 1 which contains at least one methylene(methylimino) intersugar linkage.

8. A composition comprising the oligonucleotide of claim 1 and a pharmaceutically acceptable carrier or diluent.

9. The composition of claim 8 wherein said pharmaceutically acceptable carrier or diluent comprises a lipid or liposome.

10. A method of inhibiting the expression of human tumor necrosis factor-α in cells or tissue comprising contacting said cells or tissue in vitro with the oligonucleotide of claim 1 whereby the expression of human tumor necrosis factor-α in cells or tissue is inhibited.

11. A method of reducing an inflammatory response of human cells comprising contacting said human cells in vitro with the oligonucleotide of claim 1 whereby the expression of human tumor necrosis factor-α in cells or tissue is inhibited.

12. A method of inhibiting the expression of human tumor necrosis factor-α in adipose tissue comprising contacting said adipose tissue with an antisense compound comprising an antisense oligonucleotide of claim 1 whereby the expression of human tumor necrosis factor-α in adipose tissue is inhibited.

13. A method of inhibiting the function of human tumor necrosis factor-α in adipose tissue comprising contacting said adipose tissue with an antisense compound comprising an antisense oligonucleotide of claim 1 whereby the expression of human tumor necrosis factor-α in cells or tissue is inhibited.

14. An oligonucleotide complementary to a nucleic acid molecule encoding human TNF-alpha wherein said oligonucleotide inhibits the expression of said TNF-alpha and consists of SEQ ID NO: 432.