ANTISENSE MODULATION OF INTEGRIN alpha 4 EXPRESSION

Abstract: Compositions and methods are provided for modulating the expression of integrin alpha 4. Antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding integrin alpha 4 are preferred. Methods of using these compounds for modulating integrin alpha 4 expression and for treatment of diseases associated with expression of integrin alpha 4 are also provided.

Description: ANTISENSE MODULATION OF INTEGRIN α4 EXPRESSION FIELD OF THE INVENTION The present invention provides compositions and methods for modulating the expression of integrin . In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human integrin α4. Such oligonucleotides have been shown to modulate the expression of human integrin α4. BACKGROUND OF THE INVENTION Inflammation is a localized protective response elicited by tissues in response to injury, infection, or tissue destruction resulting in the destruction of the infectious or injurious agent and isolation of the injured tissue. A typical inflammatory response proceeds as follows: recognition of an antigen as foreign or recognition of tissue damage, synthesis and release of soluble inflammatory mediators, recruitment of inflammatory cells to the site of infection or tissue damage, destruction and removal of the invading organism or damaged tissue, and deactivation of the system once the invading organism or damage has been resolved. In many human diseases with an inflammatory component, the normal, homeostatic mechanisms which attenuate the inflammatory responses are defective, resulting in damage and destruction of normal tissue. Cell-cell interactions are involved in the activation of the immune response at each of the stages described above. One of the earliest detectable events in a normal inflammatory response is adhesion of leukocytes to the vascular endothelium, followed by migration of leukocytes out of the vasculature to the site of infection or injury. The adhesion of these leukocytes, or white blood cells, to vascular endothelium is an obligate step in the migration out of the vasculature (Harlan, J. M., Blood 1985, 65, 513-525). This response is mediated by the interaction of adhesion molecules expressed on the cell surface of leukocytes and vascular endothelial cells. Very late antιgen-4 (also called VLA-4, 4βl or CD49d/CD29) is a homodimeric adhesion receptor which is composed of noncovalently linked α and β subunits and serves to mediate leukocyte adhesion to vascular cell adhesion molecule-1 (VCAM-1) which is expressed on cytokme-stimulated endothelial cells. This interaction between VCAM-1 and VLA-4 contributes to leukocyte extravasation in acute and chronic inflammatory conditions including multiple sclerosis (MS), rheumatoid arthritis, asthma, psoriasis and allergy. Fibronectin is also a ligand for VLA- . Fibronectm plays an important role in many processes including embryonic development, wound healing and tumor cell metastasis (Guan, J.-L. and Hynes, R.O., Cell 1990, 60, 53-61). VLA-4 is a heterodimer of an α4 integrin and a βl integrin. The α4 integrin can also heterodimerize with a β7 integrin chain to form integrin α4β7 which is known as a ucosal homing receptor because its primary ligand is the mucosal vascular addressing MadCAM-1. Integrin 4β7 identifies a subset of memory T cells with a tropism for the intestinal tract, whereas integrin 4βl (VLA-4) is constitutively expressed on most mononuclear leukocytes, but not on circulating neutrophils. The interaction of VCAM-1 with VLA-4 suggests that VLA-4 is a potential therapeutic target for inflammatory diseases, including atherosclerosis, allergy and asthma, arthritis, and tumor cell metastasis (Kassner, P.D., et al, Adv. Exp . Med . Biol . 1992, 323, 163-170) . VLA-4 has also been found to play a role in promoting adhesion ( i . e . , retention) of hemopoietic stem cells in the bone marrow (Papayannopoulou and Nakamoto, Proc . Na tl . Acad . Sci . U. S . A . 1993, 90, 9374-9378). Asthma is an inflammatory disease associated with eosmophil infiltration into the lung. VLA-4 is expressed on eos ophils. Metzger, W.J. (Springer Semm . Immunopa thol . 1995, 16, 467-478) used a rabbit model of asthma to demonstrate that both an antι-VLA-4 antibody and a CS-1 peptide could reduce eosmophil infiltration into the lung and reduce the development of asthma. Rheumatoid arthritis is another disease associated with inflammation. Muller-Ladner, U., et al . , ( J Rheuma tology 1997, 24, 1873-1880) found that the alternatively spliced form of fibronectm containing CS-1 was expressed in the rheumatoid synovium. Additionally, they found that not only did expression of fibronectm result in recruitment of VLA-4 expressing cells, but fibroblasts ir the rheumatoid synovium expressed VLA-4. Seiffge, D. (J. Rhe wotol ogy 1996, 23, 2086- 2091) used a rat model for arthritis to show that a monoclonal antibody to the α4 chain of VLA-^ resulted ^ an improvement of symptoms. VLA-4 also plays a role m α rumber of autoimmune diseases. Marazuela, M., et al . , ( E r J Immunol . 1994, 24, 2483-2490) found elevated expression of both tne VLA-4/VCAM-1 and LFA-l/ICAM-1, 3 pathways m Graves' disease and Hashimoto's thyroiditis, suggesting that both play a role in these diseases. VLA-4 may also play a role in multiple sclerosis. Antibodies to VLA-4 have been found to prevent experimental autoimmune encephalomyelitis (EAE) , an experimentally induced disease with similarities to multiple sclerosis (Yednock, T.A., et al., Na ture 1992, 356, 63-66). Elevated expression levels of VLA-4 were detected in a patient with systemic lupus erythematosus (Takeuchi, T., et al . , Cl m . Rheuma tology 1995, 14, 370-374) . VLA-4 is involved in cellular responses to two surgical procedures, transplantation and vascular reconstructive procedures. Allograft rejection is a common response to transplantation of a foreign tissue. CS-1 peptides have been found to prevent both acute re ection (Coito, A.J., et al., Transplan ta tion 1998, 65, 699-706) and chronic rejection (Korom, S., et al . , Transplan ta tion 1998, 65, 854-859) by blocking VLA-4 binding to fibronectm. During vascular reconstructive surgery, a common cause of failure is mtimal hyperplasia which results from the accumulation of monocytes and lymphocytes. In a baboon model, Lumsden, A.B., et al., (J. Vase . Surg. 1997, 26, 87-93) demonstrated that an antι-VLA-4 antibody reduced mtimal hyperplasia. VLA-4 also plays a role m tumor cell metastasis. In metastasis, tumor cells must cross the extracellular matrix, enter the circulatory system and invade into new tissue. Bao, L. et al . , ( Differen tia tion 1993, 52, 239-246) detected VLA-4 expression of many human tumor cell lines, including a breast carcinoma, melanoma, and renal carcinoma, and found that the presence of VLA-4 correlated well with metastatic potential. Kawaguchi, S. et al . , ( Jpn J. Cancer Res . 1992, 83, 1304-1316) transfected a cDNA encoding the α4 subunit of VLA-4 into a human fibrosarcoma cell line. These cells overexpressed VLA-4 and showed increased m vi tro invasive ability. Augmentation of metastasis by IL-i (Garofalo, A.,et al., Cancer Res . 1995, 55, 414-419) or TNF-α (Okahara, H., et al., Cancer Res . 1994, 54, 3233-3236) has been shown to involve the interaction between VLA-4 and VCAM-1. These authors suggest that a therapy directed towards inhibiting this interaction would be useful in reducing the risk of metastasis with conditions associated with high serum concentrations of TNF-α, including cachexia, sepsis, surgical stress, or TNF-α therapeutic applications. Because tumor cells often secrete IL-1 and TNF-α, such a therapy may be useful m reducing the risk of metastasis associated with such tumor cells. VLA-4 is involved m promoting retention of hemopoietic progenitor cells m the bone marrow. Antibodies to mtegrm α4 (but not mtegrm β2) have been found to selectively mobilize progenitor/stem cells into the bloodstream (Papayannopoulou and Nakamoto, Proc . Na tl . Acad . Sci . U. S . A . 1993, 90, 9374-9378) . This mobilization is of clinical relevance in the field of bone marrow transplantion as it obviates the need for marrow harvesting by making hemopoietic progenitor cells available m the circulating blood. While steroids and other antnnflammatory drugs are effective in treating inflammatory diseases and conditions, long-term usage often leads to side effects such as increased risk of infection caused by impairment of phagocytic leukocyte migration and function. There is some concern that inhibition of the function of the βl mtegrm chain may be associated with increased susceptibility to infections, as demonstrated by a βl (also called CD18) monoclonal antibody in rabbits (Foster, C.A., 1996, J. Allergy Clin . Immunol . , 98, 270-277). It is believed that selective inhibition of the 4 chain may be a more desirable approach. Inhibition of the α4 chain is believed likely to reduce levels of the VLA-4 heterodimer as well as the α4β7 heterodimer. Potential therapeutic interventions targeting VLA-4 include monoclonal antibodies, and peptide antagonists. Leger, O.J.P., et al . ( Human Antibodies 1997, 8, 3-16) describe a monoclonal antibody against VLA-4 that is in phase II clinical trials for multiple sclerosis. CS-1 peptide antagonists have been described by Jackson, D.Y., et al . (J. Med . Chem . 1997, 40, 3359-3369) . Hayashi et al . (Cell Struct . Funct . 1991, 16, 241-249) have used a vector expressing RNA complementary to chicken mtegrm βl to reduce mtegrm βl expression, resulting in altered cell attachment and shape. Antisense oligonucleotides targeted to various integrins have been used as tools to dissect the functional interactions of integrins in complex settings. Lallier and Bronner-Fraser (Science, 1993, 259, 692-695) have used phosphorothioate oligonucleotides targeted to conserved and nonconserved regions of chick βl, human α4 , rat αl and human α5 integrins to determine the effects of these integrins on cell attachment. These same oligonucleotides were also injected into cranial neural crest migratory pathways m avian embryos, and it was demonstrated that those oligonucleotides that inhibited cell attachment in vi tro also caused neural crest and/or neural tube abnormalities in vivo (Kil et al . , Devel . Biol . 1996, 179, 91-101) . EP patent applicatior ^lαs et al . ) discloses 3' derivatized oligonucleotide analogs, including one sequence targeted to the βl subunit of VLA- . Antisense oligonucleotides are believed to represent a useful means of modulating the expression of mtegrm α4 and of treating diseases associated with its expression. SUMMARY OF THE INVENTION The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding mtegrm α4 , and which modulate the expression of mtegrm α4. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of mtegrm α4 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of mtegrm α4 by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention. DETAILED DESCRIPTION OF THE INVENTION The present invention employs oligomeric antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding mtegrm α4, ultimately modulating the amount of mtegrm α4 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding mtegrm α4. As used herein, the terms "target nucleic acid" and "nucleic acid encoding mtegrm α4" encompass DNA encoding mtegrm α4 , RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as "antisense". The functions of DN to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA tc the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of mtegrm α4. In the context of the present invention, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target. It is preferred to target specific nucleic acids for antisense. "Targeting" an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding mtegrm α4. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e . g. , detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred mtragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5'-AUG (m 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 ammo acid in each instance is typically methionine (in eukaryotes) or formylmethionme (in prokaryotes) . It is also known m 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 m vivo to initiate translation of an mRNA molecule transcribed from a gene encoding mtegrm α4 , regardless of the sequence (s) of such codons. It is also known m the art that a translation termination codon (or "stop codon") of a gene may have one of three sequences, i . e . , 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively) . The terms "start codon region" and "translation initiation codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides m either direction ( i . e . , 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon . The open reading frame (ORF) or "coding region, " which is known m the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5' untranslated region (5'UTR) , known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3' untranslated region (3'UTR), known m 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 guanos e 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 "mtrons," which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., mtron- exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated m disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. It has also been found that mtrons can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA. Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i . e . , hybridize sufficiently well and with sufficient specificity, to give the desired effect. In the context of this invention, "hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. "Complementary," as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific bindmσ o? ""he antisense compound to non-target sequences uno in which specific binding is desired, i . e . , under pnysiological conditions in the case of in vivo assays or therapeutic treatment, or m the case of in vi tro assays, under conditions in which the assays are performed. Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use. The specificity and sensitivity of antisense is also harnessed by those of skill m the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states m animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 30 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 m 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, ammoalkylphosphotri- esters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates and chiral phosphonates, phosphmates , phosphoramidates including 3'-ammo phosphoramidate and ammoalkylphosphoramidates, thionophosphoramidates , thiono- alkylphosphonates, thionoalkylphosphotriesters, and borano- phosphates 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. Patents 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, each of which is herein incorporated by reference. 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 morpholmo 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; methyleneimmo and methylenehydrazmo backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, 0, 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. Patents 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; 5,470,96 "7 ; 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, each of which is herein incorporated by reference. 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 ac d 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 ammoethylglycme 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.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al . , ( Science, 1991, 254, 1497-1500) . Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH 2 -NH-0-CH 2 -, -CH 2 -N (CH 3 ) -0-CH 2 - [known as a methylene (methylimmo) or MMI backbone] , (CH ) -CH -, -CH-.-N (CH, ) - N(CH 3 )-CH 2 - and -O-N (CH 3 ) -CH 2 -CH t - [ nere the native phosphodiester backbone is represented as -0-P-0-CH 2 -] of the above referenced U.S. patent 5,489,67 "7 , and the amide backbones of the above referenced U.S. patent 5,602,240. Also preferred are oligonucleotides having morpholmo backbone structures of the above-referenced U.S. patent 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; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or 0-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C τ to C c alkyl or C- to C 10 alkenyl and alkynyl. Particularly preferred are 0[ (CH,) n O] m CH 3 , 0(CH 2 ) n OCH 3 , 0(CH 2 ) r NH., C(CH ) CH , 0(CH 2 ) n ONH., and 0 (CH 2 ) n ON [ (CH 2 ) r 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 x to C 10 lower alkyl, substituted lower alkyl, alkaryl, araikyl, O-alkaryl or 0- aralkyl, SH, SCH ^ , OCN, Cl, Br, CN, CF , OCF , SOCH ^ , S0 2 CH , 0N0 2 , N0 2 , N 3 , NH , heterocycloalkyl, heterocycloalkaryl, a moalkylammo, polyalkylammo, substituted silyl, an RNA cleaving group, a reporter group, an mtercalator, a group for improving the pharmacokmetic 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 ' -0-CH 2 CH 2 OCH 3 , also known as 2'-0-(2- methoxyethyl) or 2'-MOE) (Martin et al . , Hel v. Chim . Acta , 1995, 78, 486-504) i . e . , an alkoxyalkoxy group. A further preferred modification includes 2 ' -dimethylammooxyethoxy, i . e . , a 0 (CH 2 ) 2 ON (CH 3 ) 2 group, also known as 2'-DMAOE, as described in United States patent application Serial Number 09/016,520, filed on January 30, 1998, which is commonly owned with the instant application and the contents of which are herein incorporated by reference. Other preferred modifications include 2'-methoxy (2'-0- CH 3 ) , 2'-amιnopropoxy (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 m 2'-5' linked oligonucleotides and the 5' position of 5 terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patents 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 5,627,0531 5,639,873; 5,646,265; 5,658,873 5,670, 633 and 5,700,920, each of which is herein incorporated by reference and allowed United States patent application 08/468,037, filed on June 5, 1995, which is commonly owned with the instant application and is also herein incorporated by reference. Oligonucleotides may also include nucleobase (often referred to m the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purme bases adenme (A) and guanme (G), and the pyrimidme bases thymine (T) , cytosme (C) and uracil (U) . Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosme (5-me-C), 5- hydroxymethyl cytosme, xanthme, hypoxanthme, 2- ammoadenme, 6-methyl and other alkyl derivatives of adenme and guanme, 2-propyl and other alkyl derivatives of adenme and guanme, 2-thιouracιl, 2-thιothymme and 2-thιocytosme, 5-halouracιl and cytosme, 5-propynyl uracil and cytosme, 6- azo uracil, cytosme and thymine, 5~uracιl (pseudouracil) , 4- thiouracil, 8-halo, 8-ammo, 8-thιol, 8-thιoalkyl, 8-hydroxyl and other 8-substιtuted adenmes and guanmes, 5-halo particularly 5-bromo, 5-trιfluoromethyl and other 5- substituted uracils and cytosmes, 7-methylguanιne and 7- ethyladenme, 8-azaguanme and 8-azaadenme, 7-deazaguanme and 7-deazaadenme and 3-deazaguanme and 3-deazaadenme . Further nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in The Concise Encycl opedia Of Polymer Science And Engineering, , Kroschwitz, J.I., ed. John Wiley & Sons, 1990, 858-859 those disclosed by Englisch et al . , (Angewandte Chemie, IE, 1991, 30, 613), and those disclosed by Sanghvi, Y.S., ( An tisense Research and Applications, 15,289-302), and Crooke, S.T. and Lebleu, B., ed., (CRC Press, 1993) . Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5- substituted pyrimidmes, 6-azapyrιmιdmes and N-2, N-6 and 0-6 substituted purines, including 2-ammopropyladenme, 5- propynyluracil and 5-propynylcytosme . 5-methylcytosme 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, 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications . Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Patents 3,687,808, as well as U.S.: 4,845,205; 5,130,302; 5,134,066; 5,175,273 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121 5,596,091; 5,614,617; 5,681,941; and 5,750,692, each of which is herein incorporated by reference. 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 Let., 1994, 4, 1053-1060), a thioether, e.g., hexyi-S-trmylthioi (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, 59, 327-330; Svmarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl- ammonium 1, 2-dι-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al . , ucl. Acids Res., 1990, 18, 3777-3783), a polyamme or a polyethylene glycol chain (Manoharan et al . , Nucleosides & Nucl eotides , 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al . , Tetrahedron Let t . , 1995, 36, 3651-3654), a palmityl moiety (Mishra et al . , Biochim . Biophys . Acta , 1995, 1264, 229-237), or an octadecylamine or hexylammo-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharma col . Exp . Ther . , 1996, 277, 923-93 "^ . Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Patents 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, 39 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, 56^, 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, each of hicr is herein incorporated by reference. It is not necessary for al_ positions m a given compound to be uniformly modified, and m fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. "Chimeric" antisense compounds or "chimeras," m the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i . e. , a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA : DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA: DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known m the art. Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers . Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Patents 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference, and allowed United States patent application serial number 08/465,880, filed on June 6, 1995, which is commonly owned with the instant application and also herein incorporated by reference. The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA) . Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The antisense compounds of the invention are synthesized in vi tro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules. The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake distribution and/or absorption assisting formulations include but are not limited to, U.S. Patents 5,108,921; 5,354,844 5,416,016 5,459,127; 5,521,291; 5,543,158; 5,547,932 5,583,020 5,591,721; 4,426,330; 4,534,899; 5,013,556 5, 108, 921 5,213,804; 5,227,170; 5,264,221; 5,356,633 5,395,619 5,416,016; 5,417,978; 5,462,854; 5,469,854 5,512,295 5,527,528; 5,534,259; 5,543,152; 5,556,948 5,580,575 and 5,595,756, each of which is herein incorporated by reference. The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents . The term "prodrug" indicates a therapeutic agent that is prepared m 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-thιoethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gossel et al . , published December 9, 1993 or in WO 94/26764 to Imbach et al. The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of tne compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N, N'-dibenzylethylenediamme, cnloroprocame, cholme, diethanolam e , dicyclohexylamine, ethylenediamme, N-methylglucamme, and procame (see, for example, Berge et al. J. of Pharma Sci . , 1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility m polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a "pharmaceutical addition salt" includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochloπdes, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfomc, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succimc acid, maleic acid, hydroxymaleic acid, methylmaleic acic, fumaric acid, malic acid, tartaπc acid, lactic acid, oxalic acid, gluconic acid, glucaπc acid, glucuronic acid, citric acid, benzoic acid, cmnamic acid, mandelic acid, salicylic acid, 4-amιnosalιcylιc acid, 2-phenoxybenzoιc acid, 2-acetoxybenzoιc acid, embonic acid, mcotinic acid or lsonicotinic acid, and with ammo acids, such as the 20 alpha-ammo acids involved in tne synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also w__th phenylacetic acid, me t hane s ul f on l c acid, e t r a •-> e s u 1 f on l c acid, 2-hydroxyethanesulfonic acid, ethare-1 , 2-dιsulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfoic acid, naphthalene-2-sulfonic acid, naphthalene-1 , 5-dιsulfonic acid, 2- or 3-phosphoglycerate , glucose- 6-phosphate , N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible. For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyammes such as spermine and spermid e, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, raaleic 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, polygalacturomc acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine. The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of mtegrm α4 is treated by administering antisense compounds accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example. The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding mtegrm α4 , enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding mtegrm α4 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of mtegrm α4 in a sample may also be prepared . The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; mtratracheal, mtranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, mtraarterial , subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., mtrathecal or mtraventricular, administration. Oligonucleotides with at least one 2'-0- methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, 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 and formulations 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 and formulations for parenteral, mtrathecal or mtraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. Pharmaceutical compositions and/or formulations comprising the oligonucleotides of the present invention may also 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 . , Cri tical Reviews m Therapeutic Drug Carrier Sys tems , 1991, 8, 91-192; Muranishi, Cri ti cal Reviews m Therapeu ti c Drug Carrier Sys tems , 1990, 7, 1-33). One or more penetration enhancers from one or more of these broad categories may be included. Penetration enhancers are described in pending United States patent application 08/886,829, filed on July 1, 1997, and pending United States patent application 08/961,469, filed on October 31, 1997, both of which are commonly owned with the instant application and both of which are herein incorporated by reference. Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid, myπstic acid, palmitic acid, stearic acid, linoleic acid, lmolenic acid, dicaprate, tricaprate, recmleate, monoolem (a.k.a. 1-monooleoyl-rac-glycerol) , dilauπn, caprylic acid, aπchidonic acid, glyceryl 1-monocaprate, l-dodecylazacycloheptan-2-one, acylcarnitmes , acylcholmes, mono- and di-glyceπdes and physiologically acceptable salts thereof ( .e., oleate, laurate, caprate, mynstate, palmitate, stearate, linoleate, etc.) (Lee et al . , Cri tical Reviews in Therapeu ti c Drug Carrier Sys tems , 1991, 8, 91-192; Muranishi, Cri tica l Reviews in Therapeu tic Drug Carrier Sys tems, 1990, 7, 1-33; El-Hariri et al . , J. Pharm . Pharma col . , 1992, 44, 651-654). Examples of some presently preferred fatty acids are sodium caprate and sodium laurate, used singly or in combination at concentrations of 0.5 to 5%. Preferred penetration enhancers are disclosed in pending United States patent application 08/886,829, filed on July 1, 1997, which is commonly owned with the instant application and which is herein incorporated by reference. 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 Pharmacol ogical Basis of Therapeuti cs , 9th Ed., Hardman et al., eds., McGraw-Hill, New York, NY 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 . Preferred bile salts are described in pending United States patent application 08/886,829, filed on July 1, 1997, which is commonly owned with the instant application and which is herein incorporated by reference. A presently preferred bile salt is chenodeoxycholic acid (CDCA) (Sigma Chemical Company, St. Louis, MO), generally used at concentrations of 0.5 to 2%. 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. Preferred combinations include CDCA combined with sodium caprate or sodium laurate (generally 0.5 to 5%) . Chelating agents include, but are not limited to, disodium ethylenediammetetraacetate (EDTA) , citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalιcylate and homovanilate) , N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Lee et al., Cri tical Reviews in Therapeuti c Drug Carrier Sys tems, 1991, 8, 92-192; Muranishi, Cri ti ca l Reviews in Tnerapeutic Drug Carrier Sys tems, 1990, 7, 1-33; Buur et al., J. Con trol 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., Cri tical Reviews m Therapeutic Drug Carrier Sys tems, 1991, 8, 92-191) ; and perfluorochemical emulsions, such as FC-43 (Takahashi et al., J. Pharm . Pharmacol . , 1988, 40, 252-257). Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al . , Cri ti cal Reviews m Therapeutic Drug Carrier Systems, 1991, 8, 92-191); and non-steroidal anti-mflammatory agents such as diclofenac sodium, mdomethacm and phenylbutazone (Yamashita et al . , J. Pharm . Pharma col . , 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 m vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the oiologically active nucleic acid or promoting its removal from circulation. The coadmmistration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result a substantial reduction of the amount of nucleic acid recovered m the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioated oligonucleotide in hepatic tissue is reduced when it is coadmmistered with polymosmic acid, dextran sulfate, polycytidic acid or 4-acetamιdo-4 '-lsothiocyano-stilbene- 2 , 2'-dιsulfonιc acid (Miyao et al . , An tisense Res . Dev. , 1995, 5, 115-121; Takakura et al . , Antisense & Nucl . Acid Drug Dev. , 1996, 6, 177-183) . In 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 mmd 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., pregelatmized maize starch, polyvmyl- pyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystallme 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. Patents 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., antipruπtics, astringents, local anesthetics or anti-mflammatory agents, or may contain additional materials useful m physically formulating various dosage forms of the composition 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 antisense compounds of the invention are introduced into a patient, colloidal dispersion systems may be used as delivery vehicles to enhance the m vi vo stability of the compounds and/or to target the compounds 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-m- water emulsions, micelles, mixed micelles, liposomes and lipid: oligonucleotide complexes of uncharacteπzed 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 layer (s) made up of lipids arranged in a bilayer configuration (see, generally, Chonn et al . , Curren t Op . Bio tech . , 1995, 6, 698-708). Liposome preparation is described m pending United States patent application 08/961,469, filed on October 31, 1997, which is commonly owned with the instant application and which is herein incorporated by reference. Certain embodiments of the invention provide for liposomes and other compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include, but are not limited to, anticancer drugs such as daunorubicm, dactmomycm, doxorubicm, bleomycm, mitomycm, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurme, 6-thιoguanme, cytarabme (CA) , 5-fluorouracil (5-FU), floxuπdme (5-FUdR), methotrexate (MTX) , colchicme, vincπstme, v blastme, etoposide, teniposide, cisplatm and diethylstilbestrol (DES) . See, generally, The Merck Manual of Diagnosis and Therapy, 1987, Berkow et al . , eds., Rahway, N.J., 1206-1228. Anti-mflammatory drugs, including but not limited to nonsteroidal anti-mflammatory drugs and corticosteroids , and antiviral drugs, including but not limited to ribivirm, vidarabme, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manua l of Dia gnos i s and Therapy, 1987, Berkow et al . , eds., Rahway, N.J., pages 2499-2506 and 46-49, respectively) . Other non-antisense chemotherapeutic agents are also withm the scope of this invention. Two or more combined compounds may be used together or sequentially. In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Two or more combined compounds may be used together or sequentially. The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those m the art. Dosing is dependent on severity and responsiveness of the disease state to oe 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 ECr, 0 s found to be effective in in vi tro and m vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years. While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. EXAMPLES Example 1 Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2 '-alkoxy amidites 2'-Deoxy and 2'-methoxy beta-cyanoethyldnsopropyl phosphoramidites are purchased from commercial sources (e.g. Chemgenes, Needham MA or Glen Research, Inc. Sterling VA) . Other 2'-0-alkoxy substituted nucleoside amidites are prepared as described in U.S. Patent 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2'-alkoxy amidites, the standard cycle for unmodified oligonucleotides is utilized, except the wait step after pulse delivery of tetrazole and base is increased to 360 seconds. Oligonucleotides containing 5-methyl-2'-deoxycytιdme (5-Me-C) nucleotides are synthesized according to published methods (Sanghvi, et. al., Nucl eic Acids Research , 1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling, VA or ChemGenes, Needham, MA) . 2 ' -Fluoro amidites 2'-Fluorodeoxyadenosine amidites 2'-fluoro oligonucleotides are synthesized as described previously (Kawasaki, et. al . , J. Med . Chem . , 1993, 36, 831- 841) and United States patent 5,670,633, herein incorporated by reference. Briefly, the protected nucleoside N6-benzoyl-2' deoxy-2'-fluoroadenosme is synthesized utilizing commercially available 9-beta-D-arabmofuranosyladenine as starting material and by modifying literature procedures whereby the 2'-alpha-fluoro atom is introduced by a S 2-dιsplacement of a 2'-beta-trιtyl group. Thus N6-benzoyl-9-beta-D- arabmofuranosyladenine is selectively protected m moderate yield as the 3', 5'-dιtetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups is accomplished using standard methodologies and standard methods are used to obtain the 5'-dιmethoxytntyl- ( DMT) and 5'-DMT-3'- phosphoramidite intermediates. 2'-Fluorodeoxyguanosine The synthesis of 2'-deoxy-2'-fluoroguanosme is accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabmofuranosylguanme as starting material, and conversion to the intermediate dnsobutyryl- arabmofuranosylguanosme . Deprotection of the TPDS group is followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected arabmofuranosylguanme . Selective O-deacylation and tπflation 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'-phosphoramιdιtes . 2'-Fluorouridine Synthesis of 2'-deoxy-2'-fluorouridme is accomplished by the modification of a literature procedure in which 2,2'- anhydro-1-beta-D-arabιnofuranosyluracιl is treated with 70% hydrogen fluoride-pyridme . Standard procedures are used to obtain the 5'-DMT and 5'-DMT-3'phosphoramιdιtes . 2'-Fluorodeoxycytιdine 2'-deoxy-2'-fluorocytidme is synthesized via ammation of 2'-deoxy-2'-fluorouridme, followed by selective protection to give N4-benzoyl-2'-deoxy-2'-fluorocytidme . Standard procedures are used to obtain the 5'-DMT and 5'-DMT- 3'phosphoramιdιtes . 2'-0- (2-Methoxyethyl) modified amidites 2'-0-Methoxyethyl-substιtuted nucleoside amidites were prepared as follows, or alternatively, as per the methods of Martin, P., ( Hel veti ca Chimica Acta , 1995, 78, 486-504). 2,2'-Anhydro[l-(beta-D-arabmofuranosyl)-5- me hyluridine] 5-Methylurιdme (πbosylthymme, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M) , diphenyl- carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL) . The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released m a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L) , with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL) . The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60°C at 1 mm Hg for 24 h) to give a solid that was crushed to a light tan powder (57 g, 85% crude yield) . The NMR spectrum was consistent with the structure, contaminated with phenol as its sodium salt (ca. 5%) . The material was used as is for further reactions (or it can be purified further by column chromatography using a gradient of methanol in ethyl acetate (10-25%) to give a white solid, mp 222-4°C) . 2'-0-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 C1 2 (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product. Additional material was obtained by reworking impure fractions . 2'-0-Methoxyethyl-5'-0-dimethoxytrityl-5-methyluridine 2'-0-Methoxyethyl-5-methyluridme (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 CHC1 3 (1.5 L) and extracted with 2x500 mL of saturated NaHC0 3 and 2x500 mL of saturated NaCl. The organic phase was dried over Na.SO,,, 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 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'-0-Acetyl-2'-0-methoxyethyl-5'-0-dimethox trityl-5- methyluridine 2'-0-Methoxyethyl-5'-0-dιmethoxytrityl-5-methyluridme (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 tic sample with the addition of MeOH . Upon completion of the reaction, as judged by tic, MeOH (50 mL) was added and the mixture evaporated at 35°C. The residue was dissolved in CHC1 3 (800 mL) and extracted with 2x200 mL of saturated sodium bicarbonate and 2x200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHC1 3 . The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product) . The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/Hexane ( 4 : 1 ) . Pure product fractions were evaporated to yield 96 g (84%) . An additional 1.5 g was recovered from later fractions. 3'-0-Acetyl-2'-0-methoxyethyl-5'-0-dimethoxytrityl-5- methyl-4-triazoleuridine A first solution was prepared by dissolving 3'-0- acetyl-2 '-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH 3 CN (700 mL ) and set aside. Triethylamme (189 mL, 1.44 M) was added to a solution of tπazole (90 g, 1.3 M) in CH 3 CN (1 L) , cooled to -5°C and stirred for 0.5 h using an overhead stirrer. P0C1 3 was added dropwise, over a 30 mmute 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 mmute period, to the latter solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1x300 mL of NaHCO, and 2x300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound. 2'-0-Methoxyethyl-5'-0-dimethoxytrιtyl-5-methylcytιdine A solution of 3'-0-acetyl-2'-0-methoxyethyl-5'-0- dιmethoxytrιtyl-5-methyl-4-trιazoleurιdme (103 g, 0.141 M) in dioxane (500 mL) and NH 4 OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2x200 mL) . The residue was dissolved m 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 (tic showed complete conversion) . The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL) . The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound. N4-Benzoyl-2'-0-methoxyethyl-5'-0-dιmethoxytrityl-5- methylcytidine 2'-0-Methoxyethyl-5 ' -O-dιmethoxytrιtyl-5-methylcytιdme (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, tic 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 CHC1 3 (700 mL) and extracted with saturated NaHC0 3 (2x300 mL) and saturated NaCl (2x300 mL) , dried over MgSO,, 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. N4-Benzoyl-2'-0-methoxyethyl-5'-0-dimethoxytrityl-5- methylcytidine-3'-amidite N4-Benzoyl-2'-0-methoxyethyl-5'-0-dιmethoxytrityl-5~ methylcytidine (74 g, 0.10 M) was dissolved in CH 2 C1 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 (tic showed the reaction to be 95% complete) . The reaction mixture was extracted with saturated NaHC0 3 (1x300 mL) and saturated NaCl (3x300 mL) . The aqueous washes were back-extracted with CH 2 C1 2 (300 mL) , and the extracts were combined, dried over MgS0 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. 2'- (Aminoox ethyl) nucleoside amidites and 2'- (dimethylaminooxyethy1) nucleoside amidites Aminooxyethyl and dimethylaminooxyethyl amidites are prepared as per the methods of United States patent applications serial number 10/037,143, filed February 14, 1998, and serial number 09/016,520, filed January 30, 1998, each of which is commonly owned with the instant application and is herein incorporated by reference. Example 2 Oligonucleotide synthesis Unsubstituted and substituted phosphodiester (P=0) oligonucleotides were synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine. Phosphorothioates (P=S) were synthesized as for the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H-1 , 2-benzodιthιole- 3-one 1,1-dιoxιde in acetonitπle for the stepwise thiation of the phosphite linkages. The thiation wait step was increased to 68 sec and was followed by the capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55°C (18 hr) , the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution. Phosphmate oligonucleotides are prepared as described m U.S. Patent 5,508,270, herein incorporated by reference. Alkyl phosphonate oligonucleotides are prepared as described in U.S. Patent 4,469,863, herein incorporated by reference . 3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared as described m U.S. Patents 5,610,289 or 5,625,050, herein incorporated by reference. Phosphoramidite oligonucleotides are prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878, herein incorporated by reference. Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94 /00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference. 3'-Deoxy-3'-amιno phosphoramidate oligonucleotides are prepared as described m U.S. Patent 5,476,925, herein incorporated by reference. Phosphotriester oligonucleotides are prepared as described in U.S. Patent 5,023,243, herein incorporated by reference . Borano phosphate oligonucleotides are prepared as described in U.S. Patents 5,130,302 and 5,177,198, both herein incorporated by reference. Example 3 Oligonucleoside Synthesis Methylenemethylimmo linked oligonucleosides , also identified as MMI linked oligonucleosides, methylenedimethyl- hydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylammo linked oligonucleosides, also identified as amιde-3 linked oligonucleosides, and methyleneammocarbonyl linked oligonucleosides, also identified as amιde-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P=0 or P=S linkages are prepared as described in U.S. Patents 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference. Formacetal and thioformacetal linked oligonucleosides are prepared as described m U.S. Patents 5,264,562 and 5,264,564, herein incorporated by reference. Ethylene oxide linked oligonucleosides are prepared as described in U.S. Patent 5,223,618, herein incorporated by reference . Example 4 PNA Synthesis Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to m Peptide Nucleic Acids (PNA) : Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemis try, 1996, 4, 5-23. They may also be prepared m accordance with U.S. Patents 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference . Example 5 Synthesis of Chimeric Oligonucleotides Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the "gap" segment of linked nucleosides is positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open end" type wherein the "gap" segment is located at either the 3' or the 5' terminus of the oligomeric compound. Oligonucleotides of the first type are also known m the art as "gapmers" or gapped oligonucleotides. Oligonucleotides of the second type are also known in tne art as "hemimers" or "wmgmers" . [2'-0-Me]--[2'-deoxy]--[2'-0-Me] Chimeric Phosphorothioate Oligonucleotides Chimeric oligonucleotides having 2'-0-alkyl phosphorothioate and 2'-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 380B, as above. Oligonucleotides are synthesized using the automated synthesizer and 2'-deoxy-5'- dιmethoxytπtyl-3'-0-phosphoramιdιte for the DNA portion and 5'-dιmethoxytrιtyl-2'-0-methyl-3'-0-phosphoramιdιte for 5' and 3' wings. The standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for RNA and twice for 2'-0- methyl . The fully protected oligonucleotide is cleaved from the support and the phosphate group is deprotected in 3:1 Ammonia/Ethanol at room temperature overnight then lyophilized to dryness. Treatment in methanolic ammonia for 24 hrs at room temperature is then done to deprotect all bases and sample was again lyophilized to dryness. The pellet is resuspended in IM TBAF in THF for 24 hrs at room temperature to deprotect the 2' positions. The reaction is then quenched with IM TEAA and the sample is then reduced to 1/2 volume by rotovac before being desalted on a G25 size exclusion column. The oligo recovered is then analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry . [2'-0- (2 -Methoxyethyl) ] -- [2'-deoxy] -- [ 2 ' - O - (Methoxyethyl) ] Chimeric Phosphorothioate Oligonucleotides [2'-0- (2-methoxyethyl) ] — [2'-deoxy] -- [-2'-0- (methoxy- ethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2'-0-methyl chimeric oligonucleotide, with the substitution of 2'-0- (methoxyethyl) amidites for the 2'-0-methyl amidites. [2'-0- (2-Methoxyethyl) Phosphodiester] -- [2'-deoxy Phosphoro thioate] -- [2'-0- (2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides [2'-0- (2-methoxyethyl phosphodiester ]-- [2'-deoxy phosphorothioate] -- [2'-0- (methoxyethyl ) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2'-0-methyl chimeric oligonucleotide with the substitution of 2'-0- (methoxyethyl) amidites for the 2'-0-methyl amidites, oxidization with iodine to generate the phosphodiester mternucleotide linkages withm the wmg portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodιthιole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate mternucleotide linkages for the center gap. Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to United States Patent 5,623,065, herein incorporated by reference. Example 6 Oligonucleotide Isolation After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55°C for 18 hours, the oligonucleotides or oligonucleosides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained m synthesis were periodically checked by 31 P nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al . ( J. Biol . Chem . 1991, 266, 18162-18171) . Results obtained with HPLC- purified material are similar to those obtained with non-HPLC purified material. Example 7 Analysis of oligonucleotide inhibition of integrin α4 expression Antisense modulation of mtegrm α4 expression can be assayed m a variety of ways known in the art. For example, mtegrm α4 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR) . Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly (A) + mRNA. Methods of RNA isolation are taught , for example, Ausubel, F . M. e t al . (Current Protocols m Mol ecular Biology , 1993., 1, 4.1.1-4.2.9 and 4.5.1-4.5.3). Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F.M. et al . ( Current Protocols in Molecular Biology 1996, 1, 4.2.1-4.2.9). Realtime quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, CA and used according to manufacturer's instructions. Other methods of PCR are also known in the art. Integrin α4 protein levels can be quantitated in a variety of ways well known m the art, such as immunoprecipitation, Western blot analysis ( lmmunoblottmg) , ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to mtegrm α4 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught m, for example, Ausubel, F.M. et al . ( Curren t Protocols m Molecular Biol ogy 1997, 2, 11.12.1-11.12.9). Preparation of monoclonal antibodies is taught in, for example, Ausubel, F.M. et al . (Current Protocols m Molecular Biology 1997, 2, 11.4.1- 11.11.5) . Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F.M. et al . (Curren t Protocol s in Molecular Biology 1998, 2, 10.16.1-10.16.11). Western blot (immurioblot) analysis is standard in the art and can be found at, for example, Ausubel, F.M. et al . ( Current Protocol s in Molecular Biology 1997, 2, 10.8.1-10.8.21). Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F.M. et al . ( Current Protocols in Molecular Biology 1991, 2, 11.2.1- 11.2.22) . Example 8 Antisense inhibition of human integrin 4 expression In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human integrin α4 RNA, using published sequences (GenBank accession number L12002, incorporated herein as SEQ ID NO: 1) . The oligonucleotides are shown in Table 1. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (GenBank accession no. L12002), to which the oligonucleotide binds. All compounds in Table 1 are 18 nucleotides in length, with a phosphorothioate backbone and a centered 10-base deoxynucleotide gap flanked by 2'- methoxyethoxy (2'MOE) wings. Human A375 melanoma cells (American Type Culture Collection, Manassas, VA) were plated at 2000 cells/cm 2 two days before oligonucleotide treatment. Cells were treated with oligonucleotides at a dose of 200 nM oligonucleotide and 6 ug/ml LIPOFECTIN for four hours . Cells were incubated overnight and harvested with 2 mM EDTA in PBS.. Cells were washed in 2% bovine serum albumin, 0.2% sodium azide in D-PBS at 4°C. Cells were centrifuged at 200 x g, and the supernatant was decanted. Protein levels were determined by flow cytometry according to published methods (Condon and Bennett, J. Biol . Chem . 1996, 271, 30398-30403). The specific conjugated antibody CD49d-FITC (Immunotech, Westbrook, ME, clone HP2/1) was added at 2 uL/test well, and the control IgG (mouse IgGl-FITC, PharMmgen, San Diego, CA) was added at a concentration of ug/ L. Antibodies were incubated with the cells for 30 minutes at 4°C m the dark, under gentle agitation. Cells were washed again as above and then resuspended in 0.3 ml of FacsFlow buffer with 0.5% formaldehyde. Cells were analyzed on Becton Dickinson FACScan. Results are expressed as percentage of control expression based on mean fluorescence intensity. As shown in Table 1, oligonucleotides 24453, 24473, 24475, 24477 and 24451 gave at least about 50% inhibition of mtegrm α4 protein expression and were subjected to further study . Example 9 Dose response curves for f antisense oligonucleotides on integrin α4 protein expression A375 cells were treated with ISIS 24473, 24475, 24477 and 24451 at concentrations of 50, 100, 200 and 400 nM for 4 hours. Cells were harvested with trypsm at 48 and 72 hours after oligonucleotide treatment and stained with 2 ug/ml CD49d-FITC as in the previous example. Results are shown in Table 2. Table 1 Inhibition of human integrin α4 protein levels by antisense oligonucleotides ISIS # Sequence arget site %Control %Inhib SEQ ID region NO 24439 CTCCGTCTCTGCCTACGC 5'-UTR 0087-0104 120 -- 2 24440 CGGGTGCTCGCGCTGCTT 5'-UTR 0163-0180 101 -- 3 24441 CCTGGGATGCCGCGCACT 5'-UTR 0 1-0241 112 -- 4 24442 ATGAGGCGCAGCGTGTCC 5'-UTR -0335 82 18 5 4- 24443 CAAAGTTGCACGGGATGC 5'-UTR 0-0387 63 37 6 24444 GGAACATTCAACACTAAG AUG 00-0417 68 32 7 24445 CCCGGGTTCGCGCCTCGC coding U443-0460 73 27 8 24446 GCGCGCTCTCAGTGTCCA coding 0535-0552 58 42 9 24447 GTGGCTGTGCAGCACGAC coding 0594-0611 76 24 10 24448 ACTGAAGCGTTGGCGAGC coding 0656-0673 55 45 11 24449 GCACGTCTGGCCGGGATT coding 0714-0731 61 39 12 24451 CCACTGATTGTCTCTCTC coding 0789-0806 51 49 13 24452 GGATCCATTTTCTCCTGG coding 0831-0848 60 40 14 24453 GCTTATTTTCATTCTTTA coding 0889-0906 47 53 15 24454 TTCTTTTACTCAGTTCTG coding 0949- ■ 0966 53 47 16 24455 TCACATAATCTTGATAAC coding 0976- ■ 0993 81 19 17 24456 CCCATCACAATTAAATCC coding 1052- ■ 1069 76 24 18 24457 TTATTTGTAGTTATATTG coding 1112--1129 106 19 24458 CCTAAATAACTTCCAAAT coding 1166--1183 12 20 24459 GAAAATGACCAGCTCCGA coding 1192--1209 61 39 21 24460 TTTCATGTAAGATATTTA coding 1300--1317 94 06 22 24461 CCACAGCACAGACAGAAG coding 1351--1368 69 31 23 24462 TGGTGCTCTGCATGGGTG coding 1408 -1425 65 35 24 24463 TACACAAACACTCTTCCT coding 1436 -1453 101 25 24464 TTTGTTTCCATTGCATTC coding 1481 -1498 62 38 26 24465 TGCAGCATATTTGTCACT coding 1509 -1526 63 37 27 24466 TTGTCAATGTCGCCAAGA coding 1550 -1567 64 36 28 24467 TCATCTTCTTGTGGAGCT coding 1595 -1612 101 29 24468 CCATCTGCACGGCCATTG coding 1637 -1654 72 28 30 o σ m m Table 2 Dose response effect of antisense oligonucleotides on integrin α4 protein expression ISIS # DOSE SEQ ID NO: % CONTROL 48 % INHIB % CONTROL % INHIB 72 (nM) hr 48 hr 72 hr HR 24 4 51 50 13 63 37 ND ND 100 44 56 ND ND 200 34 66 ND ND 400 44 56 ND ND 24453 50 15 79 21 78 22 100 95 05 93 07 200 38 62 64 36 400 41 59 58 42 24473 50 35 77 23 85 35 100 49 51 77 23 200 39 61 61 39 400 42 58 56 44 24 475 50 37 60 40 69 31 100 31 69 44 56 Q 00 00 co ID r- 2 •^ CM co ID ^Γ Q CM CM r^ co Z ID r^ CD •^ m CΛ CO o O O σ o o o O m o o o CM 'T rH M ^r Γ- CM Example 10 Dose response curves for effect of antisense oligonucleotides on integrin α4 protein expression An additional series of oligonucleotides were tested for effects on integrin α4 protein expression and compared to active compounds from the previous screens. These oligonucleotides are targeted to an integrin ce4 promotor sequence (Rosen et al., Proc . Na tl . Acad . Sci . 1991, 88, 4094-4098; GenBank Accession No. M62841, provided herein as SEQ ID NO: 41) . These oligonucleotides are shown in Table 3 and their effects on integrin α4 protein expression is shown in Table . Where backbones are mixed, linkages are shown as "s" for phosphorothioate (P=S or PS) and "o" for phosphodiester (P=0 or PO) . Where target region is indicated as 'M62841,' oligonucleotides are targeted to the promotor region whose sequence is described in GenBank Accession No. M62841. Remaining oligonucleotides are targeted to the sequence described in Genbank Accession No. L12002 and their target sites on the L12002 sequence are indicated. A mouse sequence (SEQ ID NO: 47) is included for comparison. It is targeted to the same mRNA region as the human sequence 27104 (coding region just downstream from the AUG codon) and contains 3 mismatches with the human mRNA target . Table 3 Additional oligonucleotides targeted to human integrin α4 ISIS Sequence Chemistry Target Target SEQ # region site ID NO 24475 GCCCTTATATGAGAAACA PS; 2' MOE/deoxy; All coding 1929-1946 37 2' MOE C's are 5 meC 24739 TTTAGTGACAAAGACGTTAT PS; deoxy M62841 0409-0428 42 24740 GAAGGCCCCTGGGGAACATT PS; deoxy M62841 0428-0447 43 24741 AGACGTTATGGCTATTCTCT PS; deoxy M62841 0398-0417 44 24742 TTTAGTGACAAAGACGTTAT PS; 2'MOE; all C = 5meC M62841 0409-0428 42 24743 GAAGGCCCCTGGGGAACATT PS; 2'MOE; all C = 5meC M62841 0428-0447 43 24744 AGACGTTATGGCTATTCTCT PS; 2'MOE; all C = 5 meC M62841 0398-0417 44 26643 TTGCCCTTATATGAGAAACA PS; 2' MOE; all C = 5 meC coding 1929-1948 45 27104 CCCAAGCCATGCGCTCTCGG PS/2'MOE; all C = 5 meC human coding 0421-0440 46 27108 CoCoCoAsAsGsCsCsAsTs PO/PS; 2' MOE/deoxy; All C= 5meC human coding 0421-0440 46 GsCsGoCoToCoToCoGoG 17044 CCGCAGCCATGCGCTCTTGG PS; 2' MOE/deoxy; All mouse 47 mouse 2' MOE C's are 5 meC coding Cn C •H S O υ CD o ε CJ CD ID ε J Table 4 Dose response curves for effect of antisense oligonucleotides on human integrin α4 protein expression ISIS # Oligo. % Control % Inhib. SEQ ID NO. Cone (nM) 24475 10 97 03 37 30 73 27 100 84 16 300 55 45 24739 10 69 31 42 30 67 33 100 121 300 107 24740 10 122 43 30 119 100 106 300 88 24741 10 109 44 30 113 100 125 300 127 24742 10 112 42 30 107 100 123 300 128 2244774433 1100 9966 04 43 30 121 ; 100 117 ! 300 105 24744 10 121 — 44 , 30 110 — 1 100 118 -- 300 118 -- 1 26643 10 90 10 45 30 95 05 100 93 07 300 66 34 i 27104 10 69 31 46 30 64 36 ! 100 49 51 ! 300 39 61 ' 17044 10 86 14 47 mouse 30 73 27 100 60 40 300 58 42 27108 10 69 31 46 30 95 05 1 100 64 36 ! 300 49 51 27109 10 109 -- 47 30 114 -- 100 77 23 300 55 45 In this assay, oligonucleotides 24475, 27104 and 27108 demonstrated 40% inhibition or greater and are preferred. The oligonucleotide sequence (SEQ ID NO: 47) targeted to mouse integrin α4 , which has only three mismatches with the human gene target, also showed >40% inhibition at the highest dose. Example 11 Screening of additional oligonucleotide screening for inhibition of integrin 4 protein levels New oligonucleotides 26640, 26641, 26642 and 26644 were synthesized and compared against previously tested oligonucleotides. Oligonucleotides are shown in Table 5 and their effect on integrin α4 protein levels is shown in Table 6. Target sites refer to nucleotide numbers on the target sequence (GenBank Accession No. L12002) to which the oligonucleotides hybridize. Table 5 Additional oligonucleotides targeted to human integrin α.4 ISIS # Sequence 26640 CCACTGATTGTCTCTCTCTT PS 26641 GAGCTTATTTTCATTCTTTA PS; 26642 TCAGGGTGGCTTAAAGAAGC PS; 2 'MOE/deoxy; coding 1841-1858 35 All C= 5meC 26644 TTTCCATTCTCTCAATTTGA PS; 2 'MOE/deoxy; 3 ' -UTR 3537 - 3554 37 Al l C= 5meC Table 6 Dose response curves for effect of antisense oligonucleotides on human integrin α4 protein expression ISIS # O Olliiggoo.. % % CCoonnttrrooll % % IInnhhiibb . SEQ ID NO. Cone (nM) 27104 1 100 8 844 1 166 46 30 82 18 100 59 41 300 37 63 17044 1 100 9 977 03 47 30 84 16 100 68 32 300 39 61 24451 1 100 9 966 0 044 13 30 77 23 100 66 34 300 35 65 26640 1100 9911 0099 13 30 83 100 62 38 300 35 65 24453 1100 8888 1122 15 30 90 10 100 80 20 300 43 57 26641 1100 8888 1122 15 30 85 15 100 67 33 300 31 69 24473 1100 110033 —-- 35 30 92 08 100 81 19 300 47 53 2 6642 1 100 84 1 166 35 30 77 23 100 56 44 300 23 77 24477 10 83 17 39 30 80 20 100 63 37 300 33 67 26644 10 99 01 39 30 94 06 100 78 22 300 48 52 In this assay, oligonucleotides 27104, 24451, 26640, 24453, 526641, 24473, 26642, 24477 and 26644 demonstrated 40% inhibition or greater and are preferred. The oligonucleotide sequence (SEQ ID NO: 47) targeted to mouse integrin α4 , which has only three mismatches with the human gene target, also showed >40% inhibition at the highest dose. 0 Example 12 Antisense inhibition of murine integrin α4 mRNA expression in mouse P388D (IL-1) cells Because many conditions which are believed to be ameliorable by antisense oligonucleotides targeted to integrin 5 α4 are not amenable to study in humans, a series of oligonucleotides was designed to target the murine integrin 4 using the published sequence of De Miersman et al., DNA Cell Biol . 1994, 13:743-754; GenBank Accession No:L20788, provided herein as SEQ ID NO: 48). These oligonucleotides are shown in Table 7. Nucleotide bases shown in bold are 2'- methoxyethoxy (2'MOE); remaining positions are 2' deoxy. Target site refers to the first nucleotide position on the target (GenBank Accession No. L20788) to which the oligonucleotide binds . Oligonucleotides were initially screened in murine P388D (IL-1) macrophage cells (American Type Culture Collection, Manassas VA) for 4 hours at an oligonucleotide concentration of 100 nM in 3 ug/ml lipofectm. Cells were harvested and RNA was purified by cellular lysis using Catrιmox-14 solution (Iowa Biotechnology Corp, Oakdale IA. Dahle, CE. and Macfarlane, D.E., BioTechniques 1993, 15, 1-4. RNA was electrophoresed on a 1% agarose/1.1% formaldehyde gel with 18 ug RNA loaded in each lane. The mtegrm 4 probe was a PCR- labeled 1025-base mtegrm α4 fragment (positions 3681-4706) made by RT-PCR according to the method of Bednarczuk et al . (Biotechniques 1991 10,478) . Results of the screen are shown in Table 8. Table 7 Antisense oligonucleotides targeted to murine integrin α4 ISIS # Sequence Chemistry Target Target SEQ ID region site NO 15600 CACGCCCCGTTTCTGTGGCC 15601 CACGCCCCGTTTCTGTGGCC ' 15602 CACGCCCCGTTTCTGTGGCC I 15603 GGATGCTTCAGGCTCTGGCC j 15604 GGAGCGATCGTAGTGGCCAG PS; DNA 5' UTR 0992-1011 51 1 15605 CCGGTGCTGGCAGGCGACAG PS; DNA 5' UTR 1061-1080 52 ' 15606 GATGAAGTGCAGCAGCGTGT PS; DNA 5' UTR 1081-1100 53 15607 GGCCACTGACCAGAGTTGCA PS; DNA 5' UTR 1141-1160 54 15608 CCGCAGCCATGCGCTCTTGG PS; DNA AUG 1183-1202 47 i 15609 CACCTCGCTTCCGCAGCCAT PS; DNA AUG 1193-1212 55 Table 8 Antisense inhibition of murine integrin α4 mRNA expression 1 1 ISIS No. % Control % Inhib Seq ID NO: 1 ! 15600 93 07 49 15601 60 40 49 15602 48 52 49 15603 38 62 50 15604 134 -- 51 As shown in Table 8, ISIS 15607, targeted to the 5'UTR, and ISIS 15608 and 15609, both targeted to the translation initiation codon, were most active in this assay. Dose response curves for ISIS 15607, 15608 and 15609 are shown in Table 9. Results are averages of duplicate samples. Table 9 Antisense inhibition of murine integrin α.4 mRNA expression- dose responses ISIS No. Oligo % control inhib SEQ ID NO: Cone (nM) 15607 12.5 78 22 54 25 67 33 50 50 50 100 55 45 15608 12.5 59 41 47 25 29 71 50 23 77 100 39 61 15609 12.5 54 46 55 25 24 76 50 28 72 100 57 43 Example 13 Antisense inhibition of murine integrin α4 protein expression Murine IC-21 macrophage cells (American Type Culture Collection, Manassas, VA) were used for further study. Additional oligonucleotides were designed to target murine integrin α4. These are shown in Table 10; all 2' MOE nucleotides are shown in bold and remaining positions are 2' deoxy. All 2'-MOE cytosines in these compounds are 5' methylcytosines . IC-21 cells were seeded at 5 x 10 4 cells/well in 24 well plates two days before assay. Cells were treated with 3 ug/ml LIPOFECTIN for 4 hours with 25 nM oligonucleotide. Wells were triplicates. Cells were harvested with 2 mM EDTA after 20 hours and analyzed by flow cytometry as described in Example 8, using CD49d-phycoerythrin (CD49d- PE) antibody conjugate (clone Rl-2) and Rat IgG2B-PE as control. Results are shown in Table 11. Table 10 Antisense oligonucleotides targeted to murine integrin α4 16479 ACCTCCTCGCTCACGCGCTA PS; DNA Scrambled 58 16434 CACCTCGCTTCCGCAGCCAT PS; 2' MOE/deoxy AUG 1183-1202 47 16435 CACCTCGCTTCCGCAGCCAT PS; 2' MOE/deoxy AUG 1183-1202 47 16436 CACCTCGCTTCCGCAGCCAT PS; 2' MOE/deoxy AUG 1183-1202 47 16437 CGCTTCCGCAGCCATGCGCT PS; 2 OE/deoxy AUG 1188-1207 59 All 2'MOE cytosines in Table 10 are 5-me tosines Table 11 Inhibition of integrin α4 protein expression by antisense oligonucleotides ISIS 16430, 16431, 16433, 16434, 16434, 16435, 16436 and 16437 gave better than 50% inhibition in this assay and are preferred . Dose responses for effect of ISIS 16431 and 16433 on integrin 4 protein levels are shown in Table 12. Cells (triplicates) were seeded at 2.5 x 10 4 cells/well in 24-well plates the day before treatment. Oligonucleotide concentrations were 0.2, 1, 5, 25 and 50 nM in 3 ug/ml LIPOFECTIN. Cells were harvested with 2 mM EDTA for 4 minutes, then 1 ml of 2% BSA, 0.2% azide was added per contents of each well. Protein levels were analyzed by flow cytometry as described in Example 8, using CD49d-PE antibody conjugate at 2 ug/ml, and rat IgG2B at 2 ug/ml as control. ISIS 16431 and 16433 were found to have IC50s of approximately 1 nM for integrin α4 protein inhibition. Table 12 Dose response effect of 16431, 16433 on integrin α4 protein levels ISIS No. Dose (nM) % of control % inh SEQ ID NO: 16431 0 2 82 18 47 1 57 43 5 34 66 25 32 68 50 30 70 16433 0. 2 85 15 47 1 62 38 5 37 63 25 20 80 50 30 70 16478 5 95 05 57 25 87 13 50 84 16 Another dose-response experiment on ISIS 16433 was done with scramble control included. IC-21 cells were treated with 0.3, 1, 3, 10 and 30 nM oligonucleotide with 3 ug/ml LIFOFECTIN for 4 hours. Cells were incubated for 24 hours, harvested with trypsin, stained with 2 ug/ml CD49d-PE and rat IgG2B as in previous examples. The results are shown in Table 13. ISIS 16433 at 30 nM was found to inhibit protein levels to below basal levels. Table 13 Dose response effect of 16431, 16433 on integrin α4 protein levels ISIS Oligo. % of % inhib. SEQ ID NO: No. Cone. (nM) control 16433 0 3 89 11 47 1 68 32 3 25 75 10 03 97 30 -14 (100) 16478 0. 3 137 -- 57 1 131 -- 3 127 -- 10 130 -- 1 30 108 -- j 1 Another dose response experiment was done to determine the IC50 of ISIS 16433 for inhibition of integrin 4 RNA. IC- 21 cells were treated in duplicate with ISIS 16433 at 6.25, 12.5, 25 or 50 nM with 3 ug/ml LIPOFECTIN. RNA was purified with Qiagen Qiashredder/RNEASY™ Kit according to manufacturer's directions. Probes were random primed using a Prime-a-Gene Kit (Promega, Madison, WI) . The results are shown in Table 14. ISIS 16433 was shown to have an IC50 of approximately 10 nM for integrin 4 RNA reduction. Table 14 Dose response effect of ISIS 16433 on integrin α4 RNA levels ISIS Oligo Percent of Percent SEQ ID No. dose (nM) control inhib . NO: 1 12.5 46 44 25 28 72 1 50 43 57 1 1 Example 14 Additional antisense oligonucleotides targeted to murine integrin cx4-optimization Additional oligonucleotides were synthesized with the ISIS 16433 sequence (SEQ ID NO: 47, targeted to the translation initiation codon) in order to optimize the backbone chemistry and 2' methoxyethoxy gap placement. These compounds were tested for ability to inhibit integrin α4 protein expression. Compounds and results are shown m Tables 15 and 16. ISIS 17044 was found to reduce integrin α4 expression to below starting levels and was chosen for further study in animal models of disease. TABLE 15 Optimization of oligonucleotides having SEQ ID NO: 47 ISIS No, Sequence 17044 CCGCAGCCATGCGCTCTTGG 17045 CCGCAGCCATGCGCTCTTGG 17046 CCGCAGCCATGCGCTCTTGG 17047 CCGCAGCCATGCGCTCTTGG 17160 CoCsGsCsAsGsCsCsAsTsGsCsGoCoToCoToToGoG 16433 CCGCAGCCATGCGCTCTTGG 17048 GCCGACACCCGTTCGTTCGG (scrambled control; SEQ ID NO: 57) Internucleoside backbone linkages are uniform P=S except for ISIS 17160. "o" indicates phosphodiester (P=0) linkage and "s" indicates a phosphorothioate (P=S) linkage. TABLE 16 Dose response for optimization of oligonucleotides with SEQ ID NO: 47 ISIS No. Oligo. %Contro % Inhib. SEQ ID Cone (nM) 1 NO: 17044 0 3 100 0 47 1 81 19 3 32 68 10 -9 >100 17045 0 3 109 -- 47 1 65 35 3 34 66 3 64 36 10 02 98 17048 0.3 159 -- 57 (control ) 1 159 -- 3 150 -- 10 119 -- Example 15 Mouse Experimental Autoimmune Encephalomyelitis (EAE) Model for Multiple Sclerosis Experimental autoimmune encephalomyelitis (EAE) is an inflammatory, demyelmatmg central nervous system disease frequently used as an animal model for multiple sclerosis. It is inducible m genetically susceptible animals by immunization with whole spmal cord homogenate or protein components of the myelm sheath such as myelm basic protein (MBP) or proteolipid protein (PLP) , cr by transfer of MBP- or PLP- specific T cells. Myers et al . , J Immunol . 1993, 151, 2252-2260. CSJLF-1 mice (Jackson Laboratory, Bar Harbor, ME) were immunized with the pl3 peptide (HSLGKWLGHPDKF-amide, synthesized by Research Genetics, Huntsville, AL, SEQ ID NO. 60) which corresponds to residues 139-151 of PLP and is encephalitogemc m these mice. Mice were immunized essentially as described m Myers et al., ( J. Neuro immunology 1992, 41, 1-8) . Briefly, mice were injected in the hind footpads and the base of the tail with 50-100 ug of pl3 peptide, emulsified in CFA (Difco, Detroit, MI) fortified with 4 mg/ml of heat-killed H37Ra Mycoba cteπ um t ubercul osis bacteria (Difco) . At the time of footpad injections and again 2 days later, mice were also injected intravenously with 500 ng of pertussis toxm (Sigma, St. Louis, MO) . Mice were treated with the antisense oligonucleotide ISIS 17044 at various doses, beginning one day before pl3 immunization except where indicated otherwise. Oligonucleotide was formulated in 0.9% saline and was administered daily by subcutaneous injection, with dosing continuing until more than 50% of the pl3-immunized but saline-treated control group began showing symptoms of disease. Dosing was then terminated and mice were observed for effects of treatment on the course of disease. Disease severity was scored on a scale of 0 to 5 with 0 = no symptoms, 1 = flaccid tail, 2 = hind limb weakness, 3 = hind limb paralysis, 4 = hind and front limb paralysis and 5 = moribund or dead. Time until disease onset was also measured and compared to pl3-immunized control mice that received saline instead of oligonucleotide. Example 16 EAE Experiment 1 Mice were treated with ISIS 17044 as described in Example 15, at daily doses ranging from 1 mg/kg to 20 mg/kg, injected subcutaneously (SC) . Mean peak disease severity and average number of days to disease onset were measured and these are shown in Table 17. Table 17 EAE Experiment 1- effects of ISIS 17044 on EAE Group Disease Peak Days ; Incidence Severity to Onset t 1 Saline 6/7 1.57 14.5 , 17044 20mg/kg 5/7 1.43 15 17044 lOmg/kg 4/7 2.0 12.75 1 17044 5 mg/kg 6/7 1.86 17 i 17044 1 mg/kg 4/7 0.29 17.25 As shown, 17044 reduced disease severity or delayed disease onset . Example 17 EAE Experiment 2 Mice were treated with ISIS 17044 as described in Example 15, at daily doses ranging from 0.3 mg/kg to 3 mg/kg, injected subcutaneously, and 1 mg/kg injected intravenously for comparison. Mean peak disease severity and average number of days to disease onset were measured and these are shown in Table 18. Table 18 EAE Experiment 2- effects of ISIS 17044 on EAE Group Disease Peak Days to Incidence Severi -ty Onset Saline 7/7 2/43 13.7 17044 3 mg/kg SC 7/7 2.29 16.4 17044 1 mg/kg SC 4/7 1.0 • 18.25 17044 0.3 mg/kg SC 7/7 2.14 16.57 17044 1 mg/kg IV 5/7 1.42 17.2 As shown, 17044 reduced disease severity and delayed disease onset at all concentrations. Example 18 EAE Experiment 3 Mice were treated with ISIS 17044 as described in Example 15, at daily doses ranging from 0.5 mg/kg to 2.0 mg/kg, injected subcutaneously (SC) . A thrice-weekly dosing regimen was also tested, and a scrambled control (ISIS 17614, GCCGACACCCGTTCGTTCGG, 2' MOE /deoxy; P=S; SEQ ID NO: 57) was also tested. Disease severity and time until disease onset were measured and these are shown in Table 19. Table 19 EAE Experiment 3- effects of ISIS 17044 on EAE Group Disease Peak Days Incidence Severity to Onset Saline 7/7 2.29 15.14 17044 2 mg/kg 2/6 0.83 20 17044 1.5 4/7 1.5 17.5 mg/kg 17044 1.0 4/5 1.5 19.25 mg/kg 17044 0.5 5/6 1.5 21.4 mg/kg 17044 1 mg/kg 6/7 1.0 20.3 3X weekly 17614 1 mg/kg 7/7 2.14 16.86 As shown, 17044 reduced disease severity or delayed disease onset . Example 19 EAE Experiment 4- Prophylactic vs. Therapeutic Dosing Mice were treated with ISIS 17044 as described in Example 15, at daily doses of 0.01 /kg to 2.0 mg/kg, injected subcutaneously (SC) . The scrambled control oligonucleotide, ISIS 17614, was also tested at 2 mg/kg. A 1 mg/kg daily dose, given therapeutically was also tested. In this regimen, oligonucleotide treatment began on day 18, after animals had begun showing symptoms of paralysis, and continued until day 31. Disease severity and time until disease onset were measured and these are shown in Table 20. Table 20 EAE Experiment 4- effects of ISIS 17044 on EAE- Therapeutic (T) vs. Prophylactic (P) Regimen Group Disease Peak Days to Incidence Severity Onset Saline 6/7 3.29 16 17044 2.0 7/7 2.29 16.71 mg/kg SC (P) 17044 1.0 5/5 2.2 15 mg/kg SC (P) 17044 0.1 7/7 1.57 16.29 mg/kg SC (P) 17044 0.01 6/7 1.86 13.83 mg/kg (P) 17044 1 mg/kg 6/6 1.83 13.3 (T) 17614 2 mg/kg 7/7 1.86 14.14 SC (control) As shown, 17044 reduced disease severity when given therapeutically (time to disease onset is not expected to be increased because dosing begins after onset of disease) . Example 20 EAE Experiment 5- Prophylactic vs. Therapeutic Dosing Mice were treated with ISIS 17044 as described in Example 15, at daily doses of 0.01 /kg to 2.0 mg/kg, injected subcutaneously (SC). The scrambled control oligonucleotide, ISIS 17614, was also tested at 2 mg/kg. A 1 mg/kg daily dose, given therapeutically was also tested. In this regimen, oligonucleotide treatment began on day 11, after animals had begun showing symptoms of paralysis, and continued until day 27. Disease severity and time until disease onset were measured and these are shown in Table 21. Table 21 EAE Experiment 5- effects of ISIS 17044 on EAE- Therapeutic (T) vs. Prophylactic (P) Regimen Group Disease Peak Days to Incidence Severity Onset Saline 6/7 1.86 10.83 17044 2.0 mg/kg 7/7 1.86 12 SC (P) 17044 1.0 mg/kg 6/7 1.86 11.33 SC (P) 17044 0.1 mg/kg 6/7 1.14 12.67 SC (P) 17044 0.01 mg/kg 7/7 1.57 11.57 (P) 17044 1 mg/kg 7/7 1.42 12.71 (T) 17614 2 mg/kg SC 6/7 1.71 9 (control) As shown, 17044 reduced disease severity when given therapeutically (time to disease onset is not expected to be increased because dosing begins after onset of disease). Example 21 Mouse Collagen-Induced Arthritis (CIA) Model for Rheumatoid Arthritis A model for human rheumatoid arthritis has been developed wherein mice are immunized with bovine type II collagen. Anderson et al., J. Immunol . 1991 147, 1189-1193, citing Trentham et al., J. Exp . Med 1977, 146, 857. Swelling and inflammation of the joints follows in approximately 3 weeks, with joint distortion and ankylosis typical of rheumatoid arthritis. This model has been used to study the effects of the antisense oligonucleotide 17044, targeted to mouse integrin α4 , on arthritis in mice. DBA/lLacJ mice were obtained from Jackson Laboratory, Bar Harbor, ME. Female mice aged 6 to 8 weeks were used and assigned to groups, ten mice per group. On day 0 mice were immunized at the base of the tail with 100 ug of bovine type II collagen which was 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 ug of lipopolysaccharide (LPS). Weights were recorded weekly. Mice were inspected daily for the onset of CIA, which is characterized by erythema and edema. Upon the onset of the disease, paw widths and rear ankle widths of affected and unaffected joints were measured three times a week using a constant tension caliper. In addition, limbs were clinically evaluated and graded 0-4, where 0 = normal; 1 = one digit swollen; 2 = inflammation present in more than one digit; 3 = joint distortion with or without inflammation; and 4 = ankylosis, detected by joint manipulation. The progression of all measurements was recorded to day 50. At the end of the observation period for each mouse, all paws were removed and examined histologically . The oligonucleotide, the positive control drug (cyclophosphamide, 5 mg/kg) , and the vehicle were administered daily to each mouse intraperitoneally (IP) starting on day -3 and continuing for the duration of the study. Each animal received 10 mg/kg as a bolus daily dose. Example 22 Effect of Antisense Oligonucleotide Targeted to Integrin α.4 on Collagen-Induced Arthritis (CIA) In a preliminary study, a single 10 mg/kg daily dose of ISPH 17044 was administered from the time of disease induction to the conclusion of the study 50 days later. Arthritis incidence was reduced from 70 to 30% in the ISIS 17044-treated group. A summary of clinical parameters is shown in Table 22. Table 22 Effect of ISIS 17044 on Collagen-Induced Arthritis Treatment Severity Mean Day # Paws/ Peak Day of of Onset Mouse Inflamm. Vehicle 3.2 ± 1.1 19.7 ± 1.1 1.2 ± 0.4 6.7 ± 2.9 17044 2.1 ± 1.1 20.3 ± 1.9 0.7 ± 0.4 1.6 ± 0.8 10 mg/kg Cyclophos0 ± 0 — 0 ± 0 — phamide 5 mg/kg Severity = total clinical score/total number of mice in group . # Paws/Mouse = mean number of affected paws at termination/total number of mice in group. Peak Day of Inflammation = day from onset to time of maximum swelling for each joint measured. The mice that developed arthritis in the various treatment groups were compared and the results are shown in Table 23. Table 23 Comparison of Mice With Arthritis Treatment Mean # of Paws Mean Clinical Score Involved Vehicle 1.7 ± 0.4 4.6 ± 0...4 17044 10 mg/kg 2.3 ± 0.3 7.0 ± 1.0 Cyclophospha- ide 5 mg/kg Mean # Paws Involved = number paws at termination in the affected mice/number of affected mice in group. Mean Clinical Score = total clinical score of affected mice/total number of affected mice in group. Example 23 Reduction of Leukocyte Trafficking into the Central Nervous System of EAE mice by Antisense Oligonucleotide Targeted to Integrin α.4 Mice from EAE experiment 3 (Example 18 above) were sacrificed with C0 2 and the spinal cord was removed and cut into 0.5 cm pieces through lumbar and low thoracic regions. The pieces were embedded in O.C.T. tissue freezing medium (Ted Pella Inc., Redding, CA) on dry ice and methylbutane . Four- micron frozen sections were cut and air dried overnight. The sections were fixed for three minutes in ice-cold acetone and then placed on a Dako Autostainer (Dako Corporation, Carpinteria, CA) and treated in the following steps: 5 minutes in 0.03% H 2 0 2 , 5 minutes in 5% donkey serum diluted in PBS, 45 minutes in appropriately diluted primary antibody, 30 minutes in horseradish peroxidase-labeled donkey anti-rat secondary antibody (Jackson Laboratory, Bar Harbor, ME) . Sections were rinsed between treatments with PBS. All the antibodies used were rat anti-mouse from PharMingen (San Diego, CA) , except BM-8, which was from Bachem Bioscience Inc. (King of Prussia, PA) . The immunostaining was developed with DAB (Dako Corporation) and counterstained with hematoxylin. Treatment with 1 mg/kg ISIS 17044 was shown to reduce leukocyte trafficking into the spinal cords and brains of CSJLF1 mice directly immunized to develop EAE. In preliminary experiments, leukocyte trafficking as measured by the ' expression of VLA-4, of which integrin α4 is one of two subunits, CD4 (a T-cell marker), BM-8 (a macrophage marker) and CD18 (contained in the LFA-1 and MAC-1 adhesion molecules found on leukocytes) was significantly reduced. CD4+ T cells were virtually eliminated from the central nervous system of ISIS 17044-treated mice in one experiment. These cells have been shown to be critical for the induction of EAE. In a second experiment (EAE experiment 4, Example 19 above), these results were confirmed, and there appeared to be a dose- dependent effect with 1.0 mg/kg of 17044 causing a greater reduction of leukocyte influx into the central nervous system than did 0.1 mg/kg 17044. Scrambled control oligonucleotide ISIS 17614 was not effective in reducing cellular influx in this experiment. Example 24 Effect of Antisense Oligonucleotides Targeted to Integrin α.4 on Inflammatory Bowel Disease A mouse model for inflammatory bowel disease (IBD) has recently been developed Okayasu et al . ( Gastroenterology 1990, 98, 694-702) . Administration of dextran sulfate to mice induces colitis that mimics human IBD in almost every detail. Dextran sulfate-mduced IBD and human IBD have subsequently been closely compared at the histological level and the mouse model has been found to be an extremely reproducible and reliable model. It is used here to test the effect of ISIS 17044 on inflammatory bowel disease. Female Swiss Webster mice (8 weeks of age) weighing approximately 25 to 30 grams are kept under standard conditions. Mice are allowed to acclimate for at least 5 days before initiation of experimental procedures . Mice are given 5% dextran sulfate sodium in their drinking water (available ad libitum) for 5 days. Concomitantly , ISIS 17044 oligonucleotide m pharmaceutical carrier, carrier alone (negative control) or TGF-β (known to protect against dextran sulfate-mediated colitis in mice) is administered. ISIS 17044 is given as daily subcutaneous injection of 1 mg/kg to 5 mg/kg for 5 days. TGF-β is given as 1 μg/mouse mtracolonically . Mice are sacrificed on day 6 and colons are subjected to histopathologic evaluation. Until sacrifice, disease activity is monitored by observing mice for weight changes and by observing stools for evidence of colitis . Mice are weighed daily. Stools are observed daily for changes in consistency and for presence of occult or gross bleeding. A scoring system is used to develop a disease activity index by which weight loss, stool consistency and presence of bleeding are graded on a scale of 0 to 3 (0 being normal and 3 being most severely affected) and an index is calculated. Drug-induced changes m the disease activity index are analyzed statistically. The disease activity index has been shown to correlate extremely well with IBD in general. Example 25 Effect of Antisense Oligonucleotide Targeted to Integrin α.4 on Survival in Murine Heterotopic Heart Transplant Model To determine the therapeutic effects of integrin α4 antisense oligonucleotide in preventing allograft rejection, the murine integrin α4-specific oligonucleotide ISIS 17044 is 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 . , ( Circula tion 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 1 mg/kg to 5 mg/kg ISIS 17044 is expected to increase the mean survival time. Example 26 Effect of Antisense Oligonucleotide Targeted to Integrin α4 on Leukocyte Migration Leukocyte infiltration of tissues and organs is a major aspect of the inflammatory process and contributes to tissue damage resulting from inflammation. The effect of ISIS 17044 on leukocyte migration is examined using a mouse model in which carrageenan-soaked sponges are implanted subcutaneously. Carrageenan stimulates leukocyte migration and edema. Effect of oligonucleotide on leukocyte migration in inflammatory exudates is evaluated by quantitation of leukocytes infiltrating the implanted sponges. Following a four hour fast, 40 mice are assigned randomly to eight groups each containing five mice. Each mouse is anesthetized with METOFANE and a polyester sponge impregnated with 1 ml of a 20 mg/ml solution of carrageenan is implanted subcutaneously. Saline is administered intravenously to Group 1 at 10 ml/kg four hours prior to sponge implantation and this serves as the vehicle control. Indomethacm (positive control) is administered orally at 3 mg/kg at a volume of 20 ml/kg to Group 2 immediately following surgery, again 6-8 hours later and again at 21 hours post- lmplantation. ISIS 17044 is administered intravenously at a dose of 1 mg/kg to 5 mg/kg to Group 3 four hours prior to sponge implantation. ISIS 17044 is administered intravenously at a dose of 1 mg/kg to 5 mg/kg to Group 4 immediately following sponge implantation. ISIS 17044 is administered intravenously at 1 mg/kg to 5 mg/kg to Groups 5, 6, 7 and 8 at 2, 4, 8 and 18 hours following sponge implantation, respectively. Twenty- four hours after implantation, sponges are removed, immersed in EDTA and saline (5 ml) and squeezed dry. Total numbers of leukocytes in sponge exudate mixtures are determined. The oral administration of mdomethacin at 3 mg/kg generally produces a 75-80% reduction m mean leukocyte count when compared to the vehicle control group. Example 27 Experimental metastasis assay To evaluate the role of mtegrm 4 in metastasis, experimental metastasis assays are performed by injecting 1 x 10 5 C8161 cells into the lateral tail vein of athymic nude mice. C8161 is a human melanoma cell line. Treatment of C8161 cells with the cytokine TNF-α and mterferon γ has previously been shown to result m an increased number of lung metastases when cells were injected into nude mice (Miller, D.E. and Welch, D.R., Proc . Am . Assoc . Cancer Res . 1990, 13, 353). Four-week- old female athymic nude mice (Harlan Sprague Dawley) are used. Animals are maintained under the guidelines of the NIH. Groups of 4-8 mice each are tested in experimental metastasis assays. Treatment of C8161 cells with antisense oligonucleotide ISIS 27104, complementary to human mtegrm α4, is performed in the presence of the cationic lipid, LIPOFECTIN (Gibco/BRL, Gaithersburg, MD) . Cells are seeded in 60 mm tissue culture dishes at 10 6 cells/ml and incubated at 37°C for 3 days, washed with Opti-MEM (Gibco/BRL) 3 times and 100 μl of Opti-MEM medium is added to each well. 0.5 μM oligonucleotide and 15 μg/ml LIPOFECTIN are mixed at room temperature for 15 minutes. 25 μl of the oligonucleotide-LIPOFECTIN mixture is added to the appropriate dishes and incubated at 37°C for 4 hours. The oligonucleotide-LIPOFECTIN mixture is removed and replaced with DME-F12 medium containing 10% fetal calf serum. After 4 hours, 500 U/ml TNF-α is added to the appropriate wells and incubated for 18 hours at which time cells are removed from the plates, counted and injected into athymic nude mice. Alternatively, tumor cells (untreated with oligonucleotide) are implanted into athymic nude mice as above and tumor-bearing mice are treated every other day with antisense oligonucleotide at doses of 1 mg/kg to 5 mg/kg. After 4 weeks, mice are sacrificed, organs are fixed in Bourn's fixative and metastatic lesions on lungs are scored with the aid of a dissecting microscope. Treatment of C8161 cells with ISIS 27104 decreases the metastatic potential of these cells, and eliminates the enhanced metastatic ability of C8161 which results from TNF-α treatment. EXAMPLE 28 Effect of Integrin α4 Antisense Oligonucleotide in a Murine Model for Crohn's Disease SJL/J and IL10-/- mice are used in a TNBS (2,4,5,- trmitrobenzene 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 thm, flexible polyethylene tube is slowly inserted into the colon of tne mice so that the 5 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. Oligonucleotide ISIS 17044 is administered either at the first sign of symptoms 10 or simultaneously with induction of disease. Animals, in most cases, are dosed every day. Administration is by i.v., ι.p., s.q., mmipumps or mtracolonic injection. Experimental tissues are collected at the end of the treatment regimen for histochemical evaluation. 15 EXAMPLE 29 Effect of Antisense Oligonucleotide Targeted to Integrin α.4 in a Murine Model for Hepatitis Concanavalm A-mduced hepatitis is used as a murine model for hepatitis (Mizuhara, H., et al . , J. Exp Med . , 1994, 179, 201529-1537). Female Balb/c and C57BL/6 mice between the ages of 6 weeks and 3 months are used to assess the activity of antisense oligonucleotide ISIS 17044. Mice are intravenously injected with oligonucleotide. The pretreated mice are then intravenously injected with 0.3 25 mg concanavalm 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 vi tro methods. In some experiments, blood is collected from the retro-orbital vein. EXAMPLE 30 Effect of Antisense Oligonucleotide Targeted to Integrin α4 in a Murine Model for Asthma Airway inflammation is observed in patients with allergic asthma. A murine model of allergic asthma has been developed, (Hessel et al . J. Immunol . 1998, 160, 2998-3005) . Sensitization of BALB/c mice with ovalbumm induces a high level of ovalbumm- specific IgE m serum. Inhalation of ovalbumm in sensitized mice causes an immediate bronchoconstrictive response. Repeated inhalation of ovalbumm m sensitized animals induces nonspecific airway hyperresponsiveness m vivo, and infiltration of leukocytes m airway tissue. Pathogen-free male BALB/c mice (6-8 wk) are obtained from Jackson Laboratories. Active sensitization is performed by seven IP injections of 10 ug of ovalbumm (Sigma Chemical Co, St. Louis, MO, grade II) m 0.5 ml of pyrogen-free saline on alternate days, one injection per day. This produces high titers of total IgE in mouse serum of which 80% is ovalbumm- specific IgE (Hessel et al . , 1998, J. Immunol . 160: 2998-3005). Four weeks after the last injection, mice are exposed either to ovalbumm aerosols (2 mg/ml) or saline aerosols, once per day for eight days. The aerosol is generated with a nebulizer such as Medix 8001 (Sussex, UK) . Animals were exposed for 5 minutes per aerosol challenge. Antisense oligonucleotide 17044 is given to the mice during the challenge period. Thirty minutes before the first and fifth inhalation challenge, sensitized mice were injected intravenously with a 1 mg/kg to 5 mg/kg dose of ISIS 17044 m PBS. Airway responsiveness to methacholme is measured in vivo 24 hours after the last aerosol exposure using the air-overflow pressure method, in which bronchial resistance to inflation is measured. Mice are anesthetized by IP injection of urethan 2g/kg and placed on a heated blanket. The trachea is cannulated and a small polyethylene catheter is placed in the jugular vein for IV administrations. Spontaneous breathing is suppressed by IV injection of tubocurarine chloride (3.3 mg/kg) . When it stopped, the tracheal cannula is attached to a respiration pump. At intervals, increasing doses of methacholine ranging from 40 to 1280 ug/kg were given. The increase in air-overflow pressure (caused by reduction of air flow in to the lungs as a result of increasing airway tone) is measured at its peak and expressed as a percentage increase. Bronchoalveolar lavage is used to measure the leukocyte infiltration of airway tissue. Three and 24 hours after the last aerosol, mice are anesthetized by IP injection of 0.25 ml of sodium pentobarbitone (60 mg/ml) . The abdomen and chest are opened, and the abdominal aorta is excised. Below the larynx, a small incision is made, and a flexible polyethylene cannula is inserted into the trachea and fixed with a ligature. Mice are lavaged five times with 1 ml aliquots of pyrogen-free saline warmed to 37°C. The cells derived from each lavage were pooled, were washed with cold PBS and resuspended in 200 ul of cold PBS. Total numbers of cells were counted and categorized. Example 31 Effect of Antisense to Integrin α4 on Peripheralization of Hemopoietic Progenitor Cells Mobilization (peripheralization) of hemopoietic progenitor cells from the bone marrow into the blood circulation is clinically useful in stem cell transplantation. A murine model has been established to evaluate the effects of drug compounds on this process (and its reverse, selective lodgement or homing of transplanted stem cells to the recipient's bone marrow) (Papayannopoulou et al . , Proc . Na tl . Acad. Sci . 1995, 92, 9647-9651). Briefly, pathogen-free mice 3-6 months old are used and are housed in a filtered-air-flow housing unit for the duration of the experiment. Mice used as primary or secondary recipients m the homing assay are exposed to 1150-cGy irradiation delivered from a Cs source. Single cell suspensions are prepared from peripheral blood, 5 bone marrow and spleen. Peripheral blood is obtained in preservative-free heparin by cardiac puncture and nucleated cells are recovered using NH 4 C1 hemolytic buffer. Bone marrow cells are obtained by flushing femoral marrow, under sterile conditions, with Iscove's modified Dulbecco's medium (IMDM) 10 containing 10% v/v fetal bovme serum and streptomycin (50 ug/ml) and penicillin (50 U/ml) . Splenic cell suspensions are obtained by lengthwise dissection of the spleen with a scalpel and scraping of cellular contents from the capsule, followed by vigorous pipetting. All cell suspensions are washed twice 15 m IMDM with 10% fetal bov e serum. Cell counts are obtained using a he ocytometer . Culture colony-forming unit (CFU-C; and spleen colony- forming unit (CFU-S) assays are performed as described in Papayannopoulou et al . , ( Proc . Na tl . Acad. Sci . 1995, 92, 9647- 209651) . To assess hemopoietic progenitor mobilization m normal animals, the oligonucleotide is administered by daily IV injection (at dose ranges from 1 mg/kg to 5 mg/kg) for three days. Mice are sacrificed on the fourτh day. Nucleated cells 25 present m 0.25-0.5 ml of peripheral blood are plated m clonogenic progenitor cultures to assess CFU-C content, as described in Papayannopoulou et al., ( Proc . Na tl . Acad . Sci . 1995, 92, 9647-9651) . Example 32 30 Screening of additional oligonucleotides targeted to human integrin α4 An additional set of antisense oligonucleotides was designed to target human mtegrm α4 (Genbank Accession no. L12002, incorporated herein as SEQ ID NO: 1) . The oligonucleotides are shown in Table 24. Oligonucleotides were synthesized as "gapmers" consisting of a 9-nucleotιde region of deoxynucleotides flanked by three 2 ' -O-methoxyethyl (2' -MOE) 5 nucleotides on the 5 ' end and eight 2 ' -MOE nucleotides on the 3' end. 2 ' -MOE nucleotides are shown m bold in the table. Oligonucleotides were synthesized with phosphorothioate backbones throughout, and all cytosmes are 5-methylcytosιnes . Oligonucleotides were screened m the presence of lipofectm 10 in A375 human melanoma cells as described m Example 8 hereinabove. Results are also shown m Table 24. As shown in the table, oligonucleotides 107234, 107235, 107236, 107237, 107238, 107239, 107240, 107241, 107242, 107243,107244, 107245, 107246, 107248, 107249, 107251, 107252, 107253, 107254, 107255, 15107256, 107257, 107258, 107259 and 107260 (SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 81, 82, 84, 85, 86, 87, 88, 89, 90, 91, 92 and 93, respectively) gave at least about 50% inhibition of mtegrm α4 protein expression and are preferred. Of these, ISIS 107245, 107248, 107252, 107255 and 20107259 (SEQ ID NO: 78, 81, 85, 88 and 92) were selected for further study. Table 24 Additional antisense oligonucleotides targeted to human integrin α4 co v Example 33 Dose response curves for effect of antisense oligonucleotides on integrin α4 protein expression A375 cells were treated with ISIS 107245, 107248, 107252, 5 107255 and 107259 by electroporation at 175volts, r=l, , c=1000, 0.2ml Opti-MEM, 1 xl06 cells /ml, 2mm gap electrode using a BTX Electro Cell Manipulator 600 (Genetronics , San Diego CA) . Oligonucleotide concentrations were 1, 3, 10 and 30 μM. Cells were harvested 24 hours after oligonucleotide treatment and 0 stained with 2 ug/ml CD49d-FITC for flow cytometry as in the previous examples. Results are shown in Table 25. Table 25 Dose responses of additional antisense oligonucleotides targeted to human integrin α4 ISIS 107248 was compared directly to ISIS 27104 for ability to inhibit integrin α4 expression in A375 melanoma cells. The results are shown in Table 26. Oligonucleotides were electroporated into cells as in the previous example. Table 26 Comparison of ISIS 27104 and ISIS 107248 Example 34 Antisense inhibition of integrin α4 protein expression in Jurkat T-cells ISIS 107248 was tested at doses of 1, 3, 10, 20 and 30 μM for ability to inhibit integrin α4 expression in Jurkat T-lymphocyte cells according to the previous examples. Integrin α4 protein expression was reduced by approximately 65-72% at oligonucleotide doses of 3-30 μM at 24 hours after oligonucleotide treatment. At 48 hours after treatment, integrin α4 protein expression was reduced by 60% at 3 μM and by 90% at 10, 20 and 30 μM oligonucleotide concentration. Example 35 Antisense inhibition of integrin α4 RNA expression in Jurkat T-cells Quantitation of integrin α4 mRNA levels was performed by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE or FAM, obtained from either Operon Technologies Inc., Alameda, CA or PE-Applied Biosystems, Foster City, CA) is attached to the 5' end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, CA or PE-Applied Biosystems, Foster City, CA) is attached to the 3' end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3' quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5 ' -exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples. PCR reagents were obtained from PE-Applied Biosystems, Foster City, CA. RT-PCR reactions were carried out by adding 20 μL PCR cocktail (lx TAQMAN™ buffer A, 5.5 mM MgCl 2 , 300 μM each of dATP, dCTP and dGTP, 600 μM of dUTP, 400 nM each of forward primer and reverse primer (or 100 nM in the case of G3PDH) , 100 nM probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5 Units MuLV reverse transcriptase) to 5 μL (100 ng) total mRNA solution, purified using the RNEasy™ kit (Qiagen, Valencia CA) . 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) . Integrin α4 probes and primers were designed to hybridize to the human integrin α4 sequence, using published sequence information (GenBank accession number L12002), incorporated herein as SEQ ID NO:l). For integrin α4 the PCR primers were: forward primer: GACACGCTGCGCCTCAT (SEQ ID NO: 94, hybridizes at position 319-335 of Genbank accession no. L12002) reverse primer: ATTCAACACTAAGCGGCCACTG (SEQ ID NO: 95, hybridizes at position 391-412 of Genbank accession no. L12002) and the PCR probe was: FAM- CCAACCGTCGCATCCCGTGCAA -TAMRA (probe is SEQ ID NO: 96; hybridizes at position 361-382 of Genbank accession no. L12002) where FAM (PE-Applied Biosystems, Foster City, CA) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, CA) is the quencher dye. For GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 97) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 98)and the PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC- TAMRA 3' (probe is SEQ ID NO: 99) where JOE (PE-Applied Biosystems, Foster City, CA) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, CA) is the quencher dye. 24 hours after treatment of A375 melanoma cells with ISIS 107248 by electroporation (oligonucleotide concentrations of 0.3, 1, 3, 10 and 30 μM) , total RNA was isolated using the RNeasy™ kit (Qiagen, Valencia CA) and RT-PCR was performed. Integrin α4 mRNA levels were reduced by approximately 23%, 38%, 70%, 80% and 85% compared to control at oligonucleotide doses of 0.3, 1, 3, 10 and 30 μM, respectively. Example 36 Effect of antisense inhibition of integrin α4 on cell adhesion Human umbilical vein endothelial cells (HUVEC) (Clonetics, San Diego CA) were cultured in EGM-UV medium (Clonetics) and treated with TNFα for 20 hours to induce VCAM expression. A375 melanoma cells were electroporated with ISIS 107248 as described above. Using antibody to PECAM, a specific marker for endothelial cells, flow cytometry was used to quantitate the inhibition of adherence of A375 melanoma cells to HUVECs . Compared to control (TNF treated, no oligonucleotide), ISIS 107248 (10 μM) inhibited A375 melanoma cell adherence to HUVEC by approximately 85%. An antibody to integrin α4 inhibited adherence by approximately 72%. Melanoma cells represent a clinically relevant model because a statistically significant correlation has been shown between increased expression of VLA-4 expression (of which integrin α4 is one member) and time to progression and overall survival time of patients with melanoma. Schadendorf et al . , J. Na tl . Cancer Ins t . , 1995, 81 , 366-371. A similar adherence assay was performed using Jurkat T-cells instead of melanoma cells, again detecting adherence to HUVEC. Because Jurkat cells are smaller and less complex, they can be separated from HUVEC by gating on the basis of differences in forward light scatter and side scatter. For 5000 HUVEC events, adherent Jurkat cells were quantitated. At oligonucleotide concentrations of 1, 3, 10, and 30 μM ISIS 107248, adherence of Jurkat cells to HUVEC was 0, 12%, 32% and 63%, respectively. Antibody to integrin α4 gave 96% inhibition, an antibody to VCAM-1 gave 67% inhibition, and a combination of ISIS 107248 and VCAM-1 antibody gave 78% inhibition.

Claims: What is claimed is : 1. An antisense compound 8 to 30 nucleotides in length targeted to a nucleic acid molecule encoding human integrin α4 , wherein said antisense compound inhibits the expression 5 of human integrin α4. 2. The antisense compound of claim 1 which is an antisense oligonucleotide. 3. The antisense compound of claim 2 wherein the antisense oligonucleotide comprises SEQ ID NO: 13, 15, 35, 37, 1039 or 46. 4. The antisense compound of claim 2 which comprises at least one modified internucleoside linkage. 5. The antisense compound of claim 4 wherein the modified internucleoside linkage is a phosphorothioate linkage. 15 6. The antisense compound of claim 2 which comprises at least one modified sugar moiety. 7. The antisense compound of claim 6 wherein the modified sugar moiety is a 2 ' -O-methoxyethyl sugar moiety. 8. The antisense compound of claim 2 which comprises 0 at least one modified nucleobase . 9. The antisense compound of claim 8 wherein the modified nucleobase is a 5-methylcytosine . 10. The antisense compound of claim 2 which is a chimeric oligonucleotide . 11. A pharmaceutical composition comprising the antisense compound of claim 1 and a pharmaceutically acceptable carrier or diluent. 12. The pharmaceutical composition of claim 11 further comprising a colloidal dispersion system. 13. The pharmaceutical composition of claim 11 wherein the antisense compound is an antisense oligonucleotide. 14. A method of inhibiting the expression of integrin α4 in human cells or tissues comprising contacting said cells or tissues with the antisense compound of claim 1 so that expression of integrin α4 is inhibited. 15. A method of treating a human having a disease or condition associated with integrin α4 comprising administering to said animal a therapeutically or prophylactically effective amount of the antisense compound of claim 1 so that expression of integrin α4 is inhibited. 16. The method of claim 15 wherein the disease or condition is an inflammatory disease or condition or an autoimmune disease or condition. 17. The method of claim 15 wherein the disease or condition is characterized by leukocyte migration into affected tissues . 18. The method of claim 17 wherein the affected tissues are central nervous system tissues. 19. The method of claim 15 wherein the disease or condition is rheumatoid arthritis, multiple sclerosis, asthma, inflammatory bowel disease, diabetes, hepatitis, allograft rejection or tumor metastasis. 20. A method- of reducing levels of VLA-4 in human cells or tissues comprising contacting said cells or tissues with the antisense compound of claim 1 so that levels of VLA-4 are reduced. 21. A method of reducing levels of α4β7 in human cells or tissues comprising contacting said cells or tissues with the antisense compound of claim 1 so that levels of α4β7 are reduced. 22. A method of increasing mobilization of hemopoietic progenitor cells into the circulation in a human comprising contacting said human with the antisense compound of claim 1 so that mobilization of said hemopoietic progenitor cells into the circulation is increased. 23. The antisense compound of claim 2 wherein the antisense oligonucleotide comprises SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 81, 82, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93. 24. The method of claim 15 wherein the disease or condition is a metastatic disease or condition. 25. The method of claim 24 wherein the disease or condition is melanoma. 26. A method of decreasing adherence of cells of a first type to cells of a second type comprising contacting at least one of said cell types with an antisense compound of claim 1. 27. The method of claim 26 wherein said first cell type anoma cells or lymphocytes. 28. The method of claim 26 wherein said second cell type othelial cells.

Inventors: BENNETT C FRANK, CONDON THOMAS P, COWSERT LEX M, BENNETT, C., FRANK, CONDON, THOMAS, P., COWSERT, LEX, M.
Applicants: BENNETT C FRANK, CONDON THOMAS P, COWSERT LEX M, ISIS PHARMACEUTICALS INC, BENNETT, C., FRANK, CONDON, THOMAS, P., COWSERT, LEX, M., ISIS PHARMACEUTICALS, INC.
Application year: 1999
Main classification: C12Q1
IPC codes: A61K31, A61K38, A61K48, A61P1, A61P3, A61P11, A61P25, A61P29, A61P35, A61P37, A61P43, C07H21, C12N5, C12N15, C12Q1