OLIGONUCLEOTIDE MODULATION OF HER-1 EXPRESSION

Abstract: Compounds, compositions and methods are provided for inhibiting the expressionof human Her-1. The compositions comprise oligonucleotides complementary to mRNA targeted to nucleic acids encoding Her-1. Methods of using these oligonucleotides for inhibition of Her-1 expression and for treatment of diseases such as cancers associated with overexpression of Her-1 are provided.

Description: OLIGONUCLEOTIDE MODULATION OF HER-1 EXPRESSION FIELD OF THE INVENTION The present invention provides compositions and methods of modulating the expression of Her-1. Many human tumors have been found to overexpress this receptor and such enhanced expression has been shown to be correlated with poor prognosis. In particular, this invention relates to oUgonucleotides specifically hybridizable with nucleic acids encoding human Her-1. These oUgonucleotides have been found to inhibit the expression of Her-1. BACKGROUND OF THE INVENTION Her-1, also known as epidermal growth factor receptor (EGFR) , is a specific receptor for epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) . When these mitogenic polypeptides bind to Her-1, tyrosine kinase activity of the receptor is induced, and this in turn triggers a series of events which regulate cell growth. A number of malignant and non-malignant disease conditions are now believed to be associated with Her-1, particularly aberrant expression of Her-1. Aberrant expression includes both increased expression of normal Her-1 and expression of mutant Her-1. Overexpression of Her-1 is found in many human tumors including most glioblastomas and breast, lung, ovarian, colorectal, bladder, pancreatic, squamous cell and renal carcinomas. Elevated Her-1 levels correlate with poor prognosis in human tumors. Her-1 is also implicated in nonmalignant diseases, such as psoriasis. The sequence of the mRNA encoding human Her-1 is known. Ullrich et al . , Nature, 1984, 309, 418; GenBank Accession Number X00588; and Kraus et al . , Proc . Natl . Acad. Sci . USA, 1989, 86, 9193; GenBank Accession Number M29366. The gene encoding Her-1 is also known as ERBB3 or c-erb-Bl . Two Her-1 transcripts typically appear on Northern blots, one measuring 10 kb and one measuring 5.6 kb. A number of inhibitors of Her-1 have been shown to be effective in inhibiting the growth of human tumor cells. Monoclonal antibodies to Her-1 and drugs which inhibit Her- 1 tyrosine kinase activity can inhibit the growth of human cancer cell xenografts in nude mice. Normanno et al . , Clin . Cancer Res . , 1996, 2, 601. The drug PD153035, which inhibits Her-1 tyrosine kinase activity, can inhibit the growth of A431 cells in nude mice, and tyrphostins, which inhibit the activity of Her-1 as well as other tyrosine kinases, have been shown to inhibit the growth of squamous carcinoma in nude mice. Kunkel et al . , Invest . New Drugs, 1996, 13 , 295 and Yoneda et al . , Cancer Res . , 1991, 51 , 4430. Vectors expressing Her-1 nucleic acid sequences in an orientation complementary to mRNA have been used to study the effects of Her-1 on proliferation of cultured cancer cells. Transfectants of the human epidermoid carcinoma KB cell line expressing Her-1 cDNA or RNA sequences in an orientation complementary to mRNA exhibited restored serum- dependent growth and impaired colony formation and growth in agar. Moroni et al . , J. Biol . Chem . , 1992, 267, 2714. Human pancreatic carcinoma cells of the PC- 7 cell line transfected with vectors expressing Her-1 cDNA sequences in an orientation complimentary to mRNA showed inhibited cell growth, colony formation and tumorigenicity in nude mice. Liu et al . , Chinese Medical Journal , 1995, 108 , 653. Transfection of human colon cancer cell lines with Her-1 RNA expression vectors producing an oligonucleotide complementary to mRNA caused a reduction in cell proliferation and ability to grow on soft agar. Rajagopal et al . , Int . J. Cancer, 1995, 62, 661. Human rhabdomyosarcoma cells transfected with a plasmid expressing Her-1 cDNA in an orientation complementary to mRNA had greatly impaired proliferation. De Giovanni et al . , Cancer Res . , 1996, 56, 3898. Considerable research is being directed to the application of oUgonucleotides complementary to mRNA and other oligomers for therapeutic purposes. OUgonucleotides complementary to mRNA have already been employed as therapeutic moieties in the treatment of disease states in animals and man, and compositions comprising oligomers complementary to mRNA have been shown to be capable of modulating expression of genes implicated in viral, fungal and metabolic diseases. Further, oUgonucleotides complementary to mRNA have been safely administered to humans and clinical trials of approximately a dozen oligonucleotide drugs targeted to viral and cellular gene products are underway. Oligodeoxyribonucleotides complementary to mRNA targeted to Her-1 have been encapsulated into liposomes linked to folate via a polyethylene glycol linker and delivered into cultured human epidermoid carcinoma KB cells. The oUgonucleotides were a phosphodiester (P=0) 15-mer complementary to the Her-1 gene stop codon, or the same sequence with three phosphorothioate (P=S) linkages at each end. Both of these oUgonucleotides reduced KB cell proliferation by greater than 90% after treatment with 3 nM oligonucleotide in folate-PEG-liposomes . In contrast, free P=0 oligonucleotide caused almost no growth inhibition, and free P=S-capped oligonucleotide caused only a 15% growth inhibition, even at this high dosage level. Her-1 expression, measured by indirect immunofluorescence, was virtually abolished in cells treated with either of the folate-PEG-liposome-encapsulated oUgonucleotides but Her-1 expression was qualitatively similar to untreated cells after treatment with free oligonucleotide. Wang et al . , Proc . Natl . Acad . Sci . USA, 1995, 92 , 3318. A 15-mer phosphorothioate oligonucleotide complementary to the translation initiation region of Her-1 mRNA was found to inhibit cell proliferation by over 25% in A431 cells, derived from a vulval carcinoma. This activity, though dose-dependent from l-25μM, was not mediated by an antisense mechanism, as demonstrated by a lack of reduction in either Her-1 protein or mRNA after oligonucleotide treatment. In addition, an 18-mer oligonucleotide complementary to mRNA targeted to the same region had no effect even at the highest (25μM) dose, and neither oligonucleotide had any effect in the two other tumor-derived cell lines tested. Coulson et al . , Mol . Pharm . , 1996, 50 , 314. The suppression of growth of pancreatic carcinoma cell lines by undisclosed oUgonucleotides complementary to mRNA inhibiting the expression of TGF-alpha and/or the Her- 1 has been reported. Hall et al . , unpublished data, reported in Hall and Lemoine, Models of Pancreatic Cancer, in Cancer Surveys, Volume 16: The Molecular Pathology of Cancer, 1993, p.135-155. Rubenstein et al . have reported treatment of established human-derived prostate tumor xenografts in nude mice by intralesional injection of oUgonucleotides complementary to mRNA directed against mRNAs encoding TGF- alpha and Her-1. The oUgonucleotides included 39-mers complementary to 18 bases located 5' and 3' from the AUG mRNA translation initiation codon of either TGF-alpha or Her-1 sequence. The oUgonucleotides were phosphorothioated at each of three terminal bases at both the 5' and 3' ends. The oUgonucleotides were administered either alone or in combination, with the combination treatment proving most effective. J. Surg. Oncol . , 1996, 62, 194. In U.S. Patent 5,610,288, Rubenstein et al . disclose polynucleotides of about 20 to 50 nucleic acid bases, most preferably about 40 nucleic acid bases in length, which preferentially hybridize to the start codon of the mRNA encoding Her-1. A preferred embodiment is a 39-mer including 18 bases complementary to the 5' side of the translation initiation codon. This oligonucleotide inhibited PC-3 cell growth when administered in combination with an oligonucleotide complementary to mRNA targeted to TGF-alpha. Alone, the Her-1 oligonucleotide gave inhibition of cell growth equivalent to that achieved with an inverted (5' to 3 ' ) version of the same sequence. Disclosed in US Patent 5,914,269 (Bennett, et al . ) are oUgonucleotides targeting Her-1 and methods of modulating the expression of Her-1 using said oUgonucleotides. Three of these oUgonucleotides and their analogs have also been used to investigate growth inhibition of SK0V3 cells, a human ovarian cancer cell line. In these studies it was demonstrated that all three oUgonucleotides produced a significant reduction in Her-1 expression and protein production as well as in vi tro growth inhibition of SKOV3 cells. Witters et al . , Breast Cancer Research and Treatment, 1999, 53 , 41-50. Rearrangements or deletions of the Her-1 gene resulting in mutant Her-1 protein have been found in some cancers. The in-frame deletion from nucleotides 275-1075 in the Her-1 has been referred to as class I, Type I or Type III mutation. WO 96/16988 (Wong et al . ) discloses cell lines capable of overexpressing Type III mutant Her-1, vaccines for inhibiting tumor formation comprising peptides similar to a fusion junction present in mutant human Her-1, antibodies raised against a cell line overexpressing Type III mutant Her-1, and oUgonucleotides complementary to mRNA targeted to a Type III mutant Her-1 which decrease expression of a mutant Her-1. In a preferred embodiment, the oligonucleotide complementary to mRNA contains sequences from what were formerly distant portions of the normal Her-1 cDNA. The oligonucleotide must contain the sequence 5 ' -TACCTT-3 ' . An 18-mer oligonucleotide containing this sequence was found to downregulate mutant Her-1 levels when given at a 40 nM dose in cultured cells which overexpressed Type III mutant Her-1. The present invention provides new oligonucleotide compounds complementary to mRNA, as well as other oligonucleotide compounds, and compositions comprising the same together with methodologies for the use of certain compounds of the invention for interfering with translation of selected mRNA targets related to epidermal growth factor receptor. SUMMARY OF THE INVENTION The present invention is directed to oUgonucleotides, especially those from 12-25 nucleotides in length, which are complementary to a nucleic acid molecule encoding Her-1, and which inhibit the expression of Her-1. In a preferred embodiment, the oUgonucleotides comprise at least one phosphorothioate linkage. In other preferred embodiments, the oUgonucleotides may comprise at least one 5-methyl cytosine, 2 ' -0-alkyl or 2'-fluoro modification. Pharmaceutical compositions comprising the oUgonucleotides of the invention and a pharmaceutically acceptable carrier are also provided. Further provided are methods of modulating the expression of a human epidermal growth factor receptor in cells or tissues comprising contacting said cells or tissues with the oligonucleotide of the invention. These methods may be performed in vi tro, in vivo or ex vivo . Methods of treating an animal having a hyperproliterative disease or condition by administering a therapeutically effective amount of an oligonucleotide of the invention for a time sufficient to ameliorate said hyperproliterative disease or condition are provided. The hyperproliterative disease or condition may be cancer, and in preferred embodiments the cancer is lung cancer, ovarian cancer, colon cancer or prostate cancer. DETAILED DESCRIPTION OF THE INVENTION The present invention employs oUgonucleotides for use in inhibiting the function of nucleic acid molecules encoding Her-1, ultimately modulating the amount of Her-1 produced. This is accomplished by providing oUgonucleotides complementary to mRNA which specifically hybridize with mRNA or DNA encoding Her-1. Such hybridization with mRNA interferes with the normal role of mRNA and causes a modulation of its function. The functions of mRNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. The overall effect of such interference with mRNA function is modulation of the expression of Her-1.- In the context of this 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. OUgonucleotides may comprise nucleotide sequences sufficient in identity and number to effect specific hybridization with a particular nucleic acid molecule. Such oUgonucleotides are commonly described as "complementary to mRNA." OUgonucleotides may also be directed to nucleotide sequences within the genome. OUgonucleotides are commonly used as research reagents and diagnostics. For example, oUgonucleotides complementary to mRNA, 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. OUgonucleotides complementary to mRNA are also used, for example, to distinguish between functions of various members of a biological pathway. This specific inhibitory effect has, therefore, been harnessed for research use. The specificity and sensitivity of oUgonucleotides is also harnessed by those of skill in the art for therapeutic uses. OUgonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. For example, workers in the field have now identified oUgonucleotides complementary to mRNA, triplex oUgonucleotides, and other oligonucleotide compositions which are capable of modulating expression of genes implicated in viral, fungal and metabolic diseases. OUgonucleotides complementary to mRNA have been safely administered to humans and numerous clinical trials are presently underway. It is thus established that oUgonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans. It is preferred to target specific genes for attack by oUgonucleotides complementary to mRNA. "Targeting" an oligonucleotide 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 Her-1. The targeting process also includes determination of a site or sites within this gene for the oligonucleotide interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5 ' -AUG (in transcribed mRNA molecules; 5 ' -ATG in the corresponding DNA molecule) , the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon." A minority of genes have a translation initiation codon having the RNA sequence 5 ' -GUG, 5 ' -UUG or 5'-CUG, and 5 ' -AUA, 5 ' -ACG and 5'-CUG have been shown to function in vivo . Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (prokaryotes) . It is also known in the art that eukaryotic and prokayotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, "start codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding Her-1, regardless of the sequence (s) of such codons. It is also known in the art that a translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5 ' -UAG and 5 ■ -UGA (the corresponding DNA sequences are 5 ' -TAA, 5 ' -TAG and 5 ' -TGA, respectively) . The terms "start codon region" and "translation initiation codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3 ' ) from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3 ' ) from a translation termination codon. The open reading frame (ORF) or "coding region, " which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene) and the 3 ' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3 ' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3 ' end of an mRNA or corresponding nucleotides on the gene) . mRNA splice sites may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. Such rearrangements or deletions of the Her-1 gene resulting in mutant Her-1 protein are known to occur in some cancers. Once the target site has been identified, oUgonucleotides 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, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oUgonucleotides composed of naturally-occurring nucleobases, sugars and covalent intersugar (backbone) linkages as well as oUgonucleotides having non-naturally- occurring portions which function similarly. Such modified or substituted oUgonucleotides 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. A discussion of antisense oUgonucleotides and some desirable modifications can be found in De Mesmaeker et al . , Ace . Chem. Res . , 1995, 28 , 366. Specific examples of some preferred oUgonucleotides envisioned for this invention include those containing modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oUgonucleotides with phosphorothioate backbones and those with heteroatom backbones, and in particular -CH 2 -NH-0-CH 2 - , -CH 2 -N(CH 3 ) -0-CH 2 - (known as a methylene (methylimino) or MMI backbone), -CH 2 -0-N (CH 3 ) -CH 2 - , - CH 2 -N (CH 3 ) -N (CH 3 ) -CH 2 - and -0-N(CH 3 ) -CH 2 -CH 2 - backbones, wherein the native phosphodiester backbone is represented as -0-P-0-CH 2 -) . The amide backbones disclosed by De Mesmaeker et al . ( Id. ) are also preferred. Also preferred are oUgonucleotides having morpholino backbone structures, such as those described in Summerton and Weller, U.S. Patent 5,034,506. In other preferred embodiments, such as the peptide nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleobases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone. Nielsen et al . , Science, 1991, 254 , 1497. OUgonucleotides may also contain one or more substituted sugar moieties. Preferred oUgonucleotides comprise one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, 0CH 3 0CH 3 , 0CH 3 0 (CH 2 ) n CH 3 , 0(CH 2 ) n NH 2 or 0(CH 2 ) n CH 3 where n is from 1 to about 10; C x to Cι 0 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF 3 ; 0CF 3 ; O- , S-, or N-alkyl; 0- , S-, or N- alkenyl; SOCH 3 ; S0 2 CH 3 ; ON0 2 ; N0 2 ; N 3 ; NH 2 ; heterocycloalkyl ; heterocycloalkaryl ; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2 ' -methoxyethoxy (2 ' -0-CH 2 CH 2 0CH 3 , also known as 2 ' -0- (2-methoxyethyl) ) (Martin et al . , Helv. Chim. Acta, 1995, 78, 486) . Other preferred modifications include 2 ' -methoxy (2 ' -0-CH 3 ) , 2 ' -propoxy (2 ' -OCH 2 CH 2 CH 3 ) 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 and the 5' position of 5' terminal nucleotide. OUgonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group. OUgonucleotides may also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A) , guanine (G) , thymine (T) , cytosine (C) and uracil (U) . Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5 -me pyrimidines, particularly 5-methylcytosine (also referred to as 5- methyl-2 ' deoxycytosine and often referred to in the art as 5-me-C) , 5-hydroxymethylcytosine (HMC) , glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2- aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N 6 (6- aminohexyl ) adenine and 2 , 6-diaminopurine . Kornberg, A., DNA Replication, W.H. Freeman & Co., San Francisco, 1980, pp.75-77 and Gebeyehu et al . , Nuc . Acids Res . , 1987, 15, 4513. A "universal" base known in the art, e.g., inosine, may be included. 5-me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions . Another modification of the oUgonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, a cholesteryl moiety (Letsinger et al . , Proc . Natl . Acad . Sci . USA, 1989, 86, 6553), cholic acid (Manoharan et al . , Bioorg. Med . Chem . Let . , 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al . , Ann . N. Y. Acad. Sci . , 1992, 660 , 306; Manoharan et al . , Bioorg. Med . Chem . Let . , 1993, 3, 2765), a thiocholesterol (Oberhauser et al . , Nucl . Acids Res . , 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al . , EMBO J. , 1991, 10 , 111; Kabanov et al . , FEBS Lett . , 1990, 259, 327; Svinarchuk et al . , Biochimie, 1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1, 2-di-0-hexadecyl-rac-glycero-3-H- phosphonate (Manoharan et al . , Tetrahedron Lett . , 1995, 36, 3651; Shea et al . , Nucl . Acids Res . , 1990, 18 , -3777), a polyamine or a polyethylene glycol chain (Manoharan et al . , Nucleosides & Nucleotides, 1995, 14 , 969) , or adamantane acetic acid (Manoharan et al . , Tetrahedron Lett . , 1995, 36, 3651), a palmityl moiety (Mishra et al . , Biochim . Biophys . Acta, 1995, 1264 , 229), or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety (Crooke et al . , J. Pharmacol . Exp . Ther. , 1996, 277, 923), all references being incorporated herein by reference. Oligonucleotides comprising lipophilic moieties, and methods for preparing such oligonucleotides are known in the art, for example, U.S. Patents No. 5,138,045, No. 5,218,105 and No. 5,459,255, all of which are incorporated herein by reference . The oligonucleotides of the invention may be provided as prodrugs, which comprise one or more moieties which are cleaved off, generally in the body, to yield an active oligonucleotide. One example of a prodrug approach is described by Imbach et al . in WO Publication 94/26764, incorporated herein by reference. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at a single nucleoside within an oligonucleotide. The present invention also includes oligonucleotides which are chimeric oligonucleotides . "Chimeric" oligonucleotides or "chimeras," in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA: DNA or RNA: RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art . The oligonucleotides in accordance with this invention preferably comprise from about 8 to about 30 nucleotides. It is more preferred that such oligonucleotides comprise from about 12 to 25 nucleotides. As is known in the art, a nucleotide is a base-sugar combination suitably bound to an adjacent nucleotide through a phosphodiester, phosphorothioate or other covalent linkage. The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, 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 also known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives. The oligonucleotides of the present invention can be utilized as diagnostics, therapeutics 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 Her-1 is treated by administering oligonucleotides in accordance with this invention. The oligonucleotides of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an oligonucleotide to a suitable pharmaceutically acceptable diluent or carrier. Use of the oligonucleotides and methods of the invention may also be useful prophylactically, e.g., to prevent or delay tumor formation. The oligonucleotides of the present invention can be used as diagnostics for the presence of Her-1-specific nucleic acids in a cell or tissue sample. For example, radiolabeled oligonucleotides can be prepared by 32 P labeling at the 5' end with polynucleotide kinase. Sambrook et al . , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, 1989, Volume 2, pg. 10.59. Radiolabeled oligonucleotides are then contacted with cell or tissue samples suspected of containing Her-1 mRNA (and thus, Her-1), and the samples are washed to remove unbound oligonucleotide. Radioactivity remaining in the sample indicates the presence of bound oligonucleotide, which in turn indicates the presence of nucleic acids complementary to the oligonucleotide, and can be quantitated using a scintillation counter or other routine means. Expression of nucleic acids encoding these proteins is thus detected. Radiolabeled oligonucleotides of the present invention can also be used to perform autoradiography of tissues to determine the localization, distribution and quantitation of Her-1 for research, diagnostic or therapeutic purposes. In such studies, tissue sections are treated with radiolabeled oligonucleotide and washed as described above, then exposed to photographic emulsion according to routine autoradiography procedures. The emulsion, when developed, yields an image of silver grains over the regions expressing an Her-1 gene. Quantitation of the silver grains permits detection of the expression of mRNA molecules encoding Her-1 proteins and permits targeting of oligonucleotides to these areas. Analogous assays for fluorescent detection of expression of Her-1 can be developed using oligonucleotides of the present invention which are conjugated with fluorescein or other fluorescent tags instead of radiolabeling. Such conjugations are routinely accomplished during solid phase synthesis using fluorescently-labeled amidites or controlled pore glass (CPG) columns. Fluorescein-labeled amidites and CPG are available from, e.g., Glen Research, Sterling VA. Oligonucleotides of the present invention directed to Her-1 can be used in diagnostics, therapeutics, prophylaxis, and as research reagents and kits. Because these oligonucleotides hybridize to nucleic acids encoding Her-1, sandwich and other assays can easily be constructed to exploit this fact. Hybridization of the oligonucleotides of the invention with a nucleic acid encoding an Her-1 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 systems. Kits for detecting the presence or absence of Her-1 may also be prepared. In the context of this invention, "hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleotides. 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 an oligonucleotide need not be 100% complementary to its target DNA sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vi tro assays, under conditions in which the assays are performed. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g., by inhalation or insufflation, or intrathecal or intraventricular administration. For oral administration, it has been found that oligonucleotides with at least one 2 ' -substituted ribonucleotide are particularly useful because of their absortion and distribution characteristics. U.S. Patent 5,591,721 issued to Agrawal et al . Oligonucleotides with at least one 2'- methoxyethyl modification are believed to be particularly useful for oral administration. Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Compositions for oral administration include powders or granules, suspensions or solutions in water or non- aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Compositions for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC 50 s found to be effective in in vi tro and in vivo animal models. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years. 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 : Oligonucleotide synthesis Unmodified oligodeoxynucleotides were synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine, β-cyanoethyldiisopropyl- phosphoramidites were purchased from Applied Biosystems (Foster City, CA) . For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a 0.2 M solution of 3 H-1 , 2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation cycle wait step was increased to 68 seconds and was followed by the capping step. 2 ' -methoxy oligonucleotides were synthesized using 2 ' -methoxy β-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham MA) and the standard cycle for unmodified oligonucleotides, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds. Other 2 ' -alkoxy oligonucleotides were synthesized by a modification of this method, using appropriate 2 ' - modified amidites such as those available from Glen Research, Inc., Sterling, VA. 2 ' -fluoro oligonucleotides were synthesized as described in Kawasaki et al . , J " . Med. Chem. 1993, 36, 831. Briefly, the protected nucleoside N 6 -benzoyl-2 ' -deoxy-2 ' - fluoroadenosine was synthesized utilizing commercially available 9-β-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2 ' -alpha-fluoro atom is introduced by a S N 2 -displacement of a 2 ' -β-O-trifyl group. Thus N 6 -benzoyl-9-β-D- arabinofuranosyladenine was selectively protected in moderate yield as the 3 ' , 5 ' -ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N 6 -benzoyl groups was accomplished using standard methodologies and standard methods were used to obtain the 5 ' -dimethoxytrityl- (DMT) and 5 ' -DMT-3 ' -phosphoramidite intermediates. The synthesis of 2 ' -deoxy-2 ' -fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-β-D-arabinofuranosylguanine as starting material, and conversion to the intermediate diisobutyryl- arabinofuranosylguanosine . Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected arabinofuranosylguanine . Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5 ' -DMT- and 5 ' -DMT-3 ' -phosphoramidites . Synthesis of 2 ' -deoxy-2 ' -fluorouridine was accomplished by the modification of a literature procedure in which 2, 2 ' -anhydro-1-β-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine . Standard procedures were used to obtain the 5 ' -DMT and 5 ' -DMT- 3 'phosphoramidites . 2 ' -deoxy-2 ' -fluorocytidine was synthesized via amination of 2 ' -deoxy-2 ' -fluorouridine, followed by selective protection to give N 4 -benzoyl -2 ' -deoxy-2 ' - fluorocytidine. Standard procedures were used to obtain the 5'-DMT and 5 ' -DMT-3 'phosphoramidites . 2 '- (2 -methoxyethyl) -modified amidites are synthesized according to Martin, Helv. Chim . Acta, 1995, 18 , 486. For ease of synthesis, the last nucleotide was a deoxynucleotide . 2 ' -0-CH 2 CH 2 OCH 3 -cytosines may be 5-methyl cytosines, 5-methyl-2 ' -deoxycytidine (5-me-C) containing oligonucleotides were synthesized according to published methods (Sanghvi et al . , Nuc . Acids Res . , 1993, 21 , 3197) using commercially available phosphoramidites (Glen Research, Sterling VA or ChemGenes, Needham MA) . Oligonucleotides having methylene (methylimino) backbones are synthesized according to U.S. Patent 5,378,825, which is coassigned to the assignee of the present invention and is incorporated herein in its entirety. Other nitrogen-containing backbones are synthesized according to WO 92/20823 which is also coassigned to the assignee of the present invention and incorporated herein in its entirety. Oligonucleotides having amide backbones are synthesized according to De Mesmaeker et al . , Ace. Chem. Res . , 1995, 28 , 366. The amide moiety is readily accessible by simple and well-known synthetic methods and is compatible with the conditions required for solid phase synthesis of oligonucleotides. Oligonucleotides with morpholino backbones are synthesized according to U.S. Patent 5,034,506 (Summerton and Weller) . Peptide-nucleic acid (PNA) oligomers are synthesized according to P.E. Nielsen et al . , Science 1991, 254, 1497). After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55°C for 18 hours, the oligonucleotides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by 31 P nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al . , J. Biol . Chem . , 1991, 266, 18162. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material. Example 2 : Cell culture Human SK0V3 ovarian carcinoma cells were obtained from the American Type Culture Collection. They were grown in McCoy's 5A medium containing NaHC0 3 , 10% fetal bovine serum and antibiotics and routinely passaged. A549 human lung carcinoma cells were routinely passaged in DMEM medium supplemented with 10% fetal bovine serum. Example 3 : Treatment of cells with oligonucleotide SK0V3 and A549 cells were grown in T-75 flasks until 65-75% confluent. The cells were washed once with serum- free OPTI-MEM® medium (Life Technologies, Inc., Grand Island, NY) and 5 ml of the serum-free OPTI-MEM® containing 15 μg/ml of LIPOFECTIN ® reagent (a 1:1 liposome formulation of the cationic lipid DOTMA and DOPE, Life Technologies, Inc.) was added. At that time, 300 nM of oligonucleotide was added and swirled vigorously. After a 4-5 hour incubation at 37° C, the solution was removed and fresh maintenance medium containing 10% fetal bovine serum was added. The cells were again incubated overnight at 37° in 5% C0 2 , after which the cells were assayed for Her-1 mRNA expression or cell growth. Example 4: Measurement of Her-1 mRNA by Northern blot analysis Total mRNA was extracted from the cells. For SK0V3 cells, cells were washed twice with PBS and 1-2 ml of RNAZOL B® (Tel-Test, Inc., Friendswood TX) was added. An incubation at 4° C for 5-30 minutes was done and the cells were scraped into an Eppendorf tube. This solution was frozen at -80°C for 20 minutes, thawed and chloroform (200 μl/ml) was added. The solution was centrifuged at 12,000 xg for 15 minutes at 4°C and the aqueous layer was transferred to a clean Eppendorf tube. An equal volume of isopropanol was added and incubated at room temperature for 15 minutes. Another centrifugation at 12,000 x g for 15 minutes at 4°C was done. The pellet was washed with 500 μl of 75% ethanol and centrifuged at 7500 x g for 5 minutes at 4°C. As much of the supernatant as possible was removed and the pellet was resuspended in 30 μl of double distilled water. For A549 cells, total mRNA was extracted using a guanidinium/cesium chloride extraction protocol as described by Dean et al . , J " . Biol . Chem . , 1994, 269, 16416. Ten μg of this mRNA was resolved on a 1.0% agarose gel containing 3.0% formaldehyde and transferred to a nylon membrane. The membrane was hybridized with a one-way PCR- generated human Her-1 probe radiolabeled with [alpha- 32 P] - dCTP (Dupont NEN Research Products, Boston MA) generated with the GeneAMP PCR Reagent Kit (Perkin Elmer, Foster City CA) , a T7 primer and a human Her-1 transcription template (Ambion, Austin TX) . The membrane was exposed to autoradiography film at -80°C and the mRNA bands quantitated using a densitometer (Molecular Dynamics) . Blots were stripped of radioactivity by boiling and then reprobed with a 32 P-labeled control probe which hybridized either to G3PDH (Clontech Laboratories, Inc., Palo Alto CA) or β-actin (Ambion, Austin TX) . Example 5 : Metabolic labeling and immunoprecipitation of Her-1 SKOV3 cells were treated with the indicated oligonucleotides in serum- free, methionine- free OPTI-MEM ® medium for 2.5 hours . The medium was then replaced with methionine-free OPTI-MEM® containing 35 S-methionine (100 μCi/ml) and 2% dialyzed fetal bovine serum for 10 hours. Cell extracts were prepared as described by Kumar et al . , Proc . Natl . Acad . Sci . USA, 1992, 89 , 6599. Aliquots containing equal amounts of trichloroacetic acid- precipitable counts per minute were subjected to immunoprecipitation with anti-Her-1 monoclonal antibody 528/rabbit anti-mouse IgG/protein A-Sepharose conjugate. Kumar et al . , Proc . Natl . Acad . Sci . USA, 1992, 89, 6599 and Korutla et al . , Carcinogenesis, 1995, 16, 1741. Lysates containing equal amounts of protein were also resolved on a 10% SDS-polyacrylamide gel and stained with Coomassie blue. Example 6 : Analysis of antisense effects on the growth of SKOV3 cells SK0V3 cells were treated with oligonucleotides ISIS 10563, 12876 and 12877 or the scrambled control oligonucleotide ISIS 12139 as described in Example 3, or left untreated. Cells were then either allowed to grow for 3 additional days in maintenance medium with 10% fetal bovine serum or they were treated again with oligonucleotide at 24 and 48 hours, for 4-5 hours each time. Cells were counted on a Coulter counter at 24, 48 and 72 hours. Example 7 : Synthesis of Particular Oligonucleotides The following are examples in which methods and compositions of the present invention have been used effectively. The present invention is not limited to these targets or these compositions. In accordance with the present invention, a series of oligonucleotides complementary to mRNA were designed to target different regions of the human Her-1 mRNA, using published sequences (Ullrich et al . , Nature, 1984, 309, 418; GenBank Accession Number X00588 and Kraus et al . , Proc . Na tl . Acad . Sci . USA, 1989, 86, 9193; GenBank Accession Number M29366) . Several analogs of these sequences were also designed. The oligonucleotides are shown in Table 1. Target sites are indicated by source reference (Ullrich or Kraus) and nucleotide numbers, as given in the GenBank listing, to which the oligonucleotide binds. Table 1 All backbones are phosphorothioates. Oligonucleotides for which no additional modification is indicated contain only 2 ' -deoxynucleotides . Oligonucleotides for which a modification is indicated are modified at underlined nucleosides . Example 8 : Effect Of Oligonucleotides On Her-1 mRNA Levels Some of the oligonucleotides in Table 1 were tested for their ability to inhibit Her-1 mRNA expression in A549 human lung carcinoma cells and SKOV3 human ovarian carcinoma cells, both of which overexpress the Her-1. Cultured cells were treated with 300 nM of oligonucleotide in the presence of 15 μg/ml of cationic lipid (LIPOFECTIN®) Total RNA was extracted and quantitated by Northern blot analysis, using PCR-generated Her-1 cDNA probes. Bands were quantitated on a densitometer and normalized to control probe bands (G3PDH for the A549 cells and β-actin for the SK0V3 cells) . The results are shown in Table 2 as Her-1 mRNA inhibition (10 kb band) as percent of untreated control. The scrambled control, ISIS 12139 (SEQ ID NO:10), actually gave a slight increase in Her-1 mRNA compared to untreated controls (18% increase in A549 cells, 39% in SKOV3 cells) . Table 2 ISIS # % Inhibition SEQ ID NO: A549 SKOV3 1122887788 6 666 2 222 1 10561 75% 45% 2 10563 95 61 4 12876 97 66 4 12875 77 57 4 1100556655 4 433 3 333 6 12877 93 61 6 A reduction in the amount of the 5.6 kb band was also observed after treatment with each of these oligonucleotides . Three oligonucleotides, ISIS 10563 (SEQ ID NO:4, phosphorothioate deoxyoligonucleotide) , ISIS 12876 (SEQ ID NO:4, 5-methyl C) and ISIS 12877 (SEQ ID NO: 6, 2 ' -fluoro) gave the greatest amount of reduction of Her-1 mRNA expression in both cell types. These compounds are, therefore, most preferred, and were further assessed for their effects on Her-1 protein production and cell growth. ISIS 12875 was slightly less potent but is also highly preferred. ISIS 12878 and ISIS 10561 gave greater than 50% inhibition of Her-1 mRNA expression in A549 cells, and are also preferred. Example 9 : Effect Of Oligonucleotides On Her-1 Protein Synthesis ISIS 10563, ISIS 12876 and ISIS 12877 were added to exponentially growing SKOV3 cells. Cells were metabolically labeled with 35 S-methionine and newly synthesized Her-1 protein was immunoprecipitated. A 90-98% reduction of Her-1 protein production was observed in cells treated with each of these oligonucleotides compared to untreated cells. Cells treated with scrambled control oligonucleotide (ISIS 12139, SEQ ID NO: 10) had a 10% reduction in Her-1 synthesis. This inhibition is specific to Her-1 protein production, as demonstrated by the presence of an identical number of TCA-precipitable counts per minute in oligonucleotide-treated cells and control cells (either untreated or scrambled control -treated) . Coomassie blue staining confirmed that proteins other than Her-1 were not affected by treatment of cells with antisense oligonucleotides. Example 10 : Effect Of Oligonucleotide Treatment On Growth Of SKOV3 Cells Cells were treated with oligonucleotides for 4-5 hours and allowed to grow an additional three days in maintenance medium. Cell counts at 24 hours showed a reduction in cell number of 13-24% for antisense-treated cells compared to untreated or scrambled control -treated cells. Cells treated with repeated oligonucleotide doses at 24 and 48 hours showed a sustained inhibition of cell growth of approximately 30% after 72 hours. Example 11: Oligonucleotide Synthesis - 96 Well Plate Format Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a standard 96 well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base- protected beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, CA, or Pharmacia, Piscataway, NJ) . Non-standard nucleosides are synthesized as per known literature or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites. Oligonucleotides were cleaved from support and deprotected with concentrated NH 4 0H at elevated temperature (55-60°C) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors . Example 12 : Oligonucleotide Analysis - 96 Well Plate Format The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ) ' or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length. Example 13 : Cell culture and oligonucleotide treatment- 96 Well Plate Format The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following 4 cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, Ribonuclease protection assays, or RT-PCR. T-24 cells: The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, VA) . T-24 cells were routinely cultured in complete McCoy's 5A basal media (Gibco/Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, MD) , penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, MD) . Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis. For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide. A549 cells: The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, VA) . A549 cells were routinely cultured in DMEM basal media (Gibco/Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, MD) , penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, MD) . Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. NHDF cells: Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville MD) . NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville MD) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier. HEK cells: Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville MD) . HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville MD) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier. Treatment with antisense compounds: When cells reached 80% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 200 μL 0PTI-MEM™-1 reduced- serum medium (Gibco BRL) and then treated with 130 μL of 0PTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Gibco BRL) and the desired concentration of oligonucleotide. After 4- 7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment. The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 15, a 2 ' -O-methoxyethyl gapmer (2 ' -O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to human H-ras. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 16, a 2 ' -0- methoxyethyl gapmer (2 ' -O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA is then utilized as the oligonucleotide screening- concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments . Example 14 : Analysis of oligonucleotide inhibition of Her-1 expression Antisense modulation of Her-1 expression can be assayed in a variety of ways known in the art . For example, Her-1 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR) , or real-time PCR (RT-PCR) . Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly (A) + mRNA. Methods of RNA isolation are taught in, for example, Ausubel, F.AT. et al . , Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley _ Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F.M. et al . , Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. 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 . Protein levels of Her-1 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting) , ELISA or fluorescence-activated cell sorting (FACS) . Antibodies directed to Her-1 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 in, for example, Ausubel, F.M. et al . , Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F.M. et al . , Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc. , 1997. Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F.M. et al . , Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F.M. et al . , Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1- 10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F.M. et al . , Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1- 11.2.22, John Wiley _ Sons, Inc., 1991. Example 15 : Poly (A) + mRNA isolation Poly (A) + mRNA was isolated according to Miura et al . , Clin . Chem . , 1996, 42, 1758-1764. Other methods for poly (A) + mRNA isolation are taught in, for example, Ausubel, F.M. et al . , Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris- HC1, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine CA) . Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6 , 1 mM EDTA, 0.3 M NaCl) . After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70°C was added to each well, the plate was incubated on a 90°C hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate. Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions . Example 16 : Total RNA Isolation Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia CA) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water. The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia CA) . Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out. Example 17: Real-time Quantitative PCR Analysis of Her-1 mRNA Levels Quantitation of Her-1 mRNA levels was determined 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, FAM, or VIC, 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. Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be "multiplexed" with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only ("single- plexing"), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable . Other methods of PCR are also known in the art. PCR reagents were obtained from PE-Applied Biosystems, Foster City, CA. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (lx TAQMAN™ buffer A, 5.5 mM MgCl 2 , 300 μM each of dATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 μL total RNA solution. The RT reaction was carried out by incubation for 30 minutes at 48°C. Following a 10 minute incubation at 95°C to activate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carried out: 95°C for 15 seconds (denaturation) followed by 60°C for 1.5 minutes (annealing/extension). Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, OR) . GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreen™ are taught in Jones, L.J., et al, Analytical Biochemistry, 1998, 265, 368-374. In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:2865 in lOmM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 25uL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480nm and emission at 520nm. Probes and primers to human Her-1 were designed to hybridize to a human Her-1 sequence, using published sequence information (GenBank accession number X00588, incorporated herein as SEQ ID NO: 17) . For human Her-1 the PCR primers were: forward primer: GCGTCTCTTGCCGGAATGT (SEQ ID NO: 18) reverse primer: GGCTCACCCTCCAGAAGCTT (SEQ ID NO: 19) and the PCR probe was: FAM-CCACGCATTCCCTGCCTCGGX-TAMRA (SEQ ID NO: 20) where FAM (PE-Applied Biosystems, Foster City, CA) is the fluorescent reporter dye) and TAMRA (PE- Applied Biosystems, Foster City, CA) is the quencher dye. For human GAPDH the PCR primers were : forward primer: CAACGGATTTGGTCGTATTGG (SEQ ID NO: 21) reverse primer: GGCAACAATATCCACTTTACCAGAGT (SEQ ID NO: 22) and the PCR probe was: 5' JOE-CGCCTGGTCACCAGGGCTGCT- TAMRA 3' (SEQ ID NO: 23) 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. Example 18: Northern blot analysis of Her-1 mRNA levels Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST "B" Inc., Friendswood, TX) . Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, OH) . RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST "B" Inc., Friendswood, TX) . RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, CA) and then robed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, CA) using manufacturer's recommendations for stringent conditions. To detect human Her-1, a human Her-1 specific probe was prepared by PCR using the forward primer GCGTCTCTTGCCGGAATGT (SEQ ID NO: 18) and the reverse primer GGCTCACCCTCCAGAAGCTT (SEQ ID NO: 19) . To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3- phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, CA) . Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, CA) . Data was normalized to GAPDH levels in untreated controls. Example 19: Antisense inhibition of human Her-1 expression by chimeric phosphorothioate oligonucleotides having 2 ' -MOE wings and a deoxy gap In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human Her-1 RNA, using published sequences (GenBank accession number X00588, incorporated herein as SEQ ID NO: 17, nucleotides 1-163586 of GenBank accession number AC006977, incorporated herein as SEQ ID NO: 24, GenBank accession number U48722, incorporated herein as SEQ ID NO: 25, and GenBank accession number U95089, incorporated herein as SEQ ID NO: 26) . The oligonucleotides are shown in Table 3. "Target site" indicates the first (5' -most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 3 are chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap" region consisting of ten 2 ' -deoxynucleotides, which is flanked on both sides (5' and 3' directions) by five-nucleotide "wings". The wings are composed of 2 ' -methoxyethyl (2 ' -MOE) nucleotides . The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines . The compounds were analyzed for their effect on human Her-1 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments, If present, "N.D." indicates "no data". Table 3 Inhibition of human Her-1 mRNA levels by chimeric phosphorothioate oligonucleotides having 2 ' -MOE wings and a deoxy gap As shown in Table 1, SEQ ID NOs 27, 28, 29, 30, 31, 2, 33, 36, 37, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99, 100, 101, 102, 103, 105, 106, 107, 108, 109, 110, 111, 112, 114, 116, 117, 118, 119, 120, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 135, 136, 137, 138, 140, 141, 142, 143, 144, 145, 147, 148, 149, 150, 152, 154, 155, 156, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 and 182 demonstrated at least 30% inhibition of human Her-1 expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as "active sites" and are therefore preferred sites for targeting by compounds of the present invention. Example 20: Western blot analysis of Her-1 protein levels Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well) , boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to Her-1 is used, with a radiolabelled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale CA) .

Claims: What is claimed is : 1. A compound 8 to 50 nucleobases in length targeted to a nucleic acid molecule encoding epidermal growth factor receptor and comprising at least an 8 nucleobase portion of SEQ ID NO 27, 28, 29, 30, 31, 32, 33, 36, 37, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99, 100, 101, 102, 103, 105, 106, 107, 108, 109, 110, 111, 112, 114, 116, 117, 118, 119, 120, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 135, 136, 137, 138, 140, 141, 142, 143, 144, 145, 147, 148, 149, 150, 152, 154, 155, 156, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 or 182, wherein said oligonucleotide inhibits the expression of said human epidermal growth factor receptor. 2. The compound of claim 1 which is an antisense oligonucleotide . 3. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified internucleoside linkage. 4. The compound of claim 3 wherein the modified internucleoside linkage is a phosphorothioate linkage. 5. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified sugar moiety. 6. The compound of claim 5 wherein the modified sugar moiety is a 2 ' -O-methoxyethyl sugar moiety. 7. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified nucleobase. 8. The compound of claim 7 wherein the modified nucleobase is a 5-methylcytosine . 9. The compound of claim 2 wherein the antisense oligonucleotide is a chimeric oligonucleotide. 10. A compound 8 to 50 nucleobases in length which specifically hybridizes with at least an 8-nucleobase portion of an active site on a nucleic acid molecule encoding epidermal growth factor receptor. 11. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier or diluent. 12. The composition of claim 11 further comprising a colloidal dispersion system. 13. The composition of claim 11 wherein the compound is an antisense oligonucleotide. 14. A method of inhibiting the expression of epidermal growth factor receptor in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of epidermal growth factor receptor is inhibited. 15. A method of treating an animal having a disease or condition associated with epidermal growth factor receptor comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of epidermal growth factor receptor is inhibited. 16. The method of claim 15 wherein the disease or condition is a hyperproliterative disease or condition. 17. The method of claim 16 wherein the hyperproliterative disease or condition is cancer. 18. The method of claim 17 wherein the cancer is lung cancer, ovarian cancer, colon cancer or prostate cancer. 19. The method of claim 16 wherein the hyperproliferative disease or condition is psoriasis.

Inventors: BENNETT C FRANK, FREIER SUSAN M, WYATT JACQUELINE R, BENNETT, C., FRANK, FREIER, SUSAN, M., WYATT, JACQUELINE, R.
Applicants: BENNETT C FRANK, FREIER SUSAN M, ISIS PHARMACEUTICALS INC, WYATT JACQUELINE R, BENNETT, C., FRANK, FREIER, SUSAN, M., ISIS PHARMACEUTICALS, INC., WYATT, JACQUELINE, R.
Application year: 2001
Main classification: C07H21
IPC codes: A61K38, A61K48, C12N15